U.S. patent application number 12/102998 was filed with the patent office on 2008-08-28 for expression cassettes for seed-preferential expression in plants.
This patent application is currently assigned to BASF Plant Science GmbH. Invention is credited to Elke Duwenig, Karin Herbers, Helke Hillebrand, Ulrich Keetman, Linda Patricia Loyall.
Application Number | 20080209588 12/102998 |
Document ID | / |
Family ID | 36462365 |
Filed Date | 2008-08-28 |
United States Patent
Application |
20080209588 |
Kind Code |
A1 |
Keetman; Ulrich ; et
al. |
August 28, 2008 |
Expression Cassettes For Seed-Preferential Expression In Plants
Abstract
The present invention relates to expression cassettes comprising
transcription regulating sequences with seed-preferential or
seed-specific expression profiles in plants obtainable from
Arabidopsis thaliana genes At4g12910, At1g66250, At4g00820,
At2g36640, At2g34200, At3g11180, At4g00360, At2g48030, At2g38590,
At1g23000, At3g15510, At3g50870, At4g00220, or At4g26320.
Inventors: |
Keetman; Ulrich;
(Quedlinburg, DE) ; Duwenig; Elke; (Ludwigshafen,
DE) ; Loyall; Linda Patricia; (Mannheim, DE) ;
Herbers; Karin; (Neustadt, DE) ; Hillebrand;
Helke; (Mannheim, DE) |
Correspondence
Address: |
CONNOLLY BOVE LODGE & HUTZ, LLP
P O BOX 2207
WILMINGTON
DE
19899
US
|
Assignee: |
BASF Plant Science GmbH
Ludwigshafen
DE
|
Family ID: |
36462365 |
Appl. No.: |
12/102998 |
Filed: |
April 15, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11266446 |
Nov 3, 2005 |
7375209 |
|
|
12102998 |
|
|
|
|
Current U.S.
Class: |
800/278 ;
435/252.2; 435/320.1; 435/468; 435/6.13; 536/23.6; 536/24.2;
800/298 |
Current CPC
Class: |
C12N 15/8234
20130101 |
Class at
Publication: |
800/278 ;
536/23.6; 435/320.1; 435/252.2; 435/468; 435/6; 800/298;
536/24.2 |
International
Class: |
C12N 15/29 20060101
C12N015/29; C12N 15/82 20060101 C12N015/82; C12N 1/21 20060101
C12N001/21; C12Q 1/68 20060101 C12Q001/68; A01H 5/00 20060101
A01H005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 5, 2004 |
EP |
04026295.8 |
Dec 8, 2004 |
EP |
04029024.9 |
Feb 3, 2005 |
EP |
05002262.3 |
Feb 11, 2005 |
EP |
05002850.5 |
Claims
1. An expression cassette for seed-specific or seed-preferential
transcription in plants comprising i) at least one transcription
regulating nucleotide sequence of a plant gene, wherein said plant
gene is described by the GenBank Arabidopsis thaliana genome loci
At4g00820, or a functional equivalent thereof, and functionally
linked thereto ii) at least one nucleic acid sequence which is
heterologous in relation to said transcription regulating
nucleotide sequence.
2. The expression cassette of claim 1, wherein the transcription
regulating nucleotide sequence is selected from the group of
sequences consisting of i) the sequence described by SEQ ID NO: 23,
24, 25, 26, 27, or 28, ii) a fragment of at least 50 consecutive
bases of the sequence under i) which has substantially the same
promoter activity as the corresponding transcription regulating
nucleotide sequence described by SEQ ID NO: 23, 24, 25, 26, 27, or
28, iii) a nucleotide sequence having substantial similarity with a
sequence identity of at least 40% to the transcription regulating
nucleotide sequence described by SEQ ID NO: 23, 24, 25, 26, 27, or
28, iv) a nucleotide sequence capable of hybridizing under
conditions equivalent to hybridization in 7% sodium dodecyl sulfate
(SDS), 0.5 M NaPO4, 1 mM EDTA at 50.degree. C. with washing in
2.times.SSC, 0.1% SDS at 50.degree. C. to a transcription
regulating nucleotide sequence described by SEQ ID NO: 23, 24, 25,
26, 27, or 28, or the complement thereof, v) a nucleotide sequence
capable of hybridizing under conditions equivalent to hybridization
in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at
50.degree. C. with washing in 2.times.SSC, 0.1% SDS at 50.degree.
C. to a nucleic acid comprising 50 to 200 or more consecutive
nucleotides of a transcription regulating nucleotide sequence
described by SEQ ID NO: 23, 24, 25, 26, 27, or 28, or the
complement thereof, and vi) a nucleotide sequence which is the
complement or reverse complement of any of the previously mentioned
nucleotide sequences under i) to v).
3. The expression cassette of claim 1, wherein the functional
equivalent of the transcription regulating nucleotide sequence is
obtained or obtainable from plant genomic DNA from a gene encoding
a polypeptide which has at least 70% amino acid sequence identity
to the polypeptide sequence of SEQ ID NO: 30.
4. The expression cassette of claim 1, wherein expression of the
nucleic acid sequence results in expression of a protein, or
expression of an antisense RNA, a sense RNA, or a double-stranded
RNA.
5. The expression cassette of claim 1, wherein expression of the
nucleic acid sequence confers to the plant an agronomically
valuable trait.
6. A vector comprising the expression cassette of claim 1.
7. A transgenic host cell or non-human organism comprising the
expression cassette of claim 1 or a vector comprising said
expression cassette.
8. A transgenic plant comprising the expression cassette of claim
1, or a vector comprising said expression cassette, or a transgenic
cell comprising said expression cassette or said vector.
9. A method for producing a transgenic plant cell, comprising
transforming a plant cell with the expression cassette of claim
1.
10. A method for producing a transgenic plant, comprising
transforming a plant cell with the expression cassette of claim 1,
and generating from the plant cell the transgenic plant.
11. A method for identifying and/or isolating a sequence with
seed-specific or seed-preferential transcription regulating
activity comprising utilizing i) a nucleic acid sequence encoding
the amino acid sequence as described by SEQ ID NO: 30, or a part of
at least 15 bases thereof or ii) the nucleic acid sequence of SEQ
ID NO: 29, or a part of at least 15 bases thereof.
12. The method of claim 11, wherein said identification and/or
isolation is realized by polymerase chain reaction, hybridization,
or database screening.
13. A method for providing a transgenic expression cassette for
seed-specific or seed-preferential transcription in plants
comprising: I. isolating a seed-specific or seed-preferential
transcription regulating nucleotide sequence utilizing at least one
nucleic acid sequence or a part thereof, wherein said sequence is
encoding the polypeptide described by SEQ ID NO: 30, or a part of
at least 15 bases thereof, and II. functionally linking said
seed-specific or seed-preferential transcription regulating
nucleotide sequence to another nucleotide sequence of interest,
which is heterologous in relation to said seed-specific or
seed-preferential transcription regulating nucleotide sequence.
14. An expression cassette for seed-specific or seed-preferential
transcription in plants comprising i) at least one transcription
regulating nucleotide sequence of a plant gene, and ii) at least
one nucleic acid sequence which is operably linked to and
heterologous in relation to said transcription regulating
nucleotide sequence; wherein the transcription regulating
nucleotide sequence comprises: a) at least one transcription
regulating nucleotide sequence of the plant gene encoding a
polypeptide comprising the amino acid sequence of SEQ ID NO: 30, b)
the nucleotide sequence of SEQ ID NO: 23, 24, 25, 26, 27, or 28, c)
a nucleotide sequence having at least 70% identity to the
nucleotide sequence of SEQ ID NO: 23, 24, 25, 26, 27, or 28, or d)
a fragment of SEQ ID NO: 23, 24, 25, 26, 27, or 28, wherein the
transcription regulating nucleotide sequence has seed-specific or
seed-preferential expression activity.
15. The expression cassette of claim 14, wherein expression of the
nucleic acid sequence results in expression of a protein, or
expression of an antisense RNA, a sense RNA, or a double-stranded
RNA.
16. The expression cassette of claim 14, wherein expression of the
nucleic acid sequence confers to the plant an agronomically
valuable trait.
17. A vector comprising the expression cassette of claim 14.
18. A transgenic host cell or non-human organism comprising the
expression cassette of claim 14 or a vector comprising said
expression cassette.
19. A transgenic plant comprising the expression cassette of claim
14, or a vector comprising said expression cassette, or a
transgenic cell comprising said expression cassette or said
vector.
20. A method for producing a transgenic plant or plant cell,
comprising transforming a plant cell with the expression cassette
of claim 14.
Description
RELATED APPLICATIONS
[0001] The application is a divisional of application Ser. No.
11/266,446 filed Nov. 3, 2005, which claims benefit of European
application 04026295.8 filed Nov. 5, 2004, European application
04029024.9, filed Dec. 8, 2004, European application 05002262.3,
filed Feb. 3, 2005, and European application 05002850.5, filed Feb.
11, 2005. The entire content of each above-mentioned application is
hereby incorporated by reference in entirety.
SEQUENCE LISTING SUBMISSION
[0002] The Sequence Listing associated with this application is
filed in electronic format via EFS-Web and hereby incorporated by
reference into the specification in its entirety. The name of the
text file containing the Sequence Listing is
Sequence_Listing.sub.--12810.sub.--00649. The size of the text file
is 396 KB, and the text file was created on Apr. 14, 2008.
FIELD OF THE INVENTION
[0003] The present invention relates to expression cassettes
comprising transcription regulating nucleotide sequences with
seed-preferential or seed-specific expression profiles in plants
obtainable from Arabidopsis thaliana genes At4g12910, At1g66250,
At4g00820, At2g36640, At2g34200, At3g11180, At4g00360, At2g48030,
At2g38590, At1g23000, At3g15510, At3g50870, At4g00220, or
At4g26320.
BACKGROUND OF THE INVENTION
[0004] Manipulation of plants to alter and/or improve phenotypic
characteristics (such as productivity or quality) requires the
expression of heterologous genes in plant tissues. Such genetic
manipulation relies on the availability of a means to drive and to
control gene expression as required. For example, genetic
manipulation relies on the availability and use of suitable
promoters which are effective in plants and which regulate gene
expression so as to give the desired effect(s) in the transgenic
plant.
[0005] The seed-preferential or seed-specific promoters are useful
for expressing genes as well as for producing large quantities of
protein, for expressing oils or proteins of interest, e.g.,
antibodies, genes for increasing the nutritional value of the seed
and the like. It is advantageous to have the choice of a variety of
different promoters so that the most suitable promoter may be
selected for a particular gene, construct, cell, tissue, plant or
environment. Moreover, the increasing interest in cotransforming
plants with multiple plant transcription units (PTU) and the
potential problems associated with using common regulatory
sequences for these purposes merit having a variety of promoter
sequences available.
[0006] There is, therefore, a great need in the art for the
identification of novel sequences that can be used for expression
of selected transgenes in economically important plants. It is thus
an objective of the present invention to provide new and
alternative expression cassettes for seed-preferential or
seed-specific expression of transgenes in plants. The objective is
solved by the present invention.
SUMMARY OF THE INVENTION
[0007] Accordingly, a first embodiment of the invention relates to
an expression cassette for seed-specific or seed-preferential
transcription (e.g., of an operatively linked nucleic acid
sequence) in plants comprising [0008] i) at least one transcription
regulating nucleotide sequence of a plant gene, said plant gene
selected from the group of genes described by the GenBank
Arabidopsis thaliana genome loci At4g12910, At1g66250, At4g00820,
At2g36640, At2g34200, At3g11180, At4g00360, At2g48030, At2g38590,
At1g23000, At3g15510, At3g50870, At4g00220, or At4g26320, or a
functional equivalent thereof, and functionally linked thereto
[0009] ii) at least one nucleic acid sequence which is heterologous
in relation to said transcription regulating nucleotide
sequence.
[0010] Preferably, the transcription regulating nucleotide sequence
(or the functional equivalent thereof) is selected from the group
of sequences consisting of [0011] i) the sequences described by SEQ
ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19,
20, 23, 24, 25, 26, 27, 28, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86,
87, 148, 149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161,
162, 163, 164, 165, 168, 169, 170, 171, 172, 173, 174, 175, 178,
179, 180, 181, 182, 183, 184, and 185, [0012] ii) a fragment of at
least 50 consecutive bases of a sequence under i) which has
substantially the same promoter activity as the corresponding
transcription regulating nucleotide sequence described by SEQ ID
NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20,
23, 24, 25, 26, 27, 28, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86, 87,
148, 149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161, 162,
163, 164, 165, 168, 169, 170, 171, 172, 173, 174, 175, 178, 179,
180, 181, 182, 183, 184, or 185; [0013] iii) a nucleotide sequence
having substantial similarity (e.g., with a sequence identity of at
least 40, 50, 60, to 70%, e.g., preferably 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81% to 84%, at
least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, to 98% and 99%) to a transcription regulating nucleotide
sequence described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 31, 32, 33, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55,
56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 74, 75, 76,
77, 80, 81, 84, 85, 86, 87, 148, 149, 150, 151, 152, 153, 154, 155,
158, 159, 160, 161, 162, 163, 164, 165, 168, 169, 170, 171, 172,
173, 174, 175, 178, 179, 180, 181, 182, 183, 184, or 185; [0014]
iv) a nucleotide sequence capable of hybridizing under conditions
equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5
M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in
2.times.SSC, 0.1% SDS at 50.degree. C. (more desirably in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
with washing in 1.times.SSC, 0.1% SDS at 50.degree. C., more
desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M
NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in
0.5.times.SSC, 0.1% SDS at 50.degree. C., preferably in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
with washing in 0.1.times.SSC, 0.1% SDS at 50.degree. C., more
preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1
mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at
65.degree. C.) to a transcription regulating nucleotide sequence
described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15,
16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 31, 32, 33, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77, 80,
81, 84, 85, 86, 87, 148, 149, 150, 151, 152, 153, 154, 155, 158,
159, 160, 161, 162, 163, 164, 165, 168, 169, 170, 171, 172, 173,
174, 175, 178, 179, 180, 181, 182, 183, 184, or 185, or the
complement thereof; [0015] v) a nucleotide sequence capable of
hybridizing under conditions equivalent to hybridization in 7%
sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at
50.degree. C. with washing in 2.times.SSC, 0.1% SDS at 50.degree.
C. (more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M
NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 1.times.SSC,
0.1% SDS at 50.degree. C., more desirably still in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C.,
preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1
mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at
50.degree. C., more preferably in 7% sodium dodecyl sulfate (SDS),
0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in
0.1.times.SSC, 0.1% SDS at 65.degree. C.) to a nucleic acid
comprising 50 to 200 or more consecutive nucleotides of a
transcription regulating nucleotide sequence described by SEQ ID
NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20,
23, 24, 25, 26, 27, 28, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86, 87,
148, 149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161, 162,
163, 164, 165, 168, 169, 170, 171, 172, 173, 174, 175, 178, 179,
180, 181, 182, 183, 184, and 185, or the complement thereof; [0016]
vi) a nucleotide sequence which is the complement or reverse
complement of any of the previously mentioned nucleotide sequences
under i) to v).
[0017] The functional equivalent of the transcription regulating
nucleotide sequence is obtained or obtainable from plant genomic
DNA from a gene encoding a polypeptide which has at least 70% amino
acid sequence identity to a polypeptide selected from the group
described by SEQ ID NO: 14, 22, 30, 35, 49, 59, 73, 79, 83, 89,
157, 167, 177, and 187, respectively.
[0018] The expression cassette may be employed for numerous
expression purposes such as for example expression of a protein, or
expression of a antisense RNA, sense or double-stranded RNA.
Preferably, expression of the nucleic acid sequence confers to the
plant an agronomically valuable trait.
[0019] Other embodiments of the invention relate to vectors
comprising an expression cassette of the invention, and transgenic
host cell or non-human organism comprising an expression cassette
or a vector of the invention. Preferably the organism is a
plant.
[0020] Another embodiment of the invention relates to a method for
identifying and/or isolating a sequence with seed-specific or
seed-preferential transcription regulating activity characterized
that said identification and/or isolation utilizes a nucleic acid
sequence encoding a amino acid sequence as described by SEQ ID NO:
14, 22, 30, 35, 49, 59, 73, 79, 83, 89, 157, 167, 177, or 187 or a
part of at least 15 bases thereof. Preferably the nucleic acid
sequences is described by SEQ ID NO: 13, 21, 29, 34, 48, 58, 72,
78, 82, 88, 156, 166, 176, or 186 or a part of at least 15 bases
thereof. More preferably, identification and/or isolation is
realized by a method selected from polymerase chain reaction,
hybridization, and database screening.
[0021] Another embodiment of the invention relates to a method for
providing a transgenic expression cassette for seed-specific or
seed-preferential expression comprising the steps of: [0022] I.
isolating of a seed-preferential or seed-specific transcription
regulating nucleotide sequence utilizing at least one nucleic acid
sequence or a part thereof, wherein said sequence is encoding a
polypeptide described by SEQ ID NO: 14, 22, 30, 35, 49, 59, 73, 79,
83, 89, 157, 167, 177, or 187, or a part of at least 15 bases
thereof, and [0023] II. functionally linking said seed-preferential
or seed-specific transcription regulating nucleotide sequence to
another nucleotide sequence of interest, which is heterologous in
relation to said seed-preferential or seed-specific transcription
regulating nucleotide sequence.
DEFINITIONS
[0024] It is to be understood that this invention is not limited to
the particular methodology, protocols, cell lines, plant species or
genera, constructs, and reagents described as such. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims. It must be noted that as used herein and in
the appended claims, the singular forms "a," "and," and "the"
include plural reference unless the context clearly dictates
otherwise. Thus, for example, reference to "a vector" is a
reference to one or more vectors and includes equivalents thereof
known to those skilled in the art, and so forth.
[0025] The term "about" is used herein to mean approximately,
roughly, around, or in the region of. When the term "about" is used
in conjunction with a numerical range, it modifies that range by
extending the boundaries above and below the numerical values set
forth. In general, the term "about" is used herein to modify a
numerical value above and below the stated value by a variance of
20 per-cent up or down (higher or lower).
[0026] As used herein, the word "or" means any one member of a
particular list and also includes any combination of members of
that list.
[0027] The term "gene" is used broadly to refer to any segment of
nucleic acid associated with a biological function. Thus, genes
include coding sequences and/or the regulatory sequences required
for their expression. For example, gene refers to a nucleic acid
fragment that expresses mRNA or functional RNA, or encodes a
specific protein, and which includes regulatory sequences. Genes
also include non-expressed DNA segments that, for example, form
recognition sequences for other proteins. Genes can be obtained
from a variety of sources, including cloning from a source of
interest or synthesizing from known or predicted sequence
information, and may include sequences designed to have desired
parameters.
[0028] The term "native" or "wild type" gene refers to a gene that
is present in the genome of an untransformed cell, i.e., a cell not
having a known mutation.
[0029] A "marker gene" encodes a selectable or screenable
trait.
[0030] The term "chimeric gene" refers to any gene that contains
[0031] 1) DNA sequences, including regulatory and coding sequences,
that are not found together in nature, or [0032] 2) sequences
encoding parts of proteins not naturally adjoined, or [0033] 3)
parts of promoters that are not naturally adjoined.
[0034] Accordingly, a chimeric gene may comprise regulatory
sequences and coding sequences that are derived from different
sources, or comprise regulatory sequences and coding sequences
derived from the same source, but arranged in a manner different
from that found in nature.
[0035] A "transgene" refers to a gene that has been introduced into
the genome by trans-formation and is stably maintained. Transgenes
may include, for example, genes that are either heterologous or
homologous to the genes of a particular plant to be transformed.
Additionally, transgenes may comprise native genes inserted into a
non-native organism, or chimeric genes. The term "endogenous gene"
refers to a native gene in its natural location in the genome of an
organism. A "foreign" gene refers to a gene not normally found in
the host organism but that is introduced by gene transfer.
[0036] An "oligonucleotide" corresponding to a nucleotide sequence
of the invention, e.g., for use in probing or amplification
reactions, may be about 30 or fewer nucleotides in length (e.g., 9,
12, 15, 18, 20, 21 or 24, or any number between 9 and 30).
Generally specific primers are upwards of 14 nucleotides in length.
For optimum specificity and cost effectiveness, primers of 16 to 24
nucleotides in length may be preferred. Those skilled in the art
are well versed in the design of primers for use processes such as
PCR. If required, probing can be done with entire restriction
fragments of the gene disclosed herein which may be 100's or even
1000's of nucleotides in length.
[0037] The terms "protein," "peptide" and "polypeptide" are used
interchangeably herein. The nucleotide sequences of the invention
can be introduced into any plant. The genes to be introduced can be
conveniently used in expression cassettes for introduction and
expression in any plant of interest. Such expression cassettes will
comprise the transcriptional initiation region of the invention
linked to a nucleotide sequence of interest. Preferred promoters
include constitutive, tissue-specific, developmental-specific,
inducible and/or viral promoters, most preferred are the
seed-specific or seed-preferential promoters of the invention. Such
an expression cassette is provided with a plurality of restriction
sites for insertion of the gene of interest to be under the
transcriptional regulation of the regulatory regions. The
expression cassette may additionally contain selectable marker
genes. The cassette will include in the 5'-3' direction of
transcription, a transcriptional and translational initiation
region, a DNA sequence of interest, and a transcriptional and
translational termination region functional in plants. The
termination region may be native with the transcriptional
initiation region, may be native with the DNA sequence of interest,
or may be derived from another source. Convenient termination
regions are available from the Ti-plasmid of A. tumefaciens, such,
as the octopine synthase and nopaline synthase termination regions
(see also, Guerineau 1991; Proudfoot 1991; Sanfacon 1991; Mogen
1990; Munroe 1990; Ballas 1989; Joshi 1987).
[0038] "Coding sequence" refers to a DNA or RNA sequence that codes
for a specific amino acid sequence and excludes the non-coding
sequences. It may constitute an "uninterrupted coding sequence",
i.e., lacking an intron, such as in a cDNA or it may include one or
more introns bounded by appropriate splice junctions. An "intron"
is a sequence of RNA which is contained in the primary transcript
but which is removed through cleavage and religation of the RNA
within the cell to create the mature mRNA that can be translated
into a protein.
[0039] The terms "open reading frame" and "ORF" refer to the amino
acid sequence encoded between translation initiation and
termination codons of a coding sequence. The terms "initiation
codon" and "termination codon" refer to a unit of three adjacent
nucleotides (`codon`) in a coding sequence that specifies
initiation and chain termination, respectively, of protein
synthesis (mRNA translation).
[0040] A "functional RNA" refers to an antisense RNA, ribozyme, or
other RNA that is not translated.
[0041] The term "RNA transcript" refers to the product resulting
from RNA polymerase catalyzed transcription of a DNA sequence. When
the RNA transcript is a perfect complementary copy of the DNA
sequence, it is referred to as the primary transcript or it may be
a RNA sequence derived from posttranscriptional processing of the
primary transcript and is referred to as the mature RNA. "Messenger
RNA" (mRNA) refers to the RNA that is without introns and that can
be translated into protein by the cell. "cDNA" refers to a single-
or a double-stranded DNA that is complementary to and derived from
mRNA.
[0042] "Transcription regulating nucleotide sequence", "regulatory
sequences", and "suitable regulatory sequences", each refer to
nucleotide sequences influencing the transcription, RNA processing
or stability, or translation of the associated (or functionally
linked) nucleotide sequence to be transcribed. The transcription
regulating nucleotide sequence may have various localizations with
the respect to the nucleotide sequences to be transcribed. The
transcription regulating nucleotide sequence may be located
upstream (5' non-coding sequences), within, or downstream (3'
non-coding sequences) of the sequence to be transcribed (e.g., a
coding sequence). The transcription regulating nucleotide sequences
may be selected from the group comprising enhancers, promoters,
translation leader sequences, introns, 5'-untranslated sequences,
3'-untranslated sequences, and polyadenylation signal sequences.
They include natural and synthetic sequences as well as sequences,
which may be a combination of synthetic and natural sequences. As
is noted above, the term "transcription regulating nucleotide
sequence" is not limited to promoters. However, preferably a
transcription regulating nucleotide sequence of the invention
comprises at least one promoter sequence (e.g., a sequence
localized upstream of the transcription start of a gene capable to
induce transcription of the downstream sequences). In one preferred
embodiment the transcription regulating nucleotide sequence of the
invention comprises the promoter sequence of the corresponding gene
and--optionally and preferably--the native 5'-untranslated region
of said gene. Furthermore, the 3'-untranslated region and/or the
polyadenylation region of said gene may also be employed.
[0043] "5' non-coding sequence" refers to a nucleotide sequence
located 5' (upstream) to the coding sequence. It is present in the
fully processed mRNA upstream of the initiation codon and may
affect processing of the primary transcript to mRNA, mRNA stability
or translation efficiency (Turner 1995).
[0044] "3' non-coding sequence" refers to nucleotide sequences
located 3' (downstream) to a coding sequence and include
polyadenylation signal sequences and other sequences encoding
regulatory signals capable of affecting mRNA processing or gene
expression. The polyadenylation signal is usually characterized by
affecting the addition of polyadenylic acid tracts to the 3' end of
the mRNA precursor. The use of different 3' non-coding sequences is
exemplified by Ingelbrecht et al., 1989.
[0045] The term "translation leader sequence" refers to that DNA
sequence portion of a gene between the promoter and coding sequence
that is transcribed into RNA and is present in the fully processed
mRNA upstream (5') of the translation start codon. The translation
leader sequence may affect processing of the primary transcript to
mRNA, mRNA stability or translation efficiency.
[0046] "Signal peptide" refers to the amino terminal extension of a
polypeptide, which is translated in conjunction with the
polypeptide forming a precursor peptide and which is required for
its entrance into the secretory pathway. The term "signal sequence"
refers to a nucleotide sequence that encodes the signal peptide.
The term "transit peptide" as used herein refers part of a
expressed polypeptide (preferably to the amino terminal extension
of a polypeptide), which is translated in conjunction with the
polypeptide forming a precursor peptide and which is required for
its entrance into a cell organelle (such as the plastids (e.g.,
chloroplasts) or mitochondria). The term "transit sequence" refers
to a nucleotide sequence that encodes the transit peptide.
[0047] "Promoter" refers to a nucleotide sequence, usually upstream
(5') to its coding sequence, which controls the expression of the
coding sequence by providing the recognition for RNA polymerase and
other factors required for proper transcription. "Promoter"
includes a minimal promoter that is a short DNA sequence comprised
of a TATA box and other sequences that serve to specify the site of
transcription initiation, to which regulatory elements are added
for control of expression. "Promoter" also refers to a nucleotide
sequence that includes a minimal promoter plus regulatory elements
that is capable of controlling the expression of a coding sequence
or functional RNA. This type of promoter sequence consists of
proximal and more distal upstream elements, the latter elements
often referred to as enhancers. Accordingly, an "enhancer" is a DNA
sequence which can stimulate promoter activity and may be an innate
element of the promoter or a heterologous element inserted to
enhance the level or tissue specificity of a promoter. It is
capable of operating in both orientations (normal or flipped), and
is capable of functioning even when moved either upstream or
downstream from the promoter. Both enhancers and other upstream
promoter elements bind sequence-specific DNA-binding proteins that
mediate their effects. Promoters may be derived in their entirety
from a native gene, or be composed of different elements, derived
from different promoters found in nature, or even be comprised of
synthetic DNA segments. A promoter may also contain DNA sequences
that are involved in the binding of protein factors which control
the effectiveness of transcription initiation in response to
physiological or developmental conditions.
[0048] The "initiation site" is the position surrounding the first
nucleotide that is part of the transcribed sequence, which is also
defined as position +1. With respect to this site all other
sequences of the gene and its controlling regions are numbered.
Downstream sequences (i.e., further protein encoding sequences in
the 3' direction) are denominated positive, while upstream
sequences (mostly of the controlling regions in the 5' direction)
are denominated negative.
[0049] Promoter elements, particularly a TATA element, that are
inactive or that have greatly reduced promoter activity in the
absence of upstream activation are referred to as "minimal or core
promoters." In the presence of a suitable transcription factor, the
minimal promoter functions to permit transcription. A "minimal or
core promoter" thus consists only of all basal elements needed for
transcription initiation, e.g., a TATA box and/or an initiator.
[0050] "Constitutive expression" refers to expression using a
constitutive or regulated promoter. "Conditional" and "regulated
expression" refer to expression controlled by a regulated
promoter.
[0051] "Constitutive promoter" refers to a promoter that is able to
express the open reading frame (ORF) that it controls in all or
nearly all of the plant tissues during all or nearly all
developmental stages of the plant. Each of the
transcription-activating elements do not exhibit an absolute
tissue-specificity, but mediate transcriptional activation in most
plant parts at a level of at least 1% of the level reached in the
part of the plant in which transcription is most active.
[0052] "Regulated promoter" refers to promoters that direct gene
expression not constitutively, but in a temporally- and/or
spatially-regulated manner, and includes both tissue-specific and
inducible promoters. It includes natural and synthetic sequences as
well as sequences which may be a combination of synthetic and
natural sequences. Different promoters may direct the expression of
a gene in different tissues or cell types, or at different stages
of development, or in response to different environmental
conditions. New promoters of various types useful in plant cells
are constantly being discovered, numerous examples may be found in
the compilation by Okamuro et al. (1989). Typical regulated
promoters useful in plants include but are not limited to
safener-inducible promoters, promoters derived from the
tetracycline-inducible system, promoters derived from
salicylate-inducible systems, promoters derived from
alcohol-inducible systems, promoters derived from
glucocorticoid-inducible system, promoters derived from
pathogen-inducible systems, and promoters derived from
ecdysone-inducible systems.
[0053] "Tissue-specific promoter" refers to regulated promoters
that are not expressed in all plant cells but only in one or more
cell types in specific organs (such as leaves or seeds), specific
tissues (such as embryo or cotyledon), or specific cell types (such
as leaf parenchyma or seed storage cells). These also include
promoters that are temporally regulated, such as in early or late
embryogenesis, during fruit ripening in developing seeds or fruit,
in fully differentiated leaf, or at the onset of senescence.
[0054] "Inducible promoter" refers to those regulated promoters
that can be turned on in one or more cell types by an external
stimulus, such as a chemical, light, hormone, stress, or a
pathogen.
[0055] "Operably-linked" or "functionally linked" refers preferably
to the association of nucleic acid sequences on single nucleic acid
fragment so that the function of one is affected by the other. For
example, a regulatory DNA sequence is said to be "operably linked
to" or "associated with" a DNA sequence that codes for an RNA or a
polypeptide if the two sequences are situated such that the
regulatory DNA sequence affects expression of the coding DNA
sequence (i.e., that the coding sequence or functional RNA is under
the transcriptional control of the promoter). Coding sequences can
be operably-linked to regulatory sequences in sense or antisense
orientation.
[0056] "Expression" refers to the transcription and/or translation
of an endogenous gene, ORF or portion thereof or a transgene in
plants. For example, in the case of antisense constructs,
expression may refer to the transcription of the antisense DNA
only. In addition, expression refers to the transcription and
stable accumulation of sense (mRNA) or functional RNA. Expression
may also refer to the production of protein.
[0057] "Specific expression" is the expression of gene products
which is limited to one or a few plant tissues (spatial limitation)
and/or to one or a few plant developmental stages (temporal
limitation). It is acknowledged that hardly a true specificity
exists: promoters seem to be preferably switch on in some tissues,
while in other tissues there can be no or only little activity.
This phenomenon is known as leaky expression. However, with
specific expression in this invention is meant preferable
expression in one or a few plant tissues.
[0058] The "expression pattern" of a promoter (with or without
enhancer) is the pattern of expression levels which shows where in
the plant and in what developmental stage transcription is
initiated by said promoter. Expression patterns of a set of
promoters are said to be complementary when the expression pattern
of one promoter shows little overlap with the expression pattern of
the other promoter. The level of expression of a promoter can be
determined by measuring the `steady state` concentration of a
standard transcribed reporter mRNA. This measurement is indirect
since the concentration of the reporter mRNA is dependent not only
on its synthesis rate, but also on the rate with which the mRNA is
degraded. Therefore, the steady state level is the product of
synthesis rates and degradation rates.
[0059] The rate of degradation can however be considered to proceed
at a fixed rate when the transcribed sequences are identical, and
thus this value can serve as a measure of synthesis rates. When
promoters are compared in this way techniques available to those
skilled in the art are hybridization S1-RNAse analysis, northern
blots and competitive RT-PCR. This list of techniques in no way
represents all available techniques, but rather describes commonly
used procedures used to analyze transcription activity and
expression levels of mRNA.
[0060] The analysis of transcription start points in practically
all promoters has revealed that there is usually no single base at
which transcription starts, but rather a more or less clustered set
of initiation sites, each of which accounts for some start points
of the mRNA. Since this distribution varies from promoter to
promoter the sequences of the reporter mRNA in each of the
populations would differ from each other. Since each mRNA species
is more or less prone to degradation, no single degradation rate
can be expected for different reporter mRNAs. It has been shown for
various eukaryotic promoter sequences that the sequence surrounding
the initiation site (`initiator`) plays an important role in
determining the level of RNA expression directed by that specific
promoter. This includes also part of the transcribed sequences. The
direct fusion of promoter to reporter sequences would therefore
lead to suboptimal levels of transcription.
[0061] A commonly used procedure to analyze expression patterns and
levels is through determination of the `steady state` level of
protein accumulation in a cell. Commonly used candidates for the
reporter gene, known to those skilled in the art are
beta-glucuronidase (GUS), chloramphenicol acetyl transferase (CAT)
and proteins with fluorescent properties, such as green fluorescent
protein (GFP) from Aequora victoria. In principle, however, many
more proteins are suitable for this purpose, provided the protein
does not interfere with essential plant functions. For
quantification and determination of localization a number of tools
are suited. Detection systems can readily be created or are
available which are based on, e.g., immunochemical, enzymatic,
fluorescent detection and quantification. Protein levels can be
determined in plant tissue extracts or in intact tissue using in
situ analysis of protein expression.
[0062] Generally, individual transformed lines with one chimeric
promoter reporter construct will vary in their levels of expression
of the reporter gene. Also frequently observed is the phenomenon
that such transformants do not express any detectable product (RNA
or protein). The variability in expression is commonly ascribed to
`position effects`, although the molecular mechanisms underlying
this inactivity are usually not clear.
[0063] "Overexpression" refers to the level of expression in
transgenic cells or organisms that exceeds levels of expression in
normal or untransformed (non-transgenic) cells or organisms.
[0064] "Antisense inhibition" refers to the production of antisense
RNA transcripts capable of suppressing the expression of protein
from an endogenous gene or a transgene.
[0065] "Gene silencing" refers to homology-dependent suppression of
viral genes, transgenes, or endogenous nuclear genes. Gene
silencing may be transcriptional, when the suppression is due to
decreased transcription of the affected genes, or
post-transcriptional, when the suppression is due to increased
turnover (degradation) of RNA species homologous to the affected
genes (English 1996). Gene silencing includes virus-induced gene
silencing (Ruiz et al. 1998).
[0066] The terms "heterologous DNA sequence," "exogenous DNA
segment" or "heterologous nucleic acid," as used herein, each refer
to a sequence that originates from a source foreign to the
particular host cell or, if from the same source, is modified from
its original form. Thus, a heterologous gene in a host cell
includes a gene that is endogenous to the particular host cell but
has been modified through, for example, the use of DNA shuffling.
The terms also include non-naturally occurring multiple copies of a
naturally occurring DNA sequence. Thus, the terms refer to a DNA
segment that is foreign or heterologous to the cell, or homologous
to the cell but in a position within the host cell nucleic acid in
which the element is not ordinarily found. Exogenous DNA segments
are expressed to yield exogenous polypeptides. A "homologous" DNA
sequence is a DNA sequence that is naturally associated with a host
cell into which it is introduced.
[0067] "Homologous to" in the context of nucleotide sequence
identity refers to the similarity between the nucleotide sequence
of two nucleic acid molecules or between the amino acid sequences
of two protein molecules. Estimates of such homology are provided
by either DNA-DNA or DNA-RNA hybridization under conditions of
stringency as is well understood by those skilled in the art (as
described in Haines and Higgins (eds.), Nucleic Acid Hybridization,
IRL Press, Oxford, U.K.), or by the comparison of sequence
similarity between two nucleic acids or proteins.
[0068] The term "substantially similar" refers to nucleotide and
amino acid sequences that represent functional and/or structural
equivalents of Arabidopsis sequences disclosed herein.
[0069] In its broadest sense, the term "substantially similar" when
used herein with respect to a nucleotide sequence means that the
nucleotide sequence is part of a gene which encodes a polypeptide
having substantially the same structure and function as a
polypeptide encoded by a gene for the reference nucleotide
sequence, e.g., the nucleotide sequence comprises a promoter from a
gene that is the ortholog of the gene corresponding to the
reference nucleotide sequence, as well as promoter sequences that
are structurally related the promoter sequences particularly
exemplified herein, i.e., the substantially similar promoter
sequences hybridize to the complement of the promoter sequences
exemplified herein under high or very high stringency conditions.
For example, altered nucleotide sequences which simply reflect the
degeneracy of the genetic code but nonetheless encode amino acid
sequences that are identical to a particular amino acid sequence
are substantially similar to the particular sequences. The term
"substantially similar" also includes nucleotide sequences wherein
the sequence has been modified, for example, to optimize expression
in particular cells, as well as nucleotide sequences encoding a
variant polypeptide having one or more amino acid substitutions
relative to the (unmodified) polypeptide encoded by the reference
sequence, which substitution(s) does not alter the activity of the
variant polypeptide relative to the unmodified polypeptide.
[0070] In its broadest sense, the term "substantially similar" when
used herein with respect to polypeptide means that the polypeptide
has substantially the same structure and function as the reference
polypeptide. In addition, amino acid sequences that are
substantially similar to a particular sequence are those wherein
overall amino acid identity is at least 65% or greater to the
instant sequences. Modifications that result in equivalent
nucleotide or amino acid sequences are well within the routine
skill in the art. The percentage of amino acid sequence identity
between the substantially similar and the reference polypeptide is
at least 65%, 66%, 67%, 68%, 69%, 70%, e.g., 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, and even 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, up to at least 99%, wherein the reference polypeptide is
an Arabidopsis polypeptide encoded by a gene with a promoter having
any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15,
16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 31, 32, 33, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77, 80,
81, 84, 85, 86, 87, 148, 149, 150, 151, 152, 153, 154, 155, 158,
159, 160, 161, 162, 163, 164, 165, 168, 169, 170, 171, 172, 173,
174, 175, 178, 179, 180, 181, 182, 183, 184, or 185, a nucleotide
sequence comprising an open reading frame having any one of SEQ ID
NOs; 13, 21, 29, 34, 48, 58, 72, 78, 82, 88, 156, 166, 176, or 186,
which encodes one of SEQ ID NOs: 14, 22, 30, 35, 49, 59, 73, 79,
83, 89, 157, 167, 177, or 187. One indication that two polypeptides
are substantially similar to each other, besides having
substantially the same function, is that an agent, e.g., an
antibody, which specifically binds to one of the polypeptides, also
specifically binds to the other.
[0071] Sequence comparisons maybe carried out using a
Smith-Waterman sequence alignment algorithm (see e.g., Waterman
(1995)). The locals program, version 1.16, is preferably used with
following parameters: match: 1, mismatch penalty: 0.33, open-gap
penalty: 2, extended-gap penalty: 2.
[0072] Moreover, a nucleotide sequence that is "substantially
similar" to a reference nucleotide sequence is said to be
"equivalent" to the reference nucleotide sequence. The skilled
artisan recognizes that equivalent nucleotide sequences encompassed
by this invention can also be defined by their ability to
hybridize, under low, moderate and/or stringent conditions (e.g.,
0.1.times.SSC, 0.1% SDS, 65.degree. C.), with the nucleotide
sequences that are within the literal scope of the instant
claims.
[0073] What is meant by "substantially the same activity" when used
in reference to a polynucleotide or polypeptide fragment is that
the fragment has at least 65%, 66%, 67%, 68%, 69%, 70%, e.g., 71%,
72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, and even 90% or more, e.g., 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, up to at least 99% of the activity of the
full length polynucleotide or full length polypeptide.
[0074] "Target gene" refers to a gene on the replicon that
expresses the desired target coding sequence, functional RNA, or
protein. The target gene is not essential for replicon replication.
Additionally, target genes may comprise native non-viral genes
inserted into a non-native organism, or chimeric genes, and will be
under the control of suitable regulatory sequences. Thus, the
regulatory sequences in the target gene may come from any source,
including the virus. Target genes may include coding sequences that
are either heterologous or homologous to the genes of a particular
plant to be transformed. However, target genes do not include
native viral genes. Typical target genes include, but are not
limited to genes encoding a structural protein, a seed storage
protein, a protein that conveys herbicide resistance, and a protein
that conveys insect resistance. Proteins encoded by target genes
are known as "foreign proteins". The expression of a target gene in
a plant will typically produce an altered plant trait.
[0075] The term "altered plant trait" means any phenotypic or
genotypic change in a trans-genic plant relative to the wild-type
or non-transgenic plant host.
[0076] "Replication gene" refers to a gene encoding a viral
replication protein. In addition to the ORF of the replication
protein, the replication gene may also contain other overlapping or
non-overlapping ORF(s), as are found in viral sequences in nature.
While not essential for replication, these additional ORFs may
enhance replication and/or viral DNA accumulation. Examples of such
additional ORFs are AC3 and AL3 in ACMV and TGMV geminiviruses,
respectively.
[0077] "Chimeric trans-acting replication gene" refers either to a
replication gene in which the coding sequence of a replication
protein is under the control of a regulated plant promoter other
than that in the native viral replication gene, or a modified
native viral replication gene, for example, in which a site
specific sequence(s) is inserted in the 5' transcribed but
untranslated region. Such chimeric genes also include insertion of
the known sites of replication protein binding between the promoter
and the transcription start site that attenuate transcription of
viral replication protein gene.
[0078] "Chromosomally-integrated" refers to the integration of a
foreign gene or DNA construct into the host DNA by covalent bonds.
Where genes are not "chromosomally integrated" they may be
"transiently expressed." Transient expression of a gene refers to
the expression of a gene that is not integrated into the host
chromosome but functions independently, either as part of an
autonomously replicating plasmid or expression cassette, for
example, or as part of another biological system such as a
virus.
[0079] The term "transformation" refers to the transfer of a
nucleic acid fragment into the genome of a host cell, resulting in
genetically stable inheritance. Host cells containing the
transformed nucleic acid fragments are referred to as "transgenic"
cells, and organisms comprising transgenic cells are referred to as
"transgenic organisms". Examples of methods of transformation of
plants and plant cells include Agrobacterium-mediated
transformation (De Blaere 1987) and particle bombardment technology
(U.S. Pat. No. 4,945,050). Whole plants may be regenerated from
transgenic cells by methods well known to the skilled artisan (see,
for example, Fromm 1990).
[0080] "Transformed," "transgenic," and "recombinant" refer to a
host organism such as a bacterium or a plant into which a
heterologous nucleic acid molecule has been introduced. The nucleic
acid molecule can be stably integrated into the genome generally
known in the art and are disclosed (Sambrook 1989; Innis 1995;
Gelfand 1995; Innis & Gelfand 1999. Known methods of PCR
include, but are not limited to, methods using paired primers,
nested primers, single specific primers, degenerate primers,
gene-specific primers, vector-specific primers, partially
mismatched primers, and the like. For example, "transformed,"
"transformant," and "transgenic" plants or calli have been through
the transformation process and contain a foreign gene integrated
into their chromosome. The term "untransformed" refers to normal
plants that have not been through the transformation process.
[0081] "Transiently transformed" refers to cells in which
transgenes and foreign DNA have been introduced (for example, by
such methods as Agrobacterium-mediated transformation or biolistic
bombardment), but not selected for stable maintenance.
[0082] "Stably transformed" refers to cells that have been selected
and regenerated on a selection media following transformation.
[0083] "Transient expression" refers to expression in cells in
which a virus or a transgene is introduced by viral infection or by
such methods as Agrobacterium-mediated transformation,
electroporation, or biolistic bombardment, but not selected for its
stable maintenance.
[0084] "Genetically stable" and "heritable" refer to
chromosomally-integrated genetic elements that are stably
maintained in the plant and stably inherited by progeny through
successive generations.
[0085] "Primary transformant" and "T0 generation" refer to
transgenic plants that are of the same genetic generation as the
tissue which was initially transformed (i.e., not having gone
through meiosis and fertilization since transformation).
[0086] "Secondary transformants" and the "T1, T2, T3, etc.
generations" refer to transgenic plants derived from primary
transformants through one or more meiotic and fertilization cycles.
They may be derived by self-fertilization of primary or secondary
transformants or crosses of primary or secondary transformants with
other transformed or untransformed plants.
[0087] "Wild-type" refers to a virus or organism found in nature
without any known mutation.
[0088] "Genome" refers to the complete genetic material of an
organism.
[0089] The term "nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form, composed of monomers (nucleotides) containing
a sugar, phosphate and a base which is either a purine or
pyrimidine. Unless specifically limited, the term encompasses
nucleic acids containing known analogs of natural nucleotides which
have similar binding properties as the reference nucleic acid and
are metabolized in a manner similar to naturally occurring
nucleotides. Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions) and
complementary sequences as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer 1991; Ohtsuka 1985; Rossolini
1994). A "nucleic acid fragment" is a fraction of a given nucleic
acid molecule. In higher plants, deoxyribonucleic acid (DNA) is the
genetic material while ribonucleic acid (RNA) is involved in the
transfer of information contained within DNA into proteins. The
term "nucleotide sequence" refers to a polymer of DNA or RNA which
can be single- or double-stranded, optionally containing synthetic,
non-natural or altered nucleotide bases capable of incorporation
into DNA or RNA polymers. The terms "nucleic acid" or "nucleic acid
sequence" may also be used interchangeably with gene, cDNA, DNA and
RNA encoded by a gene.
[0090] The invention encompasses isolated or substantially purified
nucleic acid or protein compositions. In the context of the present
invention, an "isolated" or "purified" DNA molecule or an
"isolated" or "purified" polypeptide is a DNA molecule or
polypeptide that, by the hand of man, exists apart from its native
environment and is therefore not a product of nature. An isolated
DNA molecule or polypeptide may exist in a purified form or may
exist in a non-native environment such as, for example, a
transgenic host cell. For example, an "isolated" or "purified"
nucleic acid molecule or protein, or biologically active portion
thereof, is substantially free of other cellular material, or
culture medium when produced by recombinant techniques, or
substantially free of chemical precursors or other chemicals when
chemically synthesized. Preferably, an "isolated" nucleic acid is
free of sequences (preferably protein encoding sequences) that
naturally flank the nucleic acid (i.e., sequences located at the 5'
and 3' ends of the nucleic acid) in the genomic DNA of the organism
from which the nucleic acid is derived. For example, in various
embodiments, the isolated nucleic acid molecule can contain less
than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of
nucleotide sequences that naturally flank the nucleic acid molecule
in genomic DNA of the cell from which the nucleic acid is derived.
A protein that is substantially free of cellular material includes
preparations of protein or polypeptide having less than about 30%,
20%, 10%, 5%, (by dry weight) of contaminating protein. When the
protein of the invention, or biologically active portion thereof,
is recombinantly produced, preferably culture medium represents
less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical
precursors or non-protein of interest chemicals.
[0091] The nucleotide sequences of the invention include both the
naturally occurring sequences as well as mutant (variant) forms.
Such variants will continue to possess the desired activity, i.e.,
either promoter activity or the activity of the product encoded by
the open reading frame of the non-variant nucleotide sequence.
[0092] The term "variant" with respect to a sequence (e.g., a
polypeptide or nucleic acid sequence such as--for example--a
transcription regulating nucleotide sequence of the invention) is
intended to mean substantially similar sequences. For nucleotide
sequences comprising an open reading frame, variants include those
sequences that, because of the degeneracy of the genetic code,
encode the identical amino acid sequence of the native protein.
Naturally occurring allelic variants such as these can be
identified with the use of well-known molecular biology techniques,
as, for example, with polymerase chain reaction (PCR) and
hybridization techniques. Variant nucleotide sequences also include
synthetically derived nucleotide sequences, such as those
generated, for example, by using site-directed mutagenesis and for
open reading frames, encode the native protein, as well as those
that encode a polypeptide having amino acid substitutions relative
to the native protein. Generally, nucleotide sequence variants of
the invention will have at least 40, 50, 60, to 70%, e.g.,
preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%,
generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and
99% nucleotide sequence identity to the native (wild type or
endogenous) nucleotide sequence.
[0093] "Conservatively modified variations" of a particular nucleic
acid sequence refers to those nucleic acid sequences that encode
identical or essentially identical amino acid sequences, or where
the nucleic acid sequence does not encode an amino acid sequence,
to essentially identical sequences. Because of the degeneracy of
the genetic code, a large number of functionally identical nucleic
acids encode any given polypeptide. For instance the codons CGT,
CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine.
Thus, at every position where an arginine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded protein. Such nucleic acid
variations are "silent variations" which are one species of
"conservatively modified variations." Every nucleic acid sequence
described herein which encodes a polypeptide also describes every
possible silent variation, except where otherwise noted. One of
skill will recognize that each codon in a nucleic acid (except ATG,
which is ordinarily the only codon for methionine) can be modified
to yield a functionally identical molecule by standard techniques.
Accordingly, each "silent variation" of a nucleic acid which
encodes a polypeptide is implicit in each described sequence.
[0094] The nucleic acid molecules of the invention can be
"optimized" for enhanced expression in plants of interest (see, for
example, WO 91/16432; Perlak 1991; Murray 1989). In this manner,
the open reading frames in genes or gene fragments can be
synthesized utilizing plant-preferred codons (see, for example,
Campbell & Gowri, 1990 for a discussion of host-preferred codon
usage). Thus, the nucleotide sequences can be optimized for
expression in any plant. It is recognized that all or any part of
the gene sequence may be optimized or synthetic. That is, synthetic
or partially optimized sequences may also be used. Variant
nucleotide sequences and proteins also encompass, sequences and
protein derived from a mutagenic and recombinogenic procedure such
as DNA shuffling. With such a procedure, one or more different
coding sequences can be manipulated to create a new polypeptide
possessing the desired properties. In this manner, libraries of
recombinant polynucleotides are generated from a population of
related sequence polynucleotides comprising sequence regions that
have substantial sequence identity and can be homologously
recombined in vitro or in vivo. Strategies for such DNA shuffling
are known in the art (see, for example, Stemmer 1994; Stemmer 1994;
Crameri 1997; Moore 1997; Zhang 1997; Crameri 1998; and U.S. Pat.
Nos. 5,605,793 and 5,837,458).
[0095] By "variant" polypeptide is intended a polypeptide derived
from the native protein by deletion (so-called truncation) or
addition of one or more amino acids to the N-terminal and/or
C-terminal end of the native protein; deletion or addition of one
or more amino acids at one or more sites in the native protein; or
substitution of one or more amino acids at one or more sites in the
native protein. Such variants may result from, for example, genetic
polymorphism or from human manipulation. Methods for such
manipulations are generally known in the art.
[0096] Thus, the polypeptides may be altered in various ways
including amino acid substitutions, deletions, truncations, and
insertions. Methods for such manipulations are generally known in
the art. For example, amino acid sequence variants of the
polypeptides can be prepared by mutations in the DNA. Methods for
mutagenesis and nucleotide sequence alterations are well known in
the art (see, for example, Kunkel 1985; Kunkel 1987; U.S. Pat. No.
4,873,192; Walker & Gaastra, 1983 and the references cited
therein). Guidance as to appropriate amino acid substitutions that
do not affect biological activity of the protein of interest may be
found in the model of Dayhoff et al. (1978). Conservative
substitutions, such as exchanging one amino acid with another
having similar properties, are preferred. Individual substitutions
deletions or additions that alter, add or delete a single amino
acid or a small percentage of amino acids (typically less than 5%,
more typically less than 1%) in an encoded sequence are
"conservatively modified variations," where the alterations result
in the substitution of an amino acid with a chemically similar
amino acid. Conservative substitution tables providing functionally
similar amino acids are well known in the art. The following five
groups each contain amino acids that are conservative substitutions
for one another: Aliphatic: Glycine (G), Alanine (A), Valine (V),
Leucine (L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine
(Y), Tryptophan (W); Sulfur-containing: Methionine (M); Cysteine
(C); Basic: Arginine (R), Lysine (K); Histidine (H); Acidic:
Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine
(Q). See also, Creighton, 1984. In addition, individual
substitutions, deletions or additions which alter, add or delete a
single amino acid or a small percentage of amino acids in an
encoded sequence are also "conservatively modified variations."
[0097] "Expression cassette" as used herein means a DNA sequence
capable of directing expression of a particular nucleotide sequence
in an appropriate host cell, comprising a promoter operably linked
to a nucleotide sequence of interest, which
is--optionally--operably linked to termination signals and/or other
regulatory elements. An expression cassette may also comprise
sequences required for proper translation of the nucleotide
sequence. The coding region usually codes for a protein of interest
but may also code for a functional RNA of interest, for example
antisense RNA or a nontranslated RNA, in the sense or antisense
direction. The expression cassette comprising the nucleotide
sequence of interest may be chimeric, meaning that at least one of
its components is heterologous with respect to at least one of its
other components. The expression cassette may also be one, which is
naturally occurring but has been obtained in a recombinant form
useful for heterologous expression. An expression cassette may be
assembled entirely extracellularly (e.g., by recombinant cloning
techniques). However, an expression cassette may also be assembled
using in part endogenous components. For example, an expression
cassette may be obtained by placing (or inserting) a promoter
sequence upstream of an endogenous sequence, which thereby becomes
functionally linked and controlled by said promoter sequences.
Likewise, a nucleic acid sequence to be expressed may be placed (or
inserted) downstream of an endogenous promoter sequence thereby
forming an expression cassette. The expression of the nucleotide
sequence in the expression cassette may be under the control of a
constitutive promoter or of an inducible promoter which initiates
transcription only when the host cell is exposed to some particular
external stimulus. In the case of a multicellular organism, the
promoter can also be specific to a particular tissue or organ or
stage of development (e.g., the seed-specific or seed-preferential
promoters of the invention).
[0098] "Vector" is defined to include, inter alia, any plasmid,
cosmid, phage or Agrobacterium binary vector in double or single
stranded linear or circular form which may or may not be self
transmissible or mobilizable, and which can transform prokaryotic
or eukaryotic host either by integration into the cellular genome
or exist extrachromosomally (e.g. autonomous replicating plasmid
with an origin of replication).
[0099] Specifically included are shuttle vectors by which is meant
a DNA vehicle capable, naturally or by design, of replication in
two different host organisms, which may be selected from
actinomycetes and related species, bacteria and eukaryotic (e.g.
higher plant, mammalian, yeast or fungal cells).
[0100] Preferably the nucleic acid in the vector is under the
control of, and operably linked to, an appropriate promoter or
other regulatory elements for transcription in a host cell such as
a microbial, e.g. bacterial, or plant cell. The vector may be a
bifunctional expression vector which functions in multiple hosts.
In the case of genomic DNA, this may contain its own promoter or
other regulatory elements and in the case of cDNA this may be under
the control of an appropriate promoter or other regulatory elements
for expression in the host cell.
[0101] "Cloning vectors" typically contain one or a small number of
restriction endonuclease recognition sites at which foreign DNA
sequences can be inserted in a determinable fashion without loss of
essential biological function of the vector, as well as a marker
gene that is suitable for use in the identification and selection
of cells transformed with the cloning vector. Marker genes
typically include genes that provide tetracycline resistance,
hygromycin resistance or ampicillin resistance.
[0102] A "transgenic plant" is a plant having one or more plant
cells that contain an expression vector.
[0103] "Plant tissue" includes differentiated and undifferentiated
tissues or plants, including but not limited to roots, stems,
shoots, leaves, pollen, seeds, tumor tissue and various forms of
cells and culture such as single cells, protoplast, embryos, and
callus tissue. The plant tissue may be in plants or in organ,
tissue or cell culture.
[0104] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides.
(a) "reference sequence", (b) "comparison window", (c) "sequence
identity", (d) "percentage of sequence identity", and (e)
"substantial identity". [0105] (a) As used herein, "reference
sequence" is a defined sequence used as a basis for sequence
comparison. A reference sequence may be a subset or the entirety of
a specified sequence; for example, as a segment of a full length
cDNA or gene sequence, or the complete cDNA or gene sequence.
[0106] (b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. Generally, the
comparison window is at least 20 contiguous nucleotides in length,
and optionally can be 30, 40, 50, 100, or longer. Those of skill in
the art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide sequence a
gap penalty is typically introduced and is subtracted from the
number of matches. [0107] Methods of alignment of sequences for
comparison are well known in the art. Thus, the determination of
percent identity between any two sequences can be accomplished
using a mathematical algorithm. Preferred, non-limiting examples of
such mathematical algorithms are the algorithm of Myers and Miller,
1988; the local homology algorithm of Smith et al. 1981; the
homology alignment algorithm of Needleman and Wunsch 1970; the
search-for-similarity-method of Pearson and Lipman 1988; the
algorithm of Karlin and Altschul, 1990, modified as in Karlin and
Altschul, 1993. [0108] Computer implementations of these
mathematical algorithms can be utilized for comparison of sequences
to determine sequence identity. Such implementations include, but
are not limited to: CLUSTAL in the PC/Gene program (available from
Intelligenetics, Mountain View, Calif.); the ALIGN program (Version
2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Version 8 (available from Genetics
Computer Group (GCG), 575 Science Drive, Madison, Wis., USA).
Alignments using these programs can be performed using the default
parameters. The CLUSTAL program is well described (Higgins 1988,
1989; Corpet 1988; Huang 1992; Pearson 1994). The ALIGN program is
based on the algorithm of Myers and Miller, supra. The BLAST
programs of Altschul et al., 1990, are based on the algorithm of
Karlin and Altschul, supra. [0109] Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information. This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul 1990). These
initial neighborhood word hits act as seeds for initiating searches
to find longer HSPs containing them. The word hits are then
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when the cumulative alignment score falls off by the
quantity X from its maximum achieved value, the cumulative score
goes to zero or below due to the accumulation of one or more
negative-scoring residue alignments, or the end of either sequence
is reached. [0110] In addition to calculating percent sequence
identity, the BLAST algorithm also performs a statistical analysis
of the similarity between two sequences (see, e.g., Karlin &
Altschul (1993). One measure of similarity provided by the BLAST
algorithm is the smallest sum probability (P(N)), which provides an
indication of the probability by which a match between two
nucleotide or amino acid sequences would occur by chance. For
example, a test nucleic acid sequence is considered similar to a
reference sequence if the smallest sum probability in a comparison
of the test nucleic acid sequence to the reference nucleic acid
sequence is less than about 0.1, more preferably less than about
0.01, and most preferably less than about 0.001. [0111] To obtain
gapped alignments for comparison purposes, Gapped BLAST (in BLAST
2.0) can be utilized as described in Altschul et al. 1997.
Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an
iterated search that detects distant relationships between
molecules. See Altschul et al., supra. When utilizing BLAST, Gapped
BLAST, PSI-BLAST, the default parameters of the respective programs
(e.g. BLASTN for nucleotide sequences, BLASTX for proteins) can be
used. The BLASTN program (for nucleotide sequences) uses as
defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff
of 100, M=5, N=-4, and a comparison of both strands. For amino acid
sequences, the BLASTP program uses as defaults a wordlength (W) of
3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff & Henikoff, 1989). Alignment may also be performed
manually by inspection. [0112] For purposes of the present
invention, comparison of nucleotide sequences for determination of
percent sequence identity to the promoter sequences disclosed
herein is preferably made using the BlastN program (version 1.4.7
or later) with its default parameters or any equivalent program. By
"equivalent program" is intended any sequence comparison program
that, for any two sequences in question, generates an alignment
having identical nucleotide or amino acid residue matches and an
identical percent sequence identity when compared to the
corresponding alignment generated by the preferred program. [0113]
(c) As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences makes
reference to the residues in the two sequences that are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity." Means for making
this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif.). [0114] (d) As
used herein, "percentage of sequence identity" means the value
determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity. [0115] (e) (i) The term "substantial identity" or
"substantial similarity" of polynucleotide sequences for a protein
encoding sequence means that a polynucleotide comprises a sequence
that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or
79%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%,
and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence
identity, compared to a reference sequence using one of the
alignment programs described using standard parameters. The term
"substantial identity" or "substantial similarity" of
polynucleotide sequences for promoter sequence means (as described
above for variants) that a polynucleotide comprises a sequence that
has at least 40, 50, 60, to 70%, e.g., preferably 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g.,
81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, to 98% and 99% nucleotide sequence
identity compared to a reference sequence using one of the
alignment programs described using standard parameters. One of
skill in the art will recognize that these values can be
appropriately adjusted to determine corresponding identity of
proteins encoded by two nucleotide sequences by taking into account
codon degeneracy, amino acid similarity, reading frame positioning,
and the like. Substantial identity of amino acid sequences for
these purposes normally means sequence identity of at least 70%,
more preferably at least 80%, 90%, and most preferably at least
95%. [0116] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each other
under stringent conditions (see below). Generally, stringent
conditions are selected to be about 5.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength and pH. However, stringent conditions
encompass temperatures in the range of about 1.degree. C. to about
20.degree. C., depending upon the desired degree of stringency as
otherwise qualified herein. Nucleic acids that do not hybridize to
each other under stringent conditions are still substantially
identical if the polypeptides they encode are substantially
identical. This may occur, e.g., when a copy of a nucleic acid is
created using the maximum codon degeneracy permitted by the genetic
code. One indication that two nucleic acid sequences are
substantially identical is when the polypeptide encoded by the
first nucleic acid is immunologically cross reactive with the
polypeptide encoded by the second nucleic acid. [0117] (ii) The
term "substantial identity" in the context of a peptide indicates
that a peptide comprises a sequence with at least 70%, 71%, 72%,
73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%,
91%, 92%, 93%, or 94%, or even more preferably, 95%, 96%, 97%, 98%
or 99%, sequence identity to the reference sequence over a
specified comparison window. Preferably, optimal alignment is
conducted using the homology alignment algorithm of Needleman and
Wunsch (1970). An indication that two peptide sequences are
substantially identical is that one peptide is immunologically
reactive with antibodies raised against the second peptide. Thus, a
peptide is substantially identical to a second peptide, for
example, where the two peptides differ only by a conservative
substitution.
[0118] For sequence comparison, typically one sequence acts as a
reference sequence to which test sequences are compared. When using
a sequence comparison algorithm, test and reference sequences are
input into a computer, subsequence coordinates are designated if
necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0119] As noted above, another indication that two nucleic acid
sequences are substantially identical is that the two molecules
hybridize to each other under stringent conditions. The phrase
"hybridizing specifically to" refers to the binding, duplexing, or
hybridizing of a molecule only to a particular nucleotide sequence
under stringent conditions when that sequence is present in a
complex mixture (e.g., total cellular) DNA or RNA. "Bind(s)
substantially" refers to complementary hybridization between a
probe nucleic acid and a target nucleic acid and embraces minor
mismatches that can be accommodated by reducing the stringency of
the hybridization media to achieve the desired detection of the
target nucleic acid sequence.
[0120] "Stringent hybridization conditions" and "stringent
hybridization wash conditions" in the context of nucleic acid
hybridization experiments such as Southern and Northern
hybridization are sequence dependent, and are different under
different environmental parameters. The T.sub.m is the temperature
(under defined ionic strength and pH) at which 50% of the target
sequence hybridizes to a perfectly matched probe. Specificity is
typically the function of post-hybridization washes, the critical
factors being the ionic strength and temperature of the final wash
solution. For DNA-DNA hybrids, the T.sub.m can be approximated from
the equation of Meinkoth and Wahl, 1984:
T.sub.m=81.5.degree. C.+16.6(log.sub.10 M)+0.41(% GC)-0.61(%
form)-500/L
where M is the molarity of monovalent cations, % GC is the
percentage of guanosine and cytosine nucleotides in the DNA, % form
is the percentage of formamide in the hybridization solution, and L
is the length of the hybrid in base pairs T.sub.m is reduced by
about 1.degree. C. for each 1% of mismatching; thus, T.sub.m,
hybridization, and/or wash conditions can be adjusted to hybridize
to sequences of the desired identity. For example, if sequences
with >90% identity are sought, the T.sub.m can be decreased
10.degree. C. Generally, stringent conditions are selected to be
about 5.degree. C. lower than the thermal melting point I for the
specific sequence and its complement at a defined ionic strength
and pH. However, severely stringent conditions can utilize a
hybridization and/or wash at 1, 2, 3, or 4.degree. C. lower than
the thermal melting point I; moderately stringent conditions can
utilize a hybridization and/or wash at 6, 7, 8, 9, or 10.degree. C.
lower than the thermal melting point I; low stringency conditions
can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or
20.degree. C. lower than the thermal melting point I. Using the
equation, hybridization and wash compositions, and desired T, those
of ordinary skill will understand that variations in the stringency
of hybridization and/or wash solutions are inherently described. If
the desired degree of mismatching results in a T of less than
45.degree. C. (aqueous solution) or 32.degree. C. (formamide
solution), it is preferred to increase the SSC concentration so
that a higher temperature can be used. An extensive guide to the
hybridization of nucleic acids is found in Tijssen, 1993.
Generally, highly stringent hybridization and wash conditions are
selected to be about 5.degree. C. lower than the thermal melting
point T.sub.m for the specific sequence at a defined ionic strength
and pH.
[0121] An example of highly stringent wash conditions is 0.15 M
NaCl at 72.degree. C. for about 15 minutes. An example of stringent
wash conditions is a 0.2.times.SSC wash at 65.degree. C. for 15
minutes (see, Sambrook, infra, for a description of SSC buffer).
Often, a high stringency wash is preceded by a low stringency wash
to remove background probe signal. An example medium stringency
wash for a duplex of, e.g., more than 100 nucleotides, is
1.times.SSC at 45.degree. C. for 15 minutes. An example low
stringency wash for a duplex of, e.g., more than 100 nucleotides,
is 4 to 6.times.SSC at 40.degree. C. for 15 minutes. For short
probes (e.g., about 10 to 50 nucleotides), stringent conditions
typically involve salt concentrations of less than about 1.5 M,
more preferably about 0.01 to 1.0 M, Na ion concentration (or other
salts) at pH 7.0 to 8.3, and the temperature is typically at least
about 30.degree. C. and at least about 60.degree. C. for long robes
(e.g., >50 nucleotides). Stringent conditions may also be
achieved with the addition of destabilizing agents such as
formamide. In general, a signal to noise ratio of 2.times. (or
higher) than that observed for an unrelated probe in the particular
hybridization assay indicates detection of a specific
hybridization. Nucleic acids that do not hybridize to each other
under stringent conditions are still substantially identical if the
proteins that they encode are substantially identical. This occurs,
e.g., when a copy of a nucleic acid is created using the maximum
codon degeneracy permitted by the genetic code.
[0122] Very stringent conditions are selected to be equal to the
T.sub.m for a particular probe. An example of stringent conditions
for hybridization of complementary nucleic acids which have more
than 100 complementary residues on a filter in a Southern or
Northern blot is 50% formamide, e.g., hybridization in 50%
formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
0.1.times.SSC at 60 to 65.degree. C. Exemplary low stringency
conditions include hybridization with a buffer solution of 30 to
35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at
37.degree. C., and a wash in 1.times. to 2.times.SSC
(20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to
55.degree. C. Exemplary moderate stringency conditions include
hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at
37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to
60.degree. C.
[0123] The following are examples of sets of hybridization/wash
conditions that may be used to clone orthologous nucleotide
sequences that are substantially identical to reference nucleotide
sequences of the present invention: a reference nucleotide sequence
preferably hybridizes to the reference nucleotide sequence in 7%
sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.47 1 mM EDTA at
50.degree. C. with washing in 2.times.SSC, 0.1% SDS at 50.degree.
C., more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M
NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 1.times.SSC,
0.1% SDS at 50.degree. C., more desirably still in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C.,
preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1
mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at
50.degree. C., more preferably in 7% sodium dodecyl sulfate (SDS),
0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in
0.1.times.SSC, 0.1% SDS at 65.degree. C.
[0124] "DNA shuffling" is a method to introduce mutations or
rearrangements, preferably randomly, in a DNA molecule or to
generate exchanges of DNA sequences between two or more DNA
molecules, preferably randomly. The DNA molecule resulting from DNA
shuffling is a shuffled DNA molecule that is a non-naturally
occurring DNA molecule derived from at least one template DNA
molecule. The shuffled DNA preferably encodes a variant polypeptide
modified with respect to the polypeptide encoded by the template
DNA, and may have an altered biological activity with respect to
the polypeptide encoded by the template DNA.
[0125] "Recombinant DNA molecule" is a combination of DNA sequences
that are joined together using recombinant DNA technology and
procedures used to join together DNA sequences as described, for
example, in Sambrook et al., 1989.
[0126] The word "plant" refers to any plant, particularly to
agronomically useful plants (e.g., seed plants), and "plant cell"
is a structural and physiological unit of the plant, which
comprises a cell wall but may also refer to a protoplast. The plant
cell may be in form of an isolated single cell or a cultured cell,
or as a part of higher organized unit such as, for example, a plant
tissue, or a plant organ differentiated into a structure that is
present at any stage of a plant's development. Such structures
include one or more plant organs including, but are not limited to,
fruit, shoot, stem, leaf, flower petal, etc. Preferably, the term
"plant" includes whole plants, shoot vegetative organs/structures
(e.g. leaves, stems and tubers), roots, flowers and floral
organs/structures (e.g. bracts, sepals, petals, stamens, carpels,
anthers and ovules), seeds (including embryo, endosperm, and seed
coat) and fruits (the mature ovary), plant tissues (e.g. vascular
tissue, ground tissue, and the like) and cells (e.g. guard cells,
egg cells, trichomes and the like), and progeny of same.
[0127] The class of plants that can be used in the method of the
invention is generally as broad as the class of higher and lower
plants amenable to transformation techniques, including angiosperms
(monocotyledonous and dicotyledonous plants), gymnosperms, ferns,
and multicellular algae. It includes plants of a variety of ploidy
levels, including aneuploid, polyploid, diploid, haploid and
hemizygous. Included within the scope of the invention are all
genera and species of higher and lower plants of the plant kingdom.
Included are furthermore the mature plants, seed, shoots and
seedlings, and parts, propagation material (for example seeds and
fruit) and cultures, for example cell cultures, derived therefrom.
Preferred are plants and plant materials of the following plant
families: Amaranthaceae, Brassicaceae, Carophyllaceae,
Chenopodiaceae, Compositae, Cucurbitaceae, Labiatae, Leguminosae,
Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae,
Saxifragaceae, Scrophulariaceae, Solanaceae, Tetragoniaceae.
[0128] Annual, perennial, monocotyledonous and dicotyledonous
plants are preferred host organisms for the generation of
transgenic plants. The use of the recombination system, or method
according to the invention is furthermore advantageous in all
ornamental plants, forestry, fruit, or ornamental trees, flowers,
cut flowers, shrubs or turf. Said plant may include--but shall not
be limited to--bryophytes such as, for example, Hepaticae
(hepaticas) and Musci (mosses); pteridophytes such as ferns,
horsetail and clubmosses; gymnosperms such as conifers, cycads,
ginkgo and Gnetaeae; algae such as Chlorophyceae, Phaeophpyceae,
Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophyceae
(diatoms) and Euglenophyceae.
[0129] Plants for the purposes of the invention may comprise the
families of the Rosaceae such as rose, Ericaceae such as
rhododendrons and azaleas, Euphorbiaceae such as poinsettias and
croton, Caryophyllaceae such as pinks, Solanaceae such as petunias,
Gesneriaceae such as African violet, Balsaminaceae such as
touch-me-not, Orchidaceae such as orchids, Iridaceae such as
gladioli, iris, freesia and crocus, Compositae such as marigold,
Geraniaceae such as geraniums, Liliaceae such as Drachaena,
Moraceae such as ficus, Araceae such as philodendron and many
others.
[0130] The transgenic plants according to the invention are
furthermore selected in particular from among dicotyledonous crop
plants such as, for example, from the families of the Leguminosae
such as pea, alfalfa and soybean; the family of the Umbelliferae,
particularly the genus Daucus (very particularly the species carota
(carrot)) and Apium (very particularly the species graveolens var.
dulce (celery)) and many others; the family of the Solanaceae,
particularly the genus Lycopersicon, very particularly the species
esculentum (tomato) and the genus Solanum, very particularly the
species tuberosum (potato) and melongena (aubergine), tobacco and
many others; and the genus Capsicum, very particularly the species
annum (pepper) and many others; the family of the Leguminosae,
particularly the genus Glycine, very particularly the species max
(soybean) and many others; and the family of the Cruciferee,
particularly the genus Brassica, very particularly the species
napus (oilseed rape), campestris (beet), oleracea cv Tastie
(cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv
Emperor (broccoli); and the genus Arabidopsis, very particularly
the species thaliana and many others; the family of the Compositae,
particularly the genus Lactuca, very particularly the species
sativa (lettuce) and many others.
[0131] The transgenic plants according to the invention may be
selected among monocotyledonous crop plants, such as, for example,
cereals such as wheat, barley, sorghum and millet, rye, triticale,
maize, rice or oats, and sugarcane. Further preferred are trees
such as apple, pear, quince, plum, cherry, peach, nectarine,
apricot, papaya, mango, and other woody species including
coniferous and deciduous trees such as poplar, pine, sequoia,
cedar, oak, etc. Especially preferred are Arabidopsis thaliana,
Nicotiana tabacum, oilseed rape, soybean, corn (maize), wheat,
Linum usitatissimum (linseed and fax), Camelina sativa, Brassica
juncea, potato and tagetes.
[0132] "Significant increase" is an increase that is larger than
the margin of error inherent in the measurement technique,
preferably an increase by about 2-fold or greater.
[0133] "Significantly less" means that the decrease is larger than
the margin of error inherent in the measurement technique,
preferably a decrease by about 2-fold or greater.
DETAILED DESCRIPTION OF THE INVENTION
[0134] The present invention thus provides for isolated nucleic
acid molecules comprising a plant nucleotide sequence that directs
seed-preferential or seed-specific transcription of an operably
linked nucleic acid fragment in a plant cell.
[0135] Specifically, the present invention provides transgenic
expression cassettes for regulating seed-preferential or
seed-specific expression (or transcription) in plants comprising
[0136] i) at least one transcription regulating nucleotide sequence
of a plant gene, said plant gene selected from the group of genes
described by the GenBank Arabidopsis thaliana genome locii
At4g12910, At1g66250, At4g00820, At2g36640, At2g34200, At3g11180,
At4g00360, At2g48030, At2g38590, At1g23000, At3g15510, At3g50870,
At4g00220, or At4g26320, or a functional equivalent thereof, and
functionally linked thereto [0137] ii) at least one nucleic acid
sequence which is heterologous in relation to said transcription
regulating nucleotide sequence.
[0138] The seed-preferential or seed-specific promoters may be
useful for expressing genes as well as for producing large
quantities of protein, for expressing oils or proteins of interest,
e.g., antibodies, genes for increasing the nutritional value of the
seed and the like.
[0139] The term "seed" in the context of the inventions means a
seed of a plant in any stage of its development i.e. starting from
the fusion of pollen and oocyte, continuing over the embryo stage
and the stage of the dormant seed, until the germinating seed,
ending with early seedling organs, as e.g. cotyledons and
hypocotyl.
[0140] "Seed-specific transcription" in the context of this
invention means the transcription of a nucleic acid sequence by a
transcription regulating element in a way that transcription of
said nucleic acid sequence in seeds contribute to more than 90%,
preferably more than 95%, more preferably more than 99% of the
entire quantity of the RNA transcribed from said nucleic acid
sequence in the entire plant during any of its developmental stage.
The transcription regulating nucleotide sequences designated
pSUK19, pSUK29, pSUK298, pSUK352 and their respective shorter and
longer variants are considered to be seed-specific transcription
regulating nucleotide sequences.
[0141] "Seed-preferential transcription" in the context of this
invention means the transcription of a nucleic acid sequence by a
transcription regulating element in a way that transcription of
said nucleic acid sequence in seeds contribute to more than 50%,
preferably more than 70%, more preferably more than 80% of the
entire quantity of the RNA transcribed from said nucleic acid
sequence in the entire plant during any of its developmental stage.
The transcription regulating nucleotide sequences designated
(pSUK222, pSUK224, pSUK226), (pSUK322, pSUK324), (pSUK250,
pSUK252), (pSUK28, pSUK30), pSUK300, (pSUK292, pSUK294, pSUK296),
pSUK164, (pSUK40, pSUK42), (pSUK48, pSUK50), (pSUK96, pSUK98),
(pSUK152, pSUK154) and their respective shorter and longer variants
are considered to be seed-preferential transcription regulating
nucleotide sequences (transcription regulating nucleotide sequences
in brackets are variants from one specific gene locus).
[0142] Preferably a transcription regulating nucleotide sequence of
the invention comprises at least one promoter sequence of the
respective gene (e.g., a sequence localized upstream of the
transcription start of the respective gene capable to induce
transcription of the downstream sequences). Said transcription
regulating nucleotide sequence may comprise the promoter sequence
of said genes but may further comprise other elements such as the
5'-untranslated sequence, enhancer, introns etc. Preferably, said
promoter sequence directs seed-preferential or seed-specific
transcription of an operably linked nucleic acid segment in a plant
or plant cell e.g., a linked plant DNA comprising an open reading
frame for a structural or regulatory gene.
[0143] The following Table 1 illustrates the genes from which the
promoters of the invention are preferably isolated, the function of
said genes, the cDNA encoded by said genes, and the protein (ORF)
encoded by said genes.
TABLE-US-00001 TABLE 1 Genes from which the promoters of the
invention are preferably isolated, putative function of said genes,
cDNA and the protein encoded by said genes. Promotor mRNA locus ID
Proteine ID Gene Locus Putative function SEQ ID cDNA SEQ ID Protein
SEQ ID At4g12910 serine carboxypeptidase SEQ ID NO: NM_117360
NP_193027.2 I precursor-like protein 1, 2, 3, 4, 5, SEQ ID NO: 13
SEQ ID NO: 14 6, 7, 8, 9, 10, 11, 12 At1g66250 glycosyl hydrolase
fam- SEQ ID NO: NM_105296 NP_176799.2 ily 17 protein 15, 16, 17,
18, SEQ ID NO: 21 SEQ ID NO: 22 19, 20 At4g00820 calmodulin-binding
pro- SEQ ID NO: NM_116308 NP_567191.2 tein-related protein 23, 24,
25, 26, SEQ ID NO: 29 SEQ ID NO: 30 27, 28 At2g36640 late
embryogenesis SEQ ID NO: NM_129219 NP_181202.1 abundant protein 31,
32, 33 SEQ ID NO: 34 SEQ ID NO: 35 (ECP63)/LEA protein At2g34200
Zinc finger (C3HC4-type SEQ ID NO: NM_128971 NP_180967.2 RING
finger) family pro- 36, 37, 38, 39, SEQ ID NO: 48 SEQ ID NO: 49
tein 40, 41, 42, 43, 44, 45, 46, 47 At3g11180 oxidoreductase, 2OG-
SEQ ID NO: NM_111954 NP_187728.1 Fe(II) oxygenase family 50, 51,
52, 53, SEQ ID NO: 58 SEQ ID NO: 59 protein 54, 55, 56, 57
At4g00360 putative cytochrome SEQ ID NO: NM_116260 NP_191946.1 P450
60, 61, 62, 63, SEQ ID NO: 72 SEQ ID NO: 73 64, 65, 66, 67, 68, 69
70, 71 At2g48030 endonuclease/exonu- SEQ ID NO: NM_130370
NP_566121.1 clease/phosphatase 74, 75, 76, 77 SEQ ID NO: 78 SEQ ID
NO: 79 family protein At2g38590 F-box family protein SEQ ID NO:
NM_129416 NP_181393.1 80, 81 SEQ ID NO: 82 SEQ ID NO: 83 At1g23000
heavy-metal-associated SEQ ID NO: NM_102148 NP_173713.1
domain-containing pro- 84, 85, 86, 87 SEQ ID NO: 88 SEQ ID NO: 89
tein At3g15510 encoding Arabidopsis SEQ ID NO: NM_112419
NP_188170.1 thaliana no apical meris- 148, 149, 150, SEQ ID NO: 156
SEQ ID NO: tem (NAM) family pro- 151, 152, 153, 157 tein (NAC2)
154, 155 At3g50870 encoding Arabidopsis SEQ ID NO: NM_114947
NP_566939.1 thaliana zinc finger 158, 159, 160, SEQ ID NO: 166 SEQ
ID NO: (GATA type) family pro- 161, 162, 163, 167 tein 164, 165
At4g00220 encoding Arabidopsis SEQ ID NO: NM_116239 NP_191933.1
thaliana LOB domain 168, 169, 170, SEQ ID NO: 176 SEQ ID NO:
protein 30/lateral organ 171, 172, 173, 177 boundaries domain pro-
174, 175 tein 30 (LBD30) At4g26320 encoding Arabidopsis SEQ ID NO:
NM_118765 NP_194362.1 thaliana arabinogalac- 178, 179, 180, SEQ ID
NO: 186 SEQ ID NO: tan-protein (AGP13) 181, 182, 183, 187 184,
185
[0144] Preferably the transcription regulating nucleotide sequence
(or the functional equivalent thereof is selected from the group of
sequences consisting of [0145] i) the sequences described by SEQ ID
NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20,
23, 24, 25, 26, 27, 28, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86, 87,
148, 149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161, 162,
163, 164, 165, 168, 169, 170, 171, 172, 173, 174, 175, 178, 179,
180, 181, 182, 183, 184, and 185, [0146] ii) a fragment of at least
50 consecutive bases of a sequence under i) which has substantially
the same promoter activity as the corresponding transcription
regulating nucleotide sequence described by 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 31,
32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52,
53, 54, 55, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
74, 75, 76, 77, 80, 81, 84, 85, 86, 87, 148, 149, 150, 151, 152,
153, 154, 155, 158, 159, 160, 161, 162, 163, 164, 165, 168, 169,
170, 171, 172, 173, 174, 175, 178, 179, 180, 181, 182, 183, 184, or
185; [0147] iii) a nucleotide sequence having substantial
similarity (e.g., with a sequence identity of at least 40, 50, 60,
to 70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to
79%, generally at least 80%, e.g., 81% to 84%, at least 85%, e.g.,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%
and 99%) to a transcription regulating nucleotide sequence
described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15,
16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 31, 32, 33, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77, 80,
81, 84, 85, 86, 87, 148, 149, 150, 151, 152, 153, 154, 155, 158,
159, 160, 161, 162, 163, 164, 165, 168, 169, 170, 171, 172, 173,
174, 175, 178, 179, 180, 181, 182, 183, 184, or 185; [0148] iv) a
nucleotide sequence capable of hybridizing under conditions
equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5
M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in
2.times.SSC, 0.1% SDS at 50.degree. C. (more desirably in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
with washing in 1.times.SSC, 0.1% SDS at 50.degree. C., more
desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M
NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in
0.5.times.SSC, 0.1% SDS at 50.degree. C., preferably in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
with washing in 0.1.times.SSC, 0.1% SDS at 50.degree. C., more
preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1
mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at
65.degree. C.) to a transcription regulating nucleotide sequence
described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15,
16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 31, 32, 33, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77, 80,
81, 84, 85, 86, 87, 148, 149, 150, 151, 152, 153, 154, 155, 158,
159, 160, 161, 162, 163, 164, 165, 168, 169, 170, 171, 172, 173,
174, 175, 178, 179, 180, 181, 182, 183, 184, or 185, or the
complement thereof; [0149] v) a nucleotide sequence capable of
hybridizing under conditions equivalent to hybridization in 7%
sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at
50.degree. C. with washing in 2.times.SSC, 0.1% SDS at 50.degree.
C. (more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M
NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 1.times.SSC,
0.1% SDS at 50.degree. C., more desirably still in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C.,
preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1
mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at
50.degree. C., more preferably in 7% sodium dodecyl sulfate (SOS),
0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in
0.1.times.SSC, 0.1% SDS at 65.degree. C.) to a nucleic acid
comprising 50 to 200 or more consecutive nucleotides (preferably at
least 100, 200, or 300, more preferably 400, 500, or 600, most
preferably at least 700, 800, or 900 consecutive nucleotides) of a
transcription regulating nucleotide sequence described by SEQ ID
NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20,
23, 24, 25, 26, 27, 28, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86, 87,
148, 149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161, 162,
163, 164, 165, 168, 169, 170, 171, 172, 173, 174, 175, 178, 179,
180, 181, 182, 183, 184, or 185, or the complement thereof; [0150]
vi) a nucleotide sequence which is the complement or reverse
complement of any of the previously mentioned nucleotide sequences
under i) to v).
[0151] A functional equivalent of the transcription regulating
nucleotide sequence can also be obtained or is obtainable from
plant genomic DNA from a gene encoding a polypeptide which is
substantially similar and preferably has at least 70%, preferably
80%, more preferably 90%, most preferably 95% amino acid sequence
identity to a polypeptide encoded by an Arabidopsis thaliana gene
comprising any one of SEQ ID NOs: 14, 22, 30, 35, 49, 59, 73, 79,
83, 89, 157, 167, 177, or 187, respectively, or a fragment of said
transcription regulating nucleotide sequence which exhibits
promoter activity in a seed-preferential or seed-specific
fashion.
[0152] The activity of a transcription regulating nucleotide
sequence is considered equivalent if transcription is initiated in
a seed-preferential or seed-specific fashion (as defined above).
Such expression profile is preferably demonstrated using reporter
genes operably linked to said transcription regulating nucleotide
sequence. Preferred reporter genes (Schenborn 1999) in this context
are green fluorescence protein (GFP) (Chui 1996; Leffel 1997),
chloramphenicol transferase, luciferase (Millar 1992),
.beta.-glucuronidase or .quadrature.-galactosidase. Especially
preferred is .beta.-glucuronidase (Jefferson 1987).
[0153] Beside this the transcription regulating activity of a
function equivalent may vary from the activity of its parent
sequence, especially with respect to expression level. The
expression level may be higher or lower than the expression level
of the parent sequence. Both derivations may be advantageous
depending on the nucleic acid sequence of interest to be expressed.
Preferred are such functional equivalent sequences, which--in
comparison with its parent sequence--does, not derivate from the
expression level of said parent sequence by more than 50%,
preferably 25%, more preferably 10% (as to be preferably judged by
either mRNA expression or protein (e.g., reporter gene)
expression). Furthermore preferred are equivalent sequences which
demonstrate an increased expression in comparison to its parent
sequence, preferably an increase my at least 50%, more preferably
by at least 100%, most preferably by at least 500%.
[0154] Preferably functional equivalent of the transcription
regulating nucleotide sequence can be obtained or is obtainable
from plant genomic DNA from a gene expressing a mRNA described by a
cDNA which is substantially similar and preferably has at least
70%, preferably 80%, more preferably 90%, most preferably 95%
sequence identity to a sequence described by any one of SEQ ID NOs:
13, 21, 29, 34, 48, 58, 72, 78, 82, 88, 156, 166, 176, or 186,
respectively, or a fragment of said transcription regulating
nucleotide sequence which exhibits promoter activity in a
seed-preferential or seed-specific fashion.
[0155] Such functional equivalent of the transcription regulating
nucleotide sequence may be obtained from other plant species by
using the seed-preferential or seed-specific Arabidopsis promoter
sequences described herein as probes to screen for homologous
structural genes in other plants by hybridization under low,
moderate or stringent hybridization conditions. Regions of the
seed-preferential or seed-specific promoter sequences of the
present invention which are conserved among species could also be
used as PCR primers to amplify a segment from a species other than
Arabidopsis, and that segment used as a hybridization probe (the
latter approach permitting higher stringency screening) or in a
transcription assay to determine promoter activity. Moreover, the
seed-preferential or seed-specific promoter sequences could be
employed to identify structurally related sequences in a database
using computer algorithms.
[0156] More specifically, based on the Arabidopsis nucleic acid
sequences of the present invention, orthologs may be identified or
isolated from the genome of any desired organism, preferably from
another plant, according to well known techniques based on their
sequence similarity to the Arabidopsis nucleic acid sequences,
e.g., hybridization, PCR or computer generated sequence
comparisons. For example, all or a portion of a particular
Arabidopsis nucleic acid sequence is used as a probe that
selectively hybridizes to other gene sequences present in a
population of cloned genomic DNA fragments or cDNA fragments (i.e.,
genomic or cDNA libraries) from a chosen source organism. Further,
suitable genomic and cDNA libraries may be prepared from any cell
or tissue of an organism. Such techniques include hybridization
screening of plated DNA libraries (either plaques or colonies; see,
e.g., Sambrook 1989) and amplification by PCR using oligonucleotide
primers preferably corresponding to sequence domains conserved
among related polypeptide or subsequences of the nucleotide
sequences provided herein (see, e.g., Innis 1990). These methods
are particularly well suited to the isolation of gene sequences
from organisms closely related to the organism from which the probe
sequence is derived. The application of these methods using the
Arabidopsis sequences as probes is well suited for the isolation of
gene sequences from any source organism, preferably other plant
species. In a PCR approach, oligonucleotide primers can be designed
for use in PCR reactions to amplify corresponding DNA sequences
from cDNA or genomic DNA extracted from any plant of interest.
Methods for designing PCR primers and PCR cloning are generally
known in the art.
[0157] In hybridization techniques, all or part of a known
nucleotide sequence is used as a probe that selectively hybridizes
to other corresponding nucleotide sequences present in a population
of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or
cDNA libraries) from a chosen organism. The hybridization probes
may be genomic DNA fragments, cDNA fragments, RNA fragments, or
other oligonucleotides, and may be labeled with a detectable group
such as .sup.32P, or any other detectable marker. Thus, for
example, probes for hybridization can be made by labeling synthetic
oligonucleotides based on the sequence of the invention. Methods
for preparation of probes for hybridization and for construction of
cDNA and genomic libraries are generally known in the art and are
disclosed in Sambrook et al. (1989). In general, sequences that
hybridize to the sequences disclosed herein will have at least 40%
to 50%, about 60% to 70% and even about 80% 85%, 90%, 95% to 98% or
more identity with the disclosed sequences. That is, the sequence
similarity of sequences may range, sharing at least about 40% to
50%, about 60% to 70%, and even about 80%, 85%, 90%, 95% to 98%
sequence similarity.
[0158] The nucleic acid molecules of the invention can also be
identified by, for example, a search of known databases for genes
encoding polypeptides having a specified amino acid sequence
identity or DNA having a specified nucleotide sequence identity.
Methods of alignment of sequences for comparison are well known in
the art and are described hereinabove.
[0159] Hence, the isolated nucleic acid molecules of the invention
include the orthologs of the Arabidopsis sequences disclosed
herein, i.e., the corresponding nucleotide sequences in organisms
other than Arabidopsis, including, but not limited to, plants other
than Arabidopsis, preferably dicotyledonous plants, e.g., Brassica
napus, alfalfa, sunflower, soybean, cotton, peanut, tobacco or
sugar beet, but also cereal plants such as corn, wheat, rye,
turfgrass, sorghum, millet, sugarcane, barley and banana. An
orthologous gene is a gene from a different species that encodes a
product having the same or similar function, e.g., catalyzing the
same reaction as a product encoded by a gene from a reference
organism. Thus, an ortholog includes polypeptides having less than,
e.g., 65% amino acid sequence identity, but which ortholog encodes
a polypeptide having the same or similar function. Databases such
GenBank may be employed to identify sequences related to the
Arabidopsis sequences, e.g., orthologs in other dicotyledonous
plants such as Brassica napus and others. Alternatively,
recombinant DNA techniques such as hybridization or PCR may be
employed to identify sequences related to the Arabidopsis sequences
or to clone the equivalent sequences from different Arabidopsis
DNAs.
[0160] The transcription regulating nucleotide sequences of the
invention or their functional equivalents can be obtained or
isolated from any plant or non-plant source, or produced
synthetically by purely chemical means. Preferred sources include,
but are not limited to the plants defined in the DEFINITION section
above.
[0161] Thus, another embodiment of the invention relates to a
method for identifying and/or isolating a sequence with
seed-preferential or seed-specific transcription regulating
activity utilizing a nucleic acid sequence encoding a amino acid
sequence as described by SEQ ID NO: 14, 22, 30, 35, 49, 59, 73, 79,
83, 89, 157, 167, 177, or 187 or a part thereof. Preferred are
nucleic acid sequences described by SEQ ID NO: 13, 21, 29, 34, 48,
58, 72, 78, 82, 88, 156, 166, 176, or 186 or parts thereof. "Part"
in this context means a nucleic acid sequence of at least 15 bases
preferably at least 25 bases, more preferably at least 50 bases.
The method can be based on (but is not limited to) the methods
described above such as polymerase chain reaction, hybridization or
database screening. Preferably, this method of the invention is
based on a polymerase chain reaction, wherein said nucleic acid
sequence or its part is utilized as oligonucleotide primer. The
person skilled in the art is aware of several methods to amplify
and isolate the promoter of a gene starting from part of its coding
sequence (such as, for example, part of a cDNA). Such methods may
include but are not limited to method such as inverse PCR ("iPCR")
or "thermal asymmetric interlaced PCR" ("TAIL PCR").
[0162] Another embodiment of the invention is related to a method
for providing a trans-genic expression cassette for
seed-preferential or seed-specific expression comprising the steps
of: [0163] I. isolating of a seed-preferential or seed-specific
transcription regulating nucleotide sequence utilizing at least one
nucleic acid sequence or a part thereof, wherein said sequence is
encoding a polypeptide described by SEQ ID NO: 14, 22, 30, 35, 49,
59, 73, 79, 83, 89, 157, 167, 177, or 187, or a part of at least 15
bases thereof, and [0164] II. functionally linking said
seed-preferential or seed-specific transcription regulating
nucleotide sequence to another nucleotide sequence of interest,
which is heterologous in relation to said seed-preferential or
seed-specific transcription regulating nucleotide sequence.
[0165] Preferably, the nucleic acid sequence employed for the
isolation comprises at least 15 base, preferably at least 25 bases,
more preferably at least 50 bases of a sequence described by SEQ ID
NO: 13, 21, 29, 34, 48, 58, 72, 78, 82, 88, 156, 166, 176, or 186.
Preferably, the isolation of the seed-preferential or seed-specific
transcription regulating nucleotide sequence is realized by a
polymerase chain reaction utilizing said nucleic acid sequence as a
primer. The operable linkage can be realized by standard cloning
method known in the art such as ligation-mediated cloning or
recombination-mediated cloning.
[0166] Preferably, the transcription regulating nucleotide
sequences and promoters of the invention include a consecutive
stretch of about 25 to 2000, including 50 to 500 or 100 to 250, and
up to 1000 or 1500, contiguous nucleotides, e.g., 40 to about 743,
60 to about 743, 125 to about 743, 250 to about 743, 400 to about
743, 600 to about 743, of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27,
28, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 50,
51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86, 87, 148, 149, 150, 151,
152, 153, 154, 155, 158, 159, 160, 161, 162, 163, 164, 165, 168,
169, 170, 171, 172, 173, 174, 175, 178, 179, 180, 181, 182, 183,
184, and 185, or the promoter orthologs thereof, which include the
minimal promoter region.
[0167] In a particular embodiment of the invention said consecutive
stretch of about 25 to 2000, including 50 to 500 or 100 to 250, and
up to 1000 or 1500, contiguous nucleotides, e.g., 40 to about 743,
60 to about 743, 125 to about 743, 250 to about 743, 400 to about
743, 600 to about 743, has at least 75%, preferably 80%, more
preferably 90% and most preferably 95%, nucleic acid sequence
identity with a corresponding consecutive stretch of about 25 to
2000, including 50 to 500 or 100 to 250, and up to 1000 or 1500,
contiguous nucleotides, e.g., 40 to about 743, 60 to about 743, 125
to about 743, 250 to about 743, 400 to about 743, 600 to about 743,
of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 31, 32, 33, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56,
57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77,
80, 81, 84, 85, 86, 87, 148, 149, 150, 151, 152, 153, 154, 155,
158, 159, 160, 161, 162, 163, 164, 165, 168, 169, 170, 171, 172,
173, 174, 175, 178, 179, 180, 181, 182, 183, 184, and 185, or the
promoter orthologs thereof, which include the minimal promoter
region. The above-defined stretch of contiguous nucleotides
preferably comprises one or more promoter motifs selected from the
group consisting of TATA box, GC-box, CAAT-box and a transcription
start site.
[0168] The transcription regulating nucleotide sequences of the
invention or their functional equivalents are capable of driving
seed-preferential or seed-specific expression of a coding sequence
in a target cell, particularly in a plant cell. The promoter
sequences and methods disclosed herein are useful in regulating
seed-preferential or seed-specific expression, respectively, of any
heterologous nucleotide sequence in a host plant in order to vary
the phenotype of that plant. These promoters can be used with
combinations of enhancer, upstream elements, and/or activating
sequences from the 5' flanking regions of plant expressible
structural genes. Similarly the upstream element can be used in
combination with various plant promoter sequences.
[0169] The transcription regulating nucleotide sequences and
promoters of the invention are useful to modify the phenotype of a
plant. Various changes in the phenotype of a trans-genic plant are
desirable, i.e., modifying the fatty acid composition in a plant,
altering the amino acid content of a plant, altering a plant's
pathogen defense mechanism, and the like. These results can be
achieved by providing expression of heterologous products or
increased expression of endogenous products in plants.
Alternatively, the results can be achieved by providing for a
reduction of expression of one or more endogenous products,
particularly enzymes or cofactors in the plant. These changes
result in an alteration in the phenotype of the transformed
plant.
[0170] Generally, the transcription regulating nucleotide sequences
and promoters of the invention may be employed to express a nucleic
acid segment that is operably linked to said promoter such as, for
example, an open reading frame, or a portion thereof, an anti-sense
sequence, a sequence encoding for a double-stranded RNA sequence,
or a transgene in plants.
[0171] An operable linkage may--for example--comprise an sequential
arrangement of the transcription regulating nucleotide sequence of
the invention (for example a sequence as described by SEQ ID NO: 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23, 24,
25, 26, 27, 28, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86, 87, 148,
149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161, 162, 163,
164, 165, 168, 169, 170, 171, 172, 173, 174, 175, 178, 179, 180,
181, 182, 183, 184, or 185) with a nucleic acid sequence to be
expressed, and--optionally--additional regulatory elements such as
for example polyadenylation or transcription termination elements,
enhancers, introns etc, in a way that the transcription regulating
nucleotide sequence can fulfill its function in the process of
expression the nucleic acid sequence of interest under the
appropriate conditions. The term "appropriate conditions" mean
preferably the presence of the expression cassette in a plant cell.
Preferred are arrangements, in which the nucleic acid sequence of
interest to be expressed is placed down-stream (i.e., in
3'-direction) of the transcription regulating nucleotide sequence
of the invention in a way, that both sequences are covalently
linked. Optionally additional sequences may be inserted in-between
the two sequences. Such sequences may be for example linker or
multiple cloning sites. Furthermore, sequences can be inserted
coding for parts of fusion proteins (in case a fusion protein of
the protein encoded by the nucleic acid of interest is intended to
be expressed). Preferably, the distance between the nucleic acid
sequence of interest to be expressed and the transcription
regulating nucleotide sequence of the invention is not more than
200 base pairs, preferably not more than 100 base pairs, more
preferably no more than 50 base pairs.
[0172] An operable linkage in relation to any expression cassette
or of the invention may be realized by various methods known in the
art, comprising both in vitro and in vivo procedure. Thus, an
expression cassette of the invention or an vector comprising such
expression cassette may by realized using standard recombination
and cloning techniques well known in the art (see e.g., Maniatis
1989; Silhavy 1984; Ausubel 1987).
[0173] An expression cassette may also be assembled by inserting a
transcription regulating nucleotide sequence of the invention (for
example a sequence as described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28,
31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51,
52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 74, 75, 76, 77, 80, 81, 84, 85, 86, 87, 148, 149, 150, 151,
152, 153, 154, 155, 158, 159, 160, 161, 162, 163, 164, 165, 168,
169, 170, 171, 172, 173, 174, 175, 178, 179, 180, 181, 182, 183,
184, or 185) into the plant genome. Such insertion will result in
an operable linkage to a nucleic acid sequence of interest which as
such already existed in the genome. By the insertion the nucleic
acid of interest is expressed in a seed-preferential or
seed-specific way due to the transcription regulating properties of
the transcription regulating nucleotide sequence. The insertion may
be directed or by chance. Preferably the insertion is directed and
realized by for example homologous recombination. By this procedure
a natural promoter may be exchanged against the transcription
regulating nucleotide sequence of the invention, thereby modifying
the expression profile of an endogenous gene. The transcription
regulating nucleotide sequence may also be inserted in a way, that
antisense mRNA of an endogenous gene is expressed, thereby inducing
gene silencing.
[0174] Similar, a nucleic acid sequence of interest to be expressed
may by inserted into a plant genome comprising the transcription
regulating nucleotide sequence in its natural genomic environment
(i.e. linked to its natural gene) in a way that the inserted
sequence becomes operably linked to the transcription regulating
nucleotide sequence, thereby forming an expression cassette of the
invention.
[0175] The open reading frame to be linked to the transcription
regulating nucleotide sequence of the invention may be obtained
from an insect resistance gene, a disease resistance gene such as,
for example, a bacterial disease resistance gene, a fungal disease
resistance gene, a viral disease resistance gene, a nematode
disease resistance gene, a herbicide resistance gene, a gene
affecting grain composition or quality, a nutrient utilization
gene, a mycotoxin reduction gene, a male sterility gene, a
selectable marker gene, a screenable marker gene, a negative
selectable marker, a positive selectable marker, a gene affecting
plant agronomic characteristics, i.e., yield, standability, and the
like, or an environment or stress resistance gene, i.e., one or
more genes that confer herbicide resistance or tolerance, insect
resistance or tolerance, disease resistance or tolerance (viral,
bacterial, fungal, oomycete, or nematode), stress tolerance or
resistance (as exemplified by resistance or tolerance to drought,
heat, chilling, freezing, excessive moisture, salt stress, or
oxidative stress), increased yields, food content and makeup,
physical appearance, male sterility, drydown, standability,
prolificacy, starch properties or quantity, oil quantity and
quality, amino acid or protein composition, and the like. By
"resistant" is meant a plant, which exhibits substantially no
phenotypic changes as a consequence of agent administration,
infection with a pathogen, or exposure to stress. By "tolerant" is
meant a plant, which, although it may exhibit some phenotypic
changes as a consequence of infection, does not have a
substantially decreased reproductive capacity or substantially
altered metabolism.
[0176] Seed-preferential or seed-specific transcription regulating
nucleotide sequences (e.g., promoters) are useful for expressing a
wide variety of genes including those which alter metabolic
pathways, confer disease resistance, for protein production, e.g.,
antibody production, or to improve nutrient uptake and the like.
Seed-preferential or seed-specific transcription regulating
nucleotide sequences (e.g., promoters) may be modified so as to be
regulatable, e.g., inducible. The genes and transcription
regulating nucleotide sequences (e.g., promoters) described
hereinabove can be used to identify orthologous genes and their
transcription regulating nucleotide sequences (e.g., promoters)
which are also likely expressed in a particular tissue and/or
development manner. Moreover, the orthologous transcription
regulating nucleotide sequences (e.g., promoters) are useful to
express linked open reading frames. In addition, by aligning the
transcription regulating nucleotide sequences (e.g., promoters) of
these orthologs, novel cis elements can be identified that are
useful to generate synthetic transcription regulating nucleotide
sequences (e.g., promoters).
[0177] The expression regulating nucleotide sequences specified
above may be optionally operably linked to other suitable
regulatory sequences, e.g., a transcription terminator sequence,
operator, repressor-binding site, transcription factor binding site
and/or an enhancer.
[0178] The present invention further provides a recombinant vector
containing the expression cassette of the invention, and host cells
comprising the expression cassette or vector, e.g., comprising a
plasmid. The expression cassette or vector may augment the genome
of a transformed plant or may be maintained extra chromosomally.
The expression cassette or vector of the invention may be present
in the nucleus, chloroplast, mitochondria and/or plastid of the
cells of the plant. Preferably, the expression cassette or vector
of the invention is comprised in the chromosomal DNA of the plant
nucleus. The present invention also provides a transgenic plant
prepared by this method, a seed from such a plant and progeny
plants from such a plant including hybrids and inbreds. The
expression cassette may be operatively linked to a structural gene,
the open reading frame thereof, or a portion thereof. The
expression cassette may further comprise a Ti plasmid and be
contained in an Agrobacterium tumefaciens cell; it may be carried
on a microparticle, wherein the microparticle is suitable for
ballistic transformation of a plant cell; or it may be contained in
a plant cell or protoplast. Further, the expression cassette or
vector can be contained in a transformed plant or cells thereof and
the plant may be a dicot or a monocot. In particular, the plant may
be a dicotyledonous plant. Preferred transgenic plants are
transgenic maize, soybean, barley, alfalfa, sunflower, canola,
soybean, cotton, peanut, sorghum, tobacco, sugarbeet, rice, wheat,
rye, turfgrass, millet, sugarcane, tomato, or potato.
[0179] The invention also provides a method of plant breeding,
e.g., to prepare a crossed fertile transgenic plant. The method
comprises crossing a fertile transgenic plant comprising a
particular expression cassette of the invention with itself or with
a second plant, e.g., one lacking the particular expression
cassette, to prepare the seed of a crossed fertile transgenic plant
comprising the particular expression cassette. The seed is then
planted to obtain a crossed fertile transgenic plant. The plant may
be a monocot or a dicot. In a particular embodiment, the plant is a
dicotyledonous plant. The crossed fertile transgenic plant may have
the particular expression cassette inherited through a female
parent or through a male parent. The second plant may be an inbred
plant. The crossed fertile transgenic may be a hybrid. Also
included within the present invention are seeds of any of these
crossed fertile transgenic plants.
[0180] The transcription regulating nucleotide sequences of the
invention further comprise sequences which are complementary to one
(hereinafter "test" sequence) which hybridizes under stringent
conditions with a nucleic acid molecule as described by SEQ ID NO:
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23,
24, 25, 26, 27, 28, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86, 87,
148, 149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161, 162,
163, 164, 165, 168, 169, 170, 171, 172, 173, 174, 175, 178, 179,
180, 181, 182, 183, 184, or 185 as well as RNA which is transcribed
from the nucleic acid molecule. When the hybridization is performed
under stringent conditions, either the test or nucleic acid
molecule of invention is preferably supported, e.g., on a membrane
or DNA chip. Thus, either a denatured test or nucleic acid molecule
of the invention is preferably first bound to a support and
hybridization is effected for a specified period of time at a
temperature of, e.g., between 55 and 70.degree. C., in double
strength citrate buffered saline (SC) containing 0.1% SDS followed
by rinsing of the support at the same temperature but with a buffer
having a reduced SC concentration. Depending upon the degree of
stringency required such reduced concentration buffers are
typically single strength SC containing 0.1% SDS, half strength SC
containing 0.1% SDS and one-tenth strength SC containing 0.1% SDS.
More preferably hybridization is carried out under high stringency
conditions (as defined above).
[0181] Virtually any DNA composition may be used for delivery to
recipient plant cells, e.g., dicotyledonous cells, to ultimately
produce fertile transgenic plants in accordance with the present
invention. For example, DNA segments or fragments in the form of
vectors and plasmids, or linear DNA segments or fragments, in some
instances containing only the DNA element to be expressed in the
plant, and the like, may be employed. The construction of vectors,
which may be employed in conjunction with the present invention,
will be known to those of skill of the art in light of the present
disclosure (see, e.g., Sambrook 1989; Gelvin 1990).
[0182] Vectors, plasmids, cosmids, YACs (yeast artificial
chromosomes), BACs (bacterial artificial chromosomes) and DNA
segments for use in transforming such cells will, of course,
generally comprise the cDNA, gene or genes which one desires to
introduce into the cells. These DNA constructs can further include
structures such as promoters, enhancers, polylinkers, or even
regulatory genes as desired. The DNA segment, fragment or gene
chosen for cellular introduction will often encode a protein which
will be expressed in the resultant recombinant cells, such as will
result in a screenable or selectable trait and/or which will impart
an improved phenotype to the regenerated plant. However, this may
not always be the case, and the present invention also encompasses
transgenic plants incorporating non-expressed transgenes.
[0183] In certain embodiments, it is contemplated that one may wish
to employ replication-competent viral vectors in monocot
transformation. Such vectors include, for example, wheat dwarf
virus (WDV) "shuttle" vectors, such as pW1-11 and PW1-GUS (Ugaki
1991). These vectors are capable of autonomous replication in maize
cells as well as E. coli, and as such may provide increased
sensitivity for detecting DNA delivered to transgenic cells. A
replicating vector may also be useful for delivery of genes flanked
by DNA sequences from transposable elements such as Ac, Ds, or Mu.
It has been proposed (Laufs 1990) that transposition of these
elements within the maize genome requires DNA replication. It is
also contemplated that transposable elements would be useful for
introducing DNA segments or fragments lacking elements necessary
for selection and maintenance of the plasmid vector in bacteria,
e.g., antibiotic resistance genes and origins of DNA replication.
It is also proposed that use of a transposable element such as Ac,
Ds, or Mu would actively promote integration of the desired DNA and
hence increase the frequency of stably transformed cells. The use
of a transposable element such as Ac, Ds, or Mu may actively
promote integration of the DNA of interest and hence increase the
frequency of stably transformed cells. Transposable elements may be
useful to allow separation of genes of interest from elements
necessary for selection and maintenance of a plasmid vector in
bacteria or selection of a transformant. By use of a transposable
element, desirable and undesirable DNA sequences may be transposed
apart from each other in the genome, such that through genetic
segregation in progeny, one may identify plants with either the
desirable undesirable DNA sequences.
[0184] The nucleotide sequence of interest linked to one or more of
the transcription regulating nucleotide sequences of the invention
can, for example, code for a ribosomal RNA, an antisense RNA or any
other type of RNA that is not translated into protein. In another
preferred embodiment of the invention, said nucleotide sequence of
interest is translated into a protein product. The transcription
regulating nucleotide sequence and/or nucleotide sequence of
interest linked thereto may be of homologous or heterologous origin
with respect to the plant to be transformed. A recombinant DNA
molecule useful for introduction into plant cells includes that
which has been derived or isolated from any source, that may be
subsequently characterized as to structure, size and/or function,
chemically altered, and later introduced into plants. An example of
a nucleotide sequence or segment of interest "derived" from a
source, would be a nucleotide sequence or segment that is
identified as a useful fragment within a given organism, and which
is then chemically synthesized in essentially pure form. An example
of such a nucleotide sequence or segment of interest "isolated"
from a source, would be nucleotide sequence or segment that is
excised or removed from said source by chemical means, e.g., by the
use of restriction endonucleases, so that it can be further
manipulated, e.g., amplified, for use in the invention, by the
methodology of genetic engineering. Such a nucleotide sequence or
segment is commonly referred to as "recombinant."
[0185] Therefore a useful nucleotide sequence, segment or fragment
of interest includes completely synthetic DNA, semi-synthetic DNA,
DNA isolated from biological sources, and DNA derived from
introduced RNA. Generally, the introduced DNA is not originally
resident in the plant genotype which is the recipient of the DNA,
but it is within the scope of the invention to isolate a gene from
a given plant genotype, and to subsequently introduce multiple
copies of the gene into the same genotype, e.g., to enhance
production of a given gene product such as a storage protein or a
protein that confers tolerance or resistance to water deficit.
[0186] The introduced recombinant DNA molecule includes but is not
limited to, DNA from plant genes, and non-plant genes such as those
from bacteria, yeasts, animals or viruses. The introduced DNA can
include modified genes, portions of genes, or chimeric genes,
including genes from the same or different genotype. The term
"chimeric gene" or "chimeric DNA" is defined as a gene or DNA
sequence or segment comprising at least two DNA sequences or
segments from species which do not combine DNA under natural
conditions, or which DNA sequences or segments are positioned or
linked in a manner which does not normally occur in the native
genome of untransformed plant.
[0187] The introduced recombinant DNA molecule used for
transformation herein may be circular or linear, double-stranded or
single-stranded. Generally, the DNA is in the form of chimeric DNA,
such as plasmid DNA, that can also contain coding regions flanked
by regulatory sequences, which promote the expression of the
recombinant DNA present in the resultant plant. Generally, the
introduced recombinant DNA molecule will be relatively small, i.e.,
less than about 30 kb to minimize any susceptibility to physical,
chemical, or enzymatic degradation which is known to increase as
the size of the nucleotide molecule increases. As noted above, the
number of proteins, RNA transcripts or mixtures thereof, which is
introduced into the plant genome, is preferably preselected and
defined, e.g., from one to about 5-10 such products of the
introduced DNA may be formed.
[0188] Two principal methods for the control of expression are
known, viz.: overexpression and underexpression. Overexpression can
be achieved by insertion of one or more than one extra copy of the
selected gene. It is, however, not unknown for plants or their
progeny, originally transformed with one or more than one extra
copy of a nucleotide sequence, to exhibit the effects of
underexpression as well as overexpression. For underexpression
there are two principle methods, which are commonly referred to in
the art as "antisense downregulation" and "sense downregulation"
(sense downregulation is also referred to as "cosuppression").
Generically these processes are referred to as "gene silencing".
Both of these methods lead to an inhibition of expression of the
target gene.
[0189] Obtaining sufficient levels of transgene expression in the
appropriate plant tissues is an important aspect in the production
of genetically engineered crops. Expression of heterologous DNA
sequences in a plant host is dependent upon the presence of an
operably linked promoter that is functional within the plant host.
Choice of the promoter sequence will determine when and where
within the organism the heterologous DNA sequence is expressed.
[0190] It is specifically contemplated by the inventors that one
could mutagenize a promoter to potentially improve the utility of
the elements for the expression of transgenes in plants. The
mutagenesis of these elements can be carried out at random and the
mutagenized promoter sequences screened for activity in a
trial-by-error procedure. Alternatively, particular sequences which
provide the promoter with desirable expression characteristics, or
the promoter with expression enhancement activity, could be
identified and these or similar sequences introduced into the
sequences via mutation. It is further contemplated that one could
mutagenize these sequences in order to enhance their expression of
transgenes in a particular species.
[0191] The means for mutagenizing a DNA segment encoding a promoter
sequence of the current invention are well known to those of skill
in the art. As indicated, modifications to promoter or other
regulatory element may be made by random, or site-specific
mutagenesis procedures. The promoter and other regulatory element
may be modified by altering their structure through the addition or
deletion of one or more nucleotides from the sequence which encodes
the corresponding unmodified sequences.
[0192] Mutagenesis may be performed in accordance with any of the
techniques known in the art, such as, and not limited to,
synthesizing an oligonucleotide having one or more mutations within
the sequence of a particular regulatory region. In particular,
site-specific mutagenesis is a technique useful in the preparation
of promoter mutants, through specific mutagenesis of the underlying
DNA. The technique further provides a ready ability to pre-pare and
test sequence variants, for example, incorporating one or more of
the foregoing considerations, by introducing one or more nucleotide
sequence changes into the DNA. Site-specific mutagenesis allows the
production of mutants through the use of specific oligonucleotide
sequences which encode the DNA sequence of the desired mutation, as
well as a sufficient number of adjacent nucleotides, to provide a
primer sequence of sufficient size and sequence complexity to form
a stable duplex on both sides of the deletion junction being
traversed. Typically, a primer of about 17 to about 75 nucleotides
or more in length is preferred, with about 10 to about 25 or more
residues on both sides of the junction of the sequence being
altered.
[0193] In general, the technique of site-specific mutagenesis is
well known in the art, as exemplified by various publications. As
will be appreciated, the technique typically employs a phage
vector, which exists in both a single stranded and double stranded
form. Typical vectors useful in site-directed mutagenesis include
vectors such as the M13 phage. These phages are readily
commercially available and their use is generally well known to
those skilled in the art. Double stranded plasmids also are
routinely employed in site directed mutagenesis, which eliminates
the step of transferring the gene of interest from a plasmid to a
phage.
[0194] In general, site-directed mutagenesis in accordance herewith
is performed by first obtaining a single-stranded vector or melting
apart of two strands of a double stranded vector which includes
within its sequence a DNA sequence which encodes the promoter. An
oligonucleotide primer bearing the desired mutated sequence is
prepared, generally synthetically. This primer is then annealed
with the single-stranded vector, and subjected to DNA polymerizing
enzymes such as E. coli polymerase I Klenow fragment, in order to
complete the synthesis of the mutation-bearing strand. Thus, a
heteroduplex is formed wherein one strand encodes the original
non-mutated sequence and the second strand bears the desired
mutation. This heteroduplex vector is then used to transform or
transfect appropriate cells, such as E. coli cells, and cells are
selected which include recombinant vectors bearing the mutated
sequence arrangement. Vector DNA can then be isolated from these
cells and used for plant transformation. A genetic selection scheme
was devised by Kunkel et al. (1987) to enrich for clones
incorporating mutagenic oligonucleotides. Alternatively, the use of
PCR with commercially available thermostable enzymes such as Taq
polymerase may be used to incorporate a mutagenic oligonucleotide
primer into an amplified DNA fragment that can then be cloned into
an appropriate cloning or expression vector. The PCR-mediated
mutagenesis procedures of Tomic et al. (1990) and Upender et al.
(1995) provide two examples of such protocols. A PCR employing a
thermostable ligase in addition to a thermostable polymerase also
may be used to incorporate a phosphorylated mutagenic
oligonucleotide into an amplified DNA fragment that may then be
cloned into an appropriate cloning or expression vector. The
mutagenesis procedure described by Michael (1994) provides an
example of one such protocol.
[0195] The preparation of sequence variants of the selected
promoter-encoding DNA segments using site-directed mutagenesis is
provided as a means of producing potentially useful species and is
not meant to be limiting, as there are other ways in which sequence
variants of DNA sequences may be obtained. For example, recombinant
vectors encoding the desired promoter sequence may be treated with
mutagenic agents, such as hydroxylamine, to obtain sequence
variants.
[0196] As used herein; the term "oligonucleotide directed
mutagenesis procedure" refers to template-dependent processes and
vector-mediated propagation which result in an increase in the
concentration of a specific nucleic acid molecule relative to its
initial concentration, or in an increase in the concentration of a
detectable signal, such as amplification. As used herein, the term
"oligonucleotide directed mutagenesis procedure" also is intended
to refer to a process that involves the template-dependent
extension of a primer molecule. The term template-dependent process
refers to nucleic acid synthesis of an RNA or a DNA molecule
wherein the sequence of the newly synthesized strand of nucleic
acid is dictated by the well-known rules of complementary base
pairing (see, for example, Watson and Rarnstad, 1987). Typically,
vector mediated methodologies involve the introduction of the
nucleic acid fragment into a DNA or RNA vector, the clonal
amplification of the vector, and the recovery of the amplified
nucleic acid fragment. Examples of such methodologies are provided
by U.S. Pat. No. 4,237,224. A number of template-dependent
processes are available to amplify the target sequences of interest
present in a sample, such methods being well known in the art and
specifically disclosed herein below.
[0197] Where a clone comprising a promoter has been isolated in
accordance with the instant invention, one may wish to delimit the
essential promoter regions within the clone. One efficient,
targeted means for preparing mutagenizing promoters relies upon the
identification of putative regulatory elements within the promoter
sequence. This can be initiated by comparison with promoter
sequences known to be expressed in similar tissue-specific or
developmentally unique manner. Sequences, which are shared among
promoters with similar expression patterns, are likely candidates
for the binding of transcription factors and are thus likely
elements that confer expression patterns. Confirmation of these
putative regulatory elements can be achieved by deletion analysis
of each putative regulatory region followed by functional analysis
of each deletion construct by assay of a reporter gene, which is
functionally attached to each construct. As such, once a starting
promoter sequence is provided, any of a number of different
deletion mutants of the starting promoter could be readily
prepared.
[0198] Functionally equivalent fragments of a transcription
regulating nucleotide sequence of the invention can also be
obtained by removing or deleting non-essential sequences without
deleting the essential one. Narrowing the transcription regulating
nucleotide sequence to its essential, transcription mediating
elements can be realized in vitro by trial-and-arrow deletion
mutations, or in silico using promoter element search routines.
Regions essential for promoter activity often demonstrate clusters
of certain, known promoter elements. Such analysis can be performed
using available computer algorithms such as PLACE ("Plant
Cis-acting Regulatory DNA Elements"; Higo 1999), the B10BASE
database "Transfac" (Biologische Datenbanken GmbH, Braunschweig;
Wingender 2001) or the database PlantCARE (Lescot 2002).
[0199] Preferably, functional equivalent fragments of one of the
transcription regulating nucleotide sequences of the invention
comprises at least 100 base pairs, preferably, at least 200 base
pairs, more preferably at least 500 base pairs of a transcription
regulating nucleotide sequence as described by SEQ ID NO: 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23, 24, 25,
26, 27, 28, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86, 87, 148, 149,
150, 151, 152, 153, 154, 155, 158, 159, 160, 161, 162, 163, 164,
165, 168, 169, 170, 171, 172, 173, 174, 175, 178, 179, 180, 181,
182, 183, 184, or 185. More preferably this fragment is starting
from the 3'-end of the indicated sequences.
[0200] Especially preferred are equivalent fragments of
transcription regulating nucleotide sequences, which are obtained
by deleting the region encoding the 5'-untranslated region of the
mRNA, thus only providing the (untranscribed) promoter region. The
5'-untranslated region can be easily determined by methods known in
the art (such as 5'-RACE analysis). Accordingly, some of the
transcription regulating nucleotide sequences of the invention are
equivalent fragments of other sequences (see Table 2 below).
TABLE-US-00002 TABLE 2 Relationship of transcription regulating
nucleotide sequences of the invention Transcription regulating
Equivalent sequence sequence Equivalent fragment SEQ ID NO: 9 (3954
bp) SEQ ID NO: 10 SEQ ID NO: 1 (980 bp) (3962 bp) SEQ ID NO: 2 (994
bp) SEQ ID NO: 3 (686 bp) SEQ ID NO: 4 (698 bp) SEQ ID NO: 5 (2051
bp) SEQ ID NO: 6 (2066 bp) SEQ ID NO: 7 (1757 bp) SEQ ID NO: 8
(1770 bp) SEQ ID NO: 11 (3660 bp) SEQ ID NO: 12 (3666 bp) SEQ ID
NO: 19 SEQ ID NO: 20 SEQ ID NO: 15 (2254 bp) (3892 bp) (3083 bp)
SEQ ID NO: 16 (2259 bp) SEQ ID NO: 17 (1446 bp) SEQ ID NO: 18 (1450
bp) SEQ ID NO: 27 SEQ ID NO: 28 SEQ ID NO: 23 (1026 bp) (2352 bp)
(2168 bp) SEQ ID NO: 24 (1030 bp) SEQ ID NO: 25 (844 bp) SEQ ID NO:
26 (846 bp) SEQ ID NO: 33 -- SEQ ID NO: 31 (1201 bp) (1441 bp) SEQ
ID NO: 32 (1216 bp) SEQ ID NO: 36 SEQ ID NO: 37 SEQ ID NO: 38 (1130
bp) (1587 bp) (1602 bp) SEQ ID NO: 39 (1145 bp) SEQ ID NO: 40 (1169
bp) SEQ ID NO: 41 (1191 bp) SEQ ID NO: 42 (1130 bp) SEQ ID NO: 43
(1145 bp) SEQ ID NO: 44 (1120 bp) SEQ ID NO: 45 (1135 bp) SEQ ID
NO: 46 (663 bp) SEQ ID NO: 47 (678 bp) SEQ ID NO: 54 SEQ ID NO: 55
SEQ ID NO: 50 (1069 bp) (2012 bp) (2033 bp) SEQ ID NO: 51 (1088 bp)
SEQ ID NO: 52 (1034 bp) SEQ ID NO: 53 (1053 bp) SEQ ID NO: 56 (1977
bp) SEQ ID NO: 57 (1998 bp) SEQ ID NO: 68 SEQ ID NO: 69 SEQ ID NO:
60 (949 bp) (2996 bp) (3008 bp) SEQ ID NO: 61 (962 bp) SEQ ID NO:
62 (768 bp) SEQ ID NO: 63 (779 bp) SEQ ID NO: 64 (2040 bp) SEQ ID
NO: 65 (2053 bp) SEQ ID NO: 66 (1859 bp) SEQ ID NO: 67 (1870 bp)
SEQ ID NO: 70 (2815 bp) SEQ ID NO: 71 (2825 bp) SEQ ID NO: 74 SEQ
ID NO: 75 SEQ ID NO: 76 (1404 bp) (1564 bp) (1578 bp) SEQ ID NO: 77
(1418 bp) SEQ ID NO: 80 SEQ ID NO: 81 -- (524 bp) (539 bp) SEQ ID
NO: 84 SEQ ID NO: 85 SEQ ID NO: 86 (1776 bp) (1988 bp) (2010 bp)
SEQ ID NO: 87 (1800 bp) SEQ ID NO: 152 SEQ ID NO: 153 SEQ ID NO:
148 (1106 bp) (1845 bp) (1854 bp) SEQ ID NO: 149 (1115 bp) SEQ ID
NO: 150 (923 bp) SEQ ID NO: 151 (930 bp) SEQ ID NO: 154 (1662 bp)
SEQ ID NO: 155 (1669 bp) SEQ ID NO: 162 SEQ ID NO: 163 SEQ ID NO:
158 (1017 bp) (2017 bp) (2004 bp) SEQ ID NO: 159 (1008 bp) SEQ ID
NO: 160 (894 bp) SEQ ID NO: 161 (886 bp) SEQ ID NO: 164 (1894 bp)
SEQ ID NO: 165 (1882 bp) SEQ ID NO: 172 SEQ ID NO: 173 SEQ ID NO:
168 (1300 bp) (3232 bp) (3269 bp) SEQ ID NO: 169 (1337 bp) SEQ ID
NO: 170 (1190 bp) SEQ ID NO: 171 (1226 bp) SEQ ID NO: 174 (3123 bp)
SEQ ID NO: 175 (3158 bp) SEQ ID NO: 182 SEQ ID NO: 183 SEQ ID NO:
178 (1504 bp) (3019 bp) (3006 bp) SEQ ID NO: 179 (1492 bp) SEQ ID
NO: 180 (1450 bp) SEQ ID NO: 181 (1436 bp) SEQ ID NO: 184 (2965 bp)
SEQ ID NO: 185 (2950 bp)
[0201] As indicated above, deletion mutants, deletion mutants of
the promoter of the invention also could be randomly prepared and
then assayed. With this strategy, a series of constructs are
prepared, each containing a different portion of the clone (a
subclone), and these constructs are then screened for activity. A
suitable means for screening for activity is to attach a deleted
promoter or intron construct, which contains a deleted segment to a
selectable or screenable marker, and to isolate only those cells
expressing the marker gene. In this way, a number of different,
deleted promoter constructs are identified which still retain the
desired, or even enhanced, activity. The smallest segment, which is
required for activity, is thereby identified through comparison of
the selected constructs. This segment may then be used for the
construction of vectors for the expression of exogenous genes.
[0202] An expression cassette of the invention may comprise further
regulatory elements. The term in this context is to be understood
in the a broad meaning comprising all sequences which may influence
construction or function of the expression cassette. Regulatory
elements may for example modify transcription and/or translation in
prokaryotic or eukaryotic organism. In an preferred embodiment the
expression cassette of the invention comprised downstream (in
3'-direction) of the nucleic acid sequence to be expressed a
transcription termination sequence and--optionally additional
regulatory elements--each operably liked to the nucleic acid
sequence to be expressed (or the transcription regulating
nucleotide sequence).
[0203] Additional regulatory elements may comprise additional
promoter, minimal promoters, or promoter elements, which may modify
the expression regulating properties. For example the expression
may be made depending on certain stress factors such water stress,
abscisin (Lam 1991) or heat stress (Schoffl 1989). Furthermore
additional promoters or promoter elements may be employed, which
may realize expression in other organisms (such as E. coli or
Agrobacterium). Such regulatory elements can be found in the
promoter sequences or bacteria such as amy and SPO2 or in the
promoter sequences of yeast or fungal promoters (such as ADC1, MFa,
AC, P-60, CYC1, GAPDH, TEF, rp28, and ADH).
[0204] Furthermore, it is contemplated that promoters combining
elements from more than one promoter may be useful. For example,
U.S. Pat. No. 5,491,288 discloses combining a Cauliflower Mosaic
Virus promoter with a histone promoter. Thus, the elements from the
promoters disclosed herein may be combined with elements from other
promoters. Promoters, which are useful for plant transgene
expression include those that are inducible, viral, synthetic,
constitutive (Odell 1985), temporally regulated, spatially
regulated, tissue-specific, and spatial-temporally regulated.
[0205] Where expression in specific tissues or organs is desired
tissue-specific promoters may be used. In contrast, where gene
expression in response to a stimulus is desired, inducible
promoters are the regulatory elements of choice. Where continuous
expression is desired throughout the cells of a plant, constitutive
promoters are utilized. Additional regulatory sequences upstream
and/or downstream from the core promoter sequence may be included
in expression constructs of transformation vectors to bring about
varying levels of expression of heterologous nucleotide sequences
in a transgenic plant.
[0206] A variety of 5' and 3' transcriptional regulatory sequences
are available for use in the present invention. Transcriptional
terminators are responsible for the termination of transcription
and correct mRNA polyadenylation. The 3' nontranslated regulatory
DNA sequence preferably includes from about 50 to about 1,000, more
preferably about 100 to about 1,000, nucleotide base pairs and
contains plant transcriptional and translational termination
sequences. Appropriate transcriptional terminators and those which
are known to function in plants include the CaMV 35S terminator,
the tml terminator, the nopaline synthase terminator, the pea rbcS
E9 terminator, the terminator for the T7 transcript from the
octopine synthase gene of Agrobacterium tumefaciens, and the 3, end
of the protease inhibitor I or II genes from potato or tomato,
although other 3' elements known to those of skill in the art can
also be employed. Alternatively, one also could use a gamma coixin,
oleosin 3 or other terminator from the genus Coix.
[0207] Preferred 3' elements include those from the nopaline
synthase gene of Agrobacterium tumefaciens (Bevan 1983), the
terminator for the T7 transcript from the octopine synthase gene of
Agrobacterium tumefaciens, and the 3' end of the protease inhibitor
I or II genes from potato or tomato.
[0208] As the DNA sequence between the transcription initiation
site and the start of the coding sequence, i.e., the untranslated
leader sequence, can influence gene expression, one may also wish
to employ a particular leader sequence. Preferred leader sequences
are contemplated to include those, which include sequences,
predicted to direct optimum expression of the attached gene, i.e.,
to include a preferred consensus leader sequence, which may
increase or maintain mRNA stability and prevent inappropriate
initiation of translation. The choice of such sequences will be
known to those of skill in the art in light of the present
disclosure. Sequences that are derived from genes that are highly
expressed in plants will be most preferred.
[0209] Preferred regulatory elements also include the
5'-untranslated region, introns and the 3'-untranslated region of
genes. Such sequences that have been found to enhance gene
expression in transgenic plants include intron sequences (e.g.,
from Adh1, bronze1, actin1, actin 2 (WO 00/760067), or the sucrose
synthase intron; see: The Maize Handbook, Chapter 116, Freeling and
Walbot, Eds., Springer, New York (1994)) and viral leader sequences
(e.g., from TMV, MCMV and AMV; Gallie 1987). For example, a number
of non-translated leader sequences derived from viruses are known
to enhance expression. Specifically, leader sequences from Tobacco
Mosaic Virus (TMV), Maize Chlorotic Mottle Virus (MCMV), and
Alfalfa Mosaic Virus (AMV) have been shown to be effective in
enhancing expression (e.g., Gallie 1987; Skuzeski 1990). Other
leaders known in the art include but are not limited to:
Picornavirus leaders, for example, EMCV leader
(Encephalomyocarditis 5' noncoding region) (Elroy-Stein 1989);
Potyvirus leaders, for example, TEV leader (Tobacco Etch Virus);
MDMV leader (Maize Dwarf Mosaic Virus); Human immunoglobulin
heavy-chain binding protein (BiP) leader, (Macejak 1991);
Untranslated leader from the coat protein mRNA of alfalfa mosaic
virus (AMV RNA 4), (Jobling 1987; Tobacco mosaic virus leader
(TMV), (Gallie 1989; and Maize Chlorotic Mottle Virus leader (MCMV)
(Lommel 1991. See also, Delia-Cioppa 1987. Regulatory elements such
as Adh intron 1 (Callis 1987), sucrose synthase intron (Vasil 1989)
or TMV omega element (Gallie 1989), may further be included where
desired. Especially preferred are the 5'-untranslated region,
introns and the 3'-untranslated region from the genes described by
the GenBank Arabidopsis thaliana genome locii At4g12910, At1g66250,
At4g00820, At2g36640, At2g34200, At3g11180, At4g00360, At2g48030,
At2g38590, At1g23000, At3g15510, At3g50870, At4g00220, or
At4g26320, or of functional equivalent thereof.
[0210] Additional preferred regulatory elements are enhancer
sequences or polyadenylation sequences. Preferred polyadenylation
sequences are those from plant genes or Agrobacterium T-DNA genes
(such as for example the terminator sequences of the OCS (octopine
synthase) or NOS (nopaline synthase) genes).
[0211] Examples of enhancers include elements from the CaMV 35S
promoter, octopine synthase genes (Ellis et al., 1987), the rice
actin I gene, the maize alcohol dehydrogenase gene (Callis 1987),
the maize shrunken I gene (Vasil 1989), TMV Omega element (Gallie
1989) and promoters from non-plant eukaryotes (e.g. yeast; Ma
1988). Vectors for use in accordance with the present invention may
be constructed to include the ocs enhancer element. This element
was first identified as a 16 bp palindromic enhancer from the
octopine synthase (ocs) gene of ultilane (Ellis 1987), and is
present in at least 10 other promoters (Bouchez 1989). The use of
an enhancer element, such as the ocs elements and particularly
multiple copies of the element, will act to increase the level of
transcription from adjacent promoters when applied in the context
of plant transformation.
[0212] An expression cassette of the invention (or a vector derived
therefrom) may comprise additional functional elements, which are
to be understood in the broad sense as all elements which influence
construction, propagation, or function of an expression cassette or
a vector or a transgenic organism comprising them. Such functional
elements may include origin of replications (to allow replication
in bacteria; for the ORI of pBR322 or the P15A ori; Sambrook 1989),
or elements required for Agrobacterium T-DNA transfer (such as for
example the left and/or rights border of the T-DNA).
[0213] Ultimately, the most desirable DNA segments for introduction
into, for example, a dicot genome, may be homologous genes or gene
families which encode a desired trait (e.g., increased yield per
acre) and which are introduced under the control of novel promoters
or enhancers, etc., or perhaps even homologous or tissue specific
(e.g., root-, collar/sheath-, whorl-, stalk-, earshank-, kernel- or
leaf-specific) promoters or control elements. Indeed, it is
envisioned that a particular use of the present invention will be
the expression of a gene in a seed-preferential or seed-specific
manner.
[0214] Additionally, vectors may be constructed and employed in the
intracellular targeting of a specific gene product within the cells
of a transgenic plant or in directing a protein to the
extracellular environment. This will generally be achieved by
joining a DNA sequence encoding a transit or signal peptide
sequence to the coding sequence of a particular gene. The resultant
transit or signal peptide will transport the protein to a
particular intracellular or extracellular destination,
respectively, and will then be post-translationally removed.
Transit or signal peptides act by facilitating the transport of
proteins through intracellular membranes, e.g., vacuole, vesicle,
plastid and mitochondrial membranes, whereas signal peptides direct
proteins through the extracellular membrane.
[0215] A particular example of such a use concerns the direction of
a herbicide resistance gene, such as the EPSPS gene, to a
particular organelle such as the chloroplast rather than to the
cytoplasm. This is exemplified by the use of the rbcs transit
peptide which confers plastid-specific targeting of proteins. In
addition, it is proposed that it may be desirable to target certain
genes responsible for male sterility to the mitochondria, or to
target certain genes for resistance to phytopathogenic organisms to
the extracellular spaces, or to target proteins to the vacuole.
[0216] By facilitating the transport of the protein into
compartments inside and outside the cell, these sequences may
increase the accumulation of gene product protecting them from
proteolytic degradation. These sequences also allow for additional
mRNA sequences from highly expressed genes to be attached to the
coding sequence of the genes. Since mRNA being translated by
ribosomes is more stable than naked mRNA, the presence of
translatable mRNA in front of the gene may increase the overall
stability of the mRNA transcript from the gene and thereby increase
synthesis of the gene product. Since transit and signal sequences
are usually post-translationally removed from the initial
translation product, the use of these sequences allows for the
addition of extra translated sequences that may not appear on the
final polypeptide. Targeting of certain proteins may be desirable
in order to enhance the stability of the protein (U.S. Pat. No.
5,545,818).
[0217] It may be useful to target DNA itself within a cell. For
example, it may be useful to target introduced DNA to the nucleus
as this may increase the frequency of transformation. Within the
nucleus itself it would be useful to target a gene in order to
achieve site-specific integration. For example, it would be useful
to have a gene introduced through transformation replace an
existing gene in the cell. Other elements include those that can be
regulated by endogenous or exogenous agents, e.g., by zinc finger
proteins, including naturally occurring zinc finger proteins or
chimeric zinc finger proteins (see, e.g., U.S. Pat. No. 5,789,538,
WO 99/48909; WO 99/45132; WO 98/53060; WO 98/53057; WO 98/53058; WO
00/23464; WO 95/19431; and WO 98/54311) or myb-like transcription
factors. For example, a chimeric zinc finger protein may include
amino acid sequences, which bind to a specific DNA sequence (the
zinc finger) and amino acid sequences that activate (e.g., GAL 4
sequences) or repress the transcription of the sequences linked to
the specific DNA sequence.
[0218] It is one of the objects of the present invention to provide
recombinant DNA molecules comprising a nucleotide sequence
according to the invention operably linked to a nucleotide segment
of interest.
[0219] A nucleotide segment of interest is reflective of the
commercial markets and interests of those involved in the
development of the crop. Crops and markets of interest changes, and
as developing nations open up world markets, new crops and
technologies will also emerge. In addition, as the understanding of
agronomic traits and characteristics such as yield and heterosis
increase, the choice of genes for transformation will change
accordingly. General categories of nucleotides of interest include,
for example, genes involved in information, such as zinc fingers,
those involved in communication, such as kinases, and those
involved in housekeeping, such as heat shock proteins. More
specific categories of transgenes, for example, include genes
encoding important traits for agronomics, insect resistance,
disease resistance, herbicide resistance, sterility, grain
characteristics, and commercial products. Genes of interest
include, generally, those involved in starch, oil, carbohydrate, or
nutrient metabolism, as well as those affecting kernel size,
sucrose loading, zinc finger proteins, see, e.g., U.S. Pat. No.
5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO 98/53057; WO
98/53058; WO 00/23464; WO 95/19431; and WO 98/54311, and the
like.
[0220] One skilled in the art recognizes that the expression level
and regulation of a transgene in a plant can vary significantly
from line to line. Thus, one has to test several lines to find one
with the desired expression level and regulation. Once a line is
identified with the desired regulation specificity of a chimeric
Cre transgene, it can be crossed with lines carrying different
inactive replicons or inactive transgene for activation.
[0221] Other sequences which may be linked to the gene of interest,
which encodes a polypeptide, are those which can target to a
specific organelle, e.g., to the mitochondria, nucleus, or plastid,
within the plant cell. Targeting can be achieved by providing the
polypeptide with an appropriate targeting peptide sequence, such as
a secretory signal peptide (for secretion or cell wall or membrane
targeting, a plastid transit peptide, a chloroplast transit
peptide, e.g., the chlorophyll a/b binding protein, a mitochondrial
target peptide, a vacuole targeting peptide, or a nuclear targeting
peptide, and the like. For example, the small subunit of ribulose
bisphosphate carboxylase transit peptide, the EPSPS transit peptide
or the dihydrodipicolinic acid synthase transit peptide may be
used, for examples of plastid organelle targeting sequences (see WO
00/12732). Plastids are a class of plant organelles derived from
proplastids and include chloroplasts, leucoplasts, amyloplasts, and
chromoplasts. The plastids are major sites of biosynthesis in
plants. In addition to photosynthesis in the chloroplast, plastids
are also sites of lipid biosynthesis, nitrate reduction to
ammonium, and starch storage. And while plastids contain their own
circular, genome, most of the proteins localized to the plastids
are encoded by the nuclear genome and are imported into the
organelle from the cytoplasm.
[0222] Transgenes used with the present invention will often be
genes that direct the expression of a particular protein or
polypeptide product, but they may also be non-expressible DNA
segments, e.g., transposons such as Ds that do no direct their own
transposition. As used herein, an "expressible gene" is any gene
that is capable of being transcribed into RNA (e.g., mRNA,
antisense RNA, etc.) or translated into a protein, expressed as a
trait of interest, or the like, etc., and is not limited to
selectable, screenable or non-selectable marker genes. The
invention also contemplates that, where both an expressible gene
that is not necessarily a marker gene is employed in combination
with a marker gene, one may employ the separate genes on either the
same or different DNA segments for transformation. In the latter
case, the different vectors are delivered concurrently to recipient
cells to maximize co-transformation.
[0223] The choice of the particular DNA segments to be delivered to
the recipient cells will often depend on the purpose of the
transformation. One of the major purposes of transformation of crop
plants is to add some commercially desirable, agronomically
important traits to the plant. Such traits include, but are not
limited to, herbicide resistance or tolerance; insect resistance or
tolerance; disease resistance or tolerance (viral, bacterial,
fungal, nematode); stress tolerance and/or resistance, as
exemplified by resistance or tolerance to drought, heat, chilling,
freezing, excessive moisture, salt stress; oxidative stress;
increased yields; food content and makeup; physical appearance;
male sterility; drydown; standability; prolificacy; starch
properties; oil quantity and quality; and the like. One may desire
to incorporate one or more genes conferring any such desirable
trait or traits, such as, for example, a gene or genes encoding
pathogen resistance.
[0224] In certain embodiments, the present invention contemplates
the transformation of a recipient cell with more than one
advantageous transgene. Two or more transgenes can be supplied in a
single transformation event using either distinct
transgene-encoding vectors, or using a single vector incorporating
two or more gene coding sequences. For example, plasmids bearing
the bar and aroA expression units in either convergent, divergent,
or colinear orientation, are considered to be particularly useful.
Further preferred combinations are those of an insect resistance
gene, such as a Bt gene, along with a protease inhibitor gene such
as pinII, or the use of bar in combination with either of the above
genes. Of course, any two or more transgenes of any description,
such as those conferring herbicide, insect, disease (viral,
bacterial, fungal, nematode) or drought resistance, male sterility,
drydown, standability, prolificacy, starch properties, oil quantity
and quality, or those increasing yield or nutritional quality may
be employed as desired.
1. Exemplary Transgenes
1.1, Herbicide Resistance
[0225] The genes encoding phosphinothricin acetyltransferase (bar
and pat), glyphosate tolerant EPSP synthase genes, the glyphosate
degradative enzyme gene gox encoding glyphosate oxidoreductase, deh
(encoding a dehalogenase enzyme that inactivates dalapon),
herbicide resistant (e.g., sulfonylurea and imidazolinone)
acetolactate synthase, and bxn genes (encoding a nitrilase enzyme
that degrades bromoxynil) are good examples of herbicide resistant
genes for use in transformation. The bar and pat genes code for an
enzyme, phosphinothricin acetyltransferase (PAT), which inactivates
the herbicide phosphinothricin and prevents this compound from
inhibiting glutamine synthetase enzymes. The enzyme
5-enolpyruvylshikimate 3-phosphate synthase (EPSP Synthase), is
normally inhibited by the herbicide N-(phosphonomethyl)glycine
(glyphosate). However, genes are known that encode
glyphosate-resistant EPSP Synthase enzymes. The deh gene encodes
the enzyme dalapon dehalogenase and confers resistance to the
herbicide dalapon. The bxn gene codes for a specific nitrilase
enzyme that converts bromoxynil to a non-herbicidal degradation
product.
1.2 Insect Resistance
[0226] An important aspect of the present invention concerns the
introduction of insect resistance-conferring genes into plants.
Potential insect resistance genes, which can be introduced, include
Bacillus thuringiensis crystal toxin genes or Bt genes (Watrud
1985). Bt genes may provide resistance to lepidopteran or
coleopteran pests such as European Corn Borer (ECB) and corn
rootworm (CRW). Preferred Bt toxin genes for use in such
embodiments include the CryIA(b) and CryIA(c) genes. Endotoxin
genes from other species of B. thuringiensis, which affect insect
growth or development, may also be employed in this regard.
Protease inhibitors may also provide insect resistance (Johnson
1989), and will thus have utility in plant transformation. The use
of a protease inhibitor 11 gene, pinII, from tomato or potato is
envisioned to be particularly useful. Even more advantageous is the
use of a pinII gene in combination with a Bt toxin gene, the
combined effect of which has been discovered by the present
inventors to produce synergistic insecticidal activity. Other
genes, which encode inhibitors of the insects' digestive system, or
those that encode enzymes or co-factors that facilitate the
production of inhibitors, may also be useful. Cystatin and amylase
inhibitors, such as those from wheat and barley, may exemplify this
group.
[0227] Also, genes encoding lectins may confer additional or
alternative insecticide properties. Lectins (originally termed
phytohemagglutinins) are multivalent carbohydrate-binding proteins,
which have the ability to agglutinate red blood cells from a range
of species. Lectins have been identified recently as insecticidal
agents with activity against weevils, ECB and rootworm (Murdock
1990; Czapla & Lang, 1990). Lectin genes contemplated to be
useful include, for example, barley and wheat germ agglutinin (WGA)
and rice lectins (Gatehouse 1984), with WGA being preferred.
[0228] Genes controlling the production of large or small
polypeptides active against insects when introduced into the insect
pests, such as, e.g., lytic peptides, peptide hormones and toxins
and venoms, form another aspect of the invention. For example, it
is contemplated, that the expression of juvenile hormone esterase,
directed towards specific insect pests, may also result in
insecticidal activity, or perhaps cause cessation of metamorphosis
(Hammock 1990).
[0229] Transgenic plants expressing genes, which encode enzymes
that affect the integrity of the insect cuticle form yet another
aspect of the invention. Such genes include those encoding, e.g.,
chitinase, proteases, lipases and also genes for the production of
nikkomycin, a compound that inhibits chitin synthesis, the
introduction of any of which is contemplated to produce insect
resistant maize plants. Genes that code for activities that affect
insect molting, such those affecting the production of ecdysteroid
UDP-glucosyl transferase, also fall within the scope of the useful
transgenes of the present invention.
[0230] Genes that code for enzymes that facilitate the production
of compounds that reduce the nutritional quality of the host plant
to insect pests are also encompassed by the present invention. It
may be possible, for instance, to confer insecticidal activity on a
plant by altering its sterol composition. Sterols are obtained by
insects from their diet and are used for hormone synthesis and
membrane stability. Therefore alterations in plant sterol
composition by expression of novel genes, e.g., those that directly
promote the production of undesirable sterols or those that convert
desirable sterols into undesirable forms, could have a negative
effect on insect growth and/or development and hence endow the
plant with insecticidal activity. Lipoxygenases are naturally
occurring plant enzymes that have been shown to exhibit
anti-nutritional effects on insects and to reduce the nutritional
quality of their diet. Therefore, further embodiments of the
invention concern transgenic plants with enhanced lipoxygenase
activity which may be resistant to insect feeding.
[0231] The present invention also provides methods and compositions
by which to achieve qualitative or quantitative changes in plant
secondary metabolites. One example concerns transforming plants to
produce DIMBOA which, it is contemplated, will confer resistance to
European corn borer, rootworm and several other maize insect pests.
Candidate genes that are particularly considered for use in this
regard include those genes at the bx locus known to be involved in
the synthetic DIMBOA pathway (Dunn 1981). The introduction of genes
that can regulate the production of maysin, and genes involved in
the production of dhurrin in sorghum, is also contemplated to be of
use in facilitating resistance to earworm and rootworm,
respectively.
[0232] Tripsacum dactyloides is a species of grass that is
resistant to certain insects, including corn rootworm. It is
anticipated that genes encoding proteins that are toxic to insects
or are involved in the biosynthesis of compounds toxic to insects
will be isolated from Tripsacum and that these novel genes will be
useful in conferring resistance to insects. It is known that the
basis of insect resistance in Tripsacum is genetic, because said
resistance has been transferred to Zea mays via sexual crosses
(Branson & Guss, 1972).
[0233] Further genes encoding proteins characterized as having
potential insecticidal activity may also be used as transgenes in
accordance herewith. Such genes include, for example, the cowpea
trypsin inhibitor (CpTI; Hilder 1987) which may be used as a
rootworm deterrent; genes encoding avermectin (Campbell 1989; Ikeda
1987) which may prove particularly useful as a corn rootworm
deterrent; ribosome inactivating protein genes; and even genes that
regulate plant structures. Transgenic maize including anti-insect
antibody genes and genes that code for enzymes that can covert a
non-toxic insecticide (pro-insecticide) applied to the outside of
the plant into an insecticide inside the plant are also
contemplated.
1.3 Environment or Stress Resistance
[0234] Improvement of a plant's ability to tolerate various
environmental stresses such as, but not limited to, drought, excess
moisture, chilling, freezing, high temperature, salt, and oxidative
stress, can also be effected through expression of heterologous, or
overexpression of homologous genes. Benefits may be realized in
terms of increased resistance to freezing temperatures through the
introduction of an "antifreeze" protein such as that of the Winter
Flounder (Cutler 1989) or synthetic gene derivatives thereof.
Improved chilling tolerance may also be conferred through increased
expression of glycerol-3-phosphate acetyltransferase in
chloroptasts (Murata 1992; Wolter 1992). Resistance to oxidative
stress (often exacerbated by conditions such as chilling
temperatures in combination with high light intensities) can be
conferred by expression of superoxide dismutase (Gupta 1993), and
may be improved by glutathione reductase (Bowler 1992). Such
strategies may allow for tolerance to freezing in newly emerged
fields as well as extending later maturity higher yielding
varieties to earlier relative maturity zones.
[0235] Expression of novel genes that favorably effect plant water
content, total water potential, osmotic potential, and turgor can
enhance the ability of the plant to tolerate drought. As used
herein, the terms "drought resistance" and "drought tolerance" are
used to refer to a plants increased resistance or tolerance to
stress induced by a reduction in water availability, as compared to
normal circumstances, and the ability of the plant to function and
survive in lower-water environments, and perform in a relatively
superior manner. In this aspect of the invention it is proposed,
for example, that the expression of a gene encoding the
biosynthesis of osmotically active solutes can impart protection
against drought. Within this class of genes are DNAs encoding
mannitol dehydrogenase (Lee and Saier, 1982) and
trehalose-6-phosphate synthase (Kaasen 1992). Through the
subsequent action of native phosphatases in the cell or by the
introduction and coexpression of a specific phosphatase, these
introduced genes will result in the accumulation of either mannitol
or trehalose, respectively, both of which have been well documented
as protective compounds able to mitigate the effects of stress.
Mannitol accumulation in transgenic tobacco has been verified and
preliminary results indicate that plants expressing high levels of
this metabolite are able to tolerate an applied osmotic stress
(Tarczynski 1992).
[0236] Similarly, the efficacy of other metabolites in protecting
either enzyme function (e.g. alanopine or propionic acid) or
membrane integrity (e.g., alanopine) has been documented (Loomis
1989), and therefore expression of gene encoding the biosynthesis
of these compounds can confer drought resistance in a manner
similar to or complimentary to mannitol. Other examples of
naturally occurring metabolites that are osmotically active and/or
provide some direct protective effect during drought and/or
desiccation include sugars and sugar derivatives such as fructose,
erythritol (Coxson 1992), sorbitol, dulcitol (Karsten 1992),
glucosylglycerol (Reed 1984; Erdmann 1992), sucrose, stachyose
(Koster & Leopold 1988; Blackman 1992), ononitol and pinitol
(Vernon & Bohnert 1992), and raffinose (Bernal-Lugo &
Leopold 1992). Other osmotically active solutes, which are not
sugars, include, but are not limited to, proline and
glycine-betaine (Wyn-Jones and Storey, 1981). Continued canopy
growth and increased reproductive fitness during times of stress
can be augmented by introduction and expression of genes such as
those controlling the osmotically active compounds discussed above
and other such compounds, as represented in one exemplary
embodiment by the enzyme myoinositol 0-methyltransferase.
[0237] It is contemplated that the expression of specific proteins
may also increase drought tolerance. Three classes of Late
Embryogenic Proteins have been assigned based on structural
similarities (see Dure 1989). All three classes of these proteins
have been demonstrated in maturing (i.e., desiccating) seeds.
Within these 3 types of proteins, the Type-II (dehydrin-type) have
generally been implicated in drought and/or desiccation tolerance
in vegetative plant parts (e.g. Mundy and Chua, 1988; Piatkowski
1990; Yamaguchi-Shinozaki 1992). Recently, expression of a Type-III
LEA (HVA-1) in tobacco was found to influence plant height,
maturity and drought tolerance (Fitzpatrick, 1993). Expression of
structural genes from all three groups may therefore confer drought
tolerance. Other types of proteins induced during water stress
include thiol proteases, aldolases and transmembrane transporters
(Guerrero 1990), which may confer various protective and/or
repair-type functions during drought stress. The expression of a
gene that effects lipid biosynthesis and hence membrane composition
can also be useful in conferring drought resistance on the
plant.
[0238] Many genes that improve drought resistance have
complementary modes of action. Thus, combinations of these genes
might have additive and/or synergistic effects in improving drought
resistance in maize. Many of these genes also improve freezing
tolerance (or resistance); the physical stresses incurred during
freezing and drought are similar in nature and may be mitigated in
similar fashion. Benefit may be conferred via constitutive
expression or tissue-specific of these genes, but the preferred
means of expressing these novel genes may be through the use of a
turgor-induced promoter (such as the promoters for the
turgor-induced genes described in Guerrero et al. 1990 and Shagan
1993). Spatial and temporal expression patterns of these genes may
enable maize to better withstand stress.
[0239] Expression of genes that are involved with specific
morphological traits that allow for increased water extractions
from drying soil would be of benefit. For example, introduction and
expression of genes that alter root characteristics may enhance
water uptake. Expression of genes that enhance reproductive fitness
during times of stress would be of significant value. For example,
expression of DNAs that improve the synchrony of pollen shed and
receptiveness of the female flower parts, i.e., silks, would be of
benefit. In addition, expression of genes that minimize kernel
abortion during times of stress would increase the amount of grain
to be harvested and hence be of value. Regulation of cytokinin
levels in monocots, such as maize, by introduction and expression
of an isopentenyl transferase gene with appropriate regulatory
sequences can improve monocot stress resistance and yield (Gan
1995).
[0240] Given the overall role of water in determining yield, it is
contemplated that enabling plants to utilize water more
efficiently, through the introduction and expression of novel
genes, will improve overall performance even when soil water
availability is not limiting. By introducing genes that improve the
ability of plants to maximize water usage across a full range of
stresses relating to water availability, yield stability or
consistency of yield performance may be realized.
[0241] Improved protection of the plant to abiotic stress factors
such as drought, heat or chill, can also be achieved--for
example--by overexpressing antifreeze polypeptides from
Myoxocephalus Scorpius (WO 00/00512), Myoxocephalus
octodecemspinosus, the Arabidopsis thaliana transcription activator
CBF1, glutamate dehydrogenases (WO 97/12983, WO 98/11240),
calcium-dependent protein kinase genes (WO 98/26045), calcineurins
(WO 99/05902), casein kinase from yeast (WO 02/052012),
farnesyltransferases (WO 99/06580; Pei Z M et al. (1998) Science
282:287-290), ferritin (Deak M et al. (1999) Nature Biotechnology
17:192-196), oxalate oxidase (WO 99/04013; Dunwell J M (1998)
Biotechn Genet Eng Rev 15:1-32), DREB1A factor ("dehydration
response element B1A"; Kasuga M et al. (1999) Nature Biotech
17:276-286), genes of mannitol or trehalose synthesis such as
trehalose-phosphate synthase or trehalose-phosphate phosphatase (WO
97/42326) or by inhibiting genes such as trehalase (WO
97/50561).
1.4 Disease Resistance
[0242] It is proposed that increased resistance to diseases may be
realized through introduction of genes into plants period. It is
possible to produce resistance to diseases caused, by viruses,
bacteria, fungi, root pathogens, insects and nematodes. It is also
contemplated that control of mycotoxin producing organisms may be
realized through expression of introduced genes.
[0243] Resistance to viruses may be produced through expression of
novel genes. For example, it has been demonstrated that expression
of a viral coat protein in a transgenic plant can impart resistance
to infection of the plant by that virus and perhaps other closely
related viruses (Cuozzo 1988, Hemenway 1988, Abel 1986). It is
contemplated that expression of antisense genes targeted at
essential viral functions may impart resistance to said virus. For
example, an antisense gene targeted at the gene responsible for
replication of viral nucleic acid may inhibit said replication and
lead to resistance to the virus. It is believed that interference
with other viral functions through the use of antisense genes may
also increase resistance to viruses. Further it is proposed that it
may be possible to achieve resistance to viruses through other
approaches, including, but not limited to the use of satellite
viruses.
[0244] It is proposed that increased resistance to diseases caused
by bacteria and fungi may be realized through introduction of novel
genes. It is contemplated that genes encoding so-called "peptide
antibiotics," pathogenesis related (PR) proteins, toxin resistance,
and proteins affecting host-pathogen interactions such as
morphological characteristics will be useful. Peptide antibiotics
are polypeptide sequences, which are inhibitory to growth of
bacteria and other microorganisms. For example, the classes of
peptides referred to as cecropins and magainins inhibit growth of
many species of bacteria and fungi. It is proposed that expression
of PR proteins in plants may be useful in conferring resistance to
bacterial disease. These genes are induced following pathogen
attack on a host plant and have been divided into at least five
classes of proteins (Bol 1990). Included amongst the PR proteins
are beta-1,3-glucanases, chitinases, and osmotin and other proteins
that are believed to function in plant resistance to disease
organisms. Other genes have been identified that have antifungal
properties, e.g., UDA (stinging nettle lectin) and hevein
(Broakgert 1989; Barkai-Golan 1978). It is known that certain plant
diseases are caused by the production of phytotoxins. Resistance to
these diseases could be achieved through expression of a novel gene
that encodes an enzyme capable of degrading or otherwise
inactivating the phytotoxin. Expression novel genes that alter the
interactions between the host plant and pathogen may be useful in
reducing the ability the disease organism to invade the tissues of
the host plant, e.g., an increase in the waxiness of the leaf
cuticle or other morphological characteristics.
[0245] Plant parasitic nematodes are a cause of disease in many
plants. It is proposed that it would be possible to make the plant
resistant to these organisms through the expression of novel genes.
It is anticipated that control of nematode infestations would be
accomplished by altering the ability of the nematode to recognize
or attach to a host plant and/or enabling the plant to produce
nematicidal compounds, including but not limited to proteins.
[0246] Furthermore, a resistance to fungi, insects, nematodes and
diseases, can be achieved by targeted accumulation of certain
metabolites or proteins. Such proteins include but are not limited
to glucosinolates (defense against herbivores), chitinases or
glucanases and other enzymes which destroy the cell wall of
parasites, ribosome-inactivating proteins (RIPs) and other proteins
of the plant resistance and stress reaction as are induced when
plants are wounded or attacked by microbes, or chemically, by, for
example, salicylic acid, jasmonic acid or ethylene, or lysozymes
from nonplant sources such as, for example, T4-lysozyme or lysozyme
from a variety of mammals, insecticidal proteins such as Bacillus
thuringiensis endotoxin, a-amylase inhibitor or protease inhibitors
(cowpea trypsin inhibitor), lectins such as wheatgerm agglutinin,
RNAses or ribozymes. Further examples are nucleic acids which
encode the Trichoderma harzianum chit42 endochitinase (GenBank Acc.
No.: S78423) or the N-hydroxylating, multi-functional cytochrome
P-450 (CYP79) protein from Sorghum bicolor (GenBank Acc. No.:
U32624), or functional equivalents of these. The accumulation of
glucosinolates as protection from pests (Rask L et al. (2000) Plant
Mol Biol 42:93-113; Menard R et al. (1999) Phytochemistry
52:29-35), the expression of Bacillus thuringiensis endotoxins
(Vaeck et al. (1987) Nature 328:33-37) or the protection against
attack by fungi, by expression of chitinases, for example from
beans (Broglie et al. (1991) Science 254:1194-1197), is
advantageous. Resistance to pests such as, for example, the rice
pest Nilaparvata lugens in rice plants can be achieved by
expressing the snowdrop (Galanthus nivalls) lectin agglutinin (Rao
et al. (1998) Plant J 15(4):469-77). The expression of synthetic
cryIA(b) and cryIA(c) genes, which encode lepidoptera-specific
Bacillus thuringiensis D-endotoxins can bring about a resistance to
insect pests in various plants (Goyal R K et al. (2000) Crop
Protection 19(5):307-312). Further target genes which are suitable
for pathogen defense comprise "polygalacturonase-inhibiting
protein" (PGIP), thaumatine, invertase and antimicrobial peptides
such as lactoferrin (Lee T J et al. (2002) J Amer Soc Horticult Sci
127(2):158-164).
1.5 Mycotoxin Reduction/Elimination
[0247] Production of mycotoxins, including aflatoxin and fumonisin,
by fungi associated with plants is a significant factor in
rendering the grain not useful. These fungal organisms do not cause
disease symptoms and/or interfere with the growth of the plant, but
they produce chemicals (mycotoxins) that are toxic to animals.
Inhibition of the growth of these fungi would reduce the synthesis
of these toxic substances and, therefore, reduce grain losses due
to mycotoxin contamination. Novel genes may be introduced into
plants that would inhibit synthesis of the mycotoxin without
interfering with fungal growth. Expression of a novel gene, which
encodes an enzyme capable of rendering the mycotoxin nontoxic,
would be useful in order to achieve reduced mycotoxin contamination
of grain. The result of any of the above mechanisms would be a
reduced presence of mycotoxins on grain.
1.6 Grain Composition or Quality
[0248] Genes may be introduced into plants, particularly
commercially important cereals such as maize, wheat or rice, to
improve the grain for which the cereal is primarily grown. A wide
range of novel transgenic plants produced in this manner may be
envisioned depending on the particular end use of the grain.
[0249] For example, the largest use of maize grain is for feed or
food. Introduction of genes that alter the composition of the grain
may greatly enhance the feed or food value. The primary components
of maize grain are starch, protein, and oil. Each of these primary
components of maize grain may be improved by altering its level or
composition. Several examples may be mentioned for illustrative
purposes but in no way provide an exhaustive list of
possibilities.
[0250] The protein of many cereal grains is suboptimal for feed and
food purposes especially when fed to pigs, poultry, and humans. The
protein is deficient in several amino acids that are essential in
the diet of these species, requiring the addition of supplements to
the grain. Limiting essential amino acids may include lysine,
methionine, tryptophan, threonine, valine, arginine, and histidine.
Some amino acids become limiting only after the grain is
supplemented with other inputs for feed formulations. For example,
when the grain is supplemented with soybean meal to meet lysine
requirements, methionine becomes limiting. The levels of these
essential amino acids in seeds and grain may be elevated by
mechanisms which include, but are not limited to, the introduction
of genes to increase the biosynthesis of the amino acids, decrease
the degradation of the amino acids, increase the storage of the
amino acids in proteins, or increase transport of the amino acids
to the seeds or grain.
[0251] One mechanism for increasing the biosynthesis of the amino
acids is to introduce genes that deregulate the amino acid
biosynthetic pathways such that the plant can no longer adequately
control the levels that are produced. This may be done by
deregulating or bypassing steps in the amino acid biosynthetic
pathway that are normally regulated by levels of the amino acid end
product of the pathway. Examples include the introduction of genes
that encode deregulated versions of the enzymes aspartokinase or
dihydrodipicolinic acid (DHDP)-synthase for increasing lysine and
threonine production, and anthranilate synthase for increasing
tryptophan production. Reduction of the catabolism of the amino
acids may be accomplished by introduction of DNA sequences that
reduce or eliminate the expression of genes encoding enzymes that
catalyse steps in the catabolic pathways such as the enzyme
lysine-ketoglutarate reductase.
[0252] The protein composition of the grain may be altered to
improve the balance of amino acids in a variety of ways including
elevating expression of native proteins, decreasing expression of
those with poor composition, changing the composition of native
proteins, or introducing genes encoding entirely new proteins
possessing superior composition. DNA may be introduced that
decreases the expression of members of the zein family of storage
proteins. This DNA may encode ribozymes or antisense sequences
directed to impairing expression of zein proteins or expression of
regulators of zein expression such as the opaque-2 gene product.
The protein composition of the grain may be modified through the
phenomenon of cosuppression, i.e., inhibition of expression of an
endogenous gene through the expression of an identical structural
gene or gene fragment introduced through transformation (Goring
1991). Additionally, the introduced DNA may encode enzymes, which
degrade zeines. The decreases in zein expression that are achieved
may be accompanied by increases in proteins with more desirable
amino acid composition or increases in other major seed
constituents such as starch. Alternatively, a chimeric gene may be
introduced that comprises a coding sequence for a native protein of
adequate amino acid composition such as for one of the globulin
proteins or 10 kD zein of maize and a promoter or other regulatory
sequence designed to elevate expression of said protein. The coding
sequence of said gene may include additional or replacement codons
for essential amino acids. Further, a coding sequence obtained from
another species, or, a partially or completely synthetic sequence
encoding a completely unique peptide sequence designed to enhance
the amino acid composition of the seed may be employed.
[0253] The introduction of genes that alter the oil content of the
grain may be of value. Increases in oil content may result in
increases in metabolizable energy content and density of the seeds
for uses in feed and food. The introduced genes may encode enzymes
that remove or reduce rate-limitations or regulated steps in fatty
acid or lipid biosynthesis. Such genes may include, but are not
limited to, those that encode acetyl-CoA carboxylase,
ACP-acyltransferase, beta-ketoacyl-ACP synthase, plus other
well-known fatty acid biosynthetic activities. Other possibilities
are genes that encode proteins that do not possess enzymatic
activity such as acyl carrier protein. Additional examples include
2-acetyltransferase, oleosin pyruvate dehydrogenase complex, acetyl
CoA synthetase, ATP citrate lyase, ADP-glucose pyrophosphorylase
and genes of the carnitine-CoA-acetyl-CoA shuttles. It is
anticipated that expression of genes related to oil biosynthesis
will be targeted to the plastid, using a plastid transit peptide
sequence and preferably expressed in the seed embryo. Genes may be
introduced that alter the balance of fatty acids present in the oil
providing a more healthful or nutritive feedstuff. The introduced
DNA may also encode sequences that block expression of enzymes
involved in fatty acid biosynthesis, altering the proportions of
fatty acids present in the grain such as described below.
[0254] Genes may be introduced that enhance the nutritive value of
the starch component of the grain, for example by increasing the
degree of branching, resulting in improved utilization of the
starch in cows by delaying its metabolism.
[0255] Besides affecting the major constituents of the grain, genes
may be introduced that affect a variety of other nutritive,
processing, or other quality aspects of the grain as used for feed
or food. For example, pigmentation of the grain may be increased or
decreased. Enhancement and stability of yellow pigmentation is
desirable in some animal feeds and may be achieved by introduction
of genes that result in enhanced production of xanthophylis and
carotenes by eliminating rate-limiting steps in their production.
Such genes may encode altered forms of the enzymes phytoene
synthase, phytoene desaturase, or lycopene synthase. Alternatively,
unpigmented white corn is desirable for production of many food
products and may be produced by the introduction of DNA, which
blocks or eliminates steps in pigment production pathways.
[0256] Feed or food comprising some cereal grains possesses
insufficient quantities of vitamins and must be supplemented to
provide adequate nutritive value. Introduction of genes that
enhance vitamin biosynthesis in seeds may be envisioned including,
for example, vitamins A, E, B.sub.12, choline, and the like. For
example, maize grain also does not possess sufficient mineral
content for optimal nutritive value. Genes that affect the
accumulation or availability of compounds containing phosphorus,
sulfur, calcium, manganese, zinc, and iron among others would be
valuable. An example may be the introduction of a gene that reduced
phytic acid production or encoded the enzyme phytase, which
enhances phytic acid breakdown. These genes would increase levels
of available phosphate in the diet, reducing the need for
supplementation with mineral phosphate.
[0257] Numerous other examples of improvement of cereals for feed
and food purposes might be described. The improvements may not even
necessarily involve the grain, but may, for example, improve the
value of the grain for silage. Introduction of DNA to accomplish
this might include sequences that alter lignin production such as
those that result in the "brown midrib" phenotype associated with
superior feed value for cattle.
[0258] In addition to direct improvements in feed or food value,
genes may also be introduced which improve the processing of grain
and improve the value of the products resulting from the
processing. The primary method of processing certain grains such as
maize is via wetmilling. Maize may be improved though the
expression of novel genes that increase the efficiency and reduce
the cost of processing such as by decreasing steeping time.
[0259] Improving the value of wetmilling products may include
altering the quantity or quality of starch, oil, corn gluten meal,
or the components of corn gluten feed. Elevation of starch may be
achieved through the identification and elimination of rate
limiting steps in starch biosynthesis or by decreasing levels of
the other components of the grain resulting in proportional
increases in starch. An example of the former may be the
introduction of genes encoding ADP-glucose pyrophosphorylase
enzymes with altered regulatory activity or which are expressed at
higher level. Examples of the latter may include selective
inhibitors of, for example, protein or oil biosynthesis expressed
during later stages of kernel development.
[0260] The properties of starch may be beneficially altered by
changing the ratio of amylose to amylopectin, the size of the
starch molecules, or their branching pattern. Through these changes
a broad range of properties may be modified which include, but are
not limited to, changes in gelatinization temperature, heat of
gelatinization, clarity of films and pastes, Theological
properties, and the like. To accomplish these changes in
properties, genes that encode granule-bound or soluble starch
synthase activity or branching enzyme activity may be introduced
alone or combination. DNA such as antisense constructs may also be
used to decrease levels of endogenous activity of these enzymes.
The introduced genes or constructs may possess regulatory sequences
that time their expression to specific intervals in starch
biosynthesis and starch granule development. Furthermore, it may be
advisable to introduce and express genes that result in the in vivo
derivatization, or other modification, of the glucose moieties of
the starch molecule. The covalent attachment of any molecule may be
envisioned, limited only by the existence of enzymes that catalyze
the derivatizations and the accessibility of appropriate substrates
in the starch granule. Examples of important derivations may
include the addition of functional groups such as amines,
carboxyls, or phosphate groups, which provide sites for subsequent
in vitro derivatizations or affect starch properties through the
introduction of ionic charges. Examples of other modifications may
include direct changes of the glucose units such as loss of
hydroxyl groups or their oxidation to aldehyde or carboxyl
groups.
[0261] Oil is another product of wetmilling of corn and other
grains, the value of which may be improved by introduction and
expression of genes. The quantity of oil that can be extracted by
wetmilling may be elevated by approaches as described for feed and
food above. Oil properties may also be altered to improve its
performance in the production and use of cooking oil, shortenings,
lubricants or other oil-derived products or improvement of its
health attributes when used in the food-related applications. Novel
fatty acids may also be synthesized which upon extraction can serve
as starting materials for chemical syntheses. The changes in oil
properties may be achieved by altering the type, level, or lipid
arrangement of the fatty acids present in the oil. This in turn may
be accomplished by the addition of genes that encode enzymes that
catalyze the synthesis of novel fatty acids and the lipids
possessing them or by increasing levels of native fatty acids while
possibly reducing levels of precursors. Alternatively DNA sequences
may be introduced which slow or block steps in fatty acid
biosynthesis resulting in the increase in precursor fatty acid
intermediates. Genes that might be added include desaturases,
epoxidases, hydratases, dehydratases, and other enzymes that
catalyze reactions involving fatty acid intermediates.
Representative examples of catalytic steps that might be blocked
include the desaturations from stearic to oleic acid and oleic to
linolenic acid resulting in the respective accumulations of stearic
and oleic acids.
[0262] Improvements in the other major cereal wetmilling products,
gluten meal and gluten feed, may also be achieved by the
introduction of genes to obtain novel plants. Representative
possibilities include but are not limited to those described above
for improvement of food and feed value.
[0263] In addition it may further be considered that the plant be
used for the production or manufacturing of useful biological
compounds that were either not produced at all, or not produced at
the same level, in the plant previously. The novel plants producing
these compounds are made possible by the introduction and
expression of genes by transformation methods. The possibilities
include, but are not limited to, any biological compound which is
presently produced by any organism such as proteins, nucleic acids,
primary and intermediary metabolites, carbohydrate polymers, etc.
The compounds may be produced by the plant, extracted upon harvest
and/or processing, and used for any presently recognized useful
purpose such as pharmaceuticals, fragrances, industrial enzymes to
name a few.
[0264] Further possibilities to exemplify the range of grain traits
or properties potentially encoded by introduced genes in transgenic
plants include grain with less breakage susceptibility for export
purposes or larger grit size when processed by dry milling through
introduction of genes that enhance gamma-zein synthesis, popcorn
with improved popping, quality and expansion volume through genes
that increase pericarp thickness, corn with whiter grain for food
uses though introduction of genes that effectively block expression
of enzymes involved in pigment production pathways, and improved
quality of alcoholic beverages or sweet corn through introduction
of genes which affect flavor such as the shrunken gene (encoding
sucrose synthase) for sweet corn.
1.7 Tuber or Seed Composition or Quality
[0265] Various traits can be advantageously expressed especially in
seeds or tubers to improve composition or quality. Such traits
include but are not limited to: [0266] Expression of metabolic
enzymes for use in the food-and-feed sector, for example of
phytases and cellulases. Especially preferred are nucleic acids
such as the artificial cDNA which encodes a microbial phytase
(GenBank Acc. No.: A19451) or functional equivalents thereof.
[0267] Expression of genes which bring about an accumulation of
fine chemicals such as of tocopherols, tocotrienois or carotenoids.
An example which may be mentioned is phytoene desaturase. Preferred
are nucleic acids which encode the Narcissus pseudonarcissus
photoene desaturase (GenBank Acc. No.: X78815) or functional
equivalents thereof. [0268] Production of nutraceuticals such as,
for example, polyunsaturated fatty acids (for example arachidonic
acid, eicosapentaenoic acid or docosahexaenoic acid) by expression
of fatty acid elongases and/or desaturases, or production of
proteins with improved nutritional value such as, for example, with
a high content of essential amino acids (for example the
high-methionine 2S albumin gene of the brazil nut). Preferred are
nucleic acids which encode the Bertholletia excelsa high-methionine
2S albumin (GenBank Acc. No.: AB044391), the Physcomitrelia patens
.quadrature.6-acyl-lipid desaturase (GenBank Acc. No.: AJ222980:
Girke et al. (1998) Plant J 15:39-48), the Mortierella alpina
.quadrature.6-desaturase (Sakuradani et al. 1999 Gene 238:445-453),
the Caenorhabditis elegans .quadrature.5-desaturase (Michaelson et
al. 1998, FEBS Letters 439:215-218), the Caenorhabditis elegans
.quadrature.5-fatty acid desaturase (des-5) (GenBank Acc. No.:
AF078796), the Mortierella alpina .quadrature.5-desaturase
(Michaelson et al. JBC 273:19055-19059), the Caenorhabditis elegans
.quadrature.6-elongase (Beaudoin et al. 2000, PNAS 97:6421-6426),
the Physcomitrella patens .quadrature.6-elongase (Zank et al. 2000,
Biochemical Society Transactions 28.654-657), or functional
equivalents of these. [0269] Production of high-quality proteins
and enzymes for industrial purposes (for example enzymes, such as
lipases) or as pharmaceuticals (such as, for example, antibodies,
blood clotting factors, interferons, lymphokins, colony stimulation
factor, plasminogen activators, hormones or vaccines, as described
by Hood E E, Jilka J M (1999) Curr Opin Biotechnol 10(4):382-6; Ma
J K, Vine N D (1999) Curr Top Microbiol Immunol 236:275-92). For
example, it has been possible to produce recombinant avidin from
chicken albumen and bacterial b-glucuronidase (GUS) on a large
scale in transgenic maize plants (Hood et al. (1999) Adv Exp Med
Biol 464:127-47. Review). [0270] Obtaining an increased storability
in cells which normally comprise fewer storage proteins or storage
lipids, with the purpose of increasing the yield of these
substances, for example by expression of acetyl-CoA carboxylase.
Preferred nucleic acids are those which encode the Medicago sativa
acetyl-CoA carboxylase (ACCase) (GenBank Acc. No.: L25042), or
functional equivalents thereof. [0271] Reducing levels of
.quadrature.-glucan L-type tuber phosphorylase (GLTP) or
..quadrature.-glucan H-type tuber phosphorylase (GHTP) enzyme
activity preferably within the potato tuber (see U.S. Pat. No.
5,998,701). The conversion of starches to sugars in potato tubers,
particularly when stored at temperatures below 7.degree. C., is
reduced in tubers exhibiting reduced GLTP or GHTP enzyme activity.
Reducing cold-sweetening in potatoes allows for potato storage at
cooler temperatures, resulting in prolonged dormancy, reduced
incidence of disease, and increased storage life. Reduction of GLTP
or GHTP activity within the potato tuber may be accomplished by
such techniques as suppression of gene expression using homologous
antisense or double-stranded RNA, the use of co-suppression,
regulatory silencing sequences. A potato plant having improved
cold-storage characteristics, comprising a potato plant transformed
with an expression cassette having a TPT promoter sequence operably
linked to a DNA sequence comprising at least 20 nucleotides of a
gene encoding an .quadrature.-glucan phosphorylase selected from
the group consisting of {tilde over (.quadrature.)}glucan L-type
tuber phosphorylase (GLTP) and {tilde over (.quadrature.)}glucan
H-type phosphorylase (GHTP).
[0272] Further examples of advantageous genes are mentioned for
example in Dunwell J M, Transgenic approaches to crop improvement,
J Exp Bot. 2000; 51 Spec No; pages 487-96.
1.8 Plant Agronomic Characteristics
[0273] Two of the factors determining where plants can be grown are
the average daily temperature during the growing season and the
length of time between frosts. Within the areas where it is
possible to grow a particular plant, there are varying limitations
on the maximal time it is allowed to grow to maturity and be
harvested. The plant to be grown in a particular area is selected
for its ability to mature and dry down to harvestable moisture
content within the required period of time with maximum possible
yield. Therefore, plants of varying maturities are developed for
different growing locations. Apart from the need to dry down
sufficiently to permit harvest is the desirability of having
maximal drying take place in the field to minimize the amount of
energy required for additional drying post-harvest. Also the more
readily the grain can dry down, the more time there is available
for growth and kernel fill. Genes that influence maturity and/or
dry down can be identified and introduced into plant lines using
transformation techniques to create new varieties adapted to
different growing locations or the same growing location but having
improved yield to moisture ratio at harvest. Expression of genes
that are involved in regulation of plant development may be
especially useful, e.g., the liguleless and rough sheath genes that
have been identified in plants.
[0274] Genes may be introduced into plants that would improve
standability and other plant growth characteristics. For example,
expression of novel genes, which confer stronger stalks, improved
root systems, or prevent or reduce ear droppage would be of great
value to the corn farmer. Introduction and expression of genes that
increase the total amount of photoassimilate available by, for
example, increasing light distribution and/or interception would be
advantageous. In addition the expression of genes that increase the
efficiency of photosynthesis and/or the leaf canopy would further
increase gains in productivity. Such approaches would allow for
increased plant populations in the field.
[0275] Delay of late season vegetative senescence would increase
the flow of assimilates into the grain and thus increase yield.
Overexpression of genes within plants that are associated with
"stay green" or the expression of any gene that delays senescence
would be advantageous. For example, a non-yellowing mutant has been
identified in Festuca pratensis (Davies 1990). Expression of this
gene as well as others may prevent premature breakdown of
chlorophyll and thus maintain canopy function,
1.9 Nutrient Utilization
[0276] The ability to utilize available nutrients and minerals may
be a limiting factor in growth of many plants. It is proposed that
it would be possible to alter nutrient uptake, tolerate pH
extremes, mobilization through the plant, storage pools, and
availability for metabolic activities by the introduction of novel
genes. These modifications would allow a plant to more efficiently
utilize available nutrients. It is contemplated that an increase in
the activity of, for example, an enzyme that is normally present in
the plant and involved in nutrient utilization would increase the
availability of a nutrient. An example of such an enzyme would be
phytase. It is also contemplated that expression of a novel gene
may make a nutrient source available that was previously not
accessible, e.g., an enzyme that releases a component of nutrient
value from a more complex molecule, perhaps a macromolecule.
1.10 Male Sterility
[0277] Male sterility is useful in the production of hybrid seed.
It is proposed that male sterility may be produced through
expression of novel genes. For example, it has been shown that
expression of genes that encode proteins that interfere with
development of the male inflorescence and/or gametophyte result in
male sterility. Chimeric ribonuclease genes that express in the
anthers of transgenic tobacco and oilseed rape have been
demonstrated to lead to male sterility (Mariani 1990). For example,
a number of mutations were discovered in maize that confer
cytoplasmic male sterility. One mutation in particular, referred to
as T cytoplasm, also correlates with sensitivity to Southern corn
leaf blight. A DNA sequence, designated TURF-13 (Levings 1990), was
identified that correlates with T cytoplasm. It would be possible
through the introduction of TURF-13 via transformation to separate
male sterility from disease sensitivity. As it is necessary to be
able to restore male fertility for breeding purposes and for grain
production, it is proposed that genes encoding restoration of male
fertility may also be introduced.
1.11. Non-Protein-Expressing Sequences
1.11.1 RNA-Expressing
[0278] DNA may be introduced into plants for the purpose of
expressing RNA transcripts that function to affect plant phenotype
yet are not translated into protein. Two examples are antisense RNA
and RNA with ribozyme activity. Both may serve possible functions
in reducing or eliminating expression of native or introduced plant
genes.
[0279] Genes may be constructed or isolated, which when
transcribed, produce antisense RNA or double-stranded RNA that is
complementary to all or part(s) of a targeted messenger RNA(s). The
antisense RNA reduces production of the polypeptide product of the
messenger RNA. The polypeptide product may be any protein encoded
by the plant genome. The aforementioned genes will be referred to
as antisense genes. An antisense gene may thus be introduced into a
plant by transformation methods to produce a novel transgenic plant
with reduced expression of a selected protein of interest. For
example, the protein may be an enzyme that catalyzes a reaction in
the plant. Reduction of the enzyme activity may reduce or eliminate
products of the reaction which include any enzymatically
synthesized compound in the plant such as fatty acids, amino acids,
carbohydrates, nucleic acids and the like. Alternatively, the
protein may be a storage protein, such as a zein, or a structural
protein, the decreased expression of which may lead to changes in
seed amino acid composition or plant morphological changes
respectively. The possibilities cited above are provided only by
way of example and do not represent the full range of
applications.
[0280] Expression of antisense-RNA or double-stranded RNA by one of
the expression cassettes of the invention is especially preferred.
Also expression of sense RNA can be employed for gene silencing
(co-suppression). This RNA is preferably a non-translatable RNA.
Gene regulation by double-stranded RNA ("double-stranded RNA
interference"; dsRNAi) is well known in the art and described for
various organism including plants (e.g., Matzke 2000; Fire A et al
1998; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO
00/44895; WO 00/49035; WO 00/63364).
[0281] Genes may also be constructed or isolated, which when
transcribed produce RNA enzymes, or ribozymes, which can act as
endoribonucleases and catalyze the cleavage of RNA molecules with
selected sequences. The cleavage of selected messenger RNA's can
result in the reduced production of their encoded polypeptide
products. These genes may be used to prepare novel transgenic
plants, which possess them. The transgenic plants may possess
reduced levels of polypeptides including but not limited to the
polypeptides cited above that may be affected by antisense RNA.
[0282] It is also possible that genes may be introduced to produce
novel transgenic plants, which have reduced expression of a native
gene product, by a mechanism of cosuppression. It has been
demonstrated in tobacco, tomato, and petunia (Goring 1991; Smith
1990; Napoli 1990; van der Krol 1990) that expression of the sense
transcript of a native gene will reduce or eliminate expression of
the native gene in a manner similar to that observed for antisense
genes. The introduced gene may encode all or part of the targeted
native protein but its translation may not be required for
reduction of levels of that native protein.
1.11.2 Non-RNA-Expressing
[0283] For example, DNA elements including those of transposable
elements such as Ds, Ac, or Mu, may be, inserted into a gene and
cause mutations. These DNA elements may be inserted in order to
inactivate (or activate) a gene and thereby "tag" a particular
trait. In this instance the transposable element does not cause
instability of the tagged mutation, because the utility of the
element does not depend on its ability to move in the genome. Once
a desired trait is tagged, the introduced DNA sequence may be used
to clone the corresponding gene, e.g., using the introduced DNA
sequence as a PCR primer together with PCR gene cloning techniques
(Shapiro, 1983; Dellaporta 1988). Once identified, the entire
gene(s) for the particular trait, including control or regulatory
regions where desired may be isolated, cloned and manipulated as
desired. The utility of DNA elements introduced into an organism
for purposed of gene tagging is independent of the DNA sequence and
does not depend on any biological activity of the DNA sequence,
i.e., transcription into RNA or translation into protein. The sole
function of the DNA element is to disrupt the DNA sequence of a
gene.
[0284] It is contemplated that unexpressed DNA sequences, including
novel synthetic sequences could be introduced into cells as
proprietary "labels" of those cells and plants and seeds thereof.
It would not be necessary for a label DNA element to disrupt the
function of a gene endogenous to the host organism, as the sole
function of this DNA would be to identify the origin of the
organism. For example, one could introduce a unique DNA sequence
into a plant and this DNA element would identify all cells, plants,
and progeny of these cells as having arisen from that labeled
source. It is proposed that inclusion of label DNAs would enable
one to distinguish proprietary germplasm or germplasm derived from
such, from unlabelled germplasm.
[0285] Another possible element, which may be introduced, is a
matrix attachment region element (MAR), such as the chicken
lysozyme A element (Stief 1989), which can be positioned around an
expressible gene of interest to effect an increase in overall
expression of the gene and diminish position dependant effects upon
incorporation into the plant genome (Stief 1989; Phi-Van 1990).
[0286] Further nucleotide sequences of interest that may be
contemplated for use within the scope of the present invention in
operable linkage with the promoter sequences according to the
invention are isolated nucleic acid molecules, e.g., DNA or RNA,
comprising a plant nucleotide sequence according to the invention
comprising an open reading frame that is preferentially expressed
in a specific tissue, i.e., seed-, root, green tissue (leaf and
stem), panicle-, or pollen, or is expressed constitutively.
2. Marker Genes
[0287] In order to improve the ability to identify transformants,
one may desire to employ a selectable or screenable marker gene as,
or in addition to, the expressible gene of interest. "Marker genes"
are genes that impart a distinct phenotype to cells expressing the
marker gene and thus allow such transformed cells to be
distinguished from cells that do not have the marker. Such genes
may encode either a selectable or screenable marker, depending on
whether the marker confers a trait which one can `select` for by
chemical means, i.e., through the use of a selective agent (e.g., a
herbicide, antibiotic, or the like), or whether it is simply a
trait that one can identify through observation or testing, i.e.,
by `screening` (e.g., the R-locus trait, the green fluorescent
protein (GFP)). Of course, many examples of suitable marker genes
are known to the art and can be employed in the practice of the
invention.
[0288] Included within the terms selectable or screenable marker
genes are also genes which encode a "secretable marker" whose
secretion can be detected as a means of identifying or selecting
for transformed cells. Examples include markers, which encode a
secretable antigen that can be identified by antibody interaction,
or even secretable enzymes, which can be detected by their
catalytic activity. Secretable proteins fall into a number of
classes, including small, diffusible proteins detectable, e.g., by
ELISA; small active enzymes detectable in extracellular solution
(e.g., alpha-amylase, beta-lactamase, phosphinothricin
acetyltransferase); and proteins that are inserted or trapped in
the cell wall (e.g., proteins that include a leader sequence such
as that found in the expression unit of extensin or tobacco
PR-S).
[0289] With regard to selectable secretable markers, the use of a
gene that encodes a protein that becomes sequestered in the cell
wall, and which protein includes a unique epitope is considered to
be particularly advantageous. Such a secreted antigen marker would
ideally employ an epitope sequence that would provide low
background in plant tissue, a promoter-leader sequence that would
impart efficient expression and targeting across the plasma
membrane, and would produce protein that is bound in the cell wall
and yet accessible to antibodies. A normally secreted wall protein
modified to include a unique epitope would satisfy all such
requirements.
[0290] One example of a protein suitable for modification in this
manner is extensin, or hydroxyproline rich glycoprotein (HPRG). For
example, the maize HPRG (Steifel 1990) molecule is well
characterized in terms of molecular biology, expression and protein
structure. However, any one of a variety of ultilane and/or
glycine-rich wall proteins (Keller 1989) could be modified by the
addition of an antigenic site to create a screenable marker.
[0291] One exemplary embodiment of a secretable screenable marker
concerns the use of a maize sequence encoding the wall protein
HPRG, modified to include a 15 residue epitope from the pro-region
of murine interleukin, however, virtually any detectable epitope
may be employed in such embodiments, as selected from the extremely
wide variety of antigen-antibody combinations known to those of
skill in the art. The unique extracellular epitope can then be
straightforwardly detected using antibody labeling in conjunction
with chromogenic or fluorescent adjuncts.
[0292] Elements of the present disclosure may be exemplified in
detail through the use of the bar and/or GUS genes, and also
through the use of various other markers. Of course, in light of
this disclosure, numerous other possible selectable and/or
screenable marker genes will be apparent to those of skill in the
art in addition to the one set forth herein below. Therefore, it
will be understood that the following discussion is exemplary
rather than exhaustive. In light of the techniques disclosed herein
and the general recombinant techniques which are known in the art,
the present invention renders possible the introduction of any
gene, including marker genes, into a recipient cell to generate a
transformed plant.
2.1 Selectable Markers
[0293] Various selectable markers are known in the art suitable for
plant transformation. Such markers may include but are not limited
to:
2.1.1 Negative Selection Markers
[0294] Negative selection markers confer a resistance to a biocidal
compound such as a metabolic inhibitor (e.g.,
2-deoxyglucose-6-phosphate, WO 98/45456), antibiotics (e.g.,
kanamycin, G 418, bleomycin or hygromycin) or herbicides (e.g.,
phosphinothricin or glyphosate). Transformed plant material (e.g.,
cells, tissues or plantlets), which express marker genes, are
capable of developing in the presence of concentrations of a
corresponding selection compound (e.g., antibiotic or herbicide),
which suppresses growth of an untransformed wild type tissue.
Especially preferred negative selection markers are those, which
confer resistance to herbicides. Examples, which may be mentioned,
are: [0295] Phosphinothricin acetyltransferases (PAT; also named
Bialophos.RTM. resistance; bar; de Block 1987; Vasil 1992, 1993;
Weeks 1993; Becker 1994; Nehra 1994; Wan & Lemaux 1994; EP 0
333 033; U.S. Pat. No. 4,975,374). Preferred are the bar gene from
Streptomyces hygroscopicus or the pat gene from Streptomyces
viridochromogenes. PAT inactivates the active ingredient in the
herbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutamine
synthetase, (Murakami 1986; Twell 1989) causing rapid accumulation
of ammonia and cell death. [0296] altered
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) conferring
resistance to Glyphosate.RTM. (N-(phosphonomethyl)glycine) (Hinchee
1988; Shah 1986; Della-Cioppa 1987). Where a mutant EPSP synthase
gene is employed, additional benefit may be realized through the
incorporation of a suitable chloroplast transit peptide, CTP (EP-A1
0 218 571). [0297] Glyphosate.RTM. degrading enzymes
(Glyphosate.RTM. oxidoreductase; gox), [0298] Dalapon.RTM.
inactivating dehalogenases (deh) [0299] sulfonylurea- and/or
imidazolinone-inactivating acetolactate synthases (ahas or ALS; for
example mutated ahas/ALS variants with, for example, the S4, XI12,
XA17, and/or Hra mutation (EP-A1 154 204) [0300] Bromoxynil.RTM.
degrading nitrilases (bxn; Stalker 1988) [0301] Kanamycin- or
geneticin (G418) resistance genes (NPTII; NPT or neo; Potrykus
1985) coding e.g., for neomycin phosphotransferases (Fraley 1983;
Nehra 1994) [0302] 2-Desoxyglucose-6-phosphate phosphatase
(DOG.RTM.1-Gene product; WO 98/45456; EP 0 807 836) conferring
resistance against 2-desoxyglucose (Randez-Gil 1995). [0303]
hygromycin phosphotransferase (HPT), which mediates resistance to
hygromycin (Vanden Elzen 1985). [0304] altered dihydrofolate
reductase (Eichholtz 1987) conferring resistance against
methotrexat (Thillet 1988); [0305] mutated anthranilate synthase
genes that confers resistance to 5-methyl tryptophan.
[0306] Additional negative selectable marker genes of bacterial
origin that confer resistance to antibiotics include the aadA gene,
which confers resistance to the antibiotic spectinomycin,
gentamycin acetyl transferase, streptomycin phosphotransferase
(SPT), aminoglycoside-3-adenyl transferase and the bleomycin
resistance determinant (Hayford 1988; Jones 1987; Svab 1990; Hille
1986).
[0307] Especially preferred are negative selection markers that
confer resistance against the toxic effects imposed by D-amino
acids like e.g., D-alanine and D-serine (WO 03/060133; Erikson
2004). Especially preferred as negative selection marker in this
contest are the daoI gene (EC: 1.4.3.3: GenBank Acc.-No.: U60066)
from the yeast Rhodotorula gracilis (Rhodosporidium toruloides) and
the E. coli gene dsdA (D-serine dehydratase (D-serine deaminase)
[EC: 4.3.1.18; GenBankAcc.-No.: J01603).
[0308] Transformed plant material (e.g., cells, embryos, tissues or
plantlets) which express such marker genes are capable of
developing in the presence of concentrations of a corresponding
selection compound (e.g., antibiotic or herbicide) which suppresses
growth of an untransformed wild type tissue. The resulting plants
can be bred and hybridized in the customary fashion. Two or more
generations should be grown in order to ensure that the genomic
integration is stable and hereditary. Corresponding methods are
described (Jenes 1993; Potrykus 1991).
[0309] Furthermore, reporter genes can be employed to allow visual
screening, which may or may not (depending on the type of reporter
gene) require supplementation with a substrate as a selection
compound.
[0310] Various time schemes can be employed for the various
negative selection marker genes. In case of resistance genes (e.g.,
against herbicides or D-amino acids) selection is preferably
applied throughout callus induction phase for about 4 weeks and
beyond at least 4 weeks into regeneration. Such a selection scheme
can be applied for all selection regimes. It is furthermore
possible (although not explicitly preferred) to remain the
selection also throughout the entire regeneration scheme including
rooting.
[0311] For example, with the phosphinotricin resistance gene (bar)
as the selective marker, phosphinotricin at a concentration of from
about 1 to 50 mg/l may be included in the medium. For example, with
the dao1 gene as the selective marker, D-serine or D-alanine at a
concentration of from about 3 to 100 mg/l may be included in the
medium. Typical concentrations for selection are 20 to 40 mg/l. For
example, with the mutated ahas genes as the selective marker,
PURSUIT.TM. at a concentration of from about 3 to 100 mg/1 may be
included in the medium. Typical concentrations for selection are 20
to 40 mg/l.
2.1.2 Positive Selection Marker
[0312] Furthermore, positive selection marker can be employed.
Genes like isopentenyl-transferase from Agrobacterium tumefaciens
(strain:PO22; Genbank Acc.-No.: AB025109) may--as a key enzyme of
the cytokinin biosynthesis--facilitate regeneration of transformed
plants (e.g., by selection on cytokinin-free medium). Corresponding
selection methods are described (Ebinuma 2000a,b). Additional
positive selection markers, which confer a growth advantage to a
transformed plant in comparison with a non-transformed one, are
described e.g., in EP-A 0 601 092. Growth stimulation selection
markers may include (but shall not be limited to)
.quadrature.-Glucuronidase (in combination with e.g., a cytokinin
glucuronide), mannose-6-phosphate isomerase (in combination with
mannose), UDP-galactose-4-epimerase (in combination with e.g.,
galactose), wherein mannose-6-phosphate isomerase in combination
with mannose is especially preferred.
2.1.3 Counter-Selection Marker
[0313] Counter-selection markers are especially suitable to select
organisms with defined deleted sequences comprising said marker
(Koprek 1999). Examples for counter-selection marker comprise
thymidin kinases (TK), cytosine deaminases (Gleave 1999; Perera
1993; Stougaard 1993), cytochrom P450 proteins (Koprek 1999),
haloalkan dehalogenases (Naested 1999), iaaH gene products
(Sundaresan 1995), cytosine deaminase codA (Schlaman & Hooykaas
1997), tms2 gene products (Fedoroff & Smith 1993), or
.quadrature.-naphthalene acetamide (NAM; Depicker 1988). Counter
selection markers may be useful in the construction of transposon
tagging lines. For example, by marking an autonomous transposable
element such as Ac, Master Mu, or En/Spn with a counter selection
marker, one could select for transformants in which the autonomous
element is not stably integrated into the genome. This would be
desirable, for example, when transient expression of the autonomous
element is desired to activate in trans the transposition of a
defective transposable element, such as Ds, but stable integration
of the autonomous element is not desired. The presence of the
autonomous element may not be desired in order to stabilize the
defective element, i.e., pre-vent it from further transposing.
However, it is proposed that if stable integration of an autonomous
transposable element is desired in a plant the presence of a
negative selectable marker may make it possible to eliminate the
autonomous element during the breeding process.
2.2. Screenable Markers
[0314] Screenable markers that may be employed include, but are not
limited to, a beta-glucuronidase (GUS) or uidA gene which encodes
an enzyme for which various chromogenic substrates are known; an
R-locus gene, which encodes a product that regulates the production
of anthocyanin pigments (red color) in plant tissues (Dellaporta
1988); a beta-lactamase gene (Sutcliffe 1978), which encodes an
enzyme for which various chromogenic substrates are known (e.g.,
PADAC, a chromogenic cephalosporin); a xyIE gene (Zukowsky 1983)
which encodes a catechol dioxygenase that can convert chromogenic
catechols; an .quadrature.-amylase gene (Ikuta 1990); a tyrosinase
gene (Katz 1983) which encodes an enzyme capable of oxidizing
tyrosine to DOPA and dopaquinone which in turn condenses to form
the easily detectable compound melanin; .quadrature.-galactosidase
gene, which encodes an enzyme for which there are chromogenic
substrates; a luciferase (lux) gene (Ow 1986), which allows for
bioluminescence detection; or even an aequorin gene (Prasher 1985),
which may be employed in calcium-sensitive bioluminescence
detection, or a green fluorescent protein gene (Niedz 1995).
[0315] Genes from the maize R gene complex are contemplated to be
particularly useful as screenable markers. The R gene complex in
maize encodes a protein that acts to regulate the production of
anthocyanin pigments in most seed and plant tissue. A gene from the
R gene complex was applied to maize transformation, because the
expression of this gene in transformed cells does not harm the
cells. Thus, an R gene introduced into such cells will cause the
expression of a red pigment and, if stably incorporated, can be
visually scored as a red sector. If a maize line is dominant for
genes encoding the enzymatic intermediates in the anthocyanin
biosynthetic pathway (C2, A1, A2, Bz1 and Bz2), but carries a
recessive allele at the R locus, transformation of any cell from
that line with R will result in red pigment formation. Exemplary
lines include Wisconsin 22 which contains the rg-Stadler allele and
TR112, a K55 derivative which is r-g, b, PR. Alternatively any
genotype of maize can be utilized if the C1 and R alleles are
introduced together.
[0316] It is further proposed that R gene regulatory regions may be
employed in chimeric constructs in order to provide mechanisms for
controlling the expression of chimeric genes. More diversity of
phenotypic expression is known at the R locus than at any other
locus (Coe 1988). It is contemplated that regulatory regions
obtained from regions 5' to the structural R gene would be valuable
in directing the expression of genes, e.g., insect resistance,
drought resistance, herbicide tolerance or other protein coding
regions. For the purposes of the pre-sent invention, it is believed
that any of the various R gene family members may be successfully
employed (e.g., P, S, Lc, etc.). However, the most preferred will
generally be Sn (particularly Sn:bol3). Sn is a dominant member of
the R gene complex and is functionally similar to the R and B loci
in that Sn controls the tissue specific deposition of anthocyanin
pigments in certain seedling and plant cells, therefore, its
phenotype is similar to R.
[0317] A further screenable marker contemplated for use in the
present invention is firefly luciferase, encoded by the lux gene.
The presence of the lux gene in transformed cells may be detected
using, for example, X-ray film, scintillation counting, fluorescent
spectrophotometry, low-light video cameras, photon counting cameras
or multiwell luminometry. It is also envisioned that this system
may be developed for populational screening for bioluminescence,
such as on tissue culture plates, or even for whole plant
screening. Where use of a screenable marker gene such as lux or GFP
is desired, benefit may be realized by creating a gene fusion
between the screenable marker gene and a selectable marker gene,
for example, a GFP-NPTII gene fusion. This could allow, for
example, selection of transformed cells followed by screening of
transgenic plants or seeds.
3. Exemplary DNA Molecules
[0318] The invention provides an isolated nucleic acid molecule,
e.g., DNA or RNA, comprising a plant nucleotide sequence comprising
an open reading frame that is preferentially expressed in a
specific plant tissue, i.e., in seeds, roots, green tissue (leaf
and stem), panicles or pollen, or is expressed constitutively, or a
promoter thereof.
[0319] These promoters include, but are not limited to,
constitutive, inducible, temporally regulated, developmentally
regulated, spatially-regulated, chemically regulated,
stress-responsive, tissue-specific, viral and synthetic promoters.
Promoter sequences are known to be strong or weak. A strong
promoter provides for a high level of gene expression, whereas a
weak promoter provides for a very low level of gene expression. An
inducible promoter is a promoter that provides for the turning on
and off of gene expression in response to an exogenously added
agent, or to an environmental or developmental stimulus. A
bacterial promoter such as the P.sub.tac promoter can be induced to
varying levels of gene expression depending on the level of
isothiopropylgalactoside added to the transformed bacterial cells.
An isolated promoter sequence that is a strong promoter for
heterologous nucleic acid is advantageous because it provides for a
sufficient level of gene expression to allow for easy detection and
selection of transformed cells and provides for a high level of
gene expression when desired.
[0320] Within a plant promoter region there are several domains
that are necessary for full function of the promoter. The first of
these domains lies immediately upstream of the structural gene and
forms the "core promoter region" containing consensus sequences,
normally 70 base pairs immediately upstream of the gene. The core
promoter region contains the characteristic CAAT and TATA boxes
plus surrounding sequences, and represents a transcription
initiation sequence that defines the transcription start point for
the structural gene.
[0321] The presence of the core promoter region defines a sequence
as being a promoter: if the region is absent, the promoter is
non-functional. Furthermore, the core promoter region is
insufficient to provide full promoter activity. A series of
regulatory sequences upstream of the core constitute the remainder
of the promoter. The regulatory sequences determine expression
level, the spatial and temporal pattern of expression and, for an
important subset of promoters, expression under inductive
conditions (regulation by external factors such as light,
temperature, chemicals, hormones).
[0322] Regulated expression of the chimeric transacting viral
replication protein can be further regulated by other genetic
strategies. For example, Cre-mediated gene activation as described
by Odell et al. 1990. Thus, a DNA fragment containing 3' regulatory
sequence bound by lox sites between the promoter and the
replication protein coding sequence that blocks the expression of a
chimeric replication gene from the promoter can be removed by
Cre-mediated excision and result in the expression of the
trans-acting replication gene. In this case, the chimeric Cre gene,
the chimeric trans-acting replication gene, or both can be under
the control of tissue- and developmental-specific or inducible
promoters. An alternate genetic strategy is the use of tRNA
suppressor gene. For example, the regulated expression of a tRNA
suppressor gene can conditionally control expression of a
trans-acting replication protein coding sequence containing an
appropriate termination codon as described by Ulmasov et al. 1997.
Again, either the chimeric tRNA suppressor gene, the chimeric
transacting replication gene, or both can be under the control of
tissue- and developmental-specific or inducible promoters.
[0323] Frequently it is desirable to have continuous or inducible
expression of a DNA sequence throughout the cells of an organism in
a tissue-independent manner. For example, increased resistance of a
plant t6 infection by soil- and airborne-pathogens might be
accomplished by genetic manipulation of the plants genome to
comprise a continuous promoter operably linked to a heterologous
pathogen-resistance gene such that pathogen-resistance proteins are
continuously expressed throughout the plant's tissues.
[0324] Alternatively, it might be desirable to inhibit expression
of a native DNA sequence within the seeds of a plant to achieve a
desired phenotype. In this case, such inhibition might be
accomplished with transformation of the plant to comprise a
promoter operably linked to an antisense nucleotide sequence, such
that seed-preferential or seed-specific expression of the antisense
sequence produces an RNA transcript that interferes with
translation of the mRNA of the native DNA sequence.
[0325] To define a minimal promoter region, a DNA segment
representing the promoter region is removed from the 5' region of
the gene of interest and operably linked to the coding sequence of
a marker (reporter) gene by recombinant DNA techniques well known
to the art. The reporter gene is operably linked downstream of the
promoter, so that transcripts initiating at the promoter proceed
through the reporter gene. Reporter genes generally encode
proteins, which are easily measured, including, but not limited to,
chloramphenicol acetyl transferase (CAT), beta-glucuronidase (GUS),
green fluorescent protein (GFP), beta-galactosidase (beta-GAL), and
luciferase.
[0326] The construct containing the reporter gene under the control
of the promoter is then introduced into an appropriate cell type by
transfection techniques well known to the art. To assay for the
reporter protein, cell lysates are prepared and appropriate assays,
which are well known in the art, for the reporter protein are
performed. For example, if CAT were the reporter gene of choice,
the lysates from cells transfected with constructs containing CAT
under the control of a promoter under study are mixed with
isotopically labeled chloramphenicol and acetyl-coenzyme A
(acetyl-CoA). The CAT enzyme transfers the acetyl group from
acetyl-CoA to the 2- or 3-position of chloramphenicol. The reaction
is monitored by thin-layer chromatography, which separates
acetylated chloramphenicol from unreacted material. The reaction
products are then visualized by autoradiography.
[0327] The level of enzyme activity corresponds to the amount of
enzyme that was made, which in turn reveals the level of expression
from the promoter of interest. This level of expression can be
compared to other promoters to determine the relative strength of
the promoter under study. In order to be sure that the level of
expression is determined by the promoter, rather than by the
stability of the mRNA, the level of the reporter mRNA can be
measured directly, such as by Northern blot analysis.
[0328] Once activity is detected, mutational and/or deletional
analyses may be employed to determine the minimal region and/or
sequences required to initiate transcription. Thus, sequences can
be deleted at the 5' end of the promoter region and/or at the 3'
end of the promoter region, and nucleotide substitutions
introduced. These constructs are then introduced to cells and their
activity determined.
[0329] In one embodiment, the promoter may be a gamma zein
promoter, an oleosin ole16 promoter, a globulins promoter, an actin
I promoter, an actin cl promoter, a sucrose synthetase promoter, an
INOPS promoter, an EXMS promoter, a globulin2 promoter, a b-32,
ADPG-pyrophosphorylase promoter, an LtpI promoter, an Ltp2
promoter, an oleosin ole17 promoter, an oleosin ole18 promoter, an
actin 2 promoter, a pollen-specific protein promoter, a
pollen-specific pectate lyase promoter, an anther-specific protein
promoter, an anther-specific gene RTS2 promoter, a pollen-specific
gene promoter, a tapeturn-specific gene promoter, tapeturn-specific
gene RAB24 promoter, a anthranilate synthase alpha subunit
promoter, an alpha zein promoter, an anthranilate synthase beta
subunit promoter, a dihydrodipicolinate synthase promoter, a Thil
promoter, an alcohol dehydrogenase promoter, a cab binding protein
promoter, an H3C4 promoter, a RUBISCO SS starch branching enzyme
promoter, an ACCase promoter, an actin3 promoter, an actin7
promoter, a regulatory protein GF14-12 promoter, a ribosomal
protein L9 promoter, a cellulose biosynthetic enzyme promoter, an
S-adenosyl-L-homocysteine hydrolase promoter, a superoxide
dismutase promoter, a C-kinase receptor promoter, a
phosphoglycerate mutase promoter, a root-specific RCc3 mRNA
promoter, a glucose-6 phosphate isomerase promoter, a
pyrophosphate-fructose 6-phosphatelphosphotransferase promoter, an
ubiquitin promoter, a beta-ketoacyl-ACP synthase promoter, a 33 kDa
photosystem 11 promoter, an oxygen evolving protein promoter, a 69
kDa vacuolar ATPase subunit promoter, a metallothionein-like
protein promoter, a glyceraldehyde-3-phosphate dehydrogenase
promoter, an ABA- and ripening-inducible-like protein promoter, a
phenylalanine ammonia lyase promoter, an adenosine triphosphatase
S-adenosyl-L-homocysteine hydrolase promoter, an a-tubulin
promoter, a cab promoter, a PEPCase promoter, an R gene promoter, a
lectin promoter, a light harvesting complex promoter, a heat shock
protein promoter, a chalcone synthase promoter, a zein promoter, a
globulin-1 promoter, an ABA promoter, an auxin-binding protein
promoter, a UDP glucose flavonoid glycosyl-transferase gene
promoter, an NTI promoter, an actin promoter, an opaque 2 promoter,
a b70 promoter, an oleosin promoter, a CaMV 35S promoter, a CaMV
34S promoter, a CaMV 19S promoter, a histone promoter, a
turgor-inducible promoter, a pea small subunit RuBP carboxylase
promoter, a Ti plasmid mannopine synthase promoter, Ti plasmid
nopaline synthase promoter, a petunia chalcone isomerase promoter,
a bean glycine rich protein I promoter, a CaMV 35S transcript
promoter, a potato patatin promoter, or a S-E9 small subunit RuBP
carboxylase promoter.
4. Transformed (Transgenic) Plants of the Invention and Methods of
Preparation
[0330] Plant species may be transformed with the DNA construct of
the present invention by the DNA-mediated transformation of plant
cell protoplasts and subsequent regeneration of the plant from the
transformed protoplasts in accordance with procedures well known in
the art.
[0331] Any plant tissue capable of subsequent clonal propagation,
whether by organogenesis or embryogenesis, may be transformed with
a vector of the present invention. The term "organogenesis," as
used herein, means a process by which shoots and roots are
developed sequentially from meristematic centers; the term
"embryogenesis," as used herein, means a process by which shoots
and roots develop together in a concerted fashion (not
sequentially), whether from somatic cells or gametes. The
particular tissue chosen will vary depending on the clonal
propagation systems available for, and best suited to, the
particular species being transformed. Exemplary tissue targets
include leaf disks, pollen, embryos, cotyledons, hypocotyls,
megagametophytes, callus tissue, existing meristematic tissue
(e.g., apical meristems, axillary buds, and root meristems), and
induced meristem tissue (e.g., cotyledon meristem and ultilane
meristem).
[0332] Plants of the present invention may take a variety of forms.
The plants may be chimeras of transformed cells and non-transformed
cells; the plants may be clonal transformants (e.g., all cells
transformed to contain the expression cassette); the plants may
comprise grafts of transformed and untransformed tissues (e.g., a
transformed root stock grafted to an untransformed scion in citrus
species). The transformed plants may be propagated by a variety of
means, such as by clonal propagation or classical breeding
techniques. For example, first generation (or T1) transformed
plants may be selfed to give homozygous second generation (or T2)
transformed plants, and the T2 plants further propagated through
classical breeding techniques. A dominant selectable marker (such
as npt II) can be associated with the expression cassette to assist
in breeding.
[0333] Thus, the present invention provides a transformed
(transgenic) plant cell, in planta or ex planta, including a
transformed plastid or other organelle, e.g., nucleus, mitochondria
or chloroplast. The present invention may be used for
transformation of any plant species, including, but not limited to,
cells from the plant species specified above in the DEFINITION
section. Preferably, transgenic plants of the present invention are
crop plants and in particular cereals (for example, corn, alfalfa,
sunflower, rice, Brassica, canola, soybean, barley, soybean,
sugarbeet, cotton, safflower, peanut, sorghum, wheat, millet,
tobacco, Linum usitatissimum (linseed and fax), Camelina sativa,
Brassica juncea, etc.), and even more preferably corn, rice and
soybean. Other embodiments of the invention are related to cells,
cell cultures, tissues, parts (such as plants organs, leaves,
roots, etc.) and propagation material (such as seeds) of such
plants.
[0334] The transgenic expression cassette of the invention may not
only be comprised in plants or plant cells but may advantageously
also be containing in other organisms such for example bacteria.
Thus, another embodiment of the invention relates to transgenic
cells or non-human, transgenic organisms comprising an expression
cassette of the invention. Preferred are prokaryotic and eukaryotic
organisms. Both microorganism and higher organisms are comprised.
Preferred microorganisms are bacteria, yeast, algae, and fungi.
Preferred bacteria are those of the genus Escherichia, Erwinia,
Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus
or Cyanobacterim such as--for example--Synechocystis and other
bacteria described in Brock Biology of Microorganisms Eighth
Edition (pages A-8, A-9, A10 and A11).
[0335] Especially preferred are microorganisms capable to infect
plants and to transfer DNA into their genome, especially bacteria
of the genus Agrobacterium, preferably Agrobacterium tumefaciens
and rhizogenes. Preferred yeasts are Candida, Saccharomyces,
Hansenula and Pichia. Preferred Fungi are Aspergillus, Trichoderma,
Ashbya, Neurospora, Fusarium, and Beauveria. Most preferred are
plant organisms as defined above.
[0336] Transformation of plants can be undertaken with a single DNA
molecule or multiple DNA molecules (i.e., co-transformation), and
both these techniques are suitable for use with the expression
cassettes of the present invention. Numerous transformation vectors
are available for plant transformation, and the expression
cassettes of this invention can be used in conjunction with any
such vectors. The selection of vector will depend upon the
preferred transformation technique and the target species for
transformation.
[0337] A variety of techniques are available and known to those
skilled in the art for introduction of constructs into a plant cell
host. These techniques generally include transformation with DNA
employing A. tumefaciens or A. rhizogenes as the transforming
agent, liposomes, PEG precipitation, electroporation, DNA
injection, direct DNA uptake, microprojectile bombardment, particle
acceleration, and the like (See, for example, EP 295959 and EP
138341) (see below). However, cells other than plant cells may be
transformed with the expression cassettes of the invention. The
general descriptions of plant expression vectors and reporter
genes, and Agrobacterium and Agrobacterium-mediated gene transfer,
can be found in Gruber et al. (1993).
[0338] Expression vectors containing genomic or synthetic fragments
can be introduced into protoplasts or into intact tissues or
isolated cells. Preferably expression vectors are introduced into
intact tissue. General methods of culturing plant tissues are
provided for example by Maki et al., (1993); and by Phillips et al.
(1988). Preferably, expression vectors are introduced into maize or
other plant tissues using a direct gene transfer method such as
microprojectile-mediated delivery, DNA injection, electroporation
and the like. More preferably expression vectors are introduced
into plant tissues using the microprojectile media delivery with
the biolistic device. See, for example, Tomes et al. (1995). The
vectors of the invention can not only be used for expression of
structural genes but may also be used in exon-trap cloning, or
promoter trap procedures to detect differential gene expression in
varieties of tissues (Lindsey 1993; Auch & Reth 1990).
[0339] It is particularly preferred to use the binary type vectors
of Ti and Ri plasmids of Agrobacterium spp. Ti-derived vectors
transform a wide variety of higher plants, including
monocotyledonous and dicotyledonous plants, such as soybean,
cotton, rape, tobacco, and rice (Pacciotti 1985: Byrne 1987;
Sukhapinda 1987; Lorz 1985; Potrykus, 1985; Park 1985: Hiei 1994).
The use of T-DNA to transform plant cells has received extensive
study and is amply described (EP 120516; Hoekema, 1985; Knauf,
1983; and An 1985). For introduction into plants, the chimeric
genes of the invention can be inserted into binary vectors as
described in the examples.
[0340] Other transformation methods are available to those skilled
in the art, such as direct uptake of foreign DNA constructs (see EP
295959), techniques of electroporation (Fromm 1986) or high
velocity ballistic bombardment with metal particles coated with the
nucleic acid constructs (Kline 1987, and U.S. Pat. No. 4,945,050).
Once transformed, the cells can be regenerated by those skilled in
the art. Of particular relevance are the recently described methods
to transform foreign genes into commercially important crops, such
as rapeseed (De Block 1989), sunflower (Everett 1987), soybean
(McCabe 1988; Hinchee 1988; Chee 1989; Christou 1989; EP 301749),
rice (Hiei 1994), and corn (Gordon-Kamm 1990; Fromm 1990).
[0341] Those skilled in the art will appreciate that the choice of
method might depend on the type of plant, i.e., monocotyledonous or
dicotyledonous, targeted for transformation. Suitable methods of
transforming plant cells include, but are not limited to,
microinjection (Crossway 1986), electroporation (Riggs 1986),
Agrobacterium-mediated transformation (Hinchee 1988), direct gene
transfer (Paszkowski 1984), and ballistic particle acceleration
using devices available from Agracetus, Inc., Madison, Wis. And
BioRad, Hercules, Calif. (see, for example, U.S. Pat. No.
4,945,050; and McCabe 1988). Also see, Weissinger 1988; Sanford
1987 (onion); Christou 1988 (soybean); McCabe 1988 (soybean); Danta
1990 (rice); Klein 1988 (maize); Klein 1988 (maize); Klein 1988
(maize); Fromm 1990 (maize); and Gordon-Kamm 1990 (maize); Svab
1990 (tobacco chloroplast); Koziel 1993 (maize); Shimamoto 1989
(rice); Christou 1991 (rice); European Patent Application EP 0 332
581 (orchardgrass and other Pooideae); Vasil 1993 (wheat); Weeks
1993 (wheat).
[0342] In another embodiment, a nucleotide sequence of the present
invention is directly transformed into the plastid genome. Plastid
transformation technology is extensively described in U.S. Pat.
Nos. 5,451,513, 5,545,817, and 5,545,818, in PCT application no. WO
95116783, and in McBride et al., 1994. The basic technique for
chloroplast transformation involves introducing regions of cloned
plastid DNA flanking a selectable marker together with the gene of
interest into a suitable target tissue, e.g., using biolistics or
protoplast transformation (e.g., calcium chloride or PEG mediated
transformation). The 1 to 1.5 kb flanking regions, termed targeting
sequences, facilitate orthologous recombination with the plastid
genome and thus allow the replacement or modification of specific
regions of the plastome. Initially, point mutations in the
chloroplast 16S rRNA and rps12 genes conferring resistance to
spectinomycin and/or streptomycin are utilized as selectable
markers for transformation (Svab 1990; Staub 1992). This resulted
in stable homoplasmic transformants at a frequency of approximately
one per 100 bombardments of target leaves. The presence of cloning
sites between these markers allowed creation of a plastid-targeting
vector for introduction of foreign genes (Staub 1993). Substantial
increases in transformation frequency are obtained by replacement
of the recessive rRNA or r-protein antibiotic resistance genes with
a dominant selectable marker, the bacterial aadA gene encoding the
spectinomycin-detoxifying enzyme
aminoglycoside-3N-adenyltransferase (Svab 1993). Other selectable
markers useful for plastid transformation are known in the art and
encompassed within the scope of the invention. Typically,
approximately 15-20 cell division cycles following transformation
are required to reach a homoplastidic state. Plastid expression, in
which genes are inserted by orthologous recombination into all of
the several thousand copies of the circular plastid genome present
in each plant cell, takes advantage of the enormous copy number
advantage over nuclear-expressed genes to permit expression levels
that can readily exceed 10% of the total soluble plant protein. In
a preferred embodiment, a nucleotide sequence of the present
invention is inserted into a plastid-targeting vector and
transformed into the plastid genome of a desired plant host. Plants
homoplastic for plastid genomes containing a nucleotide sequence of
the pre-sent invention are obtained, and are preferentially capable
of high expression of the nucleotide sequence.
[0343] Agrobacterium tumefaciens cells containing a vector
comprising an expression cassette of the present invention, wherein
the vector comprises a Ti plasmid, are useful in methods of making
transformed plants. Plant cells are infected with an Agrobacterium
tumefaciens as described above to produce a transformed plant cell,
and then a plant is regenerated from the transformed plant cell.
Numerous Agrobacterium vector systems useful in carrying out the
present invention are known.
[0344] Various Agrobacterium strains can be employed, preferably
disarmed Agrobacterium tumefaciens or rhizogenes strains. In a
preferred embodiment, Agrobacterium strains for use in the practice
of the invention include octopine strains, e.g., LBA4404 or
agropine strains, e.g., EHA101 or EHA105. Suitable strains of A.
tumefaciens for DNA transfer are for example EHA101[pEHA101] (Hood
1986), EHA105[pEHA105] (Li 1992), LBA4404[pAL4404] (Hoekema 1983),
C58C1[pMP90] (Koncz & Schell 1986), and C58C1[pGV2260]
(Deblaere 1985). Other suitable strains are Agrobacterium
tumefaciens C58, a nopaline strain. Other suitable strains are A.
tumefaciens C58C1 (Van Larebeke 1974), A136 (Watson 1975) or
LBA4011 (Klapwijk 1980). In another preferred embodiment the
soil-borne bacterium is a disarmed variant of Agrobacterium
rhizogenes strain K599 (NCPPB 2659). Preferably, these strains are
comprising a disarmed plasmid variant of a Ti- or Ri-plasmid
providing the functions required for T-DNA transfer into plant
cells (e.g., the vir genes). In a preferred embodiment, the
Agrobacterium strain used to transform the plant tissue
pre-cultured with the plant phenolic compound contains a
L,L-succinamopine type Ti-plasmid, preferably disarmed, such as
pEHA101. In another preferred embodiment, the Agrobacterium strain
used to transform the plant tissue pre-cultured with the plant
phenolic compound contains an octopine-type Ti-plasmid, preferably
disarmed, such as pAL4404. Generally, when using octopine-type
Ti-plasmids or helper plasmids, it is preferred that the virF gene
be deleted or inactivated (Jarschow 1991).
[0345] The method of the invention can also be used in combination
with particular Agrobacterium strains, to further increase the
transformation efficiency, such as Agrobacterium strains wherein
the vir gene expression and/or induction thereof is altered due to
the presence of mutant or chimeric virA or virG genes (e.g. Hansen
1994; Chen and Winans 1991; Scheeren-Groot, 1994). Preferred are
further combinations of Agrobacterium tumefaciens strain LBA4404
(Hiei 1994) with super-virulent plasmids. These are preferably
pTOK246-based vectors (Ishida 1996).
[0346] A binary vector or any other vector can be modified by
common DNA recombination techniques, multiplied in E. coli, and
introduced into Agrobacterium by e.g., electroporation or other
transformation techniques (Mozo & Hooykaas 1991).
[0347] Agrobacterium is grown and used in a manner similar to that
described in Ishida (1996). The vector comprising Agrobacterium
strain may, for example, be grown for 3 days on YP medium (5 g/l
yeast extract, 10 .mu.l peptone, 5 g/l NaCl, 15 .mu.l agar, pH 6.8)
supplemented with the appropriate antibiotic (e.g., 50 mg/l
spectinomycin). Bacteria are collected with a loop from the solid
medium and resuspended. In a preferred embodiment of the invention,
Agrobacterium cultures are started by use of aliquots frozen at
-80.degree. C.
[0348] The transformation of the target tissue (e.g., an immature
embryo) by the Agrobacterium may be carried out by merely
contacting the target tissue with the Agrobacterium. The
concentration of Agrobacterium used for infection and
co-cultivation may need to be varied. For example, a cell
suspension of the Agrobacterium having a population density of
approximately from 10.sup.5-10.sup.11, preferably 10.sup.6 to
10.sup.10, more preferably about 10.sup.8 cells or cfu/ml is
prepared and the target tissue is immersed in this suspension for
about 3 to 10 minutes. The resulting target tissue is then cultured
on a solid medium for several days together with the
Agrobacterium.
[0349] Preferably, the bacterium is employed in concentration of
10.sup.6 to 10.sup.10 cfu/ml. In a preferred embodiment for the
co-cultivation step about 1 to 10 .mu.l of a suspension of the
soil-borne bacterium (e.g., Agrobacteria) in the co-cultivation
medium are directly applied to each target tissue explant and
air-dried. This is saving labor and time and is reducing unintended
Agrobacterium-mediated damage by excess Agrobacterium usage.
[0350] For Agrobacterium treatment, the bacteria are resuspended in
a plant compatible co-cultivation medium. Supplementation of the
co-culture medium with antioxidants (e.g., silver nitrate),
phenol-absorbing compounds (like polyvinylpyrrolidone, Perl 1996)
or thiol compounds (e.g., dithiothreitol, L-cysteine, Olhoft 2001)
which can decrease tissue necrosis due to plant defence responses
(like phenolic oxidation) may further improve the efficiency of
Agrobacterium-mediated transformation. In another preferred
embodiment, the co-cultivation medium of comprises least one thiol
compound, preferably selected from the group consisting of sodium
thiol sulfate, dithiotrietol (DTT) and cysteine. Preferably the
concentration is between about 1 mM and 10 mM of L-Cysteine, 0.1 mM
to 5 mM DTT, and/or 0.1 mM to 5 mM sodium thiol sulfate.
Preferably, the medium employed during co-cultivation comprises
from about 1 .mu.M to about 10 .mu.M of silver nitrate and from
about 50 mg/L to about 1,000 mg/L of L-Cystein. This results in a
highly reduced vulnerability of the target tissue against
Agrobacterium-mediated damage (such as induced necrosis) and highly
improves overall transformation efficiency.
[0351] Various vector systems can be used in combination with
Agrobacteria. Preferred are binary vector systems. Common binary
vectors are based on "broad host range"-plasmids like pRK252 (Bevan
1984) or pTJS75 (Watson 1985) derived from the P-type plasmid RK2.
Most of these vectors are derivatives of pBIN19 (Bevan 1984).
Various binary vectors are known, some of which are commercially
available such as, for example, pBI101.2 or pBIN19 (Clontech
Laboratories, Inc. USA). Additional vectors were improved with
regard to size and handling (e.g. pPZP; Hajdukiewicz 1994).
Improved vector systems are described also in WO 02/00900.
[0352] Methods using either a form of direct gene transfer or
Agrobacterium-mediated transfer usually, but not necessarily, are
undertaken with a selectable marker, which may provide resistance
to an antibiotic (e.g., kanamycin, hygromycin or methotrexate) or a
herbicide (e.g., phosphinothricin). The choice of selectable marker
for plant transformation is not, however, critical to the
invention.
[0353] For certain plant species, different antibiotic or herbicide
selection markers may be preferred. Selection markers used
routinely in transformation include the nptII gene which confers
resistance to kanamycin and related antibiotics (Messing &
Vierra, 1982; Bevan 1983), the bar gene which confers resistance to
the herbicide phosphinothricin (White 1990, Spencer 1990), the hph
gene which confers resistance to the antibiotic hygromycin
(Blochlinger & Diggelmann), and the dhfr gene, which confers
resistance to methotrexate (Bourouis 1983).
5. Production and Characterization of Stably Transformed Plants
[0354] Transgenic plant cells are then placed in an appropriate
selective medium for selection of transgenic cells, which are then
grown to callus. Shoots are grown from callus. Plantlets are
generated from the shoot by growing in rooting medium. The various
constructs normally will be joined to a marker for selection in
plant cells. Conveniently, the marker may be resistance to a
biocide (particularly an antibiotic, such as kanamycin, G418,
bleomycin, hygromycin, chloramphenicol, herbicide, or the like).
The particular marker used will allow for selection of transformed
cells as compared to cells lacking the DNA, which has been
introduced. Components of DNA constructs including transcription
cassettes of this invention may be prepared from sequences, which
are native (endogenous) or foreign (exogenous) to the host. By
"foreign" it is meant that the sequence is not found in the
wild-type host into which the construct is introduced. Heterologous
constructs will contain at least one region, which is not native to
the gene from which the transcription-initiation-region is
derived.
[0355] To confirm the presence of the transgenes in transgenic
cells and plants, a variety of assays may be performed. Such assays
include, for example, "molecular biological" assays well known to
those of skill in the art, such as Southern and Northern blotting,
in situ hybridization and nucleic acid-based amplification methods
such as PCR or RT-PCR or TaqMan; "biochemical" assays, such as
detecting the presence of a protein product, e.g., by immunological
means (ELISAs and Western blots) or by enzymatic function; plant
part assays, such as seed assays; and also, by analyzing the
phenotype of the whole regenerated plant, e.g., for disease or pest
resistance.
[0356] DNA may be isolated from cell lines or any plant parts to
determine the presence of the preselected nucleic acid segment
through the use of techniques well known to those skilled in the
art. Note that intact sequences will not always be present,
presumably due to rearrangement or deletion of sequences in the
cell.
[0357] The presence of nucleic acid elements introduced through the
methods of this invention may be determined by polymerase chain
reaction (PCR). Using these technique discreet fragments of nucleic
acid are amplified and detected by gel electrophoresis. This type
of analysis permits one to determine whether a preselected nucleic
acid segment is present in a stable transformant, but does not
prove integration of the introduced preselected nucleic acid
segment into the host cell genome. In addition, it is not possible
using PCR techniques to determine whether transformants have
exogenous genes introduced into different sites in the, genome,
i.e., whether transformants are of independent origin. It is
contemplated that using PCR techniques it would be possible to
clone fragments of the host genomic DNA adjacent to an introduced
preselected DNA segment.
[0358] Positive proof of DNA integration into the host genome and
the independent identities of transformants may be determined using
the technique of Southern hybridization. Using this technique
specific DNA sequences that were introduced into the host genome
and flanking host DNA sequences can be identified. Hence the
Southern hybridization pattern of a given transformant serves as an
identifying characteristic of that transformant. In addition it is
possible through Southern hybridization to demonstrate the presence
of introduced preselected DNA segments in high molecular weight
DNA, i.e., confirm that the introduced preselected, DNA segment has
been integrated into the host cell genome. The technique of
Southern hybridization provides information that is obtained using
PCR, e.g., the presence of a preselected DNA segment, but also
demonstrates integration into the genome and characterizes each
individual transformant.
[0359] It is contemplated that using the techniques of dot or slot
blot hybridization which are modifications of Southern
hybridization techniques one could obtain the same information that
is derived from PCR, e.g., the presence of a preselected DNA
segment.
[0360] Both PCR and Southern hybridization techniques can be used
to demonstrate transmission of a preselected DNA segment to
progeny. In most instances the characteristic Southern
hybridization pattern for a given transformant will segregate in
progeny as one or more Mendelian genes (Spencer 1992); Laursen
1994) indicating stable inheritance of the gene. The non-chimeric
nature of the callus and the parental transformants (R.sub.0) was
suggested by germline transmission and the identical Southern blot
hybridization patterns and intensities of the transforming DNA in
callus, R.sub.0 plants and R.sub.1 progeny that segregated for the
transformed gene.
[0361] Whereas DNA analysis techniques may be conducted using DNA
isolated from any part of a plant, RNA may only be expressed in
particular cells or tissue types and hence it will be necessary to
prepare RNA for analysis from these tissues. PCR techniques may
also be used for detection and quantitation of RNA produced from
introduced preselected DNA segments. In this application of PCR it
is first necessary to reverse transcribe RNA into DNA, using
enzymes such as reverse transcriptase, and then through the use of
conventional PCR techniques amplify the DNA. In most instances PCR
techniques, while useful, will not demonstrate integrity of the RNA
product. Further information about the nature of the RNA product
may be obtained by Northern blotting. This technique will
demonstrate the presence of an RNA species and give information
about the integrity of that RNA. The presence or absence of an RNA
species can also be determined using dot or slot blot Northern
hybridizations. These techniques are modifications of Northern
blotting and will only demonstrate the presence or absence of an
RNA species.
[0362] While Southern blotting and PCR may be used to detect the
preselected DNA segment in question, they do not provide
information as to whether the preselected DNA segment is being
expressed. Expression may be evaluated by specifically identifying
the protein products of the introduced preselected DNA segments or
evaluating the phenotypic changes brought about by their
expression.
[0363] Assays for the production and identification of specific
proteins may make use of physical-chemical, structural, functional,
or other properties of the proteins. Unique physical-chemical or
structural properties allow the proteins to be separated and
identified by electrophoretic procedures, such as native or
denaturing gel electrophoresis or isoelectric focusing, or by
chromatographic techniques such as ion exchange or gel exclusion
chromatography. The unique structures of individual proteins offer
opportunities for use of specific antibodies to detect their
presence in formats such as an ELISA assay. Combinations of
approaches may be employed with even greater specificity such as
Western blotting in which antibodies are used to locate individual
gene products that have been separated by electrophoretic
techniques. Additional techniques may be employed to absolutely
confirm the identity of the product of interest such as evaluation
by amino acid sequencing following purification. Although these are
among the most commonly employed, other procedures may be
additionally used.
[0364] Assay procedures may also be used to identify the expression
of proteins by their functionality, especially the ability of
enzymes to catalyze specific chemical reactions involving specific
substrates and products. These reactions may be followed by
providing and quantifying the loss of substrates or the generation
of products of the reactions by physical or chemical procedures.
Examples are as varied as the enzyme to be analyzed.
[0365] Very frequently the expression of a gene product is
determined by evaluating the phenotypic results of its expression.
These assays also may take many forms including but not limited to
analyzing changes in the chemical composition, morphology, or
physiological properties of the plant. Morphological changes may
include greater stature or thicker stalks. Most often changes in
response of plants or plant parts to imposed treatments are
evaluated under carefully controlled conditions termed
bioassays.
6. Uses of Transgenic Plants
[0366] Once an expression cassette of the invention has been
transformed into a particular plant species, it may be propagated
in that species or moved into other varieties of the same species,
particularly including commercial varieties, using traditional
breeding techniques. Particularly preferred plants of the invention
include the agronomically important crops listed above. The genetic
properties engineered into the transgenic seeds and plants
described above are passed on by sexual reproduction and can thus
be maintained and propagated in progeny plants. The present
invention also relates to a transgenic plant cell, tissue, organ,
seed or plant part obtained from the transgenic plant. Also
included within the invention are transgenic descendants of the
plant as well as transgenic plant cells, tissues, organs, seeds and
plant parts obtained from the descendants.
[0367] Preferably, the expression cassette in the transgenic plant
is sexually transmitted. In one preferred embodiment, the coding
sequence is sexually transmitted through a complete normal sexual
cycle of the R0 plant to the R1 generation. Additionally preferred,
the expression cassette is expressed in the cells, tissues, seeds
or plant of a transgenic plant in an amount that is different than
the amount in the cells, tissues, seeds or plant of a plant, which
only differs in that the expression cassette is absent.
[0368] The transgenic plants produced herein are thus expected to
be useful for a variety of commercial and research purposes.
Transgenic plants can be created for use in traditional agriculture
to possess traits beneficial to the grower (e.g., agronomic traits
such as resistance to water deficit, pest resistance, herbicide
resistance or increased yield), beneficial to the consumer of the
grain harvested from the plant (e.g., improved nutritive content in
human food or animal feed; increased vitamin, amino acid, and
antioxidant content; the production of antibodies (passive
immunization) and nutriceuticals), or beneficial to the food
processor (e.g., improved processing traits). In such uses, the
plants are generally grown for the use of their grain in human or
animal foods. Additionally, the use of root-specific promoters in
transgenic plants can provide beneficial traits that are localized
in the consumable (by animals and humans) roots of plants such as
carrots, parsnips, and beets. However, other parts of the plants,
including stalks, husks, vegetative parts, and the like, may also
have utility, including use as part of animal silage or for
ornamental purposes. Often, chemical constituents (e.g., oils or
starches) of maize and other crops are extracted for foods or
industrial use and transgenic plants may be created which have
enhanced or modified levels of such components.
[0369] Transgenic plants may also find use in the commercial
manufacture of proteins or other molecules, where the molecule of
interest is extracted or purified from plant parts, seeds, and the
like. Cells or tissue from the plants may also be cultured, grown
in vitro, or fermented to manufacture such molecules. The
transgenic plants may also be used in commercial breeding programs,
or may be crossed or bred to plants of related crop species.
Improvements encoded by the expression cassette may be transferred,
e.g., from maize cells to cells of other species, e.g., by
protoplast fusion.
[0370] The transgenic plants may have many uses in research or
breeding, including creation of new mutant plants through
insertional mutagenesis, in order to identify beneficial mutants
that might later be created by traditional mutation and selection.
An example would be the introduction of a recombinant DNA sequence
encoding a transposable element that may be used for generating
genetic variation. The methods of the invention may also be used to
create plants having unique "signature sequences" or other marker
sequences which can be used to identify proprietary lines or
varieties.
[0371] Thus, the transgenic plants and seeds according to the
invention can be used in plant breeding, which aims at the
development of plants with improved properties conferred by the
expression cassette, such as tolerance of drought, disease, or
other stresses. The various breeding steps are characterized by
well-defined human intervention such as selecting the lines to be
crossed, directing pollination of the parental lines, or selecting
appropriate descendant plants. Depending on the desired properties
different breeding measures are taken. The relevant techniques are
well known in the art and include but are not limited to
hybridization, inbreeding, backcross breeding, multilane breeding,
variety blend, interspecific hybridization, aneuploid techniques,
etc. Hybridization techniques also include the sterilization of
plants to yield male or female sterile plants by mechanical,
chemical or biochemical means. Cross-pollination of a male sterile
plant with pollen of a different line assures that the genome of
the male sterile but female fertile plant will uniformly obtain
properties of both parental lines. Thus, the transgenic seeds and
plants according to the invention can be used for the breeding of
improved plant lines, which for example increase the effectiveness
of conventional methods such as herbicide or pesticide treatment or
allow dispensing with said methods due to their modified genetic
properties. Alternatively new crops with improved stress tolerance
can be obtained which, due to their optimized genetic "equipment",
yield harvested product of better quality than products, which were
not able to tolerate comparable adverse developmental
conditions.
EXAMPLES
Materials and General Methods
[0372] Unless indicated otherwise, chemicals and reagents in the
Examples were obtained from Sigma Chemical Company (St. Louis,
Mo.), restriction endonucleases were from New England Biolabs
(Beverly, Mass.) or Roche (Indianapolis, Ind.), oligonucleotides
were synthesized by MWG Biotech Inc. (High Point, N.C.), and other
modifying enzymes or kits regarding biochemicals and molecular
biological assays were from Clontech (Palo Alto, Calif.), Pharmacia
Biotech (Piscataway, N.J.), Promega Corporation (Madison, Wis.), or
Stratagene (La Jolla, Calif.). Materials for cell culture media
were obtained from Gibco/BRL (Gaithersburg, Md.) or DIFCO (Detroit,
Mich.). The cloning steps carried out for the purposes of the
present invention, such as, for example, restriction cleavages,
agarose gel electrophoresis, purification of DNA fragments,
transfer of nucleic acids to nitrocellulose and nylon membranes,
linking DNA fragments, transformation of E. coli cells, growing
bacteria, multiplying phages and sequence analysis of recombinant
DNA, are carried out as described by Sambrook (1989). The
sequencing of recombinant DNA molecules is carried out using ABI
laser fluorescence DNA sequencer following the method of Sanger
(Sanger 1977).
Example 1
Generation of Transgenic Plants
[0373] 1.1 Generation of Transgenic Arabidopsis thaliana Plants
[0374] For generating transgenic Arabidopsis plants Agrobacterium
tumefaciens (strain C58C1[pMP90]) is transformed with the various
promoter::GUS vector constructs (see below). Resulting
Agrobacterium strains are subsequently employed to obtain
transgenic plants. For this purpose a isolated transformed
Agrobacterium colony is incubated in 4 ml culture (Medium: YEB
medium with 50 .mu.g/ml Kanamycin and 25 .mu.g/ml Rifampicin) over
night at 28.degree. C. With this culture a 400 ml culture of the
same medium is inoculated and incubated over night (28.degree. C.,
220 rpm). The bacteria a precipitated by centrifugation (GSA-Rotor,
8.000 U/min, 20 min) and the pellet is resuspended in infiltration
medium (1/2 MS-Medium; 0.5 g/l MES, pH 5.8; 50 g/l sucrose). The
suspension is placed in a plant box (Duchefa) and 100 ml SILVET
L-77 (Osi Special-ties Inc., Cat. P030196) are added to a final
concentration of 0.02%. The plant box with 8 to 12 Plants is placed
into an exsiccator for 10 to 15 min. under vacuum with subsequent,
spontaneous ventilation (expansion). This process is repeated 2-3
times. Thereafter all plants are transferred into pods with
wet-soil and grown under long daytime conditions (16 h light; day
temperature 22-24.degree. C., night temperature 19.degree. C.; 65%
rel. humidity). Seeds are harvested after 6 weeks
1.2 Generation of Transgenic Linseed
[0375] Transgenic linseed plants can be generated for example by
the method of Bell et al., 1999, In Vitro Cell. Dev. Biol.-Plant.
35(6):456-465 by means of particle bombardment.
Agrobacteria-mediated transformations can be generated for example
by the method of Mlynarova et al. (1994), Plant Cell Report 13:
282-285.
Example 2
Growth Conditions for Plants for Tissue-Specific Expression
Analysis
[0376] To obtain 4 and 7 days old seedlings, about 400 seeds
(Arabidopsis thaliana eco-type Columbia) are sterilized with a 80%
(v/v) ethanol:water solution for 2 minutes, treated with a sodium
hypochlorite solution (0.5% v/v) for 5 minutes, washed three times
with distillated water and incubated at 4.degree. C. for 4 days to
ensure a standardized germination. Subsequently, seeds are
incubated on Petri dishes with MS medium (Sigma M5519) supplemented
with 1% sucrose, 0.5 g/l MES (Sigma M8652), 0.8% Difco-BactoAgar
(Difco 0140-01), adjusted to pH 5.7. The seedlings are grown under
16 h light/8 h dark cyklus (Philips 58W/33 white light) at
22.degree. C. and harvested after 4 or 7 days, respectively.
[0377] To obtain root tissue, 100 seeds are sterilized as described
above, incubated at 4.degree. C. for 4 days, and transferred into
250 ml flasks with MS medium (Sigma M5519) supplemented with
additional 3% sucrose and 0.5 g/l MES (Sigma M8652), adjusted to pH
5.7 for further growing. The seedlings are grown at a 16 h light/8
h dark cycle (Philips 58W/33 white light) at 22.degree. C. and 120
rpm and harvested after 3 weeks. For all other plant organs
employed, seeds are sown on standard soil (Type V M, Manna-Italia,
Via S. Giacomo 42, 39050 San Giacomo/Laives, Bolzano, Italien),
incubated for 4 days at 4.degree. C. to ensure uniform germination,
and subsequently grown under a 16 h light/8 darkness regime (OSRAM
Lumi-lux Daylight 36W/12) at 22.degree. C. Young rosette leaves are
harvested at the 8-leaf stage (after about 3 weeks), mature rosette
leaves are harvested after 8 weeks briefly before stem formation.
Apices of out-shooting stems are harvested briefly after
out-shooting. Stem, stem leaves, and flower buds are harvested in
development stage 12 (Bowmann J (ed.), Arabidopsis, Atlas of
Morphology, Springer New York, 1995) prior to stamen development.
Open flowers are harvested in development stage 14 immediately
after stamen development. Wilting flow-ers are harvested in stage
15 to 16. Green and yellow shoots used for the analysis have a
length of 10 to 13 mm.
Example 3
Demonstration of Expression Profile
[0378] To demonstrate and analyze the transcription regulating
properties of a promoter of the useful to operably link the
promoter or its fragments to a reporter gene, which can be employed
to monitor its expression both qualitatively and quantitatively.
Preferably bacterial R-glucuronidase is used (Jefferson 1987).
.beta.-glucuronidase activity can be monitored in planta with
chromogenic substrates such as
5-bromo-4-Chloro-3-indolyl-.beta.-D-glucuronic acid during
corresponding activity assays (Jefferson 1987). For determination
of promoter activity and tissue specificity plant tissue is
dissected, embedded, stained and analyzed as described (e.g.,
Baumlein 1991).
[0379] For quantitative .beta.-glucuronidase activity analysis MUG
(methylumbelliferyl glucuronide) is used as a substrate, which is
converted into MU (methylumbelliferone) and glucuronic acid. Under
alkaline conditions this conversion can be quantitatively monitored
fluorometrically (excitation at 365 nm, measurement at 455 nm;
SpectroFluorimeter Thermo Life Sciences Fluoroscan) as described
(Bustos 1989).
Example 4
Cloning of the Promoter Fragments
[0380] To isolate the promoter fragments described by SEQ ID NO: 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23, 24,
25, 26, 27, 28, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86, 87, 148,
149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161, 162, 163,
164, 165, 168, 169, 170, 171, 172, 173, 174, 175, 178, 179, 180,
181, 182, 183, 184, and 185, genomic DNA is isolated from
Arabidopsis thaliana (ecotype Columbia) as described (Galbiati
2000). The isolated genomic DNA is employed as matrix DNA for a
polymerase chain reaction (PCR) mediated amplification using the
oligonucleotide primers and protocols indicated below (Table
3).
TABLE-US-00003 TABLE 3 PCR oligonucleotide primers for
amplification of the various transcription regulating nucleotide
sequences and restriction enzymes for modifying the resulting PCR
products Forward Reverse Restriction SEQ ID Promoter Primer Primer
enzymes SEQ ID NO: 1 pSUK222L SUK222for SUK222Lrev BamHI/NcoI SEQ
ID NO: 90 SEQ ID NO: 91 SEQ ID NO: 2 pSUK222LGB SUK222for
SUK222Lrev BamHI/NcoI SEQ ID NO: 90 SEQ ID NO: 91 SEQ ID NO: 3
pSUK222S SUK222for SUK222Srev BamHI/NcoI SEQ ID NO: 90 SEQ ID NO:
92 SEQ ID NO: 4 pSUK222SGB SUK222for SUK222Srev BamHI/NcoI SEQ ID
NO: 90 SEQ ID NO: 92 SEQ ID NO: 5 pSUK224L SUK224for SUK224Lrev
BamHI/NcoI SEQ ID NO: 93 SEQ ID NO: 94 SEQ ID NO: 6 pSUK224LGB
SUK224for SUK224Lrev BamHI/NcoI SEQ ID NO: 93 SEQ ID NO: 94 SEQ ID
NO: 7 pSUK224S SUK224for SUK224Srev BamHI/NcoI SEQ ID NO: 93 SEQ ID
NO: 95 SEQ ID NO: 8 pSUK224SGB SUK224for SUK224Srev BamHI/Ncol SEQ
ID NO: 93 SEQ ID NO: 95 SEQ ID NO: 9 pSUK226L SUK226for SUK226Lrev
BamHI/NcoI SEQ ID NO: 96 SEQ ID NO: 97 SEQ ID NO: 10 pSUK226LGB
SUK226for SUK226Lrev BamHI/NcoI SEQ ID NO: 96 SEQ ID NO: 97 SEQ ID
NO: 11 pSUK226S SUK226for SUK226Srev BamHI/NcoI SEQ ID NO: 96 SEQ
ID NO: 98 SEQ ID NO: 12 pSUK226SGB SUK226for SUK226Srev BamHI/NcoI
SEQ ID NO: 96 SEQ ID NO: 98 SEQ ID NO: 15 pSUK322L SUK322for
SUK322Lrev XhoI/BamHI SEQ ID NO: 99 SEQ ID NO: 100 SEQ ID NO: 16
pSUK322LGB SUK322for SUK322Lrev XhoI/BamHI SEQ ID NO: 99 SEQ ID NO:
100 SEQ ID NO: 17 pSUK322S SUK322for SUK322Srev XhoI/HindIII SEQ ID
NO: 99 SEQ ID NO: 101 SEQ ID NO: 18 pSUK322SGB SUK322for SUK322Srev
XhoI/HindIII SEQ ID NO: 99 SEQ ID NO: 101 SEQ ID NO: 19 pSUK324LGB
SUK324for SUK324Lrev EcoRI/EcoRI SEQ ID NO: 102 SEQ ID NO: 103 SEQ
ID NO: 20 pSUK324SGB SUK324for SUK324Srev EcoRI/HindIII SEQ ID NO:
102 SEQ ID NO: 104 SEQ ID NO: 23 pSUK250L SUK250for SUK250Lrev
BamHI/NcoI SEQ ID NO: 105 SEQ ID NO: 106 SEQ ID NO: 24 pSUK250LGB
SUK250for SUK250Lrev BamHI/NcoI SEQ ID NO: 105 SEQ ID NO: 106 SEQ
ID NO: 25 pSUK250S SUK250for SUK250Srev BamHI/NcoI SEQ ID NO: 105
SEQ ID NO: 107 SEQ ID NO: 26 pSUK250SGB SUK250for SUK250Srev
BamHI/NcoI SEQ ID NO: 105 SEQ ID NO: 107 SEQ ID NO: 27 pSUK252LGB
SUK252for SUK252Lrev BamHI/NcoI SEQ ID NO: 108 SEQ ID NO: 109 SEQ
ID NO: 28 pSUK252SGB SUK252for SUK252Srev BamHI/NcoI SEQ ID NO: 108
SEQ ID NO: 110 SEQ ID NO: 31 pSUK19S SUK19Sfor SUK19Srev
HindIII/XbaI SEQ ID NO: 111 SEQ ID NO: 112 SEQ ID NO: 32 pSUK19SGB
SUK19Sfor SUK19Srev HindIII/XbaI SEQ ID NO: 111 SEQ ID NO: 112 SEQ
ID NO: 33 pSUK19LGB SUK19Lfor SUK19Lrev BamHI/NcoI SEQ ID NO: 113
SEQ ID NO: 114 SEQ ID NO: 36 pSUK28L SUK28for SUK28Lrev SalI/SmaI
SEQ ID NO: 115 SEQ ID NO: 116 SEQ ID NO: 37 pSUK28LGB SUK28for
SUK28Lrev SalI/SmaI SEQ ID NO: 115 SEQ ID NO: 116 SEQ ID NO: 38
pSUK28S SUK28for SUK28Srev SalI/XhoI SEQ ID NO: 115 SEQ ID NO: 117
SEQ ID NO: 39 pSUK28SGB SUK28for SUK28Srev SalI/XhoI SEQ ID NO: 115
SEQ ID NO: 117 SEQ ID NO: 40 pSUK29L SUK29for SUK29Lrev SalI/SmaI
SEQ ID NO: 118 SEQ ID NO: 119 SEQ ID NO: 41 pSUK29LGB SUK29for
SUK29Lrev SalI/SmaI SEQ ID NO: 118 SEQ ID NO: 119 SEQ ID NO: 42
pSUK29S SUK29for SUK29Srev SalI/XhoI SEQ ID NO: 118 SEQ ID NO: 120
SEQ ID NO: 43 pSUK29SGB SUK29for SUK29Srev SalI/XhoI SEQ ID NO: 118
SEQ ID NO: 120 SEQ ID NO: 44 pSUK30L SUK30for SUK30Lrev BamHI/NcoI
SEQ ID NO: 121 SEQ ID NO: 122 SEQ ID NO: 45 pSUK30LGB SUK30for
SUK30Lrev BamHI/NcoI SEQ ID NO: 121 SEQ ID NO: 122 SEQ ID NO: 46
pSUK30S SUK30for SUK30Srev BamHI/XhoI SEQ ID NO: 121 SEQ ID NO: 123
SEQ ID NO: 47 pSUK30SGB SUK30for SUK30Srev BamHI/XhoI SEQ ID NO:
121 SEQ ID NO: 123 SEQ ID NO: 50 pSUK298L SUK298for SUK298Lrev
BamHI/NcoI SEQ ID NO: 124 SEQ ID NO: 125 SEQ ID NO: 51 pSUK298LGB
SUK298for SUK298Lrev BamHI/NcoI SEQ ID NO: 124 SEQ ID NO: 125 SEQ
ID NO: 52 pSUK298S SUK298for SUK298Srev BamHI/NcoI SEQ ID NO: 124
SEQ ID NO: 126 SEQ ID NO: 53 pSUK298SGB SUK298for SUK298Srev
BamHI/NcoI SEQ ID NO: 124 SEQ ID NO: 126 SEQ ID NO: 54 pSUK300L
SUK300for SUK300Lrev BamHI/NcoI SEQ ID NO: 127 SEQ ID NO: 128 SEQ
ID NO: 55 pSUK300LGB SUK300for SUK300Lrev BamHI/NcoI SEQ ID NO: 127
SEQ ID NO: 128 SEQ ID NO: 56 pSUK300S SUK300for SUK300Srev
BamHI/NcoI SEQ ID NO: 127 SEQ ID NO: 129 SEQ ID NO: 57 pSUK300SGB
SUK300for SUK300Srev BamHI/NcoI SEQ ID NO: 127 SEQ ID NO: 129 SEQ
ID NO: 60 pSUK292L SUK292for SUK292Lrev BamHI/NcoI SEQ ID NO: 130
SEQ ID NO: 131 SEQ ID NO: 61 pSUK292LGB SUK292for SUK292Lrev
BamHI/NcoI SEQ ID NO: 130 SEQ ID NO: 131 SEQ ID NO: 62 pSUK292S
SUK292for SUK292Srev BamHI/NcoI SEQ ID NO: 130 SEQ ID NO: 132 SEQ
ID NO: 63 pSUK292SGB SUK292for SUK292Srev BamHI/NcoI SEQ ID NO: 130
SEQ ID NO: 132 SEQ ID NO: 64 pSUK294L SUK294for SUK294Lrev
BamHI/NcoI SEQ ID NO: 133 SEQ ID NO: 134 SEQ ID NO: 65 pSUK294LGB
SUK294tor SUK294Lrev BamHI/NcoI SEQ ID NO: 133 SEQ ID NO: 134 SEQ
ID NO: 66 pSUK294S SUK294for SUK294Srev BamHI/NcoI SEQ ID NO: 133
SEQ ID NO: 135 SEQ ID NO: 67 pSUK294SGB SUK294for SUK294Srev
BamHI/NcoI SEQ ID NO: 133 SEQ ID NO: 135 SEQ ID NO: 68 pSUK296L
SUK296for SUK296Lrev BamHI/NcoI SEQ ID NO: 136 SEQ ID NO: 137 SEQ
ID NO: 69 pSUK296LGB SUK296for SUK296Lrev BamHI/NcoI SEQ ID NO: 136
SEQ ID NO: 137 SEQ ID NO: 70 pSUK296S SUK296for SUK296Srev
BamHI/NcoI SEQ ID NO: 136 SEQ ID NO: 138 SEQ ID NO: 71 pSUK296SGB
SUK296for SUK296Srev BamHI/NcoI SEQ ID NO: 136 SEQ ID NO: 138 SEQ
ID NO: 74 pSUK262L SUK262for SUK262Lrev BamHI/NcoI SEQ ID NO: 139
SEQ ID NO: 140 SEQ ID NO: 75 pSUK262LGB SUK262for SUK262Lrev
BamHI/NcoI SEQ ID NO: 139 SEQ ID NO: 140 SEQ ID NO: 76 pSUK262S
SUK262for SUK262Srev BamHI/NcoI SEQ ID NO: 139 SEQ ID NO: 141 SEQ
ID NO: 77 pSUK262SGB SUK262for SUK262Srev BamHI/NcoI SEQ ID NO: 139
SEQ ID NO: 141 SEQ ID NO: 80 pSUK164 SUK164for SUK164rev BamHI/NcoI
SEQ ID NO: 142 SEQ ID NO: 143 SEQ ID NO: 81 pSUK164GB SUK164for
SUK164rev BamHI/NcoI SEQ ID NO: 142 SEQ ID NO: 143 SEQ ID NO: 84
pSUK352L SUK352for SUK352Lrev BamHI/NcoI SEQ ID NO: 144 SEQ ID NO:
145 SEQ ID NO: 85 pSUK352LGB SUK352for SUK352Lrev BamHI/NcoI SEQ ID
NO: 144 SEQ ID NO: 145 SEQ ID NO: 86 pSUK352S SUK352for SUK352Srev
BamHI/NcoI SEQ ID NO: 144 SEQ ID NO: 146 SEQ ID NO: 87 pSUK352SGB
SUK352for SUK352Srev BamHI/NcoI SEQ ID NO: 144 SEQ ID NO: 146 SEQ
ID NO: 148 pSUK40L SUK40for SUK40Lrev BamHI/NcoI SEQ ID NO: 188 SEQ
ID NO: 189 SEQ ID NO: 149 pSUK40LGB SUK40for SUK40Lrev BamHI/NcoI
SEQ ID NO: 188 SEQ ID NO: 189 SEQ ID NO: 150 pSUK40S SUK40for
SUK40Srev BamHI/NcoI SEQ ID NO: 188 SEQ ID NO: 190 SEQ ID NO: 151
pSUK40SGB SUK40for SUK40Srev BamHI/NcoI SEQ ID NO: 188 SEQ ID NO:
190 SEQ ID NO: 152 pSUK42L SUK42for SUK42Lrev BamHI/NcoI SEQ ID NO:
191 SEQ ID NO: 192 SEQ ID NO: 153 pSUK42LGB SUK42for SUK42Lrev
BamHI/NcoI SEQ ID NO: 191 SEQ ID NO: 192 SEQ ID NO: 154 pSUK42S
SUK42for SUK42Srev BamHI/NcoI SEQ ID NO: 191 SEQ ID NO: 193 SEQ ID
NO: 155 pSUK42SGB SUK42for SUK42Srev BamHI/NcoI SEQ ID NO: 191 SEQ
ID NO: 193 SEQ ID NO: 158 pSUK48L SUK48for SUK48Lrev BamHI/NcoI SEQ
ID NO: 194 SEQ ID NO: 195 SEQ ID NO: 159 pSUK48LGB SUK48for
SUK48Lrev BamHI/NcoI SEQ ID NO: 194 SEQ ID NO: 195 SEQ ID NO: 160
pSUK48S SUK48for SUK48Srev BamHI/NcoI SEQ ID NO: 194 SEQ ID NO: 196
SEQ ID NO: 161 pSUK48SGB SUK48for SUK48Srev BamHI/NcoI SEQ ID NO:
194 SEQ ID NO: 196
SEQ ID NO: 162 pSUK50L SUK50for SUK50Lrev BamHI/NcoI SEQ ID NO: 197
SEQ ID NO: 198 SEQ ID NO: 163 pSUK50LGB SUK50for SUK50Lrev
BamHI/NcoI SEQ ID NO: 197 SEQ ID NO: 198 SEQ ID NO: 164 pSUK50S
SUK50for SUK50Srev BamHI/NcoI SEQ ID NO: 197 SEQ ID NO: 199 SEQ ID
NO: 165 pSUK50SGB SUK50for SUK50Srev BamHI/NcoI SEQ ID NO: 197 SEQ
ID NO: 199 SEQ ID NO: 168 pSUK96L SUK96for SUK96Lrev XhoI/NcoI SEQ
ID NO: 200 SEQ ID NO: 201 SEQ ID NO: 169 pSUK96LGB SUK96for
SUK96Lrev XhoI/NcoI SEQ ID NO: 200 SEQ ID NO: 201 SEQ ID NO: 170
pSUK96S SUK96for SUK96Srev XhoI/NcoI SEQ ID NO: 200 SEQ ID NO: 202
SEQ ID NO: 171 pSUK96SSGB SUK96for SUK96Srev XhoI/NcoI SEQ ID NO:
200 SEQ ID NO: 202 SEQ ID NO: 172 pSUK98L SUK98for SUK98Lrev
XhoI/NcoI SEQ ID NO: 203 SEQ ID NO: 204 SEQ ID NO: 173 pSUK98LGB
SUK98for SUK98rev XhoI/NcoI SEQ ID NO: 203 SEQ ID NO: 204 SEQ ID
NO: 174 pSUK98S SUK98for SUK98Srev XhoI/NcoI SEQ ID NO: 203 SEQ ID
NO: 205 SEQ ID NO: 175 pSUK98SGB SUK98for SUK98Srev XhoI/NcoI SEQ
ID NO: 203 SEQ ID NO: 205 SEQ ID NO: 178 pSUK152L SUK152for
SUK152Lrev BamHI/NcoI SEQ ID NO: 206 SEQ ID NO: 207 SEQ ID NO: 179
pSUK152LGB SUK152for SUK152Lrev BamHI/NcoI SEQ ID NO: 206 SEQ ID
NO: 207 SEQ ID NO: 180 pSUK152S SUK152for SUK152Srev BamHI/NcoI SEQ
ID NO: 206 SEQ ID NO: 208 SEQ ID NO: 181 pSUK152SGB SUK152for
SUK152Srev BamHI/NcoI SEQ ID NO: 206 SEQ ID NO: 208 SEQ ID NO: 182
pSUK154L SUK154for SUK154Lrev BamHI/NcoI SEQ ID NO: 209 SEQ ID NO:
210 SEQ ID NO: 183 pSUK154LGB SUK154for SUK154Lrev BamHI/NcoI SEQ
ID NO: 209 SEQ ID NO: 210 SEQ ID NO: 184 pSUK154S SUK154for
SUK154Srev BamHI/NcoI SEQ ID NO: 209 SEQ ID NO: 211 SEQ ID NO: 185
pSUK154SGB SUK154for SUK154Srev BamHI/NcoI SEQ ID NO: 209 SEQ ID
NO: 211
[0381] Amplification is carried out as follows:
100 ng genomic DNA 1.times.PCR buffer
2.5 mM MgCl.sub.2,
[0382] 200 .mu.M each of dATP, dCTP, dGTP und dTTP 10 pmol of each
oligonucleotide primers
2.5 Units Pfu DNA Polymerase (Stratagene)
[0383] in a final volume of 50 .mu.l
[0384] The following temperature program is employed for the
various amplifications (BIORAD Thermocycler).
1. 95.degree. C. for 5 min
[0385] 2. 54.degree. C. for 1 min, followed by 72.degree. C. for 5
min and 95.degree. C. for 30 sec. Repeated 25 times. 3. 54.degree.
C. for 1 min, followed by 72.degree. C. for 10 min.
4. Storage at 4.degree. C.
[0386] The resulting PCR-products are digested with the restriction
endonucleases specified in the Table above (Table 3) and cloned
into the vector pSUN0301 (SEQ ID NO: 147) (pre-digested with the
same enzymes) upstream and in operable linkage to the glucuronidase
(GUS) gene. Following stable transformation of each of these
constructs into Arabidopsis thaliana tissue specificity and
expression profile was analyzed by a histochemical and quantitative
GUS-assay, respectively.
Example 5
Expression Profile of the Various Promoter::GUS Constructs in
Stably Transformed A. thaliana Plants
[0387] 5.1 pSUK222L, pSUK222LGB, pSUK222S, pSUK222SGB, pSUK224L,
pSUK224LGB, pSUK224S, pSUK224SGB, pSUK226L, pSUK226LGB, pSUK226S,
pSUK226SGB
[0388] All promoter fragments conferred seed-preferred expression
to the reporter gene construct tested. GUS expression was not
limited to embryos but covered also seed coat and endosperm tissue.
GUS expression was detected early in seed development and continued
into maturing siliques. Promoter activity was also observed in
various organs and tissues of adult plants at varying strength,
e.g. in hydathodes of rosette and caudal leaves, vessels of stalks
and rosette leaves, and also in sepals, petals and pollen. GUS
expression in seedlings was detected in vasculature of rosette
leaves as well as in the central cylinder of roots and also in root
tips. These side activities were least apparent while expression in
seeds was strongest for the longest promoter fragment analyzed.
5.2 pSUK322L, pSUK322LGB, pSUK322S, pSUK322SGB, pSUK324LGB,
pSUK324SGB
[0389] These promoter fragments conferred seed-preferred GUS
expression. GUS activity in seeds including expression in embryos
became apparent early during silique development and continued well
into later stages. GUS expression was also detected at strongly
varying degrees in rosette leaves and lateral roots of seedlings,
as well as rosette leaves, stalks, siliques and anthers (mainly
pollen) of adult plants analyzed.
5.3 pSUK250L, pSUK250LGB, pSUK250S, pSUK250SGB, pSUK252LGB,
pSUK252SGB
[0390] Seed-preferred GUS expression conferred by the promoters
analyzed commenced early in seed development and continued well
into mature siliques. The promoters were found to be active only
partially in embryos but strongly in seed coat and endosperm. In
seedlings, side activities were mainly detected in petioles and
leaf tips as well as root tips while GUS expression in adult plants
was highly specific for seeds.
5.4 pSUK19S, pSUK19SGB, pSUK19LGB
[0391] Rather strong and seed-specific GUS expression was detected
late during seed development in maturing siliques. The promoters
were predominantly active in embryos but side activities were also
observed at cotyledons of seedlings as well as guard cells of
stalks and siliques and (rarely) in midveins of rosette leaves and
anthers.
5.5 pSUK28L, pSUK28LGB, pSUK28S, pSUK28SGB, pSUK29L, pSUK29LGB,
pSUK29S, pSUK29SGB, pSUK30L, pSUK30LGB, pSUK30S, pSUK30SGB
[0392] Embryos as well as seed coat and endosperm in late
developing to mature seeds revealed GUS expression driven by all
promoters analyzed. While pSUK28L and pSUK30L were also
characterized by promoter side activity in vascular tissue of
stalks and leaves of adult plants as well as silique and flower
bottoms and mainly vascular tissue of seedlings, pSUK29L was highly
seed-specific in its expression pattern observed.
5.6 pSUK298L, pSUK298LGB, pSUK298S, pSUK298SGB, pSUK300L,
pSUK300LGB, pSUK300S, pSUK300SGB
[0393] These promoter fragments drive expression in entire seeds,
i.e. embryos and seed coats as well as endosperm are affected.
However, promoter activity is confined to an intermediate
developmental phase of the seeds, i.e. neither young nor completely
mature seeds reveal GUS expression. Side activities occur in patchy
styles in hypocotyl, petioles and vascular leaf tissue of seedlings
as well as in stalks, siliques, rosette leaves and floral organs of
full-grown plants. These side activities are less pronounced for
the shorter promoter fragment pSUK298L compared with pSUK300L.
5.7 pSUK292L, pSUK292LGB, pSUK292S, pSUK292SGB, pSUK294L,
pSUK294LGB, pSUK294S, pSUK294SGB, pSUK296L, pSUK296LGB, pSUK296S,
pSUK296SGB
[0394] Reporter gene expression was driven by all promoter
fragments in embryos, endosperm and seed coat. GUS activity
commenced rather early in seed development and continued into
mature seeds. However, multiple side activities were observed, in
particular for the longer promoter fragments analyzed. The most
prominent side activity was obvious in guard cells, but other
organs of seedlings and adult plants transformed with reporter gene
constructs harboring the longer promoter variants also clearly
showed GUS expression (e.g. in styles, trichomes).
5.8 pSUK262L, pSUK262LGB, pSUK262S, pSUK262SGB
[0395] These weak to medium-strong promoters are active in maturing
seeds while expression was not detected in very young and
completely mature seeds. Reporter gene expression was observed in
seed coat and endosperm but could not be proven for the embryo.
Side activity is marginal compared to other promoters disclosed:
Expression apart from the seeds became only apparent for sepals,
midveins and hydathodes of adult plants and seedlings analyzed.
5.9 pSUK164, pSUK164 GB
[0396] Promoter activity was detected in embryo, endosperm and seed
coat of seeds starting early in development and continuing into the
maturing phase. Side activities were observed in cotyledons and
first rosette leaves of seedlings as well as in vascular tissue of
rosette leaves of adult plants and in pollen.
5.10 pSUK352L, pSUK352LGB, pSUK352S, pSUK352SGB
[0397] Promoter activity commences early in seed development and
ceases before full maturation. Expression is specific for embryos,
i.e. no reporter gene activity was detected in seeds coat or
endosperm. Weak to medium side activity was also observed in
cotyledons and leaves of seedlings.
5.11 pSUK40L, pSUK40LGB, pSUK40S, pSUK40SGB, pSUK42L, pSUK42LGB,
pSUK42S, pSUK42SGB
[0398] These promoters confer seed-preferential expression when
tested in Arabidopsis thaliana. Expression is strongest in young
seeds and covers the developing embryo as well as the seed coat.
Side activities are observed in root tips, rarely in vascular
tissue of pods and stalks and also in anthers. Longer promoter
fragments confer stronger expression in seeds but are also
accompanied by more pronounced side activities.
5.12 pSUK48L, pSUK48LGB, pSUK48S, pSUK48SGB, pSUK50L, pSUK50LGB,
pSUK50S, pSUK50SGB
[0399] These promoters drive seed-preferential expression starting
at rather early but maintained also in later stages of seed
development of Arabidopsis thaliana. Endosperm and seed coat
clearly revealed reporter gene activity while it could not been
shown that these promoters are also active in embryos. Whereas the
shorter promoter fragments (pSUK48 derivatives) are almost void of
side activity apart from some expression observed in vascular
tissue of stalks and rarely observed expression in root tips,
pSUK50 derivatives are also characterized by varying degrees of
reporter gene activity in various organs and tissues analyzed.
5.13 pSUK96L, pSUK96LGB, pSUK96S, pSUK96SGB, pSUK98L, pSUK98LGB,
pSUK98S, pSUK98SGB
[0400] These promoters confer seed-preferential expression in
Arabidopsis thaliana. Expression in seed coat is stronger than in
any other seed tissue. Side activity of the promoters was observed
mainly in vascular tissue of stalks, ovaries and siliques but also
in roots (particularly root tips). Those side activity but also the
expression detected in seeds was stronger with longer promoter
fragments (pSUK98 derivatives) where expression of the marker gene
was also visible in vascular tissue of flowers and leaves of a
number of plants analyzed.
5.14 pSUK152L, pSUK152LGB, pSUK152S, pSUK152SGB, pSUK154L,
pSUK154LGB, pSUK154S, pSUK154SGB
[0401] These promoters drive strong seed-preferential expression of
the marker gene analyzed in Arabidopsis thaliana. However,
expression is weaker or not detectable in very young and fully
mature seeds. The promoters are active in endosperm and embryo but
not in the seed coat. The strength and variation of side activity
does not depend on the length of the promoter analyzed. Marker gene
expression was (besides seeds) observed in roots, apical meristems
and petioles of seedlings, axillary meristems and vascular tissue
of stalks of adult plants as well as in various flower organs.
Example 6
Expression Profile of the Various Promoter::GUS Constructs in
Stably Transformed Linseed Plants
[0402] 6.1 pSUK40L
[0403] pSUK40L is a seed-specific promoter in linseed. No GUS
expression was detected in early and young embryos. Middle strong
to strong GUS expression was found in middle, late and mature
embryos.
6.2 pSUK48L, and pSUK50L
[0404] pSUK48L is a seed-specific promoter in linseed. No GUS
expression was detected in early and young embryos. Middle strong
to strong GUS expression was found in middle, late and mature
embryos.
[0405] pSUK50L is a seed-specific promoter in linseed. No GUS
expression was detected in early and young embryos. Middle strong
GUS expression was found mid-phase during embryo development.
Middle strong to strong GUS expression was found in late and mature
embryos.
6.3 pSUK96L
[0406] pSUK96L is a seed-specific promoter in linseed. No GUS
expression was detected in early and young embryos. Weak to
medium-strong expression was found in medium, late and mature
embryos.
6.4 pSUK152L
[0407] pSUK152L is a seed-specific promoter in linseed. No to very
weak GUS expression was detected in early and young embryos. Middle
strong to strong GUS expression was found in middle, late and
mature embryos. Often medium strong to strong GUS expression was
also found in the seed capsula and in the flower.
Example 7
Vector Construction for Overexpression and Gene "Knockout"
Experiments
7.1 Overexpression
[0408] Vectors used for expression of full-length "candidate genes"
of interest in plants (overexpression) are designed to overexpress
the protein of interest and are of two general types, biolistic and
binary, depending on the plant transformation method to be
used.
[0409] For biolistic transformation (biolistic vectors), the
requirements are as follows: [0410] 1. a backbone with a bacterial
selectable marker (typically, an antibiotic resistance gene) and
origin of replication functional in Escherichia coli (E. coli;
e.g., ColE1), and [0411] 2. a plant-specific portion consisting of:
[0412] a. a gene expression cassette consisting of a promoter (eg.
ZmUBlint MOD), the gene of interest (typically, a full-length cDNA)
and a transcriptional terminator (e.g., Agrobacterium tumefaciens
nos terminator); [0413] b. a plant selectable marker cassette,
consisting of a suitable promoter, selectable marker gene (e.g.,
D-amino acid oxidase; dao1) and transcriptional terminator (eg. nos
terminator).
[0414] Vectors designed for transformation by Agrobacterium
tumefaciens (A. tumefaciens; binary vectors) consist of: [0415] 1.
a backbone with a bacterial selectable marker functional in both E.
coli and A. tumefaciens (e.g., spectinomycin resistance mediated by
the aadA gene) and two origins of replication, functional in each
of aforementioned bacterial hosts, plus the A. tumefaciens virG
gene; [0416] 2. a plant-specific portion as described for biolistic
vectors above, except in this instance this portion is flanked by
A. tumefaciens right and left border sequences which mediate
transfer of the DNA flanked by these two sequences to the
plant.
7.2 Gene Silencing Vectors
[0417] Vectors designed for reducing or abolishing expression of a
single gene or of a family or related genes (gene silencing
vectors) are also of two general types corresponding to the
methodology used to downregulate gene expression: antisense or
double-stranded RNA interference (dsRNAi).
(a) Anti-Sense
[0418] For antisense vectors, a full-length or partial gene
fragment (typically, a portion of the cDNA) can be used in the same
vectors described for full-length expression, as part of the gene
expression cassette. For antisense-mediated down-regulation of gene
expression, the coding region of the gene or gene fragment will be
in the opposite orientation relative to the promoter; thus, mRNA
will be made from the non-coding (antisense) strand in planta.
(b) dsRNAi
[0419] For dsRNAi vectors, a partial gene fragment (typically, 300
to 500 basepairs long) is used in the gene expression cassette, and
is expressed in both the sense and antisense orientations,
separated by a spacer region (typically, a plant intron, eg. the
OsSH1 intron 1, or a selectable marker, eg. conferring kanamycin
resistance). Vectors of this type are designed to form a
double-stranded mRNA stem, resulting from the basepairing of the
two complementary gene fragments in planta.
[0420] Biolistic or binary vectors designed for overexpression or
knockout can vary in a number of different ways, including eg. the
selectable markers used in plant and bacteria, the transcriptional
terminators used in the gene expression and plant selectable marker
cassettes, and the methodologies used for cloning in gene or gene
fragments of interest (typically, conventional restriction
enzyme-mediated or Gateway.TM. recombinase-based cloning).
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applications are incorporated herein by reference. While in the
foregoing specification this invention has been described in
relation to certain preferred embodiments thereof, and many details
have been set forth for purposes of illustration, it will be
apparent to those skilled in the art that the invention is
susceptible to additional embodiments and that certain of the
details described herein may be varied considerably without
departing from the basic principles of the invention.
Sequence CWU 1
1
2111980DNAArabidopsis thalianapromoter(1)..(980)transcription
regulating sequence from gene At4g12910 1ttttatctgt ataataaaac
cggtagtttc ttgtacccaa ccaaattctt acttcaaaat 60tttgaaaaca taatacaacg
aactttattt atttattacc tttttcaaat tcttcccttc 120caaaactttc
tgattttatt attctttaca tatactctta taatgttata acaattccag
180agaaaacaat atgaaaagaa gaaaaactaa ctaaacaaaa tagtagtgtg
gacctggatc 240tattcttcta ttaaaatttg ctttccacta gattttagat
atataggttt aaaagctaac 300catagctttt aacctcataa atttcaaaat
gctttccaaa atacggatta tactattccc 360aataattgaa taatcagttt
attttctaca taatttaaca atcggaaagc taattcgtag 420aagttatcgt
tcaaaataaa accaaaactt tttaatcagt atgtaggaat cttttccaaa
480caaaacgcct aaaacttaac aaaaagaatt cacaaacacc agccacgtgt
caccttttgt 540taagccagtc gtcactttaa gtaaccgctt gtcactttaa
gaagcggtcc caagtcaact 600cgcgccactc aaatcaatca tgcttatgta
aataacaact aaacgcagac gcaatcacca 660ttttctttat agaaatgctc
aaaatcgtgc cttttaagga aactgttgct ttgcagaatc 720acaaaatcta
tataccaacc ataagtttcc aaattttgtt ctaaaaatca aaaaaattcg
780tatcatcttt ttgtcttttt ttaccagaat ttcatcctta tttcgtgcta
aatcattttc 840caaacattca taatactttt tgttggttta tggccaacta
acaaaatatc ctctagattt 900tcttctttgt gttagtctat ataaagcaat
accacagtga tcctcttttt taattcattc 960tgtgtttatg tcaataatca
9802994DNAArabidopsis thalianapromoter(1)..(994)transcription
regulating sequence from gene At4g12910 2aacttgttat ctttttatct
gtataataaa accggtagtt tcttgtacca aaccaaattc 60ttagttcaaa attttgaaaa
cataatacaa cgaactttat ttatttatta cctttttcaa 120attcttccct
tccaaaactt tctgatttta ttattcttta catatactct tataatgtta
180taacaattcc agagaaaaca atatgaaaag aagaaaaact aactaaacaa
aatagtagtg 240tggacctgga tctattcttc tattaaaatt tgctttccac
tagattttag atatataggt 300ttaaaagcta accatagctt ttaacctcat
aaatttcaaa atgctttcca aaatacggat 360tatactattc ccaataattg
aataatcagt ttattttcta cgtaatttaa caatgggaaa 420gctaattcgt
agaagttatc gttcaaaata aaaccaaaac tttttaatca ttatgtagga
480atcttttcca aacaaaacgc ctaaaactta acaaaaagaa ttcacaaaaa
ccagccacgt 540gtcacctttt gttaagccag tcgtcacttt aagtaaccgc
ttgtcacttt aagaagcggt 600cccaagtcaa ctcgcgccac tcaaatcaat
catgcttatg taaataacaa ctaaacgcag 660acgcaatcac cattttcttt
atagaaatgc tcaaaatcgt gccttttaag gaaactgttg 720ctttgcagaa
tcacaaaatc tatataccaa ccataagttt ccaaattttg ttctaaaaat
780caaaaaaatt catatcatct ttttgtcttt ttttaccaga atttcatcct
tatttcgtgc 840taaatcattt tccaaacatt cataatactt tttgttggtt
tatggccaac taacaaaata 900tcctctagat tttcttcttt gtgttagtct
atataaagca ataccacagt gatcctcttt 960tttaattcat tctgtgttta
tgtcaataat caca 9943686DNAArabidopsis
thalianapromoter(1)..(686)transcription regulating sequence from
gene At4g12910 3ttttatctgt ataataaaac cggtagtttc ttgtacccaa
ccaaattctt acttcaaaat 60tttgaaaaca taatacaacg aactttattt atttattacc
tttttcaaat tcttcccttc 120caaaactttc tgattttatt attctttaca
tatactctta taatgttata acaattccag 180agaaaacaat atgaaaagaa
gaaaaactaa ctaaacaaaa tagtagtgtg gacctggatc 240tattcttcta
ttaaaatttg ctttccacta gattttagat atataggttt aaaagctaac
300catagctttt aacctcataa atttcaaaat gctttccaaa atacggatta
tactattccc 360aataattgaa taatcagttt attttctaca taatttaaca
atcggaaagc taattcgtag 420aagttatcgt tcaaaataaa accaaaactt
tttaatcagt atgtaggaat cttttccaaa 480caaaacgcct aaaacttaac
aaaaagaatt cacaaacacc agccacgtgt caccttttgt 540taagccagtc
gtcactttaa gtaaccgctt gtcactttaa gaagcggtcc caagtcaact
600cgcgccactc aaatcaatca tgcttatgta aataacaact aaacgcagac
gcaatcacca 660ttttctttat agaaatgctc aaaatc 6864698DNAArabidopsis
thalianapromoter(1)..(698)transcription regulating sequence from
gene At4g12910 4aacttgttat ctttttatct gtataataaa accggtagtt
tcttgtacca aaccaaattc 60ttagttcaaa attttgaaaa cataatacaa cgaactttat
ttatttatta cctttttcaa 120attcttccct tccaaaactt tctgatttta
ttattcttta catatactct tataatgtta 180taacaattcc agagaaaaca
atatgaaaag aagaaaaact aactaaacaa aatagtagtg 240tggacctgga
tctattcttc tattaaaatt tgctttccac tagattttag atatataggt
300ttaaaagcta accatagctt ttaacctcat aaatttcaaa atgctttcca
aaatacggat 360tatactattc ccaataattg aataatcagt ttattttcta
cgtaatttaa caatgggaaa 420gctaattcgt agaagttatc gttcaaaata
aaaccaaaac tttttaatca ttatgtagga 480atcttttcca aacaaaacgc
ctaaaactta acaaaaagaa ttcacaaaaa ccagccacgt 540gtcacctttt
gttaagccag tcgtcacttt aagtaaccgc ttgtcacttt aagaagcggt
600cccaagtcaa ctcgcgccac tcaaatcaat catgcttatg taaataacaa
ctaaacgcag 660acgcaatcac cattttcttt atagaaatgc tcaaaatc
69852051DNAArabidopsis thalianapromoter(1)..(2051)transcription
regulating sequence from gene At4g12910 5ctcccttttt atgtctagaa
ttttcaagta tatctaacat ctcaaatgtg cattacatgc 60aaaaacttgc atgtctttgt
tactaactta ccataagaca cataaatgaa aagaaagaag 120gtcatatgat
aaatcatatc cacaaaacaa ataataagaa aatattcact gtaaaattag
180taacttaata agaggttagg tagcaaacat ataaactaca acacaagtaa
ttgttttagc 240aattatcaag taatgaccat taataaataa aataaactaa
tgaaagaaga agagtagaat 300gaaagtaata cttgatacat tagcatgatt
ttgtattttt tagtattttt aacaaatgaa 360ttaagagtaa gattaatgga
tgacttaatt cacaatagga gaattttggt ttttcttcaa 420ttaatgtagc
taatcttttt atttaaataa tataaaagga caaatgtcat tttttcatgt
480gataatggta tatctatatg gagaaaattg accaactttc atatatatga
ttatataagt 540aataatatgt aaagaagaaa agaaacgaaa gagtgaaaga
gtgagcaaga gaaacttgta 600taattaactt gtatatatgc gttaaaaaaa
tgagaaccga ctcttttact aaatcattta 660ttaagcagag atttagctga
cattatactt ctccatctat atgatagaaa agtataattt 720tattcaatcc
agatgattgg attttcaaaa atatattttg aaagttttcg caaaaagtaa
780gtcgaaaacg ttatcaacca attattttgc tagtaggacc aaattaaaat
gattcacatt 840ccgaaatcct attccacttt tcgatttact attgcataat
ttcttctaaa ctatgtagtt 900tatacccttc gatttaccat ttttactcca
ctaaaattta gtaaccgcat ttaaaagttt 960agaagtaatt atttttagta
ctttgaagta agaaatcaca taaattgata atggcgcagc 1020tatacacttt
attagcactt cttttttacc tttcaaaaaa acttgttatc tttttatctg
1080tataataaaa ccggtagttt cttgtaccca accaaattct tacttcaaaa
ttttgaaaac 1140ataatacaac gaactttatt tatttattac ctttttcaaa
ttcttccctt ccaaaacttt 1200ctgattttat tattctttac atatactctt
ataatgttat aacaattcca gagaaaacaa 1260tatgaaaaga agaaaaacta
actaaacaaa atagtagtgt ggacctggat ctattcttct 1320attaaaattt
gctttccact agattttaga tatataggtt taaaagctaa ccatagcttt
1380taacctcata aatttcaaaa tgctttccaa aatacggatt atactattcc
caataattga 1440ataatcagtt tattttctac ataatttaac aatcggaaag
ctaattcgta gaagttatcg 1500ttcaaaataa aaccaaaact ttttaatcag
tatgtaggaa tcttttccaa acaaaacgcc 1560taaaacttaa caaaaagaat
tcacaaacac cagccacgtg tcaccttttg ttaagccagt 1620cgtcacttta
agtaaccgct tgtcacttta agaagcggtc ccaagtcaac tcgcgccact
1680caaatcaatc atgcttatgt aaataacaac taaacgcaga cgcaatcacc
attttcttta 1740tagaaatgct caaaatcgtg ccttttaagg aaactgttgc
tttgcagaat cacaaaatct 1800atataccaac cataagtttc caaattttgt
tctaaaaatc aaaaaaattc gtatcatctt 1860tttgtctttt tttaccagaa
tttcatcctt atttcgtgct aaatcatttt ccaaacattc 1920ataatacttt
ttgttggttt atggccaact aacaaaatat cctctagatt ttcttctttg
1980tgttagtcta tataaagcaa taccacagtg atcctctttt ttaattcatt
ctgtgtttat 2040gtcaataatc a 205162066DNAArabidopsis
thalianapromoter(1)..(2066)transcription regulating sequence from
gene At4g12910 6tactttagat ctctcccttt ttatgtctag aattttcaag
tatatctaac atctcaaatg 60tgcattacgt gcaaaaactt gcatgtcttt gttactaact
taccataaga cacataaatg 120aaaagagaaa aggtcatatg ataaatcata
tccacaaaac aaataataag aaaatattca 180ctgtaaaatt agtaacttaa
taagaggtta ggtagcaaac atctaaacta caacacaagt 240aattgtttta
gcaattatca agtaatgatc attaataaat aaaaaaaact aatgaaagaa
300gaagagtaga atgaaagtaa tacttgatac attagcatga ttttgtattt
ttttagtatt 360tttaacaaat gaattaagag taagattaat ggatgactta
attcacaata ggagaatttt 420ggtttttctt caattaatgt agctaatctt
tttatttaaa taatataaaa cgacaaatgt 480cattttctca tgtgataatg
gtacacttat atggagaaaa tttaccaact ttcatatata 540tgattatata
agtaataata tgtaaagaag aaaagaaacg aaagagtgaa agagtgagca
600agagaaactt gtataattaa cttgtatata tgcgttaaaa aaatgagaac
cgactctttt 660actaaatcat ttattaagca gagatttagc tgacattata
cttctccatc tatatgatag 720aaaagtataa ttttattcaa tccagatgat
tggattttca aaaatatatt ttgaaagttt 780tcgcaaaaag taagtcgaaa
acgttatcaa ccaattattt tgctagtagg accaaattaa 840aatgattcac
attccgaaat cctattccac ttttcgattt actattgcat aatttcttct
900aaactatgta gtttataccc ttcgatttac catttttact ccactaaaat
ttagtaaccg 960catttaaaag tttagaagta attattttta gtactttgaa
gtaagaaatc acataaattg 1020ataatggcgc agctatacac tttattagca
cttctttttt acctttcaaa aaaacttgtt 1080atctttttat ctgtataata
aaaccggtag tttcttgtac caaaccaaat tcttagttca 1140aaattttgaa
aacataatac aacgaacttt atttatttat tacctttttc aaattcttcc
1200cttccaaaac tttctgattt tattattctt tacatatact cttataatgt
tataacaatt 1260ccagagaaaa caatatgaaa agaagaaaaa ctaactaaac
aaaatagtag tgtggacctg 1320gatctattct tctattaaaa tttgctttcc
actagatttt agatatatag gtttaaaagc 1380taaccatagc ttttaacctc
ataaatttca aaatgctttc caaaatacgg attatactat 1440tcccaataat
tgaataatca gtttattttc tacgtaattt aacaatggga aagctaattc
1500gtagaagtta tcgttcaaaa taaaaccaaa actttttaat cattatgtag
gaatcttttc 1560caaacaaaac gcctaaaact taacaaaaag aattcacaaa
aaccagccac gtgtcacctt 1620ttgttaagcc agtcgtcact ttaagtaacc
gcttgtcact ttaagaagcg gtcccaagtc 1680aactcgcgcc actcaaatca
atcatgctta tgtaaataac aactaaacgc agacgcaatc 1740accattttct
ttatagaaat gctcaaaatc gtgcctttta aggaaactgt tgctttgcag
1800aatcacaaaa tctatatacc aaccataagt ttccaaattt tgttctaaaa
atcaaaaaaa 1860ttcatatcat ctttttgtct ttttttacca gaatttcatc
cttatttcgt gctaaatcat 1920tttccaaaca ttcataatac tttttgttgg
tttatggcca actaacaaaa tatcctctag 1980attttcttct ttgtgttagt
ctatataaag caataccaca gtgatcctct tttttaattc 2040attctgtgtt
tatgtcaata atcaca 206671757DNAArabidopsis
thalianapromoter(1)..(1757)transcription regulating sequence from
gene At4g12910 7ctcccttttt atgtctagaa ttttcaagta tatctaacat
ctcaaatgtg cattacatgc 60aaaaacttgc atgtctttgt tactaactta ccataagaca
cataaatgaa aagaaagaag 120gtcatatgat aaatcatatc cacaaaacaa
ataataagaa aatattcact gtaaaattag 180taacttaata agaggttagg
tagcaaacat ataaactaca acacaagtaa ttgttttagc 240aattatcaag
taatgaccat taataaataa aataaactaa tgaaagaaga agagtagaat
300gaaagtaata cttgatacat tagcatgatt ttgtattttt tagtattttt
aacaaatgaa 360ttaagagtaa gattaatgga tgacttaatt cacaatagga
gaattttggt ttttcttcaa 420ttaatgtagc taatcttttt atttaaataa
tataaaagga caaatgtcat tttttcatgt 480gataatggta tatctatatg
gagaaaattg accaactttc atatatatga ttatataagt 540aataatatgt
aaagaagaaa agaaacgaaa gagtgaaaga gtgagcaaga gaaacttgta
600taattaactt gtatatatgc gttaaaaaaa tgagaaccga ctcttttact
aaatcattta 660ttaagcagag atttagctga cattatactt ctccatctat
atgatagaaa agtataattt 720tattcaatcc agatgattgg attttcaaaa
atatattttg aaagttttcg caaaaagtaa 780gtcgaaaacg ttatcaacca
attattttgc tagtaggacc aaattaaaat gattcacatt 840ccgaaatcct
attccacttt tcgatttact attgcataat ttcttctaaa ctatgtagtt
900tatacccttc gatttaccat ttttactcca ctaaaattta gtaaccgcat
ttaaaagttt 960agaagtaatt atttttagta ctttgaagta agaaatcaca
taaattgata atggcgcagc 1020tatacacttt attagcactt cttttttacc
tttcaaaaaa acttgttatc tttttatctg 1080tataataaaa ccggtagttt
cttgtaccca accaaattct tacttcaaaa ttttgaaaac 1140ataatacaac
gaactttatt tatttattac ctttttcaaa ttcttccctt ccaaaacttt
1200ctgattttat tattctttac atatactctt ataatgttat aacaattcca
gagaaaacaa 1260tatgaaaaga agaaaaacta actaaacaaa atagtagtgt
ggacctggat ctattcttct 1320attaaaattt gctttccact agattttaga
tatataggtt taaaagctaa ccatagcttt 1380taacctcata aatttcaaaa
tgctttccaa aatacggatt atactattcc caataattga 1440ataatcagtt
tattttctac ataatttaac aatcggaaag ctaattcgta gaagttatcg
1500ttcaaaataa aaccaaaact ttttaatcag tatgtaggaa tcttttccaa
acaaaacgcc 1560taaaacttaa caaaaagaat tcacaaacac cagccacgtg
tcaccttttg ttaagccagt 1620cgtcacttta agtaaccgct tgtcacttta
agaagcggtc ccaagtcaac tcgcgccact 1680caaatcaatc atgcttatgt
aaataacaac taaacgcaga cgcaatcacc attttcttta 1740tagaaatgct caaaatc
175781770DNAArabidopsis thalianapromoter(1)..(1770)transcription
regulating sequence from gene At4g12910 8tactttagat ctctcccttt
ttatgtctag aattttcaag tatatctaac atctcaaatg 60tgcattacgt gcaaaaactt
gcatgtcttt gttactaact taccataaga cacataaatg 120aaaagagaaa
aggtcatatg ataaatcata tccacaaaac aaataataag aaaatattca
180ctgtaaaatt agtaacttaa taagaggtta ggtagcaaac atctaaacta
caacacaagt 240aattgtttta gcaattatca agtaatgatc attaataaat
aaaaaaaact aatgaaagaa 300gaagagtaga atgaaagtaa tacttgatac
attagcatga ttttgtattt ttttagtatt 360tttaacaaat gaattaagag
taagattaat ggatgactta attcacaata ggagaatttt 420ggtttttctt
caattaatgt agctaatctt tttatttaaa taatataaaa cgacaaatgt
480cattttctca tgtgataatg gtacacttat atggagaaaa tttaccaact
ttcatatata 540tgattatata agtaataata tgtaaagaag aaaagaaacg
aaagagtgaa agagtgagca 600agagaaactt gtataattaa cttgtatata
tgcgttaaaa aaatgagaac cgactctttt 660actaaatcat ttattaagca
gagatttagc tgacattata cttctccatc tatatgatag 720aaaagtataa
ttttattcaa tccagatgat tggattttca aaaatatatt ttgaaagttt
780tcgcaaaaag taagtcgaaa acgttatcaa ccaattattt tgctagtagg
accaaattaa 840aatgattcac attccgaaat cctattccac ttttcgattt
actattgcat aatttcttct 900aaactatgta gtttataccc ttcgatttac
catttttact ccactaaaat ttagtaaccg 960catttaaaag tttagaagta
attattttta gtactttgaa gtaagaaatc acataaattg 1020ataatggcgc
agctatacac tttattagca cttctttttt acctttcaaa aaaacttgtt
1080atctttttat ctgtataata aaaccggtag tttcttgtac caaaccaaat
tcttagttca 1140aaattttgaa aacataatac aacgaacttt atttatttat
tacctttttc aaattcttcc 1200cttccaaaac tttctgattt tattattctt
tacatatact cttataatgt tataacaatt 1260ccagagaaaa caatatgaaa
agaagaaaaa ctaactaaac aaaatagtag tgtggacctg 1320gatctattct
tctattaaaa tttgctttcc actagatttt agatatatag gtttaaaagc
1380taaccatagc ttttaacctc ataaatttca aaatgctttc caaaatacgg
attatactat 1440tcccaataat tgaataatca gtttattttc tacgtaattt
aacaatggga aagctaattc 1500gtagaagtta tcgttcaaaa taaaaccaaa
actttttaat cattatgtag gaatcttttc 1560caaacaaaac gcctaaaact
taacaaaaag aattcacaaa aaccagccac gtgtcacctt 1620ttgttaagcc
agtcgtcact ttaagtaacc gcttgtcact ttaagaagcg gtcccaagtc
1680aactcgcgcc actcaaatca atcatgctta tgtaaataac aactaaacgc
agacgcaatc 1740accattttct ttatagaaat gctcaaaatc
177093954DNAArabidopsis thalianapromoter(1)..(3954)transcription
regulating sequence from gene At4g12910 9actagtcatc tatttcttga
gaatatccct aagcttgacg tcttaatcat ctgaatttat 60acacatacaa tttgttgctt
gcttgtttgc tttctcttta tttattttta cttattgatt 120tagtttagtc
tcgatactat aaccttctgt gtgaacaaaa aagttttgtg gaaatcgatc
180cttaagtatt acaactggcc tcttatttga gagagtagtc taggttaatt
tgatcctata 240ttagttatcg tccatatgtg taccacacca tcacacaaga
aaatgacatc attaccaagg 300attaaattgc gagaggttct agattctctc
atccgacaaa ggcaaaaaga gaacttcgag 360ccaaactccc cagaatggag
tactttccaa ccctagccct aatatgcccg ttctagtaaa 420atccgaaccg
ttgctatact cgttgcatcc aaaatacaaa catgcaacat tttgaactca
480ctatcaagtg tatataatgt aagcatttct tgcacaagag ttcctttcat
gttgttctcg 540tctacataat gttgtcggta ggttattttc ttatcaaatg
atcaatgtga gatacatatt 600tgattttgga ttaggatttc cacacatcgc
attgtagttt gttattgaag ctaagagcat 660ttgaataaac cttatggtca
taatagtagg agcaagggaa gcattgagtg cagtgaggtt 720gctccccata
ttcatgtgat gagtttatcg cttttgagga gatttaaaat gaatttcagc
780cataaatgct atatctcggt gggagtggat cgagtgacga gagaggtgga
aaacaacttt 840gttgtcgtaa aaactttcca ctgtgcatat atggatatat
agttaaacaa cttttttttt 900tattcggcca ttatggaata atgactattg
ataaaaaaat aaaaataata agtagagtag 960aatgaaagta gtgtttgata
cattattatg attttttttt cattaaatgt ttttagaaaa 1020tcatttaaga
gtaagattaa tagagaattt aacataacaa aagacttttg atttttcctc
1080aattagtttt gtttatcatt ttatttaatt aatgtagcat gacacatgcc
attttcttgt 1140gtgatgatat gttacttata tggagagagt tgaccaattt
ttatatatat gattaattaa 1200aagacggagg aaagaaaaac aagaaaagtc
tatgtgattt tcatttatat tttttttttt 1260actgattaat taaggatgtg
atttgtagta aaatgtgatg attttaaagt agtgaacgaa 1320agatttgtgg
aagtaagagg taagttggtt taatggttaa agagtgaggg aagaaagaaa
1380aaatgagtta cttagtagaa attttgttgg tagtgtatgt tgggatataa
tttggtttga 1440tgtggggata tgaaagtatg taataaaaat atatgtaaat
catctaatat catatgaata 1500agggtatttt gaaaattgct aacaaaacaa
atatttatgg ttattattgt tatttatgtt 1560catttttgtg ttattgtgaa
aataataaac tcaaatctat ctcatatagc tgcaaatttt 1620atgattagat
tcatctttga ttcgaaatat caattatacg ttttatgtac aaatctaaat
1680ttttatacat ctaaaatcct aaatttttac tatcatgaat acatagaaca
acattataag 1740atatattgat acaatagtat ataattatgt tacttatatg
gtttgaaact tgcataattt 1800gattctgaac aaacattttt ttgttccacg
tactcttttc tcatctacta ctctaaccat 1860atcctgatat gctattaatt
taagtcaata cttttttaga tctctcccta tttatgtcta 1920gaattttcaa
gtatatctaa catctcaaat gtgcattaca tgcaaaaact tgcatgtctt
1980tgttactaac ttaccataag acacataaat gaaaagaaag aaggtcatat
gataaatcat 2040atccacaaaa caaataataa gaaaatattc actgtaaaat
tagtaactta ataagaggtt 2100aggtagcaaa catataaact acaacacaag
taattgtttt agcaattatc aagtaatgac 2160cattaataaa taaaataaac
taatgaaaga agaagagtag aatgaaagta atacttgata 2220cattagcatg
attttgtatt ttttagtatt tttaacaaat gaattaagag taagattaat
2280ggatgactta attcacaata ggagaatttt ggtttttctt caattaatgt
agctaatctt 2340tttatttaaa taatataaaa ggacaaatgt cattttttca
tgtgataatg gtatatctat 2400atggagaaaa ttgaccaact ttcatatata
tgattatata agtaataata tgtaaagaag 2460aaaagaaacg aaagagtgaa
agagtgagca agagaaactt gtataattaa cttgtatata 2520tgcgttaaaa
aaatgagaac cgactctttt actaaatcat ttattaagca gagatttagc
2580tgacattata cttctccatc tatatgatag aaaagtataa ttttattcaa
tccagatgat 2640tggattttca aaaatatatt ttgaaagttt tcgcaaaaag
taagtcgaaa acgttatcaa 2700ccaattattt tgctagtagg accaaattaa
aatgattcac attccgaaat cctattccac 2760ttttcgattt actattgcat
aatttcttct aaactatgta gtttataccc
ttcgatttac 2820catttttact ccactaaaat ttagtaaccg catttaaaag
tttagaagta attattttta 2880gtactttgaa gtaagaaatc acataaattg
ataatggcgc agctatacac tttattagca 2940cttctttttt acctttcaaa
aaaacttgtt atctttttat ctgtataata aaaccggtag 3000tttcttgtac
ccaaccaaat tcttacttca aaattttgaa aacataatac aacgaacttt
3060atttatttat tacctttttc aaattcttcc cttccaaaac tttctgattt
tattattctt 3120tacatatact cttataatgt tataacaatt ccagagaaaa
caatatgaaa agaagaaaaa 3180ctaactaaac aaaatagtag tgtggacctg
gatctattct tctattaaaa tttgctttcc 3240actagatttt agatatatag
gtttaaaagc taaccatagc ttttaacctc ataaatttca 3300aaatgctttc
caaaatacgg attatactat tcccaataat tgaataatca gtttattttc
3360tacataattt aacaatcgga aagctaattc gtagaagtta tcgttcaaaa
taaaaccaaa 3420actttttaat cagtatgtag gaatcttttc caaacaaaac
gcctaaaact taacaaaaag 3480aattcacaaa caccagccac gtgtcacctt
ttgttaagcc agtcgtcact ttaagtaacc 3540gcttgtcact ttaagaagcg
gtcccaagtc aactcgcgcc actcaaatca atcatgctta 3600tgtaaataac
aactaaacgc agacgcaatc accattttct ttatagaaat gctcaaaatc
3660gtgcctttta aggaaactgt tgctttgcag aatcacaaaa tctatatacc
aaccataagt 3720ttccaaattt tgttctaaaa atcaaaaaaa ttcgtatcat
ctttttgtct ttttttacca 3780gaatttcatc cttatttcgt gctaaatcat
tttccaaaca ttcataatac tttttgttgg 3840tttatggcca actaacaaaa
tatcctctag attttcttct ttgtgttagt ctatataaag 3900caataccata
gtgatcctct tttttaattc attctgtgtt tatgtcaaga atca
3954103962DNAArabidopsis thalianapromoter(1)..(3962)transcription
regulating sequence from gene At4g12910 10ataattagat ctactagtca
tctatttctt gagaatatcc ctaagcttga cgtcttaatc 60atctgaattt atacacatac
aatttgttgc ttgcttattt gctttctctt tatttatttt 120tacttattga
tttagtttag tctcgatact ataaccttct gtgtgaacaa aaaagtttcg
180tggaaatcga tccttaagta ttacaactgg cctcttattt gagagagtag
tctaggttaa 240tttgatccta tatcagttat cgtccatatg tgtaccacac
catcacacaa gaaaatgaca 300tcattaccaa ggattaaatt gtgagaggtt
ctagattctc tcatccgacc aaggcaaaaa 360gagaacttcg agccaaactc
cccagaatgg agtactttcc aaccctagcc ctaatatgcc 420cgttctagta
aaatccgaac cgttgctata ctcgttgcat ccaaaataca aacatgcaac
480attttgaact cactatcaag tgtatataat gtaagcattt cttgcacaag
agttcctttc 540atgttgttct cgtctacata atgttgtcgg taggttattt
tcttatcaaa tgatcaatgt 600gagatacata tttgattttg gattaggatt
tccacacatc gcattgtagt ttgttattga 660agctaagagc atttgaataa
accttatggt cataatagta ggagcaaggg aagcattgag 720tgcagtgaag
ttgctcccca tattcatgtg atgagtttat cgcttttgag gagatttaaa
780atgaatttca gccataaatg ctatatctcg gtgggagtgg agtgacgaga
gaggaggaaa 840acaactttgt tgtcgtaaaa actttccatt gtgcatatat
ggatatatag ttaaacaact 900ttttttttta ttcggccatt atggaataat
gactattgat aaaaaaataa aaataataag 960tagagtagaa tgaaagtagt
gcttgataca ttattatgat ttttttttca ttaaatgttt 1020ttagaaaatc
atttaagagt aagattaata gagaatttaa cataacaaaa gacttttgat
1080ttttcctcaa ttagttttgt taatcatttt atttaattaa tgtagcatga
cacatgccat 1140tttcttgtgt gatgatatgt tacttatatg gagagagttg
accaattttt atatatatga 1200ttaattaaaa gacggaggaa agaaaaacaa
gaaaagtcaa tgtgattttc atttatatta 1260aattttttga ctgattaatt
aaggatgtga tttgtagtaa aatgtgatga ttttaaagta 1320gtgaacgaaa
gatttgtgga agtaagaggt aagttgggtt aatggttaaa gagtgaggga
1380agaaagaaaa aatgagttac ttagtagaaa ttttgttggt agtgtatgtt
gggatataat 1440ttggtttgat gtggggatat gaaagtatgt aataaaaata
tatgtaaatc atctaatatc 1500atatgaataa gggtattttg aaaattgcta
acaaaacaaa tatttatggt tattattgtt 1560atttatgttc atttttgtgt
tattgtgaaa ataataaact caaatctatc tcatatagct 1620gcaaatttta
tgattagatt catctttgat tcgaaatatc aattatacgt tttatgtaca
1680aatctaaatt tttatacatc taaaatccta aatttttact atcatgaata
catagaataa 1740cattataaga tatattgata caatagtata taattatgtt
acttatatgg tttgaaactt 1800gcataatttg ttctgaacaa acattttttt
gttccacgta ctcttttctc atctactact 1860ctaaccatat cctgatatgc
tattaattta agtcaatact ttagatctct ccctttttat 1920gtctagaatt
ttcaagtata tctaacatct caaatgtgca ttacgtgcaa aaacttgcat
1980gtctttgtta ctaacttacc ataagacaca taaatgaaaa gagaaaaggt
catatgataa 2040atcatatcca caaaacaaat aataagaaaa tattcactgt
aaaattagta acttaataag 2100aggttaggta gcaaacatct aaactacaac
acaagtaatt gttttagcaa ttatcaagta 2160atgatcatta ataaataaaa
aaaactaatg aaagaagaag agtagaatga aagtaatact 2220tgatacatta
gcatgatttt gtattttttt agtattttta acaaatgaat taagagtaag
2280attaatggat gacttaattc acaataggag aattttggtt tttcttcaat
taatgtagct 2340aatcttttta tttaaataat ataaaacgac aaatgtcatt
ttctcatgtg ataatggtac 2400acttatatgg agaaaattta ccaactttca
tatatatgat tatataagta ataatatgta 2460aagaagaaaa gaaacgaaag
agtgaaagag tgagcaagag aaacttgtat aattaacttg 2520tatatatgcg
ttaaaaaaat gagaaccgac tcttttacta aatcatttat taagcagaga
2580tttagctgac attatacttc tccatctata tgatagaaaa gtataatttt
attcaatcca 2640gatgattgga ttttcaaaaa tatattttga aagttttcgc
aaaaagtaag tcgaaaacgt 2700tatcaaccaa ttattttgct agtaggacca
aattaaaatg attcacattc cgaaatccta 2760ttccactttt cgatttacta
ttgcataatt tcttctaaac tatgtagttt atacccttcg 2820atttaccatt
tttactccac taaaatttag taaccgcatt taaaagttta gaagtaatta
2880tttttagtac tttgaagtaa gaaatcacat aaattgataa tggcgcagct
atacacttta 2940ttagcacttc ttttttacct ttcaaaaaaa cttgttatct
ttttatctgt ataataaaac 3000cggtagtttc ttgtaccaaa ccaaattctt
agttcaaaat tttgaaaaca taatacaacg 3060aactttattt atttattacc
tttttcaaat tcttcccttc caaaactttc tgattttatt 3120attctttaca
tatactctta taatgttata acaattccag agaaaacaat atgaaaagaa
3180gaaaaactaa ctaaacaaaa tagtagtgtg gacctggatc tattcttcta
ttaaaatttg 3240ctttccacta gattttagat atataggttt aaaagctaac
catagctttt aacctcataa 3300atttcaaaat gctttccaaa atacggatta
tactattccc aataattgaa taatcagttt 3360attttctacg taatttaaca
atgggaaagc taattcgtag aagttatcgt tcaaaataaa 3420accaaaactt
tttaatcatt atgtaggaat cttttccaaa caaaacgcct aaaacttaac
3480aaaaagaatt cacaaaaacc agccacgtgt caccttttgt taagccagtc
gtcactttaa 3540gtaaccgctt gtcactttaa gaagcggtcc caagtcaact
cgcgccactc aaatcaatca 3600tgcttatgta aataacaact aaacgcagac
gcaatcacca ttttctttat agaaatgctc 3660aaaatcgtgc cttttaagga
aactgttgct ttgcagaatc acaaaatcta tataccaacc 3720ataagtttcc
aaattttgtt ctaaaaatca aaaaaattca tatcatcttt ttgtcttttt
3780ttaccagaat ttcatcctta tttcgtgcta aatcattttc caaacattca
taatactttt 3840tgttggttta tggccaacta acaaaatatc ctctagattt
tcttctttgt gttagtctat 3900ataaagcaat accacagtga tcctcttttt
taattcattc tgtgtttatg tcaataatca 3960ca 3962113660DNAArabidopsis
thalianapromoter(1)..(3660)transcription regulating sequence from
gene At4g12910 11actagtcatc tatttcttga gaatatccct aagcttgacg
tcttaatcat ctgaatttat 60acacatacaa tttgttgctt gcttgtttgc tttctcttta
tttattttta cttattgatt 120tagtttagtc tcgatactat aaccttctgt
gtgaacaaaa aagttttgtg gaaatcgatc 180cttaagtatt acaactggcc
tcttatttga gagagtagtc taggttaatt tgatcctata 240ttagttatcg
tccatatgtg taccacacca tcacacaaga aaatgacatc attaccaagg
300attaaattgc gagaggttct agattctctc atccgacaaa ggcaaaaaga
gaacttcgag 360ccaaactccc cagaatggag tactttccaa ccctagccct
aatatgcccg ttctagtaaa 420atccgaaccg ttgctatact cgttgcatcc
aaaatacaaa catgcaacat tttgaactca 480ctatcaagtg tatataatgt
aagcatttct tgcacaagag ttcctttcat gttgttctcg 540tctacataat
gttgtcggta ggttattttc ttatcaaatg atcaatgtga gatacatatt
600tgattttgga ttaggatttc cacacatcgc attgtagttt gttattgaag
ctaagagcat 660ttgaataaac cttatggtca taatagtagg agcaagggaa
gcattgagtg cagtgaggtt 720gctccccata ttcatgtgat gagtttatcg
cttttgagga gatttaaaat gaatttcagc 780cataaatgct atatctcggt
gggagtggat cgagtgacga gagaggtgga aaacaacttt 840gttgtcgtaa
aaactttcca ctgtgcatat atggatatat agttaaacaa cttttttttt
900tattcggcca ttatggaata atgactattg ataaaaaaat aaaaataata
agtagagtag 960aatgaaagta gtgtttgata cattattatg attttttttt
cattaaatgt ttttagaaaa 1020tcatttaaga gtaagattaa tagagaattt
aacataacaa aagacttttg atttttcctc 1080aattagtttt gtttatcatt
ttatttaatt aatgtagcat gacacatgcc attttcttgt 1140gtgatgatat
gttacttata tggagagagt tgaccaattt ttatatatat gattaattaa
1200aagacggagg aaagaaaaac aagaaaagtc tatgtgattt tcatttatat
tttttttttt 1260actgattaat taaggatgtg atttgtagta aaatgtgatg
attttaaagt agtgaacgaa 1320agatttgtgg aagtaagagg taagttggtt
taatggttaa agagtgaggg aagaaagaaa 1380aaatgagtta cttagtagaa
attttgttgg tagtgtatgt tgggatataa tttggtttga 1440tgtggggata
tgaaagtatg taataaaaat atatgtaaat catctaatat catatgaata
1500agggtatttt gaaaattgct aacaaaacaa atatttatgg ttattattgt
tatttatgtt 1560catttttgtg ttattgtgaa aataataaac tcaaatctat
ctcatatagc tgcaaatttt 1620atgattagat tcatctttga ttcgaaatat
caattatacg ttttatgtac aaatctaaat 1680ttttatacat ctaaaatcct
aaatttttac tatcatgaat acatagaaca acattataag 1740atatattgat
acaatagtat ataattatgt tacttatatg gtttgaaact tgcataattt
1800gattctgaac aaacattttt ttgttccacg tactcttttc tcatctacta
ctctaaccat 1860atcctgatat gctattaatt taagtcaata cttttttaga
tctctcccta tttatgtcta 1920gaattttcaa gtatatctaa catctcaaat
gtgcattaca tgcaaaaact tgcatgtctt 1980tgttactaac ttaccataag
acacataaat gaaaagaaag aaggtcatat gataaatcat 2040atccacaaaa
caaataataa gaaaatattc actgtaaaat tagtaactta ataagaggtt
2100aggtagcaaa catataaact acaacacaag taattgtttt agcaattatc
aagtaatgac 2160cattaataaa taaaataaac taatgaaaga agaagagtag
aatgaaagta atacttgata 2220cattagcatg attttgtatt ttttagtatt
tttaacaaat gaattaagag taagattaat 2280ggatgactta attcacaata
ggagaatttt ggtttttctt caattaatgt agctaatctt 2340tttatttaaa
taatataaaa ggacaaatgt cattttttca tgtgataatg gtatatctat
2400atggagaaaa ttgaccaact ttcatatata tgattatata agtaataata
tgtaaagaag 2460aaaagaaacg aaagagtgaa agagtgagca agagaaactt
gtataattaa cttgtatata 2520tgcgttaaaa aaatgagaac cgactctttt
actaaatcat ttattaagca gagatttagc 2580tgacattata cttctccatc
tatatgatag aaaagtataa ttttattcaa tccagatgat 2640tggattttca
aaaatatatt ttgaaagttt tcgcaaaaag taagtcgaaa acgttatcaa
2700ccaattattt tgctagtagg accaaattaa aatgattcac attccgaaat
cctattccac 2760ttttcgattt actattgcat aatttcttct aaactatgta
gtttataccc ttcgatttac 2820catttttact ccactaaaat ttagtaaccg
catttaaaag tttagaagta attattttta 2880gtactttgaa gtaagaaatc
acataaattg ataatggcgc agctatacac tttattagca 2940cttctttttt
acctttcaaa aaaacttgtt atctttttat ctgtataata aaaccggtag
3000tttcttgtac ccaaccaaat tcttacttca aaattttgaa aacataatac
aacgaacttt 3060atttatttat tacctttttc aaattcttcc cttccaaaac
tttctgattt tattattctt 3120tacatatact cttataatgt tataacaatt
ccagagaaaa caatatgaaa agaagaaaaa 3180ctaactaaac aaaatagtag
tgtggacctg gatctattct tctattaaaa tttgctttcc 3240actagatttt
agatatatag gtttaaaagc taaccatagc ttttaacctc ataaatttca
3300aaatgctttc caaaatacgg attatactat tcccaataat tgaataatca
gtttattttc 3360tacataattt aacaatcgga aagctaattc gtagaagtta
tcgttcaaaa taaaaccaaa 3420actttttaat cagtatgtag gaatcttttc
caaacaaaac gcctaaaact taacaaaaag 3480aattcacaaa caccagccac
gtgtcacctt ttgttaagcc agtcgtcact ttaagtaacc 3540gcttgtcact
ttaagaagcg gtcccaagtc aactcgcgcc actcaaatca atcatgctta
3600tgtaaataac aactaaacgc agacgcaatc accattttct ttatagaaat
gctcaaaatc 3660123666DNAArabidopsis
thalianapromoter(1)..(3666)transcription regulating sequence from
gene At4g12910 12ataattagat ctactagtca tctatttctt gagaatatcc
ctaagcttga cgtcttaatc 60atctgaattt atacacatac aatttgttgc ttgcttattt
gctttctctt tatttatttt 120tacttattga tttagtttag tctcgatact
ataaccttct gtgtgaacaa aaaagtttcg 180tggaaatcga tccttaagta
ttacaactgg cctcttattt gagagagtag tctaggttaa 240tttgatccta
tatcagttat cgtccatatg tgtaccacac catcacacaa gaaaatgaca
300tcattaccaa ggattaaatt gtgagaggtt ctagattctc tcatccgacc
aaggcaaaaa 360gagaacttcg agccaaactc cccagaatgg agtactttcc
aaccctagcc ctaatatgcc 420cgttctagta aaatccgaac cgttgctata
ctcgttgcat ccaaaataca aacatgcaac 480attttgaact cactatcaag
tgtatataat gtaagcattt cttgcacaag agttcctttc 540atgttgttct
cgtctacata atgttgtcgg taggttattt tcttatcaaa tgatcaatgt
600gagatacata tttgattttg gattaggatt tccacacatc gcattgtagt
ttgttattga 660agctaagagc atttgaataa accttatggt cataatagta
ggagcaaggg aagcattgag 720tgcagtgaag ttgctcccca tattcatgtg
atgagtttat cgcttttgag gagatttaaa 780atgaatttca gccataaatg
ctatatctcg gtgggagtgg agtgacgaga gaggaggaaa 840acaactttgt
tgtcgtaaaa actttccatt gtgcatatat ggatatatag ttaaacaact
900ttttttttta ttcggccatt atggaataat gactattgat aaaaaaataa
aaataataag 960tagagtagaa tgaaagtagt gcttgataca ttattatgat
ttttttttca ttaaatgttt 1020ttagaaaatc atttaagagt aagattaata
gagaatttaa cataacaaaa gacttttgat 1080ttttcctcaa ttagttttgt
taatcatttt atttaattaa tgtagcatga cacatgccat 1140tttcttgtgt
gatgatatgt tacttatatg gagagagttg accaattttt atatatatga
1200ttaattaaaa gacggaggaa agaaaaacaa gaaaagtcaa tgtgattttc
atttatatta 1260aattttttga ctgattaatt aaggatgtga tttgtagtaa
aatgtgatga ttttaaagta 1320gtgaacgaaa gatttgtgga agtaagaggt
aagttgggtt aatggttaaa gagtgaggga 1380agaaagaaaa aatgagttac
ttagtagaaa ttttgttggt agtgtatgtt gggatataat 1440ttggtttgat
gtggggatat gaaagtatgt aataaaaata tatgtaaatc atctaatatc
1500atatgaataa gggtattttg aaaattgcta acaaaacaaa tatttatggt
tattattgtt 1560atttatgttc atttttgtgt tattgtgaaa ataataaact
caaatctatc tcatatagct 1620gcaaatttta tgattagatt catctttgat
tcgaaatatc aattatacgt tttatgtaca 1680aatctaaatt tttatacatc
taaaatccta aatttttact atcatgaata catagaataa 1740cattataaga
tatattgata caatagtata taattatgtt acttatatgg tttgaaactt
1800gcataatttg ttctgaacaa acattttttt gttccacgta ctcttttctc
atctactact 1860ctaaccatat cctgatatgc tattaattta agtcaatact
ttagatctct ccctttttat 1920gtctagaatt ttcaagtata tctaacatct
caaatgtgca ttacgtgcaa aaacttgcat 1980gtctttgtta ctaacttacc
ataagacaca taaatgaaaa gagaaaaggt catatgataa 2040atcatatcca
caaaacaaat aataagaaaa tattcactgt aaaattagta acttaataag
2100aggttaggta gcaaacatct aaactacaac acaagtaatt gttttagcaa
ttatcaagta 2160atgatcatta ataaataaaa aaaactaatg aaagaagaag
agtagaatga aagtaatact 2220tgatacatta gcatgatttt gtattttttt
agtattttta acaaatgaat taagagtaag 2280attaatggat gacttaattc
acaataggag aattttggtt tttcttcaat taatgtagct 2340aatcttttta
tttaaataat ataaaacgac aaatgtcatt ttctcatgtg ataatggtac
2400acttatatgg agaaaattta ccaactttca tatatatgat tatataagta
ataatatgta 2460aagaagaaaa gaaacgaaag agtgaaagag tgagcaagag
aaacttgtat aattaacttg 2520tatatatgcg ttaaaaaaat gagaaccgac
tcttttacta aatcatttat taagcagaga 2580tttagctgac attatacttc
tccatctata tgatagaaaa gtataatttt attcaatcca 2640gatgattgga
ttttcaaaaa tatattttga aagttttcgc aaaaagtaag tcgaaaacgt
2700tatcaaccaa ttattttgct agtaggacca aattaaaatg attcacattc
cgaaatccta 2760ttccactttt cgatttacta ttgcataatt tcttctaaac
tatgtagttt atacccttcg 2820atttaccatt tttactccac taaaatttag
taaccgcatt taaaagttta gaagtaatta 2880tttttagtac tttgaagtaa
gaaatcacat aaattgataa tggcgcagct atacacttta 2940ttagcacttc
ttttttacct ttcaaaaaaa cttgttatct ttttatctgt ataataaaac
3000cggtagtttc ttgtaccaaa ccaaattctt agttcaaaat tttgaaaaca
taatacaacg 3060aactttattt atttattacc tttttcaaat tcttcccttc
caaaactttc tgattttatt 3120attctttaca tatactctta taatgttata
acaattccag agaaaacaat atgaaaagaa 3180gaaaaactaa ctaaacaaaa
tagtagtgtg gacctggatc tattcttcta ttaaaatttg 3240ctttccacta
gattttagat atataggttt aaaagctaac catagctttt aacctcataa
3300atttcaaaat gctttccaaa atacggatta tactattccc aataattgaa
taatcagttt 3360attttctacg taatttaaca atgggaaagc taattcgtag
aagttatcgt tcaaaataaa 3420accaaaactt tttaatcatt atgtaggaat
cttttccaaa caaaacgcct aaaacttaac 3480aaaaagaatt cacaaaaacc
agccacgtgt caccttttgt taagccagtc gtcactttaa 3540gtaaccgctt
gtcactttaa gaagcggtcc caagtcaact cgcgccactc aaatcaatca
3600tgcttatgta aataacaact aaacgcagac gcaatcacca ttttctttat
agaaatgctc 3660aaaatc 3666131914DNAArabidopsis
thalianaCDS(282)..(1736)encoding serine carboxypeptidase I
precursor- like protein 13gtgcctttta aggaaactgt tgctttgcag
aatcacaaaa tctatatacc aaccataagt 60ttccaaattt tgttctaaaa atcaaaaaaa
ttcatatcat ctttttgtct ttttttacca 120gaatttcatc cttatttcgt
gctaaatcat tttccaaaca ttcataatac tttttgttgg 180tttatggcca
actaacaaaa tatcctctag attttcttct ttgtgttagt ctatataaag
240caataccaca gtgatcctct tttttaattc attctgtgtt t atg tca ata atc
aca 296 Met Ser Ile Ile Thr 1 5atg gtt tgg tta atg aaa gtt ttt gtc
ttt gtg aca tta ctc tct tta 344Met Val Trp Leu Met Lys Val Phe Val
Phe Val Thr Leu Leu Ser Leu 10 15 20gtg ttt gta ata aca gaa tca gct
cct gaa tct gct ctt atc acc aaa 392Val Phe Val Ile Thr Glu Ser Ala
Pro Glu Ser Ala Leu Ile Thr Lys 25 30 35ctt cct ggt ttc gaa ggc act
ttt cct tcg aaa cat tac tcc ggg tat 440Leu Pro Gly Phe Glu Gly Thr
Phe Pro Ser Lys His Tyr Ser Gly Tyr 40 45 50gtg aca att gat aag gaa
cat gga aag aat cta tgg tat tac ttt att 488Val Thr Ile Asp Lys Glu
His Gly Lys Asn Leu Trp Tyr Tyr Phe Ile 55 60 65gaa tcg gag aag aat
cca tcg aaa gat ccg gtg gtg ctt tgg ctc aat 536Glu Ser Glu Lys Asn
Pro Ser Lys Asp Pro Val Val Leu Trp Leu Asn70 75 80 85ggt ggt cca
ggt tgt tca agc atg gat gga ttt gtg tat gag cat ggt 584Gly Gly Pro
Gly Cys Ser Ser Met Asp Gly Phe Val Tyr Glu His Gly 90 95 100cca
ttc aat ttc gaa cta cca aag aaa aac aat agt ctc cca ctc ttg 632Pro
Phe Asn Phe Glu Leu Pro Lys Lys Asn Asn Ser Leu Pro Leu Leu 105 110
115cat ctt aat cct tat agt tgg tcc aag gtc tct aac att ata tac ctt
680His Leu Asn Pro Tyr Ser Trp Ser Lys Val Ser Asn Ile Ile Tyr Leu
120 125 130gat tct cct gtt ggt gtg gga ttc tct tac tca aac aat aag
tct gat 728Asp Ser Pro Val Gly Val Gly Phe Ser Tyr Ser Asn Asn Lys
Ser Asp 135 140 145tac ata acc ggt gat att aaa acc gcg gtt gac tct
cac gcg ttc ctt 776Tyr Ile Thr Gly Asp Ile Lys Thr Ala Val Asp Ser
His Ala Phe Leu150 155 160 165ctc aag tgg ttc caa atg ttt cct gag
ttt caa tca aat cct ttt ttc 824Leu Lys Trp Phe Gln Met
Phe Pro Glu Phe Gln Ser Asn Pro Phe Phe 170 175 180atc tct gga gaa
tct tac gcc gga gtt tat gtc cca act ctt gct tca 872Ile Ser Gly Glu
Ser Tyr Ala Gly Val Tyr Val Pro Thr Leu Ala Ser 185 190 195gaa gtt
gtc ata ggg aac aaa aat gga gta aag ccg gct ctt aac ttc 920Glu Val
Val Ile Gly Asn Lys Asn Gly Val Lys Pro Ala Leu Asn Phe 200 205
210aag ggg tat ttg gtt gga aat gga gtt gcg gat ccg aag ttt gat gga
968Lys Gly Tyr Leu Val Gly Asn Gly Val Ala Asp Pro Lys Phe Asp Gly
215 220 225aat gct ttt gtc ccg ttt gca cat ggg atg gga cta atc tct
gat gaa 1016Asn Ala Phe Val Pro Phe Ala His Gly Met Gly Leu Ile Ser
Asp Glu230 235 240 245ctc ttt gag aac gtt aca aaa gct tgc aag gga
aat ttc tat gaa ata 1064Leu Phe Glu Asn Val Thr Lys Ala Cys Lys Gly
Asn Phe Tyr Glu Ile 250 255 260gag gga ctc gag tgc gag gaa cag tat
acg aaa gtt aac gat gat act 1112Glu Gly Leu Glu Cys Glu Glu Gln Tyr
Thr Lys Val Asn Asp Asp Thr 265 270 275aac cag ttg aat ata tac aac
att ctt gag cca tgt tac cac gga aca 1160Asn Gln Leu Asn Ile Tyr Asn
Ile Leu Glu Pro Cys Tyr His Gly Thr 280 285 290tcg cta tct gca ttc
gac att aga tcg ctt cct tca agt ctc ctt cag 1208Ser Leu Ser Ala Phe
Asp Ile Arg Ser Leu Pro Ser Ser Leu Leu Gln 295 300 305cta ggc aaa
act gaa aaa cgg tta cct ata aga aaa aga atg ttt ggt 1256Leu Gly Lys
Thr Glu Lys Arg Leu Pro Ile Arg Lys Arg Met Phe Gly310 315 320
325cga gcg tgg cca gtt cgt gcg cct gtt cat cca ggc att gtc cct agc
1304Arg Ala Trp Pro Val Arg Ala Pro Val His Pro Gly Ile Val Pro Ser
330 335 340tgg tct caa ctt ctt gcc gac gtc act gtt cct tgc att gat
gat aga 1352Trp Ser Gln Leu Leu Ala Asp Val Thr Val Pro Cys Ile Asp
Asp Arg 345 350 355gtt gca aca gcg tgg ttg aac gat cca gag att agg
aaa gct att cat 1400Val Ala Thr Ala Trp Leu Asn Asp Pro Glu Ile Arg
Lys Ala Ile His 360 365 370act aaa gag gag agc gag att gga agg tgg
gaa ctt tgc agt ggg aaa 1448Thr Lys Glu Glu Ser Glu Ile Gly Arg Trp
Glu Leu Cys Ser Gly Lys 375 380 385ctc tca ttc tat cac gac gca gga
agc atg atc gat ttc cat aga aac 1496Leu Ser Phe Tyr His Asp Ala Gly
Ser Met Ile Asp Phe His Arg Asn390 395 400 405ctt aca cta agt gga
tat aga gct ctc att tac agc ggg gat cac gat 1544Leu Thr Leu Ser Gly
Tyr Arg Ala Leu Ile Tyr Ser Gly Asp His Asp 410 415 420atg tgt gtt
ccg ttt act ggc tcg gaa gct tgg aca aaa tct ctt gga 1592Met Cys Val
Pro Phe Thr Gly Ser Glu Ala Trp Thr Lys Ser Leu Gly 425 430 435tac
aaa gtt att gat gaa tgg agg gca tgg ata tca aat gac caa gtc 1640Tyr
Lys Val Ile Asp Glu Trp Arg Ala Trp Ile Ser Asn Asp Gln Val 440 445
450gcg ggg tat acg caa gga tat gca aac aat ctc aca ttt tta acc atc
1688Ala Gly Tyr Thr Gln Gly Tyr Ala Asn Asn Leu Thr Phe Leu Thr Ile
455 460 465aag ggt gca gga cat acg gtt cct gag aca aac cgc ggg agg
ctt tag 1736Lys Gly Ala Gly His Thr Val Pro Glu Thr Asn Arg Gly Arg
Leu470 475 480acttctatag ccgatttcta gaaggaagca agatttaaga
gagctgtctc atcagtttca 1796tattctgtgc tgtatttaaa agtgatcatc
ttatagcaat atagaatctt gattgatttg 1856aataagaaag atatattctt
caactcatta ttcaacatag taaaaatcat ttacctct 191414484PRTArabidopsis
thaliana 14Met Ser Ile Ile Thr Met Val Trp Leu Met Lys Val Phe Val
Phe Val1 5 10 15Thr Leu Leu Ser Leu Val Phe Val Ile Thr Glu Ser Ala
Pro Glu Ser 20 25 30Ala Leu Ile Thr Lys Leu Pro Gly Phe Glu Gly Thr
Phe Pro Ser Lys 35 40 45His Tyr Ser Gly Tyr Val Thr Ile Asp Lys Glu
His Gly Lys Asn Leu 50 55 60Trp Tyr Tyr Phe Ile Glu Ser Glu Lys Asn
Pro Ser Lys Asp Pro Val65 70 75 80Val Leu Trp Leu Asn Gly Gly Pro
Gly Cys Ser Ser Met Asp Gly Phe 85 90 95Val Tyr Glu His Gly Pro Phe
Asn Phe Glu Leu Pro Lys Lys Asn Asn 100 105 110Ser Leu Pro Leu Leu
His Leu Asn Pro Tyr Ser Trp Ser Lys Val Ser 115 120 125Asn Ile Ile
Tyr Leu Asp Ser Pro Val Gly Val Gly Phe Ser Tyr Ser 130 135 140Asn
Asn Lys Ser Asp Tyr Ile Thr Gly Asp Ile Lys Thr Ala Val Asp145 150
155 160Ser His Ala Phe Leu Leu Lys Trp Phe Gln Met Phe Pro Glu Phe
Gln 165 170 175Ser Asn Pro Phe Phe Ile Ser Gly Glu Ser Tyr Ala Gly
Val Tyr Val 180 185 190Pro Thr Leu Ala Ser Glu Val Val Ile Gly Asn
Lys Asn Gly Val Lys 195 200 205Pro Ala Leu Asn Phe Lys Gly Tyr Leu
Val Gly Asn Gly Val Ala Asp 210 215 220Pro Lys Phe Asp Gly Asn Ala
Phe Val Pro Phe Ala His Gly Met Gly225 230 235 240Leu Ile Ser Asp
Glu Leu Phe Glu Asn Val Thr Lys Ala Cys Lys Gly 245 250 255Asn Phe
Tyr Glu Ile Glu Gly Leu Glu Cys Glu Glu Gln Tyr Thr Lys 260 265
270Val Asn Asp Asp Thr Asn Gln Leu Asn Ile Tyr Asn Ile Leu Glu Pro
275 280 285Cys Tyr His Gly Thr Ser Leu Ser Ala Phe Asp Ile Arg Ser
Leu Pro 290 295 300Ser Ser Leu Leu Gln Leu Gly Lys Thr Glu Lys Arg
Leu Pro Ile Arg305 310 315 320Lys Arg Met Phe Gly Arg Ala Trp Pro
Val Arg Ala Pro Val His Pro 325 330 335Gly Ile Val Pro Ser Trp Ser
Gln Leu Leu Ala Asp Val Thr Val Pro 340 345 350Cys Ile Asp Asp Arg
Val Ala Thr Ala Trp Leu Asn Asp Pro Glu Ile 355 360 365Arg Lys Ala
Ile His Thr Lys Glu Glu Ser Glu Ile Gly Arg Trp Glu 370 375 380Leu
Cys Ser Gly Lys Leu Ser Phe Tyr His Asp Ala Gly Ser Met Ile385 390
395 400Asp Phe His Arg Asn Leu Thr Leu Ser Gly Tyr Arg Ala Leu Ile
Tyr 405 410 415Ser Gly Asp His Asp Met Cys Val Pro Phe Thr Gly Ser
Glu Ala Trp 420 425 430Thr Lys Ser Leu Gly Tyr Lys Val Ile Asp Glu
Trp Arg Ala Trp Ile 435 440 445Ser Asn Asp Gln Val Ala Gly Tyr Thr
Gln Gly Tyr Ala Asn Asn Leu 450 455 460Thr Phe Leu Thr Ile Lys Gly
Ala Gly His Thr Val Pro Glu Thr Asn465 470 475 480Arg Gly Arg
Leu152254DNAArabidopsis thalianapromoter(1)..(2254)transcription
regulating sequence from gene At1g66250 15tacctcgagc tcgtcatgga
ctcatggcag tggtaatttg gtcaccaaaa tctggctgcg 60agactccaaa gacgattaca
tgtgtcggca tcccctaaag ttatgtcatg tatgctcagt 120gtttcctcgc
attactctcc aatttttgcc ttgcaagata cttttgtgca tgggcatggc
180ttgtcacttg tgtcgcgaac cttgtcttca catatttcct cgatatattt
caatctcttt 240ttctagtata ttctttaatc cttccagata taatctgaaa
cttcattgaa ttaacaagtt 300cataaccaag atcgtctatc atttaaaggc
cttaattaaa aaagaaaagt caataacaaa 360gaacactttg ttaatatcct
cacattaaat taattaataa tcatttataa atatatcatt 420aaccaacaga
cccgttattc atttttaaaa cttgattaat tctctcgcaa ccacccacaa
480aagtgataaa agcataagta taaaaatgtc aagagatttg acaaaatttt
gtaggccgag 540ttggattgac ttaggttcgt cccaaacggc taaatcactt
gtccatgctt accaagatgc 600caaaatacac gacatattcc acaacaaaca
aattccgcta tctgataatt cccgaaaata 660tgttccaaaa atatatagaa
atgatggaaa caaaataata ttttaaaatc tttaatattc 720attaactaat
gatttatgaa aaattattaa aaaatgattt gaatatttta taaatctatt
780aagacgaaaa caagccatgt ttattctctc ttataagcta tgtatagtat
ttgtaagtta 840ctattagtat gggtaacaat tataagaaaa cgaatcaatt
tattaataga taaattaaga 900taaaatcaca taaaagtaag aaaaaggacc
aaatgtaata aagagtaaga gaaatacaga 960gaaacaaaca aaagagtcgc
aacggctatt tcgtagttac caacttattt tgctgtccaa 1020tgcacaactt
gttcttacac caaaagttgt gctctttaaa cttgcattta ttctacccaa
1080aaaaaaaaaa attgcacaat atatatacaa atatatacca tagtttatca
ctaacaatca 1140gtagtactat gtttcacact aatatactca aaattaatca
tcaaatcata ttaatattag 1200tgattaacaa acaacaaaga agctaattaa
gacttttctt tcccacagta caactctctg 1260actgtgtaac tcactctgtc
aactacaaga ttacaaaaga ccgttagatt tcatctttga 1320ccatttgaaa
tcttaagatc aaatctcagc cgtccattcc accgttacca atctctctct
1380ttcgatataa tcaacaaatt ttctaacttt ttctcggtgt cctttctcaa
tcagaagctt 1440taccctattt tctctatacc tcccacgcaa tttctctcat
tcctttcttt gtgggttttt 1500gtttattctt cacaaagtta acatttttag
actcaaaact cattcttttg cccttcctct 1560gtttctcctc tcttggggtt
tatcttagat tctccaaaaa agtctctctc tttccatggc 1620ttctcttctc
catcttcttc tcctttctct ttctctttta gttcttgctt cagcttctcc
1680ttctcctcca gctgacgaag gtgagattct tgtatctgat tcaatcttgt
actttgagtt 1740tcagattcaa tctttactcc caaatttgaa agttttgatt
tttgcatgtg ttaatgttct 1800tctagaaaac caaatgttcc aattttttat
gtagtaataa ttccactgtc agtgattaat 1860gaaaatagct ttaatataaa
gtaattaaag acaagtcctt ttgtatgtat gtgttggatt 1920tgattcattg
agtatgtttg agagtgtggt aggttatgat aataaatgtc agtatgtgtg
1980tttggcaggt gggtttgttt ttaaatgctc actaagaaac aagaaatggt
gaattttcat 2040gtccccaaat atttattaca agacaaataa ttcaatgtct
aatgttttga ccagagacat 2100agttcttttt agttaagtct atgaatccaa
atctttgagt attgtacact cttttggact 2160agttctagat atgttgatac
taatggttta gttgaaatca atatattttt tgtaggttca 2220tatattggag
taaacatagg gactgatctt tctg 2254162259DNAArabidopsis
thalianapromoter(1)..(2259)transcription regulating sequence from
gene At1g66250 16taaaagctcg agctcgtcat ggactcatgg cagtggtaat
ttggtcacca aaatctggct 60gcgagactcc aaagacgatt acatgtgtcg gcatccccta
aagttatgtc atgtatgctc 120agtgtttcct cgcattactc tccaattttt
gccttgcaag atacttttgt gcatgggcat 180ggcttgtcac ttgtgtcgcg
aaccttgtct tcacatattt cctcgatata tttcaatctc 240tttttctagt
atattcttta atccttccag atataatctg aaacttcatt gaattaacaa
300gttcataacc aagatcgtct atcatttaaa ggccttaatt aaaaaagaaa
agtcaataac 360aaagaacact ttgttaatat cctcacatta aattaattaa
taatcatcta taaatatatc 420attaaccaac agacccgtta ttcattttta
aaacttgatt aattctctcg caaccaccca 480caaaagtgat aaaagcataa
gtataaaaat gtcaagagat ttgacaaaat tttgtaggcc 540gagttagatt
gacttaggtt cgtcccaaac ggctaaatca cttgtccatg cttaccaaga
600tgccaaaata cacgacatat tccacaacaa acaaattccg ctatctgata
attcccgaaa 660atatgttcca aaaatatata gaaatgatgg aaacaaaata
atattttaaa atctttaata 720ttcattaact aatgatttat gaaaaattat
taaaaaatga tttgaatatt ttataaatct 780attaagacga aaacaagcca
tgtttattct ctcttataag ctatgtatag tatttgtaag 840ttactattag
tatgggtaac aattataaga aaacgaatca atttattaat agataaatta
900agataaaatc acataaaagt aagaaaaagg accaaatgta ataaagagta
agagaaatac 960agagaaacaa acaaaagagt cgcaacggct atttcgtagt
taccaactta ttttgctgtc 1020caatgcacaa cttgttctta caccaaaagt
tgtgctcttt aaacttgcat ttattctacc 1080caaaaaaaaa aaaaattgct
caatatatat acaaatatat accatagttt atcactaaca 1140atcagtagta
ttatgtttca cactaatata ctcaaaatta atcatcaaat catattaata
1200ttagtgatta acaaacaaca aagaagctaa ttaagacttt tctttcccac
agtacaactc 1260tctgactgtg taactcactc tgtcaactac aagattacaa
aagaccgtta gatttcatct 1320ttgaccattt gaaatcttaa gatcaaatct
cagccgtcca ttccaccgtt accaatctct 1380ctctttcgat ataatcaaca
aattttctaa ctttttctcg gtgtcctttc tcaatcagaa 1440gctttaccct
attttctcta tacctcccac gcaatttctc tcattccttt ctttgtgggt
1500ttttgtttat tcttcacaaa gttaacattt ttagactcaa aactcattct
tttgcccttc 1560ctctgtttct cctctcttgg ggtttatctt agattctcca
aaaaagtctc tctctttcca 1620tggcttctct tctccatctt cttctccttt
ctctttctct tttagttctt gcttcagctt 1680ctccttctcc tccagctgat
gaaggtgaga ttcttgtatc tgattcaatc ttgtactttg 1740agtttcagat
tcaatcttta ctcccaaatt tgaaagtttt gatttttgca tgtgttaatg
1800ttcttctaga aaaccaaatg ttccattttt tatgtagtaa taattccact
gtcagtgatt 1860aatgaaaata gctttaatat aaagtaatta aagacaagtc
cttttgtatg tatgtgttgg 1920atttgattca ttgagtatgt ttgagagtgt
ggtaggttat gataataaat gtcagtatgt 1980gtgtttggca ggtgggtttg
tttttaaatg ctcactaaga aacaagaaat ggtgaatttt 2040catgtcccca
aatatttatt acaagacaaa taattcaatg tctaatgttt tgaccagaga
2100catagttctt tttagttaag tctatgaatc caaatctttg agtattgtac
actcttttgg 2160actagttcta gatatgttga tactaatggt ttagttgaaa
tcaatatatt ttttgtaggt 2220tcatatattg gagtaaacat agggactgat
ctttctgat 2259171446DNAArabidopsis
thalianapromoter(1)..(1446)transcription regulating sequence from
gene At1g66250 17tacctcgagc tcgtcatgga ctcatggcag tggtaatttg
gtcaccaaaa tctggctgcg 60agactccaaa gacgattaca tgtgtcggca tcccctaaag
ttatgtcatg tatgctcagt 120gtttcctcgc attactctcc aatttttgcc
ttgcaagata cttttgtgca tgggcatggc 180ttgtcacttg tgtcgcgaac
cttgtcttca catatttcct cgatatattt caatctcttt 240ttctagtata
ttctttaatc cttccagata taatctgaaa cttcattgaa ttaacaagtt
300cataaccaag atcgtctatc atttaaaggc cttaattaaa aaagaaaagt
caataacaaa 360gaacactttg ttaatatcct cacattaaat taattaataa
tcatttataa atatatcatt 420aaccaacaga cccgttattc atttttaaaa
cttgattaat tctctcgcaa ccacccacaa 480aagtgataaa agcataagta
taaaaatgtc aagagatttg acaaaatttt gtaggccgag 540ttggattgac
ttaggttcgt cccaaacggc taaatcactt gtccatgctt accaagatgc
600caaaatacac gacatattcc acaacaaaca aattccgcta tctgataatt
cccgaaaata 660tgttccaaaa atatatagaa atgatggaaa caaaataata
ttttaaaatc tttaatattc 720attaactaat gatttatgaa aaattattaa
aaaatgattt gaatatttta taaatctatt 780aagacgaaaa caagccatgt
ttattctctc ttataagcta tgtatagtat ttgtaagtta 840ctattagtat
gggtaacaat tataagaaaa cgaatcaatt tattaataga taaattaaga
900taaaatcaca taaaagtaag aaaaaggacc aaatgtaata aagagtaaga
gaaatacaga 960gaaacaaaca aaagagtcgc aacggctatt tcgtagttac
caacttattt tgctgtccaa 1020tgcacaactt gttcttacac caaaagttgt
gctctttaaa cttgcattta ttctacccaa 1080aaaaaaaaaa attgcacaat
atatatacaa atatatacca tagtttatca ctaacaatca 1140gtagtactat
gtttcacact aatatactca aaattaatca tcaaatcata ttaatattag
1200tgattaacaa acaacaaaga agctaattaa gacttttctt tcccacagta
caactctctg 1260actgtgtaac tcactctgtc aactacaaga ttacaaaaga
ccgttagatt tcatctttga 1320ccatttgaaa tcttaagatc aaatctcagc
cgtccattcc accgttacca atctctctct 1380ttcgatataa tcaacaaatt
ttctaacttt ttctcggtgt cctttctcaa tcagaagctt 1440taccct
1446181450DNAArabidopsis thalianapromoter(1)..(1450)transcription
regulating sequence from gene At1g66250 18taaaagctcg agctcgtcat
ggactcatgg cagtggtaat ttggtcacca aaatctggct 60gcgagactcc aaagacgatt
acatgtgtcg gcatccccta aagttatgtc atgtatgctc 120agtgtttcct
cgcattactc tccaattttt gccttgcaag atacttttgt gcatgggcat
180ggcttgtcac ttgtgtcgcg aaccttgtct tcacatattt cctcgatata
tttcaatctc 240tttttctagt atattcttta atccttccag atataatctg
aaacttcatt gaattaacaa 300gttcataacc aagatcgtct atcatttaaa
ggccttaatt aaaaaagaaa agtcaataac 360aaagaacact ttgttaatat
cctcacatta aattaattaa taatcatcta taaatatatc 420attaaccaac
agacccgtta ttcattttta aaacttgatt aattctctcg caaccaccca
480caaaagtgat aaaagcataa gtataaaaat gtcaagagat ttgacaaaat
tttgtaggcc 540gagttagatt gacttaggtt cgtcccaaac ggctaaatca
cttgtccatg cttaccaaga 600tgccaaaata cacgacatat tccacaacaa
acaaattccg ctatctgata attcccgaaa 660atatgttcca aaaatatata
gaaatgatgg aaacaaaata atattttaaa atctttaata 720ttcattaact
aatgatttat gaaaaattat taaaaaatga tttgaatatt ttataaatct
780attaagacga aaacaagcca tgtttattct ctcttataag ctatgtatag
tatttgtaag 840ttactattag tatgggtaac aattataaga aaacgaatca
atttattaat agataaatta 900agataaaatc acataaaagt aagaaaaagg
accaaatgta ataaagagta agagaaatac 960agagaaacaa acaaaagagt
cgcaacggct atttcgtagt taccaactta ttttgctgtc 1020caatgcacaa
cttgttctta caccaaaagt tgtgctcttt aaacttgcat ttattctacc
1080caaaaaaaaa aaaaattgct caatatatat acaaatatat accatagttt
atcactaaca 1140atcagtagta ttatgtttca cactaatata ctcaaaatta
atcatcaaat catattaata 1200ttagtgatta acaaacaaca aagaagctaa
ttaagacttt tctttcccac agtacaactc 1260tctgactgtg taactcactc
tgtcaactac aagattacaa aagaccgtta gatttcatct 1320ttgaccattt
gaaatcttaa gatcaaatct cagccgtcca ttccaccgtt accaatctct
1380ctctttcgat ataatcaaca aattttctaa ctttttctcg gtgtcctttc
tcaatcagaa 1440gctttaccct 1450193892DNAArabidopsis
thalianapromoter(1)..(3892)transcription regulating sequence from
gene At1g66250 19caataagaat tcggttagtt ttctaatata catgatgtag
acatgtagtt tggtttatat 60atgttaaata tatgtataaa ttgttaccga attaatagtt
tgtaaccaaa taattacatt 120gctagattta tttaattcat taaaaaaaca
ccttaataat gttgcggcag gatatcatta 180tgcataggta ggtattccga
caaaaaatga ccttttaagt tctaaaatta gaataaacca 240ctggaactca
aaaatgtagt tcgttaatcc aaatatacat attaaaagta ggattgccaa
300gaaaatcttc aaattctggt aaaattggca cgtaggatag agttttgttg
ttgtcgtaaa 360ttcatgttaa atcttggttt gatggttgta aagtgtgttg
tttagaagtt aaaaccgcgt 420tttataatta acaacatttt aaatgacagc
aaataaacaa aacgaattac ttcttttggg 480tgttcattat cttttataaa
taactttttc ttcatttttc ttaaattttt cattagccta 540taagtagttc
tcgggaacaa agttttctag tacttaaata tataaatctt acatatctta
600tgttacttgt tgatatcaaa agaaaagttg aaaatgaaga agattacaaa
aaagctatca 660atgaaatacg tgaagctcca taagcaaaaa aagctccgcc
aagacgggtc catgaagtca 720gaggaagacg gtggagagcc tttccgggaa
tctcattcgc cggattatgc
ggaaagtaat 780gaaacaaagg agactcctaa ggttacaaag attatggagt
ccatgcatcg taagctgatg 840ctaaaagaca aggcaaataa aaagaaaatt
cacgtggacg atcaatccca aatgtcagaa 900cgttcggtta ggaatggtgg
tcgtgatcgc aaggaccagc ttgatgaagg ctcggttaga 960aacatgaatc
gcgatggtac ggaccagctt gtaggctcag ttaaaaacag tcatatggat
1020catcttgaag atttgattag gggtcgtgat cgtatggaag gttccaatag
gaatggtgct 1080cgtaatcgtg tggaccagct tgaaggtttt agcaaaaacg
gtgagaaaca tcagcatgaa 1140gaaaatactg ttaaaacaag cgatcaaata
gatcaatcag aaaaatcaac taaaagtcca 1200tctgattttg cttcaaaaaa
agattatctt gattggattg aatatgtgga aggatcgaat 1260catcattgtt
tcgatcgatc cgaagattcc gaaaaatctt atataagaga ggatatcgat
1320catgacgcga taagcgttgg gatatcagaa ggatccatag aggggaacaa
cgatgagagt 1380ctattactca agagttctaa gtatagaaaa aacgagaaat
ttgaagattc aaaaggttat 1440aagaaaggaa aaggtagcaa ggctaaagat
tcactgaaat gtcaattagt tgactaaatg 1500ttgaatgtgc attttagtcc
taacaaaata aatgtttctt tcataatgta taatatgatc 1560attgatcatt
tagttagtat gaacaactac tctgtatgat cattactttc aatgaaaatt
1620gtggttttct tattaaaagc tcgagctcgt catggactca tggcagtggt
aatttggtca 1680ccaaaatctg gctgcgagac tccaaagacg attacatgtg
tcggcatccc ctaaagttat 1740gtcatgtatg ctcagtgttt cctcgcatta
ctctccaatt tttgccttgc aagatacttt 1800tgtgcatggg catggcttgt
cacttgtgtc gcgaaccttg tcttcacata tttcctcgat 1860atatttcaat
ctctttttct agtatattct ttaatccttc cagatataat ctgaaacttc
1920attgaattaa caagttcata accaagatcg tctatcattt aaaggcctta
attaaaaaag 1980aaaagtcaat aacaaagaac actttgttaa tatcctcaca
ttaaattaat taataatcat 2040ctataaatat atcattaacc aacagacccg
ttattcattt ttaaaacttg attaattctc 2100tcgcaaccac ccacaaaagt
gataaaagca taagtataaa aatgtcaaga gatttgacaa 2160aattttgtag
gccgagttag attgacttag gttcgtccca aacggctaaa tcacttgtcc
2220atgcttacca agatgccaaa atacacgaca tattccacaa caaacaaatt
ccgctatctg 2280ataattcccg aaaatatgtt ccaaaaatat atagaaatga
tggaaacaaa ataatatttt 2340aaaatcttta atattcatta actaatgatt
tatgaaaaat tattaaaaaa tgatttgaat 2400attttataaa tctattaaga
cgaaaacaag ccatgtttat tctctcttat aagctatgta 2460tagtatttgt
aagttactat tagtatgggt aacaattata agaaaacgaa tcaatttatt
2520aatagataaa ttaagataaa atcacataaa agtaagaaaa aggaccaaat
gtaataaaga 2580gtaagagaaa tacagagaaa caaacaaaag agtcgcaacg
gctatttcgt agttaccaac 2640ttattttgct gtccaatgca caacttgttc
ttacaccaaa agttgtgctc tttaaacttg 2700catttattct acccaaaaaa
aaaaaaaatt gctcaatata tatacaaata tataccatag 2760tttatcacta
acaatcagta gtattatgtt tcacactaat atactcaaaa ttaatcatca
2820aatcatatta atattagtga ttaacaaaca acaaagaagc taattaagac
ttttctttcc 2880cacagtacaa ctctctgact gtgtaactca ctctgtcaac
tacaagatta caaaagaccg 2940ttagatttca tctttgacca tttgaaatct
taagatcaaa tctcagccgt ccattccacc 3000gttaccaatc tctctctttc
gatataatca acaaattttc taactttttc tcggtgtcct 3060ttctcaatca
gaagctttac cctattttct ctatacctcc cacgcaattt ctctcattcc
3120tttctttgtg ggtttttgtt tattcttcac aaagttaaca tttttagact
caaaactcat 3180tcttttgccc ttcctctgtt tctcctctct tggggtttat
cttagattct ccaaaaaagt 3240ctctctcttt ccatggcttc tcttctccat
cttcttctcc tttctctttc tcttttagtt 3300cttgcttcag cttctccttc
tcctccagct gatgaaggtg agattcttgt atctgattca 3360atcttgtact
ttgagtttca gattcaatct ttactcccaa atttgaaagt tttgattttt
3420gcatgtgtta atgttcttct agaaaaccaa atgttccatt ttttatgtag
taataattcc 3480actgtcagtg attaatgaaa atagctttaa tataaagtaa
ttaaagacaa gtccttttgt 3540atgtatgtgt tggatttgat tcattgagta
tgtttgagag tgtggtaggt tatgataata 3600aatgtcagta tgtgtgtttg
gcaggtgggt ttgtttttaa atgctcacta agaaacaaga 3660aatggtgaat
tttcatgtcc ccaaatattt attacaagac aaataattca atgtctaatg
3720ttttgaccag agacatagtt ctttttagtt aagtctatga atccaaatct
ttgagtattg 3780tacactcttt tggactagtt ctagatatgt tgatactaat
ggtttagttg aaatcaatat 3840attttttgta ggttcatata ttggagtaaa
catagggact gatctttctg at 3892203083DNAArabidopsis
thalianapromoter(1)..(3083)transcription regulating sequence from
gene At1g66250 20caataagaat tcggttagtt ttctaatata catgatgtag
acatgtagtt tggtttatat 60atgttaaata tatgtataaa ttgttaccga attaatagtt
tgtaaccaaa taattacatt 120gctagattta tttaattcat taaaaaaaca
ccttaataat gttgcggcag gatatcatta 180tgcataggta ggtattccga
caaaaaatga ccttttaagt tctaaaatta gaataaacca 240ctggaactca
aaaatgtagt tcgttaatcc aaatatacat attaaaagta ggattgccaa
300gaaaatcttc aaattctggt aaaattggca cgtaggatag agttttgttg
ttgtcgtaaa 360ttcatgttaa atcttggttt gatggttgta aagtgtgttg
tttagaagtt aaaaccgcgt 420tttataatta acaacatttt aaatgacagc
aaataaacaa aacgaattac ttcttttggg 480tgttcattat cttttataaa
taactttttc ttcatttttc ttaaattttt cattagccta 540taagtagttc
tcgggaacaa agttttctag tacttaaata tataaatctt acatatctta
600tgttacttgt tgatatcaaa agaaaagttg aaaatgaaga agattacaaa
aaagctatca 660atgaaatacg tgaagctcca taagcaaaaa aagctccgcc
aagacgggtc catgaagtca 720gaggaagacg gtggagagcc tttccgggaa
tctcattcgc cggattatgc ggaaagtaat 780gaaacaaagg agactcctaa
ggttacaaag attatggagt ccatgcatcg taagctgatg 840ctaaaagaca
aggcaaataa aaagaaaatt cacgtggacg atcaatccca aatgtcagaa
900cgttcggtta ggaatggtgg tcgtgatcgc aaggaccagc ttgatgaagg
ctcggttaga 960aacatgaatc gcgatggtac ggaccagctt gtaggctcag
ttaaaaacag tcatatggat 1020catcttgaag atttgattag gggtcgtgat
cgtatggaag gttccaatag gaatggtgct 1080cgtaatcgtg tggaccagct
tgaaggtttt agcaaaaacg gtgagaaaca tcagcatgaa 1140gaaaatactg
ttaaaacaag cgatcaaata gatcaatcag aaaaatcaac taaaagtcca
1200tctgattttg cttcaaaaaa agattatctt gattggattg aatatgtgga
aggatcgaat 1260catcattgtt tcgatcgatc cgaagattcc gaaaaatctt
atataagaga ggatatcgat 1320catgacgcga taagcgttgg gatatcagaa
ggatccatag aggggaacaa cgatgagagt 1380ctattactca agagttctaa
gtatagaaaa aacgagaaat ttgaagattc aaaaggttat 1440aagaaaggaa
aaggtagcaa ggctaaagat tcactgaaat gtcaattagt tgactaaatg
1500ttgaatgtgc attttagtcc taacaaaata aatgtttctt tcataatgta
taatatgatc 1560attgatcatt tagttagtat gaacaactac tctgtatgat
cattactttc aatgaaaatt 1620gtggttttct tattaaaagc tcgagctcgt
catggactca tggcagtggt aatttggtca 1680ccaaaatctg gctgcgagac
tccaaagacg attacatgtg tcggcatccc ctaaagttat 1740gtcatgtatg
ctcagtgttt cctcgcatta ctctccaatt tttgccttgc aagatacttt
1800tgtgcatggg catggcttgt cacttgtgtc gcgaaccttg tcttcacata
tttcctcgat 1860atatttcaat ctctttttct agtatattct ttaatccttc
cagatataat ctgaaacttc 1920attgaattaa caagttcata accaagatcg
tctatcattt aaaggcctta attaaaaaag 1980aaaagtcaat aacaaagaac
actttgttaa tatcctcaca ttaaattaat taataatcat 2040ctataaatat
atcattaacc aacagacccg ttattcattt ttaaaacttg attaattctc
2100tcgcaaccac ccacaaaagt gataaaagca taagtataaa aatgtcaaga
gatttgacaa 2160aattttgtag gccgagttag attgacttag gttcgtccca
aacggctaaa tcacttgtcc 2220atgcttacca agatgccaaa atacacgaca
tattccacaa caaacaaatt ccgctatctg 2280ataattcccg aaaatatgtt
ccaaaaatat atagaaatga tggaaacaaa ataatatttt 2340aaaatcttta
atattcatta actaatgatt tatgaaaaat tattaaaaaa tgatttgaat
2400attttataaa tctattaaga cgaaaacaag ccatgtttat tctctcttat
aagctatgta 2460tagtatttgt aagttactat tagtatgggt aacaattata
agaaaacgaa tcaatttatt 2520aatagataaa ttaagataaa atcacataaa
agtaagaaaa aggaccaaat gtaataaaga 2580gtaagagaaa tacagagaaa
caaacaaaag agtcgcaacg gctatttcgt agttaccaac 2640ttattttgct
gtccaatgca caacttgttc ttacaccaaa agttgtgctc tttaaacttg
2700catttattct acccaaaaaa aaaaaaaatt gctcaatata tatacaaata
tataccatag 2760tttatcacta acaatcagta gtattatgtt tcacactaat
atactcaaaa ttaatcatca 2820aatcatatta atattagtga ttaacaaaca
acaaagaagc taattaagac ttttctttcc 2880cacagtacaa ctctctgact
gtgtaactca ctctgtcaac tacaagatta caaaagaccg 2940ttagatttca
tctttgacca tttgaaatct taagatcaaa tctcagccgt ccattccacc
3000gttaccaatc tctctctttc gatataatca acaaattttc taactttttc
tcggtgtcct 3060ttctcaatca gaagctttac cct 3083211786DNAArabidopsis
thalianaCDS(170)..(1687)encoding glycosyl hydrolase family 17
protein 21attttctcta tacctcccac gcaatttctc tcattccttt ctttgtgggt
ttttgtttat 60tcttcacaaa gttaacattt ttagactcaa aactcattct tttgcccttc
ctctgtttct 120cctctcttgg ggtttatctt agattctcca aaaaagtctc tctctttcc
atg gct tct 178 Met Ala Ser 1ctt ctc cat ctt ctt ctc ctt tct ctt
tct ctt tta gtt ctt gct tca 226Leu Leu His Leu Leu Leu Leu Ser Leu
Ser Leu Leu Val Leu Ala Ser 5 10 15gct tct cct tct cct cca gct gat
gaa ggt tca tat att gga gta aac 274Ala Ser Pro Ser Pro Pro Ala Asp
Glu Gly Ser Tyr Ile Gly Val Asn20 25 30 35ata ggg act gat ctt tct
gat atg cca cat cca aca caa gtt gtt gct 322Ile Gly Thr Asp Leu Ser
Asp Met Pro His Pro Thr Gln Val Val Ala 40 45 50cta cta aaa gct caa
gaa atc cga cat att cga tta tac aac gct gat 370Leu Leu Lys Ala Gln
Glu Ile Arg His Ile Arg Leu Tyr Asn Ala Asp 55 60 65ccc ggg ttg ttg
att gct cta gca aac act ggt atc aaa gtt ata atc 418Pro Gly Leu Leu
Ile Ala Leu Ala Asn Thr Gly Ile Lys Val Ile Ile 70 75 80tcc att cct
aat gac cag ctt ctt ggt ata ggt caa tcc aat tca acc 466Ser Ile Pro
Asn Asp Gln Leu Leu Gly Ile Gly Gln Ser Asn Ser Thr 85 90 95gca gcg
aat tgg gtt aaa cgc aat gtc att gca cat tac cct gca acg 514Ala Ala
Asn Trp Val Lys Arg Asn Val Ile Ala His Tyr Pro Ala Thr100 105 110
115atg ata acc gcg gtt tct gtt ggc tct gag gtg cta acc agt ctc tct
562Met Ile Thr Ala Val Ser Val Gly Ser Glu Val Leu Thr Ser Leu Ser
120 125 130aat gca gca cct gtt cta gtc agt gct ata aag aac gtt cat
gct gct 610Asn Ala Ala Pro Val Leu Val Ser Ala Ile Lys Asn Val His
Ala Ala 135 140 145tta ctc tcg gcg aat ctt gac aag ttg ata aaa gtt
tct act cct tta 658Leu Leu Ser Ala Asn Leu Asp Lys Leu Ile Lys Val
Ser Thr Pro Leu 150 155 160tca act tcc ttg att ctt gat cct ttt cct
cca tcg caa gcg ttc ttt 706Ser Thr Ser Leu Ile Leu Asp Pro Phe Pro
Pro Ser Gln Ala Phe Phe 165 170 175aac cgt tct ttg aat gcg gtt ata
gtt ccg tta cta agc ttc ttg cag 754Asn Arg Ser Leu Asn Ala Val Ile
Val Pro Leu Leu Ser Phe Leu Gln180 185 190 195tca aca aac tca tac
ctg atg gtg aat gtg tat cca tac att gac tat 802Ser Thr Asn Ser Tyr
Leu Met Val Asn Val Tyr Pro Tyr Ile Asp Tyr 200 205 210atg caa tca
aac ggt gtg att ccg tta gac tac gcg ttg ttc aaa ccg 850Met Gln Ser
Asn Gly Val Ile Pro Leu Asp Tyr Ala Leu Phe Lys Pro 215 220 225ata
cct cca aac aaa gaa gct gtt gat gca aac act ctt gtt cgt tac 898Ile
Pro Pro Asn Lys Glu Ala Val Asp Ala Asn Thr Leu Val Arg Tyr 230 235
240tca aac gct ttt gat gca atg gtt gat gca acg tac ttc gct atg gcg
946Ser Asn Ala Phe Asp Ala Met Val Asp Ala Thr Tyr Phe Ala Met Ala
245 250 255ttc ctc aac ttc aca aac att ccg gtt tta gta acc gaa tca
ggc tgg 994Phe Leu Asn Phe Thr Asn Ile Pro Val Leu Val Thr Glu Ser
Gly Trp260 265 270 275cct tcg aaa gga gaa acc aat gag cct gat gct
aca ctt gac aac gcc 1042Pro Ser Lys Gly Glu Thr Asn Glu Pro Asp Ala
Thr Leu Asp Asn Ala 280 285 290aac act tac aac agt aat ctc att aga
cat gtt ctt aac aag acc gga 1090Asn Thr Tyr Asn Ser Asn Leu Ile Arg
His Val Leu Asn Lys Thr Gly 295 300 305act ccg aaa cgc ccg ggg atc
gct gtg agt act tat att tac gag ctt 1138Thr Pro Lys Arg Pro Gly Ile
Ala Val Ser Thr Tyr Ile Tyr Glu Leu 310 315 320tac aat gaa gat aca
aaa gca ggt tta tca gag aag aat tgg ggg ttg 1186Tyr Asn Glu Asp Thr
Lys Ala Gly Leu Ser Glu Lys Asn Trp Gly Leu 325 330 335ttt aat gca
aat ggt gaa ccg gtt tac gta ctt cgg ttg act aac tcg 1234Phe Asn Ala
Asn Gly Glu Pro Val Tyr Val Leu Arg Leu Thr Asn Ser340 345 350
355ggc tcg gtt cta gct aac gat aca acg aac cag act tat tgt act gca
1282Gly Ser Val Leu Ala Asn Asp Thr Thr Asn Gln Thr Tyr Cys Thr Ala
360 365 370aga gaa ggt gct gat aca aag atg ctt caa gct gct ctt gat
tgg gct 1330Arg Glu Gly Ala Asp Thr Lys Met Leu Gln Ala Ala Leu Asp
Trp Ala 375 380 385tgt ggt cct gga aag att gat tgt tcg cct att aag
caa gga gag act 1378Cys Gly Pro Gly Lys Ile Asp Cys Ser Pro Ile Lys
Gln Gly Glu Thr 390 395 400tgt tat gaa ccg gat aat gtc gtc gcg cat
gct aac tac gcg ttt gat 1426Cys Tyr Glu Pro Asp Asn Val Val Ala His
Ala Asn Tyr Ala Phe Asp 405 410 415act tat tat cat cag act ggg aat
aat cct gat gct tgc aac ttc aat 1474Thr Tyr Tyr His Gln Thr Gly Asn
Asn Pro Asp Ala Cys Asn Phe Asn420 425 430 435ggt gtt gct agt atc
act acc aca gat cca agt cat ggt aca tgt gtg 1522Gly Val Ala Ser Ile
Thr Thr Thr Asp Pro Ser His Gly Thr Cys Val 440 445 450ttt gct ggg
agc cgc ggt aat ggt aga aac ggg acg tct gtg aac ata 1570Phe Ala Gly
Ser Arg Gly Asn Gly Arg Asn Gly Thr Ser Val Asn Ile 455 460 465aca
gcg cca tcg gca aac tca aca act tct tct gga ata cgg agt gat 1618Thr
Ala Pro Ser Ala Asn Ser Thr Thr Ser Ser Gly Ile Arg Ser Asp 470 475
480ttg tat tac agt cgg ggt ata tgg agt atc ttg aca gta atg att ttg
1666Leu Tyr Tyr Ser Arg Gly Ile Trp Ser Ile Leu Thr Val Met Ile Leu
485 490 495aac gtt gct aat atc ttg tga gaaatctttg ggacataaca
atgttagttt 1717Asn Val Ala Asn Ile Leu500 505ctgttccgag aagttttttt
aactttctta ggtgggattg gagttagaat tgaggccgat 1777tgtgttgga
178622505PRTArabidopsis thaliana 22Met Ala Ser Leu Leu His Leu Leu
Leu Leu Ser Leu Ser Leu Leu Val1 5 10 15Leu Ala Ser Ala Ser Pro Ser
Pro Pro Ala Asp Glu Gly Ser Tyr Ile 20 25 30Gly Val Asn Ile Gly Thr
Asp Leu Ser Asp Met Pro His Pro Thr Gln 35 40 45Val Val Ala Leu Leu
Lys Ala Gln Glu Ile Arg His Ile Arg Leu Tyr 50 55 60Asn Ala Asp Pro
Gly Leu Leu Ile Ala Leu Ala Asn Thr Gly Ile Lys65 70 75 80Val Ile
Ile Ser Ile Pro Asn Asp Gln Leu Leu Gly Ile Gly Gln Ser 85 90 95Asn
Ser Thr Ala Ala Asn Trp Val Lys Arg Asn Val Ile Ala His Tyr 100 105
110Pro Ala Thr Met Ile Thr Ala Val Ser Val Gly Ser Glu Val Leu Thr
115 120 125Ser Leu Ser Asn Ala Ala Pro Val Leu Val Ser Ala Ile Lys
Asn Val 130 135 140His Ala Ala Leu Leu Ser Ala Asn Leu Asp Lys Leu
Ile Lys Val Ser145 150 155 160Thr Pro Leu Ser Thr Ser Leu Ile Leu
Asp Pro Phe Pro Pro Ser Gln 165 170 175Ala Phe Phe Asn Arg Ser Leu
Asn Ala Val Ile Val Pro Leu Leu Ser 180 185 190Phe Leu Gln Ser Thr
Asn Ser Tyr Leu Met Val Asn Val Tyr Pro Tyr 195 200 205Ile Asp Tyr
Met Gln Ser Asn Gly Val Ile Pro Leu Asp Tyr Ala Leu 210 215 220Phe
Lys Pro Ile Pro Pro Asn Lys Glu Ala Val Asp Ala Asn Thr Leu225 230
235 240Val Arg Tyr Ser Asn Ala Phe Asp Ala Met Val Asp Ala Thr Tyr
Phe 245 250 255Ala Met Ala Phe Leu Asn Phe Thr Asn Ile Pro Val Leu
Val Thr Glu 260 265 270Ser Gly Trp Pro Ser Lys Gly Glu Thr Asn Glu
Pro Asp Ala Thr Leu 275 280 285Asp Asn Ala Asn Thr Tyr Asn Ser Asn
Leu Ile Arg His Val Leu Asn 290 295 300Lys Thr Gly Thr Pro Lys Arg
Pro Gly Ile Ala Val Ser Thr Tyr Ile305 310 315 320Tyr Glu Leu Tyr
Asn Glu Asp Thr Lys Ala Gly Leu Ser Glu Lys Asn 325 330 335Trp Gly
Leu Phe Asn Ala Asn Gly Glu Pro Val Tyr Val Leu Arg Leu 340 345
350Thr Asn Ser Gly Ser Val Leu Ala Asn Asp Thr Thr Asn Gln Thr Tyr
355 360 365Cys Thr Ala Arg Glu Gly Ala Asp Thr Lys Met Leu Gln Ala
Ala Leu 370 375 380Asp Trp Ala Cys Gly Pro Gly Lys Ile Asp Cys Ser
Pro Ile Lys Gln385 390 395 400Gly Glu Thr Cys Tyr Glu Pro Asp Asn
Val Val Ala His Ala Asn Tyr 405 410 415Ala Phe Asp Thr Tyr Tyr His
Gln Thr Gly Asn Asn Pro Asp Ala Cys 420 425 430Asn Phe Asn Gly Val
Ala Ser Ile Thr Thr Thr Asp Pro Ser His Gly 435 440 445Thr Cys Val
Phe Ala Gly Ser Arg Gly Asn Gly Arg Asn Gly Thr Ser 450 455 460Val
Asn Ile Thr Ala Pro Ser Ala Asn Ser Thr Thr Ser Ser Gly Ile465 470
475 480Arg Ser Asp Leu Tyr Tyr Ser Arg Gly Ile Trp Ser Ile Leu Thr
Val 485 490 495Met Ile Leu Asn Val Ala Asn Ile Leu 500
505231026DNAArabidopsis thalianapromoter(1)..(1026)transcription
regulating sequence from gene At4g00820 23tatagaatta aattgtattt
gtattgaaaa tttggattgt atttgaattg tatacaatcg 60aattaaattg
tattctatta
tgataaaaaa aaatatttaa attgtattgt aaaatcgaat 120ttgtaagatt
gaaatttatt ttaaagaaat actaaattaa atactgaaaa aaaatatgag
180ttactattta aaattcaaat atcatcaata agttggagtg tatagagtta
aaagacagtt 240tattatgata aaagaaatag ttaaaatgtt tagtaaaatt
gaatgtatat gattgaaatt 300ttatttaatt caaaaattac taaattaaac
attagaaaag tgtggatcac tatttactat 360tcaaatttta aatattatca
atacatattt gaataaattt atatcttaca cccgcctatt 420taggcgggcc
ttatctagta tatatataag tagaataata ttcatgcata tcttaagaaa
480acccaaaaat tagaatgtat gaaatagaat tgcataatcg aaccaaggga
aagaagaatt 540gttttgcata atgaaataga attagtttat attgtatttg
tttaaaatta actctcaaaa 600caacaaaact ctatctttca atttaacagc
aacaaatctt ctagaattga atgtacaaca 660aatgcaagta ggtacatgat
ataataataa taataatgtg atattactag tttagtgaaa 720tgagttaaac
aaaaataaca aaataaatga agtggagaaa gtaaatgaag aggaagagtc
780atcgctgtca tggtcgcagt taaagaggtg gtcagagtca gaagtctccc
ttaagcaacc 840ttccaaattt attgacattt ctgtttccca agatccaccc
accactctcc tctctctctt 900cctcatttct gcgcaagaat ctaaaccaaa
tctctcatcc tctcttctat gtcccaaaac 960aggaacataa ctactactac
taccatgtct tgactctgtt gagaatctta aatcctacct 1020aacctc
1026241030DNAArabidopsis thalianapromoter(1)..(1030)transcription
regulating sequence from gene At4g00820 24taagatggag tgtatagaat
taaattgtat ttgtattgaa aatttggatt gtatttgaat 60tgtatacaat cgaattaaat
tgtattctat tatgataaaa aataatattt aaattgtatt 120gtaaaatcga
agttgtaaga ttaaaattta ttttaaagaa atactaaatt aaatactgaa
180aaaaatatga gttactattt aaaattcaaa tatcatcaat aagttggagt
gtatagagtt 240aaaaggcagt ttattatgat aaaagaaata gttaaaatgt
atagtaaaat tgaatgtata 300tgattgaaat tttatttaat tcaaaaatta
ctaaatttaa cattagaaaa gtgtggatca 360ctatttacca ttcaaatttt
aaatattatc aatacatatt tgaataaatt tatatcttag 420gcgggcctta
tctagtatat atatatatat ataagtagaa taatattcat gcatatctta
480agaaaaccca aaaattagaa tgtatgaaat ataattgcat aatcgaacca
agggaaagaa 540gaattgtttt gcataatgaa atagaattag tttatattgt
atttgtttaa aattaactct 600caaaacaaca aaactctatc tttcaattta
acagcaacaa atcttctaga attgaatgta 660caacaaatgc aagtaggtac
atgatataat aataataatg tgatataact agtttagtga 720aatgagttaa
acaaaaataa caaaataaat gaagtggaga aagtaaatga agaggaagag
780tcatcgctgt catggtcgca gttaaagagg tggtcagagt cagaagtctc
ccttaagcaa 840ccttccaaat ttattgacat ttctgtttcc caagatccac
ccaccactct cctctctctc 900ttcctcattt ctgcgcaaga atctaaacca
aatctctcat cctctcttct atgtcccaaa 960acaggaacat aactactact
actaccatgt cttgactctg ttgagaatct taaatcctac 1020ctaacctcca
103025844DNAArabidopsis thalianapromoter(1)..(844)transcription
regulating sequence from gene At4g00820 25tatagaatta aattgtattt
gtattgaaaa tttggattgt atttgaattg tatacaatcg 60aattaaattg tattctatta
tgataaaaaa aaatatttaa attgtattgt aaaatcgaat 120ttgtaagatt
gaaatttatt ttaaagaaat actaaattaa atactgaaaa aaaatatgag
180ttactattta aaattcaaat atcatcaata agttggagtg tatagagtta
aaagacagtt 240tattatgata aaagaaatag ttaaaatgtt tagtaaaatt
gaatgtatat gattgaaatt 300ttatttaatt caaaaattac taaattaaac
attagaaaag tgtggatcac tatttactat 360tcaaatttta aatattatca
atacatattt gaataaattt atatcttaca cccgcctatt 420taggcgggcc
ttatctagta tatatataag tagaataata ttcatgcata tcttaagaaa
480acccaaaaat tagaatgtat gaaatagaat tgcataatcg aaccaaggga
aagaagaatt 540gttttgcata atgaaataga attagtttat attgtatttg
tttaaaatta actctcaaaa 600caacaaaact ctatctttca atttaacagc
aacaaatctt ctagaattga atgtacaaca 660aatgcaagta ggtacatgat
ataataataa taataatgtg atattactag tttagtgaaa 720tgagttaaac
aaaaataaca aaataaatga agtggagaaa gtaaatgaag aggaagagtc
780atcgctgtca tggtcgcagt taaagaggtg gtcagagtca gaagtctccc
ttaagcaacc 840ttcc 84426846DNAArabidopsis
thalianapromoter(1)..(846)transcription regulating sequence from
gene At4g00820 26taagatggag tgtatagaat taaattgtat ttgtattgaa
aatttggatt gtatttgaat 60tgtatacaat cgaattaaat tgtattctat tatgataaaa
aataatattt aaattgtatt 120gtaaaatcga agttgtaaga ttaaaattta
ttttaaagaa atactaaatt aaatactgaa 180aaaaatatga gttactattt
aaaattcaaa tatcatcaat aagttggagt gtatagagtt 240aaaaggcagt
ttattatgat aaaagaaata gttaaaatgt atagtaaaat tgaatgtata
300tgattgaaat tttatttaat tcaaaaatta ctaaatttaa cattagaaaa
gtgtggatca 360ctatttacca ttcaaatttt aaatattatc aatacatatt
tgaataaatt tatatcttag 420gcgggcctta tctagtatat atatatatat
ataagtagaa taatattcat gcatatctta 480agaaaaccca aaaattagaa
tgtatgaaat ataattgcat aatcgaacca agggaaagaa 540gaattgtttt
gcataatgaa atagaattag tttatattgt atttgtttaa aattaactct
600caaaacaaca aaactctatc tttcaattta acagcaacaa atcttctaga
attgaatgta 660caacaaatgc aagtaggtac atgatataat aataataatg
tgatataact agtttagtga 720aatgagttaa acaaaaataa caaaataaat
gaagtggaga aagtaaatga agaggaagag 780tcatcgctgt catggtcgca
gttaaagagg tggtcagagt cagaagtctc ccttaagcaa 840ccttcc
846272352DNAArabidopsis thalianapromoter(1)..(2352)transcription
regulating sequence from gene At4g00820 27ctgtcgacat tgtctctgca
aaaccaagta atcacaaaaa ccataaacaa cagattcaaa 60tctcaacaac aacaagaaat
caaacgagaa atttcctaaa tagccactaa atttactaat 120atatgcttat
cgtacgagca caataaatcc gaagattggt gagaaatctg taacgaataa
180gcctttaggt gttggaaatg tttgaggatc gagaaaaata cctcagacta
cgattatgaa 240gcttcggctc aaatttcttt ggtggcgatg ataaataaac
ctaatctcca gaaatattgg 300gctgggaata atccttgaaa ggcccattgg
aaacttttca cttatataca attgggcctt 360tgataaacaa aaaaaaagta
gggtttgatg taaggaagcc cagatgagag tgttgttgac 420cagatgtttg
actgttaaat gtaaaaaggc cacgtgtatg gttgtaggga agaaatggac
480taccgtggtc catcatgcac ctgatgatgt ctctgtctga ctggctccat
gcacgtgata 540acacgagact tcaccagttc gttttaaaaa atttcttctt
tttttagttt gttatttatt 600ggccaataag caaagcttga aagctgtcat
tttcgactta aatcattggt ctggtttaat 660atgcttaata aatatatgat
aatgatatcc agctttttcc taaggtaaaa gatgaataat 720tggtttacgc
attcgatggt ttaaacttta ttaatttttc aaaacatgaa tcacaatcgt
780cattcgtcag tattgaaaac ataataacct tacgctcaca agtttatatt
tgaaggtata 840ttcaataatc atgcgtacgt ataatagggt tcgtccgtat
gcatagatca tagctaggtg 900cctaggtctg tctacattgt ttgttcgtct
atatatttat gcttatatat aaaaaaaata 960tatatatata tatatatata
aaaacatgta ttgtatacac tttgtacatt tatatgatac 1020ttattaaaat
catatttaaa aaagtaaact agtaatgata gtttaatgtt attgtcgatc
1080taataaatat atatatatat atatatgaaa catataagca tatatcttat
tatataaagt 1140acagttttga aagttactaa ctcacgagat catgacacgt
gtcaggtcta tcattgagat 1200gttgacatat gtcaaaaaaa attctttaaa
aaataaataa acatatttac atatactaat 1260ttatatttct atatctaaaa
atttaactta taattttatt aattaaaaag gaaaactaac 1320aataagatgg
agtgtataga attaaattgt atttgtattg aaaatttgga ttgtatttga
1380attgtataca atcgaattaa attgtattct attatgataa aaaataatat
ttaaattgta 1440ttgtaaaatc gaagttgtaa gattaaaatt tattttaaag
aaatactaaa ttaaatactg 1500aaaaaaatat gagttactat ttaaaattca
aatatcatca ataagttgga gtgtatagag 1560ttaaaaggca gtttattatg
ataaaagaaa tagttaaaat gtatagtaaa attgaatgta 1620tatgattgaa
attttattta attcaaaaat tactaaattt aacattagaa aagtgtggat
1680cactatttac cattcaaatt ttaaatatta tcaatacata tttgaataaa
tttatatctt 1740aggcgggcct tatctagtat atatatatat atataagtag
aataatattc atgcatatct 1800taagaaaacc caaaaattag aatgtatgaa
atataattgc ataatcgaac caagggaaag 1860aagaattgtt ttgcataatg
aaatagaatt agtttatatt gtatttgttt aaaattaact 1920ctcaaaacaa
caaaactcta tctttcaatt taacagcaac aaatcttcta gaattgaatg
1980tacaacaaat gcaagtaggt acatgatata ataataataa tgtgatataa
ctagtttagt 2040gaaatgagtt aaacaaaaat aacaaaataa atgaagtgga
gaaagtaaat gaagaggaag 2100agtcatcgct gtcatggtcg cagttaaaga
ggtggtcaga gtcagaagtc tcccttaagc 2160aaccttccaa atttattgac
atttctgttt cccaagatcc acccaccact ctcctctctc 2220tcttcctcat
ttctgcgcaa gaatctaaac caaatctctc atcctctctt ctatgtccca
2280aaacaggaac ataactacta ctactaccat gtcttgactc tgttgagaat
cttaaatcct 2340acctaacctc ca 2352282168DNAArabidopsis
thalianapromoter(1)..(2168)transcription regulating sequence from
gene At4g00820 28ctgtcgacat tgtctctgca aaaccaagta atcacaaaaa
ccataaacaa cagattcaaa 60tctcaacaac aacaagaaat caaacgagaa atttcctaaa
tagccactaa atttactaat 120atatgcttat cgtacgagca caataaatcc
gaagattggt gagaaatctg taacgaataa 180gcctttaggt gttggaaatg
tttgaggatc gagaaaaata cctcagacta cgattatgaa 240gcttcggctc
aaatttcttt ggtggcgatg ataaataaac ctaatctcca gaaatattgg
300gctgggaata atccttgaaa ggcccattgg aaacttttca cttatataca
attgggcctt 360tgataaacaa aaaaaaagta gggtttgatg taaggaagcc
cagatgagag tgttgttgac 420cagatgtttg actgttaaat gtaaaaaggc
cacgtgtatg gttgtaggga agaaatggac 480taccgtggtc catcatgcac
ctgatgatgt ctctgtctga ctggctccat gcacgtgata 540acacgagact
tcaccagttc gttttaaaaa atttcttctt tttttagttt gttatttatt
600ggccaataag caaagcttga aagctgtcat tttcgactta aatcattggt
ctggtttaat 660atgcttaata aatatatgat aatgatatcc agctttttcc
taaggtaaaa gatgaataat 720tggtttacgc attcgatggt ttaaacttta
ttaatttttc aaaacatgaa tcacaatcgt 780cattcgtcag tattgaaaac
ataataacct tacgctcaca agtttatatt tgaaggtata 840ttcaataatc
atgcgtacgt ataatagggt tcgtccgtat gcatagatca tagctaggtg
900cctaggtctg tctacattgt ttgttcgtct atatatttat gcttatatat
aaaaaaaata 960tatatatata tatatatata aaaacatgta ttgtatacac
tttgtacatt tatatgatac 1020ttattaaaat catatttaaa aaagtaaact
agtaatgata gtttaatgtt attgtcgatc 1080taataaatat atatatatat
atatatgaaa catataagca tatatcttat tatataaagt 1140acagttttga
aagttactaa ctcacgagat catgacacgt gtcaggtcta tcattgagat
1200gttgacatat gtcaaaaaaa attctttaaa aaataaataa acatatttac
atatactaat 1260ttatatttct atatctaaaa atttaactta taattttatt
aattaaaaag gaaaactaac 1320aataagatgg agtgtataga attaaattgt
atttgtattg aaaatttgga ttgtatttga 1380attgtataca atcgaattaa
attgtattct attatgataa aaaataatat ttaaattgta 1440ttgtaaaatc
gaagttgtaa gattaaaatt tattttaaag aaatactaaa ttaaatactg
1500aaaaaaatat gagttactat ttaaaattca aatatcatca ataagttgga
gtgtatagag 1560ttaaaaggca gtttattatg ataaaagaaa tagttaaaat
gtatagtaaa attgaatgta 1620tatgattgaa attttattta attcaaaaat
tactaaattt aacattagaa aagtgtggat 1680cactatttac cattcaaatt
ttaaatatta tcaatacata tttgaataaa tttatatctt 1740aggcgggcct
tatctagtat atatatatat atataagtag aataatattc atgcatatct
1800taagaaaacc caaaaattag aatgtatgaa atataattgc ataatcgaac
caagggaaag 1860aagaattgtt ttgcataatg aaatagaatt agtttatatt
gtatttgttt aaaattaact 1920ctcaaaacaa caaaactcta tctttcaatt
taacagcaac aaatcttcta gaattgaatg 1980tacaacaaat gcaagtaggt
acatgatata ataataataa tgtgatataa ctagtttagt 2040gaaatgagtt
aaacaaaaat aacaaaataa atgaagtgga gaaagtaaat gaagaggaag
2100agtcatcgct gtcatggtcg cagttaaaga ggtggtcaga gtcagaagtc
tcccttaagc 2160aaccttcc 2168292032DNAArabidopsis
thalianaCDS(185)..(1789)encoding calmodulin-binding protein-related
protein 29aaatttattg acatttctgt ttcccaagat ccacccacca ctctcctctc
tctcttcctc 60atttctgcgc aagaatctaa accaaatctc tcatcctctc ttctatgtcc
caaaacagga 120acataactac tactactacc atgtcttgac tctgttgaga
atcttaaatc ctacctaacc 180tcca atg ggt aag aag agc ggt tct tct tct
tct tgg ctc act gcc gtg 229 Met Gly Lys Lys Ser Gly Ser Ser Ser Ser
Trp Leu Thr Ala Val 1 5 10 15aaa cga gct ttc cga tct ccc acc aag
aaa gaa cac aac aac aat gct 277Lys Arg Ala Phe Arg Ser Pro Thr Lys
Lys Glu His Asn Asn Asn Ala 20 25 30cat ggt aat gaa gtc gac gaa gat
gaa gac aag aag aaa gag aag aga 325His Gly Asn Glu Val Asp Glu Asp
Glu Asp Lys Lys Lys Glu Lys Arg 35 40 45cgt tgg tta ttt aga aaa tct
acg aat cat gac tct ccg gtg aag acc 373Arg Trp Leu Phe Arg Lys Ser
Thr Asn His Asp Ser Pro Val Lys Thr 50 55 60tcc ggc gtc gga aaa gac
gct ccg gcg cag aaa tcc aca gaa aca acg 421Ser Gly Val Gly Lys Asp
Ala Pro Ala Gln Lys Ser Thr Glu Thr Thr 65 70 75acg atc atc aac cca
acc gtt tta tcc tct gtt aca gaa cag agg tac 469Thr Ile Ile Asn Pro
Thr Val Leu Ser Ser Val Thr Glu Gln Arg Tyr80 85 90 95gac gca tct
aca ccg ccg gcc acc gtc tcc gcc gca tcg gaa act cat 517Asp Ala Ser
Thr Pro Pro Ala Thr Val Ser Ala Ala Ser Glu Thr His 100 105 110cct
cct tcg aca acg aag gag tta cca aat ctt aca aga cgt act tat 565Pro
Pro Ser Thr Thr Lys Glu Leu Pro Asn Leu Thr Arg Arg Thr Tyr 115 120
125acc gca aga gaa gat tac gca gct gtt gta atc caa act ggt ttc aga
613Thr Ala Arg Glu Asp Tyr Ala Ala Val Val Ile Gln Thr Gly Phe Arg
130 135 140ggc tat ttg gca aga aga gca tta aga gca ttg aaa ggg cta
gtg aag 661Gly Tyr Leu Ala Arg Arg Ala Leu Arg Ala Leu Lys Gly Leu
Val Lys 145 150 155tta caa gca cta gtg aga ggt cac aat gtg agg aag
caa gca aag atg 709Leu Gln Ala Leu Val Arg Gly His Asn Val Arg Lys
Gln Ala Lys Met160 165 170 175act tta agg tgt atg caa gct cta gtt
cga gtc caa tct cgt gtg ctt 757Thr Leu Arg Cys Met Gln Ala Leu Val
Arg Val Gln Ser Arg Val Leu 180 185 190gac caa cgg aaa cgc ttg tct
cac gat ggt agt cgc aaa tct gcc ttc 805Asp Gln Arg Lys Arg Leu Ser
His Asp Gly Ser Arg Lys Ser Ala Phe 195 200 205agc gac act caa agt
gtg ctc gaa tct cgt tat ctt caa gaa ata tca 853Ser Asp Thr Gln Ser
Val Leu Glu Ser Arg Tyr Leu Gln Glu Ile Ser 210 215 220gac aga aga
tcc atg tca aga gaa gga agt agc att gcg gaa gat tgg 901Asp Arg Arg
Ser Met Ser Arg Glu Gly Ser Ser Ile Ala Glu Asp Trp 225 230 235gat
gat cga cca cac acg att gag gaa gtg aaa gca atg ttg caa caa 949Asp
Asp Arg Pro His Thr Ile Glu Glu Val Lys Ala Met Leu Gln Gln240 245
250 255aga cga gac aat gcg ttg aga cgt gag agt aac aat agt ata tca
caa 997Arg Arg Asp Asn Ala Leu Arg Arg Glu Ser Asn Asn Ser Ile Ser
Gln 260 265 270gct ttc tct cac cag gtt cgg aga aca aga ggt agt tat
tct aca gga 1045Ala Phe Ser His Gln Val Arg Arg Thr Arg Gly Ser Tyr
Ser Thr Gly 275 280 285gac gag tat gaa gaa gag aga ccg aaa tgg tta
gac aga tgg atg gct 1093Asp Glu Tyr Glu Glu Glu Arg Pro Lys Trp Leu
Asp Arg Trp Met Ala 290 295 300tct aaa ccg tgg gat aaa cga gct tca
acg gat caa aga gta cca ccg 1141Ser Lys Pro Trp Asp Lys Arg Ala Ser
Thr Asp Gln Arg Val Pro Pro 305 310 315gtt tac aaa acc gtg gag atc
gat act tct caa ccg tat tta acc cgc 1189Val Tyr Lys Thr Val Glu Ile
Asp Thr Ser Gln Pro Tyr Leu Thr Arg320 325 330 335ggt aac tcg aga
acc ggt gca agt cca agc cgt agc caa agg cct agt 1237Gly Asn Ser Arg
Thr Gly Ala Ser Pro Ser Arg Ser Gln Arg Pro Ser 340 345 350tca cca
tca agg acc agc cac cat tac caa caa cac aat ttc tca tca 1285Ser Pro
Ser Arg Thr Ser His His Tyr Gln Gln His Asn Phe Ser Ser 355 360
365gct aca cca tct ccg gct aaa tct aga ccg ata caa att cga tcc gct
1333Ala Thr Pro Ser Pro Ala Lys Ser Arg Pro Ile Gln Ile Arg Ser Ala
370 375 380agt ccg cgg atc caa aga gat gat cgg tca gcg tac aac tac
aca tca 1381Ser Pro Arg Ile Gln Arg Asp Asp Arg Ser Ala Tyr Asn Tyr
Thr Ser 385 390 395aac aca cct agc ttg aga tct aac tat agt ttc aca
gca aga agt ggt 1429Asn Thr Pro Ser Leu Arg Ser Asn Tyr Ser Phe Thr
Ala Arg Ser Gly400 405 410 415tat agt gtt tgt acc act act act act
gct aca aat gct gca ttg cca 1477Tyr Ser Val Cys Thr Thr Thr Thr Thr
Ala Thr Asn Ala Ala Leu Pro 420 425 430aac tac atg gcg att acg gaa
tct gct aag gct aga atc cgg tct cag 1525Asn Tyr Met Ala Ile Thr Glu
Ser Ala Lys Ala Arg Ile Arg Ser Gln 435 440 445agt gca cca agg caa
cgg cct tca aca ccc gag aaa gaa cgt atc agt 1573Ser Ala Pro Arg Gln
Arg Pro Ser Thr Pro Glu Lys Glu Arg Ile Ser 450 455 460tca gct aga
aaa cgg ctt tcg ttt cca gtt cca ccg ctg ccg cag caa 1621Ser Ala Arg
Lys Arg Leu Ser Phe Pro Val Pro Pro Leu Pro Gln Gln 465 470 475atg
gat ggt cag agt tta agg agt cca agt ttc aag agt ata ggt ggc 1669Met
Asp Gly Gln Ser Leu Arg Ser Pro Ser Phe Lys Ser Ile Gly Gly480 485
490 495tca caa ttg ggt gca ttg gaa caa caa tca aat tac tct tct tgt
tgt 1717Ser Gln Leu Gly Ala Leu Glu Gln Gln Ser Asn Tyr Ser Ser Cys
Cys 500 505 510act gag tct ctt ggt ggt ggt gga gag ata tca cct gct
tct act agc 1765Thr Glu Ser Leu Gly Gly Gly Gly Glu Ile Ser Pro Ala
Ser Thr Ser 515 520 525gat tat agg cga tgg tta aga tga ttccataatc
caaccaaatc aaccggtcat 1819Asp Tyr Arg Arg Trp Leu Arg 530gatttttttc
ctcccttttt tttttttgtt atatataaat aaaaacaatt tgacaaaaag
1879aaccataact ttttcagttt ttattgatga tctttgagat tggtgtgtaa
ttttgtttgt 1939gtttatgttt gtgtgtgtga atttgtagct tataattgga
agtgtttact aactaatctc 1999tatataaata tgtatgctca tatttttatc tat
203230534PRTArabidopsis thaliana 30Met Gly Lys Lys Ser Gly Ser Ser
Ser Ser Trp Leu Thr Ala Val Lys1 5 10 15Arg Ala Phe Arg Ser Pro Thr
Lys Lys Glu His Asn Asn Asn Ala His 20 25 30Gly Asn
Glu Val Asp Glu Asp Glu Asp Lys Lys Lys Glu Lys Arg Arg 35 40 45Trp
Leu Phe Arg Lys Ser Thr Asn His Asp Ser Pro Val Lys Thr Ser 50 55
60Gly Val Gly Lys Asp Ala Pro Ala Gln Lys Ser Thr Glu Thr Thr Thr65
70 75 80Ile Ile Asn Pro Thr Val Leu Ser Ser Val Thr Glu Gln Arg Tyr
Asp 85 90 95Ala Ser Thr Pro Pro Ala Thr Val Ser Ala Ala Ser Glu Thr
His Pro 100 105 110Pro Ser Thr Thr Lys Glu Leu Pro Asn Leu Thr Arg
Arg Thr Tyr Thr 115 120 125Ala Arg Glu Asp Tyr Ala Ala Val Val Ile
Gln Thr Gly Phe Arg Gly 130 135 140Tyr Leu Ala Arg Arg Ala Leu Arg
Ala Leu Lys Gly Leu Val Lys Leu145 150 155 160Gln Ala Leu Val Arg
Gly His Asn Val Arg Lys Gln Ala Lys Met Thr 165 170 175Leu Arg Cys
Met Gln Ala Leu Val Arg Val Gln Ser Arg Val Leu Asp 180 185 190Gln
Arg Lys Arg Leu Ser His Asp Gly Ser Arg Lys Ser Ala Phe Ser 195 200
205Asp Thr Gln Ser Val Leu Glu Ser Arg Tyr Leu Gln Glu Ile Ser Asp
210 215 220Arg Arg Ser Met Ser Arg Glu Gly Ser Ser Ile Ala Glu Asp
Trp Asp225 230 235 240Asp Arg Pro His Thr Ile Glu Glu Val Lys Ala
Met Leu Gln Gln Arg 245 250 255Arg Asp Asn Ala Leu Arg Arg Glu Ser
Asn Asn Ser Ile Ser Gln Ala 260 265 270Phe Ser His Gln Val Arg Arg
Thr Arg Gly Ser Tyr Ser Thr Gly Asp 275 280 285Glu Tyr Glu Glu Glu
Arg Pro Lys Trp Leu Asp Arg Trp Met Ala Ser 290 295 300Lys Pro Trp
Asp Lys Arg Ala Ser Thr Asp Gln Arg Val Pro Pro Val305 310 315
320Tyr Lys Thr Val Glu Ile Asp Thr Ser Gln Pro Tyr Leu Thr Arg Gly
325 330 335Asn Ser Arg Thr Gly Ala Ser Pro Ser Arg Ser Gln Arg Pro
Ser Ser 340 345 350Pro Ser Arg Thr Ser His His Tyr Gln Gln His Asn
Phe Ser Ser Ala 355 360 365Thr Pro Ser Pro Ala Lys Ser Arg Pro Ile
Gln Ile Arg Ser Ala Ser 370 375 380Pro Arg Ile Gln Arg Asp Asp Arg
Ser Ala Tyr Asn Tyr Thr Ser Asn385 390 395 400Thr Pro Ser Leu Arg
Ser Asn Tyr Ser Phe Thr Ala Arg Ser Gly Tyr 405 410 415Ser Val Cys
Thr Thr Thr Thr Thr Ala Thr Asn Ala Ala Leu Pro Asn 420 425 430Tyr
Met Ala Ile Thr Glu Ser Ala Lys Ala Arg Ile Arg Ser Gln Ser 435 440
445Ala Pro Arg Gln Arg Pro Ser Thr Pro Glu Lys Glu Arg Ile Ser Ser
450 455 460Ala Arg Lys Arg Leu Ser Phe Pro Val Pro Pro Leu Pro Gln
Gln Met465 470 475 480Asp Gly Gln Ser Leu Arg Ser Pro Ser Phe Lys
Ser Ile Gly Gly Ser 485 490 495Gln Leu Gly Ala Leu Glu Gln Gln Ser
Asn Tyr Ser Ser Cys Cys Thr 500 505 510Glu Ser Leu Gly Gly Gly Gly
Glu Ile Ser Pro Ala Ser Thr Ser Asp 515 520 525Tyr Arg Arg Trp Leu
Arg 530311201DNAArabidopsis
thalianapromoter(1)..(1201)transcription regulating sequence from
gene At2g36640 31tctttttgtt gtggaggata catgtcttgt aagattattc
ttttccctta actttttgtt 60gagtttttac aataagataa ttaattgaga tcggttgctt
tttgaccatt gcttttgctt 120gcttactcgt ttgggcatcg gattctccac
ttacacgaca tacgtacgtg tgtactcata 180ctcacacgta cctacacatt
atgaaagcca tgatgctagc tagaagttcc atatattgtt 240atatggttga
tcaaatcgaa actcaaacta agtgtttctg atttgatctg atcttaacta
300tatacttgag atcttaaggt actcacgtta gtgtatatca tatttacgac
aaatcatgta 360gaaaacgtga ccgatttgga tgattcggca tcaaatgcgc
gtcaacaaag ttgtcggaga 420aataataaaa aaaggacgta cctaaataag
tataaaagta atctatgtga gaacatgatt 480aacgtgattt atttattttt
gataaaaaaa aaagttaagg tgactaatag agatgtttaa 540caaaaaagag
agttgtctat tcggttaaga caatagactc atgtgctaac ctgaattagc
600tggcaaatta gagaaaattt ggaaataaat ccaaactatt gcaatgaact
tttttgtata 660ccctcccaag aaccgtcaaa aactcaaaat tatattgata
accgacttga tcaataaacg 720gctctatcca atgattaact tgcgtcctta
tttttctaaa atgcttgttt ccaagttttt 780gatgttgagg caataacttc
caataaactg tgtggcaata actaagtaat ctttcatatc 840tctaaattgc
ttgtttccaa gttatgtgcc tcagtaagtt tgaaatggcg aatgtgatct
900tcacctaaca ttttcctcga ccaacacaaa ttatgatgtc aaaagatttg
atacgaccga 960ccatcgatgc ttgcttgctt atgtcttagc tcttaagtca
tgccatgaac actcaagaat 1020catgatccat gcatgaaaag tccacgtgtc
ataccacgtc cgtagaagat aaagctaagt 1080aaattgtacg tggcagctgt
tgcgtccctc tatctaccac gtagcgtctc aacagaaaca 1140gagcattact
ataagaagta acaaaagcag ctcaactgat agcacaagtt aaagaattct 1200a
1201321216DNAArabidopsis thalianapromoter(1)..(1216)transcription
regulating sequence from gene At2g36640 32acagtcctct ttttgttgtg
gaggatacat gtcttgtaag attattcttt tcccttaact 60ttttattgag tttttacaat
aagataatta attgagatcg gttgcttttt gaccattgct 120tttgcttgct
tactcgtttg ggcatcggat tctccactta cacgacatac gtacgtgtgt
180actcatactc acacgtacct acacattatg aaagccatga tgctagctag
aagttccata 240tattgttata tggttgatca aatcgaaact caaactaagt
gtttctgatt tgatctgatc 300ttaactatat acttgagatc ttaaggtact
cacgttagtg tatatcatat ttacgacaaa 360tcatgtagaa aacgtgaccg
atttggatga ttcggcatca aatgcgcgtc aacaaagttg 420tcggagaaat
aataaaaaaa ggacgtacct aattaagtat aaaagtaatc tatgtgagaa
480catgattaac gtgatttatt tatttttgat aaaaaaaaaa agttaaggtg
actaatagag 540atgtttaaca aaaaagagag ttgtctattc ggttaagaca
atagactcat gtgctaacct 600gaattagctg gcaaattaga gaaaatttgg
aaataaatcc aaactattgc aatgaacttt 660tttgtatacc ctcccaagaa
ccgtcaaaaa ctcaaaatta tattgataac cgacttgatc 720aataaacggc
tctatccaat gattaacttg cgtccttatt tttctaaaat gcttgtttcc
780aagtttttga tgttgaggca ataacttcca ataaactgtg tggcaataac
taagtaatct 840ttcatatctc taaattgctt gtttccaagt tatgtgcctc
agtaagtttg aaatggcgaa 900tgtgatcttc acctaacatt ttcctcgacc
aacacaaatt atgatatcaa aagatttgat 960acgaccgacc atcgatgctt
gcttgcttat gtcttagctc ttaagtcatg ccatgaacac 1020tcaagaatca
tgatccatgc atgaaaagtc cacgtgtcat accacgtccg tagaagataa
1080agctaagtaa attgtacgtg gcagctgttg cgtccctcta tctaccacgt
agcgtctcaa 1140cagaaacaga gcattactat aagaagtaac aaaagcagct
caactgatag cacaagttaa 1200agaattctaa aatcgt
1216331441DNAArabidopsis thalianapromoter(1)..(1441)transcription
regulating sequence from gene At2g36640 33taatctctta tgttgctttc
aactccaaaa atctttcttt ctgactaaaa aagtttcctt 60tcggttatgt gaccgaaaca
gagactcgag aattgattga tcaatcttgt gagggtttga 120tcaaatttaa
atggtttata cgaatcattg gtctcatgaa aaaaatagtt ttatacagtc
180ctctttttgt tgtggaggat acatgtcttg taagattatt cttttccctt
aactttttat 240tgagttttta caataagata attaattgag atcggttgct
ttttgaccat tgcttttgct 300tgcttactcg tttgggcatc ggattctcca
cttacacgac atacgtacgt gtgtactcat 360actcacacgt acctacacat
tatgaaagcc atgatgctag ctagaagttc catatattgt 420tatatggttg
atcaaatcga aactcaaact aagtgtttct gatttgatct gatcttaact
480atatacttga gatcttaagg tactcacgtt agtgtatatc atatttacga
caaatcatgt 540agaaaacgtg accgatttgg atgattcggc atcaaatgcg
cgtcaacaaa gttgtcggag 600aaataataaa aaaaggacgt acctaattaa
gtataaaagt aatctatgtg agaacatgat 660taacgtgatt tatttatttt
tgataaaaaa aaaaagttaa ggtgactaat agagatgttt 720aacaaaaaag
agagttgtct attcggttaa gacaatagac tcatgtgcta acctgaatta
780gctggcaaat tagagaaaat ttggaaataa atccaaacta ttgcaatgaa
cttttttgta 840taccctccca agaaccgtca aaaactcaaa attatattga
taaccgactt gatcaataaa 900cggctctatc caatgattaa cttgcgtcct
tatttttcta aaatgcttgt ttccaagttt 960ttgatgttga ggcaataact
tccaataaac tgtgtggcaa taactaagta atctttcata 1020tctctaaatt
gcttgtttcc aagttatgtg cctcagtaag tttgaaatgg cgaatgtgat
1080cttcacctaa cattttcctc gaccaacaca aattatgata tcaaaagatt
tgatacgacc 1140gaccatcgat gcttgcttgc ttatgtctta gctcttaagt
catgccatga acactcaaga 1200atcatgatcc atgcatgaaa agtccacgtg
tcataccacg tccgtagaag ataaagctaa 1260gtaaattgta cgtggcagct
gttgcgtccc tctatctacc acgtagcgtc tcaacagaaa 1320cagagcatta
ctataagaag taacaaaagc agctcaactg atagcacaag ttaaagaatt
1380ctaaaatcgt aaagttctaa agcggttttt catcaatttt caagcaatcg
agaaaaaagc 1440a 1441341572DNAArabidopsis
thalianaCDS(61)..(1407)encoding late embryogenesis abundant protein
(ECP63) / LEA protein 34taaaatcgta aagttctaaa gcggtttttc atcaattttc
aagcaatcga gaaaaaagca 60atg gcg tca gac aaa caa aag gcg gag aga gcc
gag gtt gcg gcg agg 108Met Ala Ser Asp Lys Gln Lys Ala Glu Arg Ala
Glu Val Ala Ala Arg1 5 10 15cta gcg gct gag gac ttg cat gac att aac
aaa tcc ggt ggt gct gat 156Leu Ala Ala Glu Asp Leu His Asp Ile Asn
Lys Ser Gly Gly Ala Asp 20 25 30gtc aca atg tat aag gtg acg gag aga
aca act gaa cat cca ccg gag 204Val Thr Met Tyr Lys Val Thr Glu Arg
Thr Thr Glu His Pro Pro Glu 35 40 45caa gat agg ccc ggt gtg ata gga
tca gtg ttc agg gct gtc caa gga 252Gln Asp Arg Pro Gly Val Ile Gly
Ser Val Phe Arg Ala Val Gln Gly 50 55 60acg tat gag cat gcg aga gac
gct gta gtt gga aaa acc cac gaa gcg 300Thr Tyr Glu His Ala Arg Asp
Ala Val Val Gly Lys Thr His Glu Ala65 70 75 80gct gag tct acc aaa
gaa gga gct cag ata gct tca gag aaa gcg gtt 348Ala Glu Ser Thr Lys
Glu Gly Ala Gln Ile Ala Ser Glu Lys Ala Val 85 90 95gga gca aag gac
gca acc gtc gag aaa gct aag gaa acc gct gat tat 396Gly Ala Lys Asp
Ala Thr Val Glu Lys Ala Lys Glu Thr Ala Asp Tyr 100 105 110act gcg
gag aag gtg ggt gag tat aaa gac tat acg gtt gat aaa gct 444Thr Ala
Glu Lys Val Gly Glu Tyr Lys Asp Tyr Thr Val Asp Lys Ala 115 120
125aaa gag gct aag gac aca act gca gag aag gcg aag gag act gct aat
492Lys Glu Ala Lys Asp Thr Thr Ala Glu Lys Ala Lys Glu Thr Ala Asn
130 135 140tat act gcg gat aag gcg gtg gaa gca aag gat aag acg gcg
gag aag 540Tyr Thr Ala Asp Lys Ala Val Glu Ala Lys Asp Lys Thr Ala
Glu Lys145 150 155 160att ggt gag tac aaa gac tat gcg gtg gat aag
gca gta gaa gct aaa 588Ile Gly Glu Tyr Lys Asp Tyr Ala Val Asp Lys
Ala Val Glu Ala Lys 165 170 175gat aag aca gcg gag aag gcg aag gag
act gcg aat tat acg gcg gat 636Asp Lys Thr Ala Glu Lys Ala Lys Glu
Thr Ala Asn Tyr Thr Ala Asp 180 185 190aag gct aaa gag gct aag gac
aag acg gct gag aag gtt ggt gag tat 684Lys Ala Lys Glu Ala Lys Asp
Lys Thr Ala Glu Lys Val Gly Glu Tyr 195 200 205aag gat tac acg gtg
gac aag gcc gtg gaa gct agg gat tac aca gcg 732Lys Asp Tyr Thr Val
Asp Lys Ala Val Glu Ala Arg Asp Tyr Thr Ala 210 215 220gag aag gct
att gaa gca aag gat aag aca gct gag aag act gga gag 780Glu Lys Ala
Ile Glu Ala Lys Asp Lys Thr Ala Glu Lys Thr Gly Glu225 230 235
240tat aag gac tat acg gtg gag aag gcg acg gag ggg aaa gat gtt acg
828Tyr Lys Asp Tyr Thr Val Glu Lys Ala Thr Glu Gly Lys Asp Val Thr
245 250 255gtg agt aag cta gga gag ctg aag gat agt gcc gtt gag aca
gcg aag 876Val Ser Lys Leu Gly Glu Leu Lys Asp Ser Ala Val Glu Thr
Ala Lys 260 265 270aga gct atg ggt ttc ttg tcg ggg aag aca gag gag
gcc aaa gga aaa 924Arg Ala Met Gly Phe Leu Ser Gly Lys Thr Glu Glu
Ala Lys Gly Lys 275 280 285gct gtg gag acc aaa gat act gcc aag gaa
aac atg gag aaa gct gga 972Ala Val Glu Thr Lys Asp Thr Ala Lys Glu
Asn Met Glu Lys Ala Gly 290 295 300gaa gta aca aga caa aag atg gag
gaa atg aga ttg gaa ggt aaa gag 1020Glu Val Thr Arg Gln Lys Met Glu
Glu Met Arg Leu Glu Gly Lys Glu305 310 315 320ctc aaa gaa gaa gct
gga gca aaa gcc caa gag gca tct caa aag act 1068Leu Lys Glu Glu Ala
Gly Ala Lys Ala Gln Glu Ala Ser Gln Lys Thr 325 330 335agg gag agt
act gag tcg gga gct caa aaa gcc gaa gag acc aaa gat 1116Arg Glu Ser
Thr Glu Ser Gly Ala Gln Lys Ala Glu Glu Thr Lys Asp 340 345 350tct
gct gcc gtg agg gga aat gaa gcg aaa ggg act att ttt ggt gca 1164Ser
Ala Ala Val Arg Gly Asn Glu Ala Lys Gly Thr Ile Phe Gly Ala 355 360
365tta ggg aat gta acg gaa gca ata aag agc aaa ctg aca atg cca tca
1212Leu Gly Asn Val Thr Glu Ala Ile Lys Ser Lys Leu Thr Met Pro Ser
370 375 380gac att gtg gag gaa aca cgc gcg gca cgt gag cat gga ggg
acg ggt 1260Asp Ile Val Glu Glu Thr Arg Ala Ala Arg Glu His Gly Gly
Thr Gly385 390 395 400agg act gtg gtt gaa gtc aag gtc gag gat tca
aag ccg ggt aag gtg 1308Arg Thr Val Val Glu Val Lys Val Glu Asp Ser
Lys Pro Gly Lys Val 405 410 415gcg act tca ctg aag gcg tcg gat caa
atg acc ggt caa aca ttc aac 1356Ala Thr Ser Leu Lys Ala Ser Asp Gln
Met Thr Gly Gln Thr Phe Asn 420 425 430gac gtt gga cgg atg gat gat
gat gct cgg aaa gat aag gga aag ctg 1404Asp Val Gly Arg Met Asp Asp
Asp Ala Arg Lys Asp Lys Gly Lys Leu 435 440 445tga gaatactaga
aaaatgacaa tgttttttgg gccttttgtt ctggatgaat 1457atctctgttg
ttttttgggc cttttgtttt ggatacgtct gttatagcag ataacgtttt 1517
ctgatagttt ctatgtttgt attcttgttt tggaatagga aagctttctt cagtt
157235448PRTArabidopsis thaliana 35Met Ala Ser Asp Lys Gln Lys Ala
Glu Arg Ala Glu Val Ala Ala Arg1 5 10 15Leu Ala Ala Glu Asp Leu His
Asp Ile Asn Lys Ser Gly Gly Ala Asp 20 25 30Val Thr Met Tyr Lys Val
Thr Glu Arg Thr Thr Glu His Pro Pro Glu 35 40 45Gln Asp Arg Pro Gly
Val Ile Gly Ser Val Phe Arg Ala Val Gln Gly 50 55 60Thr Tyr Glu His
Ala Arg Asp Ala Val Val Gly Lys Thr His Glu Ala65 70 75 80Ala Glu
Ser Thr Lys Glu Gly Ala Gln Ile Ala Ser Glu Lys Ala Val 85 90 95Gly
Ala Lys Asp Ala Thr Val Glu Lys Ala Lys Glu Thr Ala Asp Tyr 100 105
110Thr Ala Glu Lys Val Gly Glu Tyr Lys Asp Tyr Thr Val Asp Lys Ala
115 120 125Lys Glu Ala Lys Asp Thr Thr Ala Glu Lys Ala Lys Glu Thr
Ala Asn 130 135 140Tyr Thr Ala Asp Lys Ala Val Glu Ala Lys Asp Lys
Thr Ala Glu Lys145 150 155 160Ile Gly Glu Tyr Lys Asp Tyr Ala Val
Asp Lys Ala Val Glu Ala Lys 165 170 175Asp Lys Thr Ala Glu Lys Ala
Lys Glu Thr Ala Asn Tyr Thr Ala Asp 180 185 190Lys Ala Lys Glu Ala
Lys Asp Lys Thr Ala Glu Lys Val Gly Glu Tyr 195 200 205Lys Asp Tyr
Thr Val Asp Lys Ala Val Glu Ala Arg Asp Tyr Thr Ala 210 215 220Glu
Lys Ala Ile Glu Ala Lys Asp Lys Thr Ala Glu Lys Thr Gly Glu225 230
235 240Tyr Lys Asp Tyr Thr Val Glu Lys Ala Thr Glu Gly Lys Asp Val
Thr 245 250 255Val Ser Lys Leu Gly Glu Leu Lys Asp Ser Ala Val Glu
Thr Ala Lys 260 265 270Arg Ala Met Gly Phe Leu Ser Gly Lys Thr Glu
Glu Ala Lys Gly Lys 275 280 285Ala Val Glu Thr Lys Asp Thr Ala Lys
Glu Asn Met Glu Lys Ala Gly 290 295 300Glu Val Thr Arg Gln Lys Met
Glu Glu Met Arg Leu Glu Gly Lys Glu305 310 315 320Leu Lys Glu Glu
Ala Gly Ala Lys Ala Gln Glu Ala Ser Gln Lys Thr 325 330 335Arg Glu
Ser Thr Glu Ser Gly Ala Gln Lys Ala Glu Glu Thr Lys Asp 340 345
350Ser Ala Ala Val Arg Gly Asn Glu Ala Lys Gly Thr Ile Phe Gly Ala
355 360 365Leu Gly Asn Val Thr Glu Ala Ile Lys Ser Lys Leu Thr Met
Pro Ser 370 375 380Asp Ile Val Glu Glu Thr Arg Ala Ala Arg Glu His
Gly Gly Thr Gly385 390 395 400Arg Thr Val Val Glu Val Lys Val Glu
Asp Ser Lys Pro Gly Lys Val 405 410 415Ala Thr Ser Leu Lys Ala Ser
Asp Gln Met Thr Gly Gln Thr Phe Asn 420 425 430Asp Val Gly Arg Met
Asp Asp Asp Ala Arg Lys Asp Lys Gly Lys Leu 435 440
445361587DNAArabidopsis thalianapromoter(1)..(1587)transcription
regulating sequence from gene At2g34200 36tttatctaat ggataaccac
cacccattag gatcaccata aaggtcatat tttggagcat 60tccaatatca taacacttta
ctcttaaggg tctaatttga gttggtggca
acatgtcata 120tagacttcag agctgcttcc aaaatgggtt tccaatctac
accatttatt acagattctt 180ttttgctttt cgtatttgga tagtttacga
gtttagtttt tacttttttt agattcagga 240agtctttaat gggtttcacc
ggtttagtag gttcaccttt atcggccaac aagaattaca 300tttctctcta
tttatgacga ctgccaccaa taattcaaaa caagtttaca tattaagttt
360tcttaaacta ctcctatata ttaagtctaa gattatatta atcttattta
tataatttaa 420ttcaactaaa tttttagttt gtttatttac atgatatacc
atggtcctgt tattttagct 480aaagtatata ttaaatttat tactttttta
tttcaaagat tttttctatt gttttttatt 540ttgttttctt ttcatttaag
cttatattat tattattata cacacacatt taaggtttat 600atttatattt
cttgcaatgc aaacatgtgt caatattagt tggtttaaga tacaaaaata
660ttaaggacta aatggttgag ataatactac tttgatatat atatatataa
ctatattcta 720taaacttaat ttatttgaga gggtaaggat tagatgacca
agaaattggt aaaaattgga 780aaaataatta tagacattat tattttacaa
gatattctca aatggctaga gatttgtgaa 840gttctgtttt ttttttcctc
aaagttcaag tgtaatattt ttcgatgaca atgattattc 900caaatttatt
tttttgttaa tcgtaaacag tgaattttgc ataattggtt tgaattagag
960ttgataatag ttgttctcat catcaccaaa cattcttctg cttaccaaaa
gtaagggact 1020ttcattagct aaaacaaaaa tcatacacaa atgggttttg
gttgtactgt ccaaagtgga 1080acacacagtt ttatagaaag acaaaactga
aacggcaagt gagagttaaa gtaaaaggaa 1140attgcaatcg agagccagct
gagattttta gatattttgc aggtaactct taacgccgag 1200catacatacg
tttctcaaag acataaagct gattgtgttt ctccatttgc tgaaagatct
1260gagccataaa gctgaaatat aaaccttttt tgtgttttgt tgttccccag
attctctgac 1320gctgcttaaa ccttaaagtt cgtagctttt tcttgctagt
gggtcttttg aggtggatct 1380ggattctggg taaggttttg ttttttatat
ctttaccagt aaattgaagt tcttatggtt 1440tcaagaaatt ttacagggtt
ttcaattatt ggaatgttgt ttctgttggc tctttatctg 1500aatacaatct
tactaagagg ttacggatct tggactttgt aggaacttga gcaaaagcca
1560tcctcttttg ttgtatatat gaattaa 1587371602DNAArabidopsis
thalianapromoter(1)..(1602)transcription regulating sequence from
gene At2g34200 37ttttttcttt atctaatgga taaccaccac ccattaggat
caccataaag atcatatttt 60ggagcattcc aatatcataa cactttactc ttaagggtct
aatttgagtt ggtggcaaca 120tgtcatatag acttcagagc tgcttccaaa
atgggtttcc aatctacacc atttattaca 180gattcatttt tgcttttcgt
atttggttag tttaggagtt tagtttttac tttttttaga 240ttcaggaagt
ctttaatggg tttcaccggt ttagtaggtt cacctttatc ggccaacaag
300aattacattt ctctctattt atgacgactg ccaccaataa ttcaaaacaa
gtttacatat 360taagttttct taaactactc ctatatatta agtctaagat
tatattaatc ttatttatat 420aatttaattc aactaaattt ttagtttgtt
tatttacatg atataccatg gtcctgttat 480tttagctaaa gtatatatta
aatttattac ttttttattt caaagatttt ttctattgtt 540ttttattttg
ttttcttttc atttaagctt atattattat tattatacac acacatttaa
600ggtttatatt tatatttctt gcaatgcaaa catgtgtcaa tattagttga
tttaagatac 660aaaaatatta aggactaaat ggttgagata atactacttt
gatatatata tatatatata 720tataactata ttctataaac ttaatttatt
tgagagggta aggattagat gaccaagaaa 780ttggtaaaaa ttggaaaaat
aattatagac attattattt tacaagatat tctcaaatgg 840ctagagattt
gtgaagttct gttttttttt tcctcaaagt tcaagtgtaa tatttttcga
900tgacaatgat tattccaaat ttattttttt gttaatcgta aacagtgaat
tttgcataat 960tggtttgaat tagagttgat aatagttgtt ctcatcatca
ccaaacattc ttctgcttac 1020caaaagtaag ggactttcat tagcaaaaac
aaaaatcata cacaaatggg ttttggttgt 1080actgtccaaa atggaacaca
cagttttata gaaagacaaa actgaaacgg caagtgagag 1140ttaaagtaaa
aggaaattgc aatcgagagc cagctgagat ttttagatat tttgcaggta
1200actcttaacg ccgagcatac atacgtttct caaagacata aagctgattg
tgtttctcca 1260tttgctgaaa gatctgagcc ataaagctga aatagaaacc
ttttttgtgt tttgttgttc 1320cccagattct ctgacgctgc ttaaacctta
aagttcgtag ctttttcttg ctagtgggtc 1380ttttgaggtg gatctggatt
ctgggtaagg ttttgttttt tatatcttta ccagtaaatt 1440gaagttctta
tggtttcaag aaattttaca gggttttcaa ttattggaat gttgtttctg
1500ttggctcttt atctgaatac aatcttacta agaggttacg gatcttggac
tttgtaggaa 1560cttgagcaaa agccatcctc ttttgttgta tatatgaatt aa
1602381130DNAArabidopsis thalianapromoter(1)..(1130)transcription
regulating sequence from gene At2g34200 38tttatctaat ggataaccac
cacccattag gatcaccata aaggtcatat tttggagcat 60tccaatatca taacacttta
ctcttaaggg tctaatttga gttggtggca acatgtcata 120tagacttcag
agctgcttcc aaaatgggtt tccaatctac accatttatt acagattctt
180ttttgctttt cgtatttgga tagtttacga gtttagtttt tacttttttt
agattcagga 240agtctttaat gggtttcacc ggtttagtag gttcaccttt
atcggccaac aagaattaca 300tttctctcta tttatgacga ctgccaccaa
taattcaaaa caagtttaca tattaagttt 360tcttaaacta ctcctatata
ttaagtctaa gattatatta atcttattta tataatttaa 420ttcaactaaa
tttttagttt gtttatttac atgatatacc atggtcctgt tattttagct
480aaagtatata ttaaatttat tactttttta tttcaaagat tttttctatt
gttttttatt 540ttgttttctt ttcatttaag cttatattat tattattata
cacacacatt taaggtttat 600atttatattt cttgcaatgc aaacatgtgt
caatattagt tggtttaaga tacaaaaata 660ttaaggacta aatggttgag
ataatactac tttgatatat atatatataa ctatattcta 720taaacttaat
ttatttgaga gggtaaggat tagatgacca agaaattggt aaaaattgga
780aaaataatta tagacattat tattttacaa gatattctca aatggctaga
gatttgtgaa 840gttctgtttt ttttttcctc aaagttcaag tgtaatattt
ttcgatgaca atgattattc 900caaatttatt tttttgttaa tcgtaaacag
tgaattttgc ataattggtt tgaattagag 960ttgataatag ttgttctcat
catcaccaaa cattcttctg cttaccaaaa gtaagggact 1020ttcattagct
aaaacaaaaa tcatacacaa atgggttttg gttgtactgt ccaaagtgga
1080acacacagtt ttatagaaag acaaaactga aacggcaagt gagagttaaa
1130391145DNAArabidopsis thalianapromoter(1)..(1145)transcription
regulating sequence from gene At2g34200 39ttttttcttt atctaatgga
taaccaccac ccattaggat caccataaag atcatatttt 60ggagcattcc aatatcataa
cactttactc ttaagggtct aatttgagtt ggtggcaaca 120tgtcatatag
acttcagagc tgcttccaaa atgggtttcc aatctacacc atttattaca
180gattcatttt tgcttttcgt atttggttag tttaggagtt tagtttttac
tttttttaga 240ttcaggaagt ctttaatggg tttcaccggt ttagtaggtt
cacctttatc ggccaacaag 300aattacattt ctctctattt atgacgactg
ccaccaataa ttcaaaacaa gtttacatat 360taagttttct taaactactc
ctatatatta agtctaagat tatattaatc ttatttatat 420aatttaattc
aactaaattt ttagtttgtt tatttacatg atataccatg gtcctgttat
480tttagctaaa gtatatatta aatttattac ttttttattt caaagatttt
ttctattgtt 540ttttattttg ttttcttttc atttaagctt atattattat
tattatacac acacatttaa 600ggtttatatt tatatttctt gcaatgcaaa
catgtgtcaa tattagttga tttaagatac 660aaaaatatta aggactaaat
ggttgagata atactacttt gatatatata tatatatata 720tataactata
ttctataaac ttaatttatt tgagagggta aggattagat gaccaagaaa
780ttggtaaaaa ttggaaaaat aattatagac attattattt tacaagatat
tctcaaatgg 840ctagagattt gtgaagttct gttttttttt tcctcaaagt
tcaagtgtaa tatttttcga 900tgacaatgat tattccaaat ttattttttt
gttaatcgta aacagtgaat tttgcataat 960tggtttgaat tagagttgat
aatagttgtt ctcatcatca ccaaacattc ttctgcttac 1020caaaagtaag
ggactttcat tagcaaaaac aaaaatcata cacaaatggg ttttggttgt
1080actgtccaaa atggaacaca cagttttata gaaagacaaa actgaaacgg
caagtgagag 1140ttaaa 1145401169DNAArabidopsis
thalianapromoter(1)..(1169)transcription regulating sequence from
gene At2g34200 40tttatctaat ggataaccac cacccattag gatcaccata
aagatcatat tttggagcat 60tccaatatca taacacttta ctcttaaggg tctaatttga
gttggtggca acatgtcata 120tagacttcag agctgcttcc aaaatgggtt
tccaatctac accatttatt acagattctt 180ttttgctttt cgtatttgga
tagtttacga gtttagtttt tacttttttt agattcagga 240agtctttaat
gggtttcacc ggtttagtag gttcaccttt atcggccaac aagaattaca
300tttctctcta tttatgacga ctgccaccaa taattcaaaa caagtttaca
tattaagttt 360tcttaaacta ctcctatata ttaagtctaa gattatatta
atcttattta tataatttaa 420ttcaactaaa tttttagttt gtttatttac
atgatatacc atggtcctgt tattttagct 480aaagtatata ttaaatttat
tactttttta tttcaaagat tttttctatt gttttttatt 540ttgttttctt
ttcatttaag cttatattat tattattata cacacacatt taaggtttat
600atttatattt cttgcaatgc aaacatgtgt caatattagt tggtttaaga
tacaaaaata 660ttaaggacta aatggttgag ataatactac tttgatatat
atatatataa ctatattcta 720taaacttaat ttatttgaga gggtaaggat
tagatgacca agaaattggt aaaaattgga 780aaaataatta tagacattat
tattttacaa gatattctca aatggctaga gatttgtgaa 840gttctgtttt
ttttttcctc aaagttcaag tgtaatattt ttcgatgaca atgattattc
900caaatttatt tttttgttaa tcgtaaacag tgaattttgc ataattggtt
tgaattagag 960ttgataatag ttgttctcat catcaccaaa cattcttctg
cttaccaaaa gtaagggact 1020ttcattagct aaaacaaaaa tcatacacaa
atgggttttg gttgtactgt ccaaaatgga 1080acacacagtt ttatagaaag
acaaaactga aacggcaagt gagagttaaa gtaaaaggaa 1140attgcaatcg
agagccagct gagattttt 1169411191DNAArabidopsis
thalianapromoter(1)..(1191)transcription regulating sequence from
gene At2g34200 41ttttttcttt atctaatgga taaccaccac ccattaggat
caccataaag atcatatttt 60ggagcattcc aatatcataa cactttactc ttaagggtct
aatttgagtt ggtggcaaca 120tgtcatatag acttcagagc tgcttccaaa
atgggtttcc aatctacacc atttattaca 180gattcatttt tgcttttcgt
atttggttag tttaggagtt tagtttttac tttttttaga 240ttcaggaagt
ctttaatggg tttcaccggt ttagtaggtt cacctttatc ggccaacaag
300aattacattt ctctctattt atgacgactg ccaccaataa ttcaaaacaa
gtttacatat 360taagttttct taaactactc ctatatatta agtctaagat
tatattaatc ttatttatat 420aatttaattc aactaaattt ttagtttgtt
tatttacatg atataccatg gtcctgttat 480tttagctaaa gtatatatta
aatttattac ttttttattt caaagatttt ttctattgtt 540ttttattttg
ttttcttttc atttaagctt atattattat tattatacac acacatttaa
600ggtttatatt tatatttctt gcaatgcaaa catgtgtcaa tattagttga
tttaagatac 660aaaaatatta aggactaaat ggttgagata atactacttt
gatatatata tatatatata 720tataactata ttctataaac ttaatttatt
tgagagggta aggattagat gaccaagaaa 780ttggtaaaaa ttggaaaaat
aattatagac attattattt tacaagatat tctcaaatgg 840ctagagattt
gtgaagttct gttttttttt tcctcaaagt tcaagtgtaa tatttttcga
900tgacaatgat tattccaaat ttattttttt gttaatcgta aacagtgaat
tttgcataat 960tggtttgaat tagagttgat aatagttgtt ctcatcatca
ccaaacattc ttctgcttac 1020caaaagtaag ggactttcat tagcaaaaac
aaaaatcata cacaaatggg ttttggttgt 1080actgtccaaa atggaacaca
cagttttata gaaagacaaa actgaaacgg caagtgagag 1140ttaaagtaaa
aggaaattgc aatcgagagc cagctgagat ttttagatat t
1191421130DNAArabidopsis thalianapromoter(1)..(1130)transcription
regulating sequence from gene At2g34200 42tttatctaat ggataaccac
cacccattag gatcaccata aagatcatat tttggagcat 60tccaatatca taacacttta
ctcttaaggg tctaatttga gttggtggca acatgtcata 120tagacttcag
agctgcttcc aaaatgggtt tccaatctac accatttatt acagattctt
180ttttgctttt cgtatttgga tagtttacga gtttagtttt tacttttttt
agattcagga 240agtctttaat gggtttcacc ggtttagtag gttcaccttt
atcggccaac aagaattaca 300tttctctcta tttatgacga ctgccaccaa
taattcaaaa caagtttaca tattaagttt 360tcttaaacta ctcctatata
ttaagtctaa gattatatta atcttattta tataatttaa 420ttcaactaaa
tttttagttt gtttatttac atgatatacc atggtcctgt tattttagct
480aaagtatata ttaaatttat tactttttta tttcaaagat tttttctatt
gttttttatt 540ttgttttctt ttcatttaag cttatattat tattattata
cacacacatt taaggtttat 600atttatattt cttgcaatgc aaacatgtgt
caatattagt tggtttaaga tacaaaaata 660ttaaggacta aatggttgag
ataatactac tttgatatat atatatataa ctatattcta 720taaacttaat
ttatttgaga gggtaaggat tagatgacca agaaattggt aaaaattgga
780aaaataatta tagacattat tattttacaa gatattctca aatggctaga
gatttgtgaa 840gttctgtttt ttttttcctc aaagttcaag tgtaatattt
ttcgatgaca atgattattc 900caaatttatt tttttgttaa tcgtaaacag
tgaattttgc ataattggtt tgaattagag 960ttgataatag ttgttctcat
catcaccaaa cattcttctg cttaccaaaa gtaagggact 1020ttcattagct
aaaacaaaaa tcatacacaa atgggttttg gttgtactgt ccaaaatgga
1080acacacagtt ttatagaaag acaaaactga aacggcaagt gagagttaaa
1130431145DNAArabidopsis thalianapromoter(1)..(1145)transcription
regulating sequence from gene At2g34200 43ttttttcttt atctaatgga
taaccaccac ccattaggat caccataaag atcatatttt 60ggagcattcc aatatcataa
cactttactc ttaagggtct aatttgagtt ggtggcaaca 120tgtcatatag
acttcagagc tgcttccaaa atgggtttcc aatctacacc atttattaca
180gattcatttt tgcttttcgt atttggttag tttaggagtt tagtttttac
tttttttaga 240ttcaggaagt ctttaatggg tttcaccggt ttagtaggtt
cacctttatc ggccaacaag 300aattacattt ctctctattt atgacgactg
ccaccaataa ttcaaaacaa gtttacatat 360taagttttct taaactactc
ctatatatta agtctaagat tatattaatc ttatttatat 420aatttaattc
aactaaattt ttagtttgtt tatttacatg atataccatg gtcctgttat
480tttagctaaa gtatatatta aatttattac ttttttattt caaagatttt
ttctattgtt 540ttttattttg ttttcttttc atttaagctt atattattat
tattatacac acacatttaa 600ggtttatatt tatatttctt gcaatgcaaa
catgtgtcaa tattagttga tttaagatac 660aaaaatatta aggactaaat
ggttgagata atactacttt gatatatata tatatatata 720tataactata
ttctataaac ttaatttatt tgagagggta aggattagat gaccaagaaa
780ttggtaaaaa ttggaaaaat aattatagac attattattt tacaagatat
tctcaaatgg 840ctagagattt gtgaagttct gttttttttt tcctcaaagt
tcaagtgtaa tatttttcga 900tgacaatgat tattccaaat ttattttttt
gttaatcgta aacagtgaat tttgcataat 960tggtttgaat tagagttgat
aatagttgtt ctcatcatca ccaaacattc ttctgcttac 1020caaaagtaag
ggactttcat tagcaaaaac aaaaatcata cacaaatggg ttttggttgt
1080actgtccaaa atggaacaca cagttttata gaaagacaaa actgaaacgg
caagtgagag 1140ttaaa 1145441120DNAArabidopsis
thalianapromoter(1)..(1120)transcription regulating sequence from
gene At2g34200 44tgttatttta gctaaagtat atattaaatt tattactttt
ttatttcaaa gattttttct 60attgtttttt attttgtttt cttttcattt aagcttatat
tattattatt atacacacac 120atttaaggtt tatatttata tttcttgcaa
tgcaaacatg tgtcaatatt agttggttta 180agatacaaaa atattaagga
ctaaatggtt gagataatac tactttgata tatatatata 240taactatatt
ctataaactt aatttatttg agagggtaag gattagatga ccaagaaatt
300ggtaaaaatt ggaaaaataa ttatagacat tattatttta caagatattc
tcaaatggct 360agagatttgt gaagttctgt tttttttttc ctcaaagttc
aagtgtaata tttttcgatg 420acaatgatta ttccaaattt atttttttgt
taatcgtaaa cagtgaattt tgcataattg 480gtttgaatta gagttgataa
tagttgttct catcatcacc aaacattctt ctgcttacca 540aaagtaaggg
actttcatta gctaaaacaa aaatcataca caaatgggtt ttggttgtac
600tgtccaaaat ggaacacaca gttttataga aagacaaaac tgaaacggca
agtgagagtt 660aaagtaaaag gaaattgcaa tcgagagcca gctgagattt
ttagatattt tgcaggtaac 720tcttaacgcc gagcatacat acgtttctca
aagacataaa gctgattgtg tttctccatt 780tgctgaaaga tctgagccat
aaagctgaaa tataaacctt ttttgtgttt tgttgttccc 840cagattctct
gacgctgctt aaaccttaaa gttcgtagct ttttcttgct agtgggtctt
900ttgaggtgga tctggattct gggtaaggtt ttgtttttta tatctttacc
agtaaattga 960agttcttatg gtttcaagaa attttacagg gttttcaatt
attggaatgt tgtttctgtt 1020ggctctttat ctgaatacaa tcttactaag
aggttacgga tcttggactt tgtaggaact 1080tgagcaaaag ccatcctctt
ttgttgtata tatgaattaa 1120451135DNAArabidopsis
thalianapromoter(1)..(1135)transcription regulating sequence from
gene At2g34200 45atggtcctgt tattttagct aaagtatata ttaaatttat
tactttttta tttcaaagat 60tttttctatt gttttttatt ttgttttctt ttcatttaag
cttatattat tattattata 120cacacacatt taaggtttat atttatattt
cttgcaatgc aaacatgtgt caatattagt 180tgatttaaga tacaaaaata
ttaaggacta aatggttgag ataatactac tttgatatat 240atatatatat
atatataact atattctata aacttaattt atttgagagg gtaaggatta
300gatgaccaag aaattggtaa aaattggaaa aataattata gacattatta
ttttacaaga 360tattctcaaa tggctagaga tttgtgaagt tctgtttttt
ttttcctcaa agttcaagtg 420taatattttt cgatgacaat gattattcca
aatttatttt tttgttaatc gtaaacagtg 480aattttgcat aattggtttg
aattagagtt gataatagtt gttctcatca tcaccaaaca 540ttcttctgct
taccaaaagt aagggacttt cattagcaaa aacaaaaatc atacacaaat
600gggttttggt tgtactgtcc aaaatggaac acacagtttt atagaaagac
aaaactgaaa 660cggcaagtga gagttaaagt aaaaggaaat tgcaatcgag
agccagctga gatttttaga 720tattttgcag gtaactctta acgccgagca
tacatacgtt tctcaaagac ataaagctga 780ttgtgtttct ccatttgctg
aaagatctga gccataaagc tgaaatagaa accttttttg 840tgttttgttg
ttccccagat tctctgacgc tgcttaaacc ttaaagttcg tagctttttc
900ttgctagtgg gtcttttgag gtggatctgg attctgggta aggttttgtt
ttttatatct 960ttaccagtaa attgaagttc ttatggtttc aagaaatttt
acagggtttt caattattgg 1020aatgttgttt ctgttggctc tttatctgaa
tacaatctta ctaagaggtt acggatcttg 1080gactttgtag gaacttgagc
aaaagccatc ctcttttgtt gtatatatga attaa 113546663DNAArabidopsis
thalianapromoter(1)..(663)transcription regulating sequence from
gene At2g34200 46tgttatttta gctaaagtat atattaaatt tattactttt
ttatttcaaa gattttttct 60attgtttttt attttgtttt cttttcattt aagcttatat
tattattatt atacacacac 120atttaaggtt tatatttata tttcttgcaa
tgcaaacatg tgtcaatatt agttggttta 180agatacaaaa atattaagga
ctaaatggtt gagataatac tactttgata tatatatata 240taactatatt
ctataaactt aatttatttg agagggtaag gattagatga ccaagaaatt
300ggtaaaaatt ggaaaaataa ttatagacat tattatttta caagatattc
tcaaatggct 360agagatttgt gaagttctgt tttttttttc ctcaaagttc
aagtgtaata tttttcgatg 420acaatgatta ttccaaattt atttttttgt
taatcgtaaa cagtgaattt tgcataattg 480gtttgaatta gagttgataa
tagttgttct catcatcacc aaacattctt ctgcttacca 540aaagtaaggg
actttcatta gctaaaacaa aaatcataca caaatgggtt ttggttgtac
600tgtccaaaat ggaacacaca gttttataga aagacaaaac tgaaacggca
agtgagagtt 660aaa 66347678DNAArabidopsis
thalianapromoter(1)..(678)transcription regulating sequence from
gene At2g34200 47atggtcctgt tattttagct aaagtatata ttaaatttat
tactttttta tttcaaagat 60tttttctatt gttttttatt ttgttttctt ttcatttaag
cttatattat tattattata 120cacacacatt taaggtttat atttatattt
cttgcaatgc aaacatgtgt caatattagt 180tgatttaaga tacaaaaata
ttaaggacta aatggttgag ataatactac tttgatatat 240atatatatat
atatataact atattctata aacttaattt atttgagagg gtaaggatta
300gatgaccaag aaattggtaa aaattggaaa aataattata gacattatta
ttttacaaga 360tattctcaaa tggctagaga tttgtgaagt tctgtttttt
ttttcctcaa agttcaagtg 420taatattttt cgatgacaat gattattcca
aatttatttt tttgttaatc gtaaacagtg 480aattttgcat aattggtttg
aattagagtt gataatagtt gttctcatca
tcaccaaaca 540ttcttctgct taccaaaagt aagggacttt cattagcaaa
aacaaaaatc atacacaaat 600gggttttggt tgtactgtcc aaaatggaac
acacagtttt atagaaagac aaaactgaaa 660cggcaagtga gagttaaa
67848968DNAArabidopsis thalianaCDS(177)..(845)encoding zinc finger
(C3HC4-type RING finger) family protein) 48gtaaaaggaa attgcaatcg
agagccagct gagattttta gatattttgc agattctctg 60acgctgctta aaccttaaag
ttcgtagctt tttcttgcta gtgggtcttt tgaggtggat 120ctggattctg
ggaacttgag caaaagccat cctcttttgt tgtatatatg aattaa atg 179 Met 1gac
cct aaa agt tgt gag aat tcg agt gat gtt aag ggt cag acc agt 227Asp
Pro Lys Ser Cys Glu Asn Ser Ser Asp Val Lys Gly Gln Thr Ser 5 10
15gac tct gta tca aaa aag gtg ttg att gag gaa gag gaa gat gta aag
275Asp Ser Val Ser Lys Lys Val Leu Ile Glu Glu Glu Glu Asp Val Lys
20 25 30aaa cca caa cag gga aaa gaa aat gat tca aga atg gcc aaa gat
gtt 323Lys Pro Gln Gln Gly Lys Glu Asn Asp Ser Arg Met Ala Lys Asp
Val 35 40 45gtt agt tgc agt agt aac att tca gct cat gtt gtt cat gag
gaa gtt 371Val Ser Cys Ser Ser Asn Ile Ser Ala His Val Val His Glu
Glu Val50 55 60 65gcg gat aat gtt act gcg gta agc tgc aac gag gca
gag agt gat ata 419Ala Asp Asn Val Thr Ala Val Ser Cys Asn Glu Ala
Glu Ser Asp Ile 70 75 80tcg aaa gca aag gcg aag gag ttc cac act att
gat ttg agt ggt gta 467Ser Lys Ala Lys Ala Lys Glu Phe His Thr Ile
Asp Leu Ser Gly Val 85 90 95gga gaa aga atc tgt agg att tgt cat ttt
ggt tct gat caa tca cct 515Gly Glu Arg Ile Cys Arg Ile Cys His Phe
Gly Ser Asp Gln Ser Pro 100 105 110gag gct tct ggt gat gat aag tca
gta agt ccg gag ttg att gag att 563Glu Ala Ser Gly Asp Asp Lys Ser
Val Ser Pro Glu Leu Ile Glu Ile 115 120 125ggt tgc aaa tgt aaa aac
gag ctt ggc ctt gca cat ttc cat tgc gct 611Gly Cys Lys Cys Lys Asn
Glu Leu Gly Leu Ala His Phe His Cys Ala130 135 140 145gaa gcc tgg
ttc aag tta aga gga aac agt gtg tgt gaa atc tgc ggt 659Glu Ala Trp
Phe Lys Leu Arg Gly Asn Ser Val Cys Glu Ile Cys Gly 150 155 160tgc
aca gcg aag aat gtt aca gta agg ttg atg gag gat tgg agc ggc 707Cys
Thr Ala Lys Asn Val Thr Val Arg Leu Met Glu Asp Trp Ser Gly 165 170
175gaa aga gac aat aca ttg gat ggg aga aga aga cga gga aga gga caa
755Glu Arg Asp Asn Thr Leu Asp Gly Arg Arg Arg Arg Gly Arg Gly Gln
180 185 190tct tgc tgc atc ttt atg gtt ttt ctg ctc act atc ctt ttg
ctt cat 803Ser Cys Cys Ile Phe Met Val Phe Leu Leu Thr Ile Leu Leu
Leu His 195 200 205tgg ttt ttc aaa aag att agt ggt tac tac caa aac
act tga 845Trp Phe Phe Lys Lys Ile Ser Gly Tyr Tyr Gln Asn Thr210
215 220atatgtatat ctgttggtaa ctttatcatt tgattctgaa tctagattat
atggaaaaga 905gatgatagaa tcaaaattct tagatcaaat tctgattcat
agtccaacag atggacaatt 965tag 96849222PRTArabidopsis thaliana 49Met
Asp Pro Lys Ser Cys Glu Asn Ser Ser Asp Val Lys Gly Gln Thr1 5 10
15Ser Asp Ser Val Ser Lys Lys Val Leu Ile Glu Glu Glu Glu Asp Val
20 25 30Lys Lys Pro Gln Gln Gly Lys Glu Asn Asp Ser Arg Met Ala Lys
Asp 35 40 45Val Val Ser Cys Ser Ser Asn Ile Ser Ala His Val Val His
Glu Glu 50 55 60Val Ala Asp Asn Val Thr Ala Val Ser Cys Asn Glu Ala
Glu Ser Asp65 70 75 80Ile Ser Lys Ala Lys Ala Lys Glu Phe His Thr
Ile Asp Leu Ser Gly 85 90 95Val Gly Glu Arg Ile Cys Arg Ile Cys His
Phe Gly Ser Asp Gln Ser 100 105 110Pro Glu Ala Ser Gly Asp Asp Lys
Ser Val Ser Pro Glu Leu Ile Glu 115 120 125Ile Gly Cys Lys Cys Lys
Asn Glu Leu Gly Leu Ala His Phe His Cys 130 135 140Ala Glu Ala Trp
Phe Lys Leu Arg Gly Asn Ser Val Cys Glu Ile Cys145 150 155 160Gly
Cys Thr Ala Lys Asn Val Thr Val Arg Leu Met Glu Asp Trp Ser 165 170
175Gly Glu Arg Asp Asn Thr Leu Asp Gly Arg Arg Arg Arg Gly Arg Gly
180 185 190Gln Ser Cys Cys Ile Phe Met Val Phe Leu Leu Thr Ile Leu
Leu Leu 195 200 205His Trp Phe Phe Lys Lys Ile Ser Gly Tyr Tyr Gln
Asn Thr 210 215 220501069DNAArabidopsis
thalianapromoter(1)..(1069)transcription regulating sequence from
gene At3g11180 50ggtgagtgct acgttttata ttagtacagt actatgtcta
gaaatatgtc tacttttgta 60atattttact caaaagagag acgattgaag aaaatagata
attacaagtt acagccattg 120ttactctctt cttacaagaa ctatgcatac
agtttatttc accatctatt gaactatata 180atttcatatt attgccttta
tagaattgta caattccaaa gagcatcctc ttccttgcgt 240aggttatcat
tttgaactat taagagcact ttgataactg aagtaagtta gaaatcactt
300tacatgtagt aaagtatcaa taaaattagt gcaattctga gataaggaag
ttaaagttaa 360atattctttt aaaaatgttg aagtcgtcag attcttttag
tatatatctc aaggcattaa 420atatttcaag gttgagtacg tcgttaagct
acccaaacat tctcaaacag aagaaaatca 480taccgatctt aatttatatt
attagggtta aaacatctac gaactttcac agctaataac 540aaaaaaatga
actccacgtg atagctaaaa ttatttgcaa ggaagctaca aaacttttca
600ttggaaaatg cttcgtactt ttcacatttg gaattattat caaatacatc
cacgtcattt 660tcaatctttt ttaccacgta tcaaaaaaaa cattttttta
tcacgttttc gtgaactaac 720aagcaacaaa ctttaatcgt atggactcta
ttcgacttcc tcatcggtta caattacgga 780ttcctacata cgttacgatt
ttcttcagtt acttattaca aaggaagata attaatattt 840tcgattcacg
agaatagtct gaaacatgtc ggtccgagtt tgattttatt tgtgtatgat
900agttaatatt cacaattcat ttatttatgt taatccaaat cttcttccct
ttctctctct 960ctttctctcc ctctaaccat ataaaccaac tatatataac
agcacttcac aacttctcat 1020gaacaagtag aaaaaaagag caagactaat
aagccatata taagagaca 1069511088DNAArabidopsis
thalianapromoter(1)..(1088)transcription regulating sequence from
gene At3g11180 51agagatggaa ctggtgagtg ctacgtttta tattagtaca
gtactatgtc tagaaatatg 60tctacttttg taatatttta ctcaaaagag agacgattga
agaaaataga taattacaag 120ttacagccat tgttactctc ttcttacaag
aactatgcat acagtttatt tcaccatcta 180ttgaactata taatttcata
ttattgcctt tatagaattg tacaattcca aagagcatcc 240tcttccttgc
gtaggttatc attttgaact attaagagca ctttgataac tgaagtaagt
300tagaaatcac tttacatgta gtaaagtatc aataaaatta gtgcaattct
gagataagga 360agttaaagtt aaatattatt ttaaaatgtt gaagtcgtca
gattctttta gtatatatct 420caaggcatta aatatttcaa ggttgagtac
gtcgttaagc tacccaaaca ttctcaaaca 480gaagaaaatc ataccgatct
taatttatat tgttagggtt aaaacatcta cgaactttca 540cagctaataa
caaaaaaatg aactccacgt gatagctaaa attatttgta aggaagctac
600aaaacttttc attgcaaaat gcttcgtagt tttcacattt ggaattatta
tcaaatacat 660ccacgtcatt ttcaatcttt tttaccacgt atcagaaaaa
acatttttta tcacgttttc 720ttgaactaac aagcaacaaa ctttaatcgt
atggactcta ttcgacttcc tcatcggtta 780caaacttaca attacggatt
cctacatacg ttacgatttt cttcagttac ttattacaaa 840ggaagataat
taatattttc gattcacgag aatagtctga aacatgtcgg tccgagtttg
900attttatttg tgtatgatag ttaatattca caattcattt atttatgtta
atccaaatct 960tgttcccttt ctctctctct ttctctccct ctaaccatat
aaaccaacta tatataacag 1020cacttcacaa cttctcatga acaagtagaa
aaaaaagagc aagactaata agccatataa 1080gagacaac
1088521034DNAArabidopsis thalianapromoter(1)..(1034)transcription
regulating sequence from gene At3g11180 52ggtgagtgct acgttttata
ttagtacagt actatgtcta gaaatatgtc tacttttgta 60atattttact caaaagagag
acgattgaag aaaatagata attacaagtt acagccattg 120ttactctctt
cttacaagaa ctatgcatac agtttatttc accatctatt gaactatata
180atttcatatt attgccttta tagaattgta caattccaaa gagcatcctc
ttccttgcgt 240aggttatcat tttgaactat taagagcact ttgataactg
aagtaagtta gaaatcactt 300tacatgtagt aaagtatcaa taaaattagt
gcaattctga gataaggaag ttaaagttaa 360atattctttt aaaaatgttg
aagtcgtcag attcttttag tatatatctc aaggcattaa 420atatttcaag
gttgagtacg tcgttaagct acccaaacat tctcaaacag aagaaaatca
480taccgatctt aatttatatt attagggtta aaacatctac gaactttcac
agctaataac 540aaaaaaatga actccacgtg atagctaaaa ttatttgcaa
ggaagctaca aaacttttca 600ttggaaaatg cttcgtactt ttcacatttg
gaattattat caaatacatc cacgtcattt 660tcaatctttt ttaccacgta
tcaaaaaaaa cattttttta tcacgttttc gtgaactaac 720aagcaacaaa
ctttaatcgt atggactcta ttcgacttcc tcatcggtta caattacgga
780ttcctacata cgttacgatt ttcttcagtt acttattaca aaggaagata
attaatattt 840tcgattcacg agaatagtct gaaacatgtc ggtccgagtt
tgattttatt tgtgtatgat 900agttaatatt cacaattcat ttatttatgt
taatccaaat cttcttccct ttctctctct 960ctttctctcc ctctaaccat
ataaaccaac tatatataac agcacttcac aacttctcat 1020gaacaagtag aaaa
1034531053DNAArabidopsis thalianapromoter(1)..(1053)transcription
regulating sequence from gene At3g11180 53agagatggaa ctggtgagtg
ctacgtttta tattagtaca gtactatgtc tagaaatatg 60tctacttttg taatatttta
ctcaaaagag agacgattga agaaaataga taattacaag 120ttacagccat
tgttactctc ttcttacaag aactatgcat acagtttatt tcaccatcta
180ttgaactata taatttcata ttattgcctt tatagaattg tacaattcca
aagagcatcc 240tcttccttgc gtaggttatc attttgaact attaagagca
ctttgataac tgaagtaagt 300tagaaatcac tttacatgta gtaaagtatc
aataaaatta gtgcaattct gagataagga 360agttaaagtt aaatattatt
ttaaaatgtt gaagtcgtca gattctttta gtatatatct 420caaggcatta
aatatttcaa ggttgagtac gtcgttaagc tacccaaaca ttctcaaaca
480gaagaaaatc ataccgatct taatttatat tgttagggtt aaaacatcta
cgaactttca 540cagctaataa caaaaaaatg aactccacgt gatagctaaa
attatttgta aggaagctac 600aaaacttttc attgcaaaat gcttcgtagt
tttcacattt ggaattatta tcaaatacat 660ccacgtcatt ttcaatcttt
tttaccacgt atcagaaaaa acatttttta tcacgttttc 720ttgaactaac
aagcaacaaa ctttaatcgt atggactcta ttcgacttcc tcatcggtta
780caaacttaca attacggatt cctacatacg ttacgatttt cttcagttac
ttattacaaa 840ggaagataat taatattttc gattcacgag aatagtctga
aacatgtcgg tccgagtttg 900attttatttg tgtatgatag ttaatattca
caattcattt atttatgtta atccaaatct 960tgttcccttt ctctctctct
ttctctccct ctaaccatat aaaccaacta tatataacag 1020cacttcacaa
cttctcatga acaagtagaa aaa 1053542012DNAArabidopsis
thalianapromoter(1)..(2012)transcription regulating sequence from
gene At3g11180 54ctgaatacga tcgatcagat ggaaacaaca aatttgtttg
cgatactgaa gctatatata 60ccatacattg cattatctta gatacattta aattctagca
aaaagtataa aagaagatca 120ttacgtgagc gacgaaggag atgttgtata
tatactacaa attaagcaga tccaaatctc 180tatgtttccg atactgaagc
tttatactaa taaaatatac attgcaatat atattatttt 240agattcatcc
ttctgaaggt ttaataccaa taattttagg gtataattaa gaaaatagtt
300ttctactttt ttcacattta aaatcaatca tagttattct tcctaacctc
actaaatttg 360tttgattaat taatttaaaa atgggattta aataagtatt
tgttacacaa tatgtagttg 420gatagtatcc acatgtgcct tccaaattca
gtgaaccatg tttctattat tgggaagtcg 480tcggccttct atttagatgg
tcaacggacc atttactaat acaagttttg cccataatat 540atttaaatcg
actctccttc ggaattttta catttaaatt gtttaaaagc tctacttgaa
600taatccaaca tacaataatc ttgaactaat ttttttttta ataatgtagt
tattagaatt 660ataaacgaaa agtgtttgat tggtatgttg aatataagcg
tacgcttaat ttgctattgc 720atgacacata aataatcatc ttttaacact
cttgaatttt taaaaattat aattgcgatt 780ctcaaattta gtcataaatt
cagttaacta tcatcgctaa gtggcataaa taatgtagtt 840agatagtcac
cgtccgttgc taatacagaa taaaagtcac cgtccaatta cacacacgta
900tgtaaggaaa ttatacataa cttaatttta tagagatgga actggtgagt
gctacgtttt 960atattagtac agtactatgt ctagaaatat gtctactttt
gtaatatttt actcaaaaga 1020gagacgattg aagaaaatag ataattacaa
gttacagcca ttgttactct cttcttacaa 1080gaactatgca tacagtttat
ttcaccatct attgaactat ataatttcat attattgcct 1140ttatagaatt
gtacaattcc aaagagcatc ctcttccttg cgtaggttat cattttgaac
1200tattaagagc actttgataa ctgaagtaag ttagaaatca ctttacatgt
agtaaagtat 1260caataaaatt agtgcaattc tgagataagg aagttaaagt
taaatattct tttaaaaatg 1320ttgaagtcgt cagattcttt tagtatatat
ctcaaggcat taaatatttc aaggttgagt 1380acgtcgttaa gctacccaaa
cattctcaaa cagaagaaaa tcataccgat cttaatttat 1440attattaggg
ttaaaacatc tacgaacttt cacagctaat aacaaaaaaa tgaactccac
1500gtgatagcta aaattatttg caaggaagct acaaaacttt tcattggaaa
atgcttcgta 1560cttttcacat ttggaattat tatcaaatac atccacgtca
ttttcaatct tttttaccac 1620gtatcaaaaa aaacattttt ttatcacgtt
ttcgtgaact aacaagcaac aaactttaat 1680cgtatggact ctattcgact
tcctcatcgg ttacaattac ggattcctac atacgttacg 1740attttcttca
gttacttatt acaaaggaag ataattaata ttttcgattc acgagaatag
1800tctgaaacat gtcggtccga gtttgatttt atttgtgtat gatagttaat
attcacaatt 1860catttattta tgttaatcca aatcttcttc cctttctctc
tctctttctc tccctctaac 1920catataaacc aactatatat aacagcactt
cacaacttct catgaacaag tagaaaaaaa 1980gagcaagact aataagccat
atataagaga ca 2012552033DNAArabidopsis
thalianapromoter(1)..(2033)transcription regulating sequence from
gene At3g11180 55gagttagtat ctctgaatac gatcgatcag atggaaacaa
caaatttgtt tgcgatactg 60aagctatata taccatacat tgcattatct tagatacatt
taaattctag caaaaagtat 120aaaagaagat cattacgtga gcgacgaagg
agatgttgta tatatactac aaattaagca 180gatccaaatc tctatgtttc
cgatactgaa gctttatact aataaaatat acattgcaat 240atatattatt
ttagattcat ccttctgaag gtttatatac caataatttt agggtataat
300taagaaaata gttttctact tttttcacat ttaaaatcaa tcatagttat
tcttcctaac 360ctcactaaat ttgtttgatt aattaactta aaaatgggat
ttaaataagt atttgttaca 420caatatgtag ttggatagta tccacatgtg
ccttccaaat tcagtgaacc atgtttctat 480tattgggaag tcgtcggcct
tctatttaga tggtcaacgg accatttact aatacaagtt 540ttgcccataa
tatatttaaa tcgactctcc ttcggaattt ttacatttaa attgtttaaa
600agctctactt gaataatcca acatacaata atcttgaact aatttttttt
tttaataatg 660tagttattag aattataaac gaaaagtgtt tgattggtat
gttgaatata agcgtacgcc 720taatttgcta ttgcatgaca cataaataat
catcttttaa cactcttgaa tttttaaaaa 780ttataattgc gattctcaaa
tttagtcata aattcagtta actatcatcg ctaagtggca 840taaataatgt
agttagatag tcaccgtccg ttgctaatac agaataaaag tcaccgtcca
900attacacata cgtatgtaag gaaattatac ataacttaat tttatagaga
tggaactggt 960gagtgctacg ttttatatta gtacagtact atgtctagaa
atatgtctac ttttgtaata 1020ttttactcaa aagagagacg attgaagaaa
atagataatt acaagttaca gccattgtta 1080ctctcttctt acaagaacta
tgcatacagt ttatttcacc atctattgaa ctatataatt 1140tcatattatt
gcctttatag aattgtacaa ttccaaagag catcctcttc cttgcgtagg
1200ttatcatttt gaactattaa gagcactttg ataactgaag taagttagaa
atcactttac 1260atgtagtaaa gtatcaataa aattagtgca attctgagat
aaggaagtta aagttaaata 1320ttattttaaa atgttgaagt cgtcagattc
ttttagtata tatctcaagg cattaaatat 1380ttcaaggttg agtacgtcgt
taagctaccc aaacattctc aaacagaaga aaatcatacc 1440gatcttaatt
tatattgtta gggttaaaac atctacgaac tttcacagct aataacaaaa
1500aaatgaactc cacgtgatag ctaaaattat ttgtaaggaa gctacaaaac
ttttcattgc 1560aaaatgcttc gtagttttca catttggaat tattatcaaa
tacatccacg tcattttcaa 1620tcttttttac cacgtatcag aaaaaacatt
ttttatcacg ttttcttgaa ctaacaagca 1680acaaacttta atcgtatgga
ctctattcga cttcctcatc ggttacaaac ttacaattac 1740ggattcctac
atacgttacg attttcttca gttacttatt acaaaggaag ataattaata
1800ttttcgattc acgagaatag tctgaaacat gtcggtccga gtttgatttt
atttgtgtat 1860gatagttaat attcacaatt catttattta tgttaatcca
aatcttgttc cctttctctc 1920tctctttctc tccctctaac catataaacc
aactatatat aacagcactt cacaacttct 1980catgaacaag tagaaaaaaa
agagcaagac taataagcca tataagagac aac 2033561977DNAArabidopsis
thalianapromoter(1)..(1977)transcription regulating sequence from
gene At3g11180 56ctgaatacga tcgatcagat ggaaacaaca aatttgtttg
cgatactgaa gctatatata 60ccatacattg cattatctta gatacattta aattctagca
aaaagtataa aagaagatca 120ttacgtgagc gacgaaggag atgttgtata
tatactacaa attaagcaga tccaaatctc 180tatgtttccg atactgaagc
tttatactaa taaaatatac attgcaatat atattatttt 240agattcatcc
ttctgaaggt ttaataccaa taattttagg gtataattaa gaaaatagtt
300ttctactttt ttcacattta aaatcaatca tagttattct tcctaacctc
actaaatttg 360tttgattaat taatttaaaa atgggattta aataagtatt
tgttacacaa tatgtagttg 420gatagtatcc acatgtgcct tccaaattca
gtgaaccatg tttctattat tgggaagtcg 480tcggccttct atttagatgg
tcaacggacc atttactaat acaagttttg cccataatat 540atttaaatcg
actctccttc ggaattttta catttaaatt gtttaaaagc tctacttgaa
600taatccaaca tacaataatc ttgaactaat ttttttttta ataatgtagt
tattagaatt 660ataaacgaaa agtgtttgat tggtatgttg aatataagcg
tacgcttaat ttgctattgc 720atgacacata aataatcatc ttttaacact
cttgaatttt taaaaattat aattgcgatt 780ctcaaattta gtcataaatt
cagttaacta tcatcgctaa gtggcataaa taatgtagtt 840agatagtcac
cgtccgttgc taatacagaa taaaagtcac cgtccaatta cacacacgta
900tgtaaggaaa ttatacataa cttaatttta tagagatgga actggtgagt
gctacgtttt 960atattagtac agtactatgt ctagaaatat gtctactttt
gtaatatttt actcaaaaga 1020gagacgattg aagaaaatag ataattacaa
gttacagcca ttgttactct cttcttacaa 1080gaactatgca tacagtttat
ttcaccatct attgaactat ataatttcat attattgcct 1140ttatagaatt
gtacaattcc aaagagcatc ctcttccttg cgtaggttat cattttgaac
1200tattaagagc actttgataa ctgaagtaag ttagaaatca ctttacatgt
agtaaagtat 1260caataaaatt agtgcaattc tgagataagg aagttaaagt
taaatattct tttaaaaatg 1320ttgaagtcgt cagattcttt tagtatatat
ctcaaggcat taaatatttc aaggttgagt 1380acgtcgttaa gctacccaaa
cattctcaaa cagaagaaaa tcataccgat cttaatttat 1440attattaggg
ttaaaacatc tacgaacttt cacagctaat aacaaaaaaa
tgaactccac 1500gtgatagcta aaattatttg caaggaagct acaaaacttt
tcattggaaa atgcttcgta 1560cttttcacat ttggaattat tatcaaatac
atccacgtca ttttcaatct tttttaccac 1620gtatcaaaaa aaacattttt
ttatcacgtt ttcgtgaact aacaagcaac aaactttaat 1680cgtatggact
ctattcgact tcctcatcgg ttacaattac ggattcctac atacgttacg
1740attttcttca gttacttatt acaaaggaag ataattaata ttttcgattc
acgagaatag 1800tctgaaacat gtcggtccga gtttgatttt atttgtgtat
gatagttaat attcacaatt 1860catttattta tgttaatcca aatcttcttc
cctttctctc tctctttctc tccctctaac 1920catataaacc aactatatat
aacagcactt cacaacttct catgaacaag tagaaaa 1977571998DNAArabidopsis
thalianapromoter(1)..(1998)transcription regulating sequence from
gene At3g11180 57gagttagtat ctctgaatac gatcgatcag atggaaacaa
caaatttgtt tgcgatactg 60aagctatata taccatacat tgcattatct tagatacatt
taaattctag caaaaagtat 120aaaagaagat cattacgtga gcgacgaagg
agatgttgta tatatactac aaattaagca 180gatccaaatc tctatgtttc
cgatactgaa gctttatact aataaaatat acattgcaat 240atatattatt
ttagattcat ccttctgaag gtttatatac caataatttt agggtataat
300taagaaaata gttttctact tttttcacat ttaaaatcaa tcatagttat
tcttcctaac 360ctcactaaat ttgtttgatt aattaactta aaaatgggat
ttaaataagt atttgttaca 420caatatgtag ttggatagta tccacatgtg
ccttccaaat tcagtgaacc atgtttctat 480tattgggaag tcgtcggcct
tctatttaga tggtcaacgg accatttact aatacaagtt 540ttgcccataa
tatatttaaa tcgactctcc ttcggaattt ttacatttaa attgtttaaa
600agctctactt gaataatcca acatacaata atcttgaact aatttttttt
tttaataatg 660tagttattag aattataaac gaaaagtgtt tgattggtat
gttgaatata agcgtacgcc 720taatttgcta ttgcatgaca cataaataat
catcttttaa cactcttgaa tttttaaaaa 780ttataattgc gattctcaaa
tttagtcata aattcagtta actatcatcg ctaagtggca 840taaataatgt
agttagatag tcaccgtccg ttgctaatac agaataaaag tcaccgtcca
900attacacata cgtatgtaag gaaattatac ataacttaat tttatagaga
tggaactggt 960gagtgctacg ttttatatta gtacagtact atgtctagaa
atatgtctac ttttgtaata 1020ttttactcaa aagagagacg attgaagaaa
atagataatt acaagttaca gccattgtta 1080ctctcttctt acaagaacta
tgcatacagt ttatttcacc atctattgaa ctatataatt 1140tcatattatt
gcctttatag aattgtacaa ttccaaagag catcctcttc cttgcgtagg
1200ttatcatttt gaactattaa gagcactttg ataactgaag taagttagaa
atcactttac 1260atgtagtaaa gtatcaataa aattagtgca attctgagat
aaggaagtta aagttaaata 1320ttattttaaa atgttgaagt cgtcagattc
ttttagtata tatctcaagg cattaaatat 1380ttcaaggttg agtacgtcgt
taagctaccc aaacattctc aaacagaaga aaatcatacc 1440gatcttaatt
tatattgtta gggttaaaac atctacgaac tttcacagct aataacaaaa
1500aaatgaactc cacgtgatag ctaaaattat ttgtaaggaa gctacaaaac
ttttcattgc 1560aaaatgcttc gtagttttca catttggaat tattatcaaa
tacatccacg tcattttcaa 1620tcttttttac cacgtatcag aaaaaacatt
ttttatcacg ttttcttgaa ctaacaagca 1680acaaacttta atcgtatgga
ctctattcga cttcctcatc ggttacaaac ttacaattac 1740ggattcctac
atacgttacg attttcttca gttacttatt acaaaggaag ataattaata
1800ttttcgattc acgagaatag tctgaaacat gtcggtccga gtttgatttt
atttgtgtat 1860gatagttaat attcacaatt catttattta tgttaatcca
aatcttgttc cctttctctc 1920tctctttctc tccctctaac catataaacc
aactatatat aacagcactt cacaacttct 1980catgaacaag tagaaaaa
1998581577DNAArabidopsis thalianaCDS(36)..(1238)encoding
oxidoreductase, 2OG-Fe(II) oxygenase family protein 58aaagagcaag
actaataagc catataagag acaac atg aac aac cta gac gag 53 Met Asn Asn
Leu Asp Glu 1 5atc aag atc gag agc aag acc tgc ctc aac gat caa gaa
caa gaa gtc 101Ile Lys Ile Glu Ser Lys Thr Cys Leu Asn Asp Gln Glu
Gln Glu Val 10 15 20aaa ata gac aac atg cac atg agc gac caa gac aag
aac aag atc gaa 149Lys Ile Asp Asn Met His Met Ser Asp Gln Asp Lys
Asn Lys Ile Glu 25 30 35atc aag aac aag agt ggc ctc ggc gaa aag tgg
cca gaa ccc atc gtc 197Ile Lys Asn Lys Ser Gly Leu Gly Glu Lys Trp
Pro Glu Pro Ile Val 40 45 50cga gtc caa tct cta gcc gag agc aac ctc
act agt ctc cct gac cgt 245Arg Val Gln Ser Leu Ala Glu Ser Asn Leu
Thr Ser Leu Pro Asp Arg55 60 65 70tac atc aag ccg ccg tct caa cgc
cct caa acc acc atc atc gac cac 293Tyr Ile Lys Pro Pro Ser Gln Arg
Pro Gln Thr Thr Ile Ile Asp His 75 80 85caa ccg gaa gta gct gac ata
aat ata ccg atc ata gac cta gac agt 341Gln Pro Glu Val Ala Asp Ile
Asn Ile Pro Ile Ile Asp Leu Asp Ser 90 95 100cta ttc tct ggc aat
gaa gac gac aag aag agg ata tcc gag gca tgc 389Leu Phe Ser Gly Asn
Glu Asp Asp Lys Lys Arg Ile Ser Glu Ala Cys 105 110 115cgt gaa tgg
gga ttc ttc cag gta atc aac cat ggc gtg aag ccg gag 437Arg Glu Trp
Gly Phe Phe Gln Val Ile Asn His Gly Val Lys Pro Glu 120 125 130ctg
atg gac gca gct aga gaa act tgg aag agc ttc ttt aat ttg cct 485Leu
Met Asp Ala Ala Arg Glu Thr Trp Lys Ser Phe Phe Asn Leu Pro135 140
145 150gtt gaa gcc aaa gaa gtt tac tca aac tcc cca aga acc tat gaa
gga 533Val Glu Ala Lys Glu Val Tyr Ser Asn Ser Pro Arg Thr Tyr Glu
Gly 155 160 165tat gga agc aga ttg ggt gtg gaa aaa gga gcc att ctt
gat tgg aat 581Tyr Gly Ser Arg Leu Gly Val Glu Lys Gly Ala Ile Leu
Asp Trp Asn 170 175 180gat tat tac tat ctc cat ttt ctt cct ctt gcc
ttg aag gat ttc aac 629Asp Tyr Tyr Tyr Leu His Phe Leu Pro Leu Ala
Leu Lys Asp Phe Asn 185 190 195aaa tgg cct tct tta cct tcc aac att
aga gaa atg aat gat gag tac 677Lys Trp Pro Ser Leu Pro Ser Asn Ile
Arg Glu Met Asn Asp Glu Tyr 200 205 210ggt aag gaa cta gtg aag cta
ggt ggg aga cta atg acg atc tta tcg 725Gly Lys Glu Leu Val Lys Leu
Gly Gly Arg Leu Met Thr Ile Leu Ser215 220 225 230tca aat ttg ggg
cta aga gca gaa caa ctt caa gaa gca ttt ggt gga 773Ser Asn Leu Gly
Leu Arg Ala Glu Gln Leu Gln Glu Ala Phe Gly Gly 235 240 245gaa gac
gtt ggt gca tgt ttg agg gtt aat tat tac cca aag tgc cct 821Glu Asp
Val Gly Ala Cys Leu Arg Val Asn Tyr Tyr Pro Lys Cys Pro 250 255
260caa ccg gag ctt gcc ctc ggc ctc tcc cct cat tct gat ccc ggc ggc
869Gln Pro Glu Leu Ala Leu Gly Leu Ser Pro His Ser Asp Pro Gly Gly
265 270 275atg acc atc ctc ttg ccg gac gat caa gtc gtc ggc ctt cag
gtc cgt 917Met Thr Ile Leu Leu Pro Asp Asp Gln Val Val Gly Leu Gln
Val Arg 280 285 290cac ggt gac acg tgg atc act gtc aat cct ctc cgc
cac gct ttt atc 965His Gly Asp Thr Trp Ile Thr Val Asn Pro Leu Arg
His Ala Phe Ile295 300 305 310gtc aat atc ggc gat caa att cag ata
cta agc aat tcg aaa tac aag 1013Val Asn Ile Gly Asp Gln Ile Gln Ile
Leu Ser Asn Ser Lys Tyr Lys 315 320 325agc gtg gaa cat cga gtg ata
gtg aat tcg gaa aaa gaa agg gtt tca 1061Ser Val Glu His Arg Val Ile
Val Asn Ser Glu Lys Glu Arg Val Ser 330 335 340cta gca ttc ttc tat
aac cct aag agt gac att ccg atc caa cca atg 1109Leu Ala Phe Phe Tyr
Asn Pro Lys Ser Asp Ile Pro Ile Gln Pro Met 345 350 355caa caa ctt
gtc acc tct acg atg cct ccc ttg tat cct ccc atg acc 1157Gln Gln Leu
Val Thr Ser Thr Met Pro Pro Leu Tyr Pro Pro Met Thr 360 365 370ttt
gat caa tat aga ctc ttc att aga acg caa ggt cca cgt gga aaa 1205Phe
Asp Gln Tyr Arg Leu Phe Ile Arg Thr Gln Gly Pro Arg Gly Lys375 380
385 390tcc cac gtt gag tct cat ata tct cct cgt taa ttgatatcta
cttcataaaa 1258Ser His Val Glu Ser His Ile Ser Pro Arg 395
400tgtctgataa atagaataat tgataccgaa gctatggaag cttaaagaac
tacctagaga 1318ttccaaaaga ttggtgaaga atgaagatat atatacatat
atattgagat caaagtgaat 1378cgactaaaga ccgagaacta tcaaacattt
gtttaagtta aattactttg tagtcaccct 1438tgtagtgaat tatatatgtt
tataattagt tcttttaggt tctattgtat ttctattatg 1498caaaatcaat
atccgtgatt tatgttgaag catgtctatc aataattatg cctcctctat
1558ctctatgatt agtaacatt 157759400PRTArabidopsis thaliana 59Met Asn
Asn Leu Asp Glu Ile Lys Ile Glu Ser Lys Thr Cys Leu Asn1 5 10 15Asp
Gln Glu Gln Glu Val Lys Ile Asp Asn Met His Met Ser Asp Gln 20 25
30Asp Lys Asn Lys Ile Glu Ile Lys Asn Lys Ser Gly Leu Gly Glu Lys
35 40 45Trp Pro Glu Pro Ile Val Arg Val Gln Ser Leu Ala Glu Ser Asn
Leu 50 55 60Thr Ser Leu Pro Asp Arg Tyr Ile Lys Pro Pro Ser Gln Arg
Pro Gln65 70 75 80Thr Thr Ile Ile Asp His Gln Pro Glu Val Ala Asp
Ile Asn Ile Pro 85 90 95Ile Ile Asp Leu Asp Ser Leu Phe Ser Gly Asn
Glu Asp Asp Lys Lys 100 105 110Arg Ile Ser Glu Ala Cys Arg Glu Trp
Gly Phe Phe Gln Val Ile Asn 115 120 125His Gly Val Lys Pro Glu Leu
Met Asp Ala Ala Arg Glu Thr Trp Lys 130 135 140Ser Phe Phe Asn Leu
Pro Val Glu Ala Lys Glu Val Tyr Ser Asn Ser145 150 155 160Pro Arg
Thr Tyr Glu Gly Tyr Gly Ser Arg Leu Gly Val Glu Lys Gly 165 170
175Ala Ile Leu Asp Trp Asn Asp Tyr Tyr Tyr Leu His Phe Leu Pro Leu
180 185 190Ala Leu Lys Asp Phe Asn Lys Trp Pro Ser Leu Pro Ser Asn
Ile Arg 195 200 205Glu Met Asn Asp Glu Tyr Gly Lys Glu Leu Val Lys
Leu Gly Gly Arg 210 215 220Leu Met Thr Ile Leu Ser Ser Asn Leu Gly
Leu Arg Ala Glu Gln Leu225 230 235 240Gln Glu Ala Phe Gly Gly Glu
Asp Val Gly Ala Cys Leu Arg Val Asn 245 250 255Tyr Tyr Pro Lys Cys
Pro Gln Pro Glu Leu Ala Leu Gly Leu Ser Pro 260 265 270His Ser Asp
Pro Gly Gly Met Thr Ile Leu Leu Pro Asp Asp Gln Val 275 280 285Val
Gly Leu Gln Val Arg His Gly Asp Thr Trp Ile Thr Val Asn Pro 290 295
300Leu Arg His Ala Phe Ile Val Asn Ile Gly Asp Gln Ile Gln Ile
Leu305 310 315 320Ser Asn Ser Lys Tyr Lys Ser Val Glu His Arg Val
Ile Val Asn Ser 325 330 335Glu Lys Glu Arg Val Ser Leu Ala Phe Phe
Tyr Asn Pro Lys Ser Asp 340 345 350Ile Pro Ile Gln Pro Met Gln Gln
Leu Val Thr Ser Thr Met Pro Pro 355 360 365Leu Tyr Pro Pro Met Thr
Phe Asp Gln Tyr Arg Leu Phe Ile Arg Thr 370 375 380Gln Gly Pro Arg
Gly Lys Ser His Val Glu Ser His Ile Ser Pro Arg385 390 395
40060949DNAArabidopsis thalianapromoter(1)..(949)transcription
regulating sequence from gene At4g00360 60atcaattcac tctttaacac
ttctttatac cattgaagaa attagatgaa agagtcacga 60gttgcttacc aaatccctca
caagaattga gaactgataa accaaattga gaagattaaa 120tatcacgtct
ccttttgatc tctattataa ttaatcgaaa ataaaaataa gagttttcaa
180caaaacgtga tcattggttt acgatcactt gcaaagtcag acctaaaacg
tagcattagt 240acactaacct taaatattaa ttatatcatg caaaccctaa
tgtcattacc taactataca 300tgtgtaatgt gttcaacaga tcttcttaac
ccacattaga tcaatattaa acaataaaaa 360agattcttat atattctact
acttacttct tcttattccc atccatattt ttctgtgcct 420taaggttctc
aactaatctc atttaattta gctagcacac agagaaacac acacgtatat
480aaataatatg ataacacaca aaaagactca tatatataaa taattagagt
cattaaatgt 540ggattcatca ttaaatgaaa caactcttct tctctgtaca
atttctcttc acaccttcac 600caaattcttt gacttcaaaa atcttataaa
atttatatat ctccaaaacc ataaaaccaa 660aacgagtttt cacaaataaa
ttacttagtt gaaatttcaa atctcattca attagggtac 720actctctcaa
caatccacat taatgagggt tgctgcttct gatggctagc agtaacagtt
780ttatcgcctc cacttcttaa tgccatcttt ttcctcttcc ctctccttct
ctatatatat 840tttctgactc tgcaaaacct taattcatcc atctctcaaa
caccattttt ggaaacacca 900tttcacattc cttaaacttt tccattttag
tatcatttca tattcattg 94961962DNAArabidopsis
thalianapromoter(1)..(962)transcription regulating sequence from
gene At4g00360 61cttgtgggat taatcaattc actctttaac acttctttat
accattgaag aaattagatg 60aaagagtcac gagttgctta ccaaatccct cacaagaatt
gagaactgat aaaccaaatt 120gagaagatta aatatcacgt ctccttttga
tctctattat aattaatcga aaataaaaat 180aagagttttc aacaaaacgt
gatcattggt ttacgatcac ttgcaaagtc aaacctaaaa 240cgtagcatta
gtacactaac cttaaatact aattatatca tgcaaaccct aatgtcatta
300cctaactata catgtgtaat gtgttcaaca gatcttctta acccacatta
gatcaatatt 360aaacaataaa aagattctta tatattctac tacttacttc
ttcttattcc catccatatt 420tttctgtgcc tttaggttct caactaatct
catttaattt agctagcaca cagagaaaca 480cacacgtata taaataatat
gataacacac aaaaagactc atatatataa ataattagag 540tcattaaatg
tggattcatc attaaatgaa acaactcttc ttctctgtac aatttctctt
600cacaccttca ccaaattctt tgacttcaaa aatcttataa aatttatata
tctccaaaac 660cataaaacca aaacgagttt tcacaaataa attacttagt
tgaaatttca aatctcattc 720aattagggta cactctctca acaatccaca
ttaatgaggg ttgctgcttc tgatggctag 780cagtaacagt tttatcgcct
ccacttctta atgccatctt tttcctcttc cctctccttc 840tctatatata
ttttctgact ctgcaaaacc ttaattcatc catctctcaa acaccatttt
900tggaaacacc atttcatatt ccttaaactt ttccatttta gtatcatttc
atattcattg 960at 96262768DNAArabidopsis
thalianapromoter(1)..(768)transcription regulating sequence from
gene At4g00360 62atcaattcac tctttaacac ttctttatac cattgaagaa
attagatgaa agagtcacga 60gttgcttacc aaatccctca caagaattga gaactgataa
accaaattga gaagattaaa 120tatcacgtct ccttttgatc tctattataa
ttaatcgaaa ataaaaataa gagttttcaa 180caaaacgtga tcattggttt
acgatcactt gcaaagtcag acctaaaacg tagcattagt 240acactaacct
taaatattaa ttatatcatg caaaccctaa tgtcattacc taactataca
300tgtgtaatgt gttcaacaga tcttcttaac ccacattaga tcaatattaa
acaataaaaa 360agattcttat atattctact acttacttct tcttattccc
atccatattt ttctgtgcct 420taaggttctc aactaatctc atttaattta
gctagcacac agagaaacac acacgtatat 480aaataatatg ataacacaca
aaaagactca tatatataaa taattagagt cattaaatgt 540ggattcatca
ttaaatgaaa caactcttct tctctgtaca atttctcttc acaccttcac
600caaattcttt gacttcaaaa atcttataaa atttatatat ctccaaaacc
ataaaaccaa 660aacgagtttt cacaaataaa ttacttagtt gaaatttcaa
atctcattca attagggtac 720actctctcaa caatccacat taatgagggt
tgctgcttct gatggcta 76863779DNAArabidopsis
thalianapromoter(1)..(779)transcription regulating sequence from
gene At4g00360 63cttgtgggat taatcaattc actctttaac acttctttat
accattgaag aaattagatg 60aaagagtcac gagttgctta ccaaatccct cacaagaatt
gagaactgat aaaccaaatt 120gagaagatta aatatcacgt ctccttttga
tctctattat aattaatcga aaataaaaat 180aagagttttc aacaaaacgt
gatcattggt ttacgatcac ttgcaaagtc aaacctaaaa 240cgtagcatta
gtacactaac cttaaatact aattatatca tgcaaaccct aatgtcatta
300cctaactata catgtgtaat gtgttcaaca gatcttctta acccacatta
gatcaatatt 360aaacaataaa aagattctta tatattctac tacttacttc
ttcttattcc catccatatt 420tttctgtgcc tttaggttct caactaatct
catttaattt agctagcaca cagagaaaca 480cacacgtata taaataatat
gataacacac aaaaagactc atatatataa ataattagag 540tcattaaatg
tggattcatc attaaatgaa acaactcttc ttctctgtac aatttctctt
600cacaccttca ccaaattctt tgacttcaaa aatcttataa aatttatata
tctccaaaac 660cataaaacca aaacgagttt tcacaaataa attacttagt
tgaaatttca aatctcattc 720aattagggta cactctctca acaatccaca
ttaatgaggg ttgctgcttc tgatggcta 779642040DNAArabidopsis
thalianapromoter(1)..(2040)transcription regulating sequence from
gene At4g00360 64aaaaagagag tcaacattgc gttttaaatg cattttgagg
tttggcaaac tatctacaag 60ctatctgaca tctacagact acgtacagtt gtgttccatt
tgttacaatt acaacaatca 120gcttgtttgt ttttggcaaa ttaagagtca
tgagtgtgta tacgaaatta ctttttggtg 180gtctgaattt atgaatgcag
ttacactaac ccttcttcaa aaataaaaat gaatgcggtt 240atactaaact
atcaaatata tctactagca aatatgaaac tacattcagt aacagtgatc
300tctctttttg gatacaagat gaaacattgt tctcttctca tgctctaaat
agaaaatact 360ctacgaaatg aaaatgtatg tcatcttata agaactatac
atttttactc cgatggttaa 420ttctttgaag gattctcgac agtttagaag
tctgcaaaaa atataatctc ataagaaaaa 480aattctaagt ctcttgatac
aatgcatata gaaactgaca aataatcgaa gaaattgtac 540ttgagcccat
tacgactatt gaaaattctg attttggatg aattcagcgg aaactacaaa
600tttaagagta ctttatgtgt ataatatgaa gcctatatat atagaagtac
taatgtaata 660aaataagaac cggtgaaaag ggggacaaga tcacaaggtt
ttcgcattca gtgcctttac 720agagttatat atttgatgat gttattgctt
acttgcctaa tagtactata ctactatata 780tatctaaggt aacacatgta
tatatatgtc acatagacat tactagtata tattatgtac 840ttctatcata
tatttatgat attgcagttg cagcgtacac aagtcagctc cttttgactt
900ttacatctca tgaatgcatt gccatgacat ctaatcttac tcgagatttg
tgcatgcaca 960ttattcactt ttgtcttttg caatttttgt tattgtaaaa
aaaggaaaaa caaatgtaaa 1020agagagagag accagaaagg tctaactaaa
cctaaagagt caatgaaatg tgtttctttc 1080ttgtgggatt aatcaattca
ctctttaaca cttctttata ccattgaaga aattagatga 1140aagagtcacg
agttgcttac caaatccctc acaagaattg agaactgata aaccaaattg
1200agaagattaa atatcacgtc tccttttgat ctctattata attaatcgaa
aataaaaata 1260agagttttca acaaaacgtg atcattggtt tacgatcact
tgcaaagtca gacctaaaac 1320gtagcattag tacactaacc ttaaatatta
attatatcat gcaaacccta
gtgtcattac 1380ctaactatac atgtgtaatg tgttcaacag atcttcttaa
cccacattag atcaatatta 1440aacaataaaa aagattctta tatattctac
tacttacttc ttcttattcc catccatatt 1500tttctgtgcc ttaaggttct
caactaatct catttaattt agctagcaca cagagaaaca 1560cacacgtata
taaataatat gataacacac aaaaagactc atatatataa ataattagag
1620tcattaaatg tggattcatc attaaatgaa acaactcttc ttctctgtac
aatttctctt 1680cacaccttca ccaaattctt tgacttcaaa aatcttataa
aatttatata tctccaaaac 1740cataaaacca aaacgagttt tcacaaataa
attacttagt tgaaatttca aatctcattc 1800aattagggta cactctctca
acaatccaca ttaatgaggg ttgctgcttc tgatggctag 1860cagtaacagt
tttatcgcct ccacttctta atgccatctt tttcctcttc cctctccttc
1920tctatatata ttttctgact ctgcaaaacc ttaattcatc catctctcaa
acaccatttt 1980tggaaacacc atttcacatt ccttaaactt ttccatttta
gtatcatttc atattcattg 2040652053DNAArabidopsis
thalianapromoter(1)..(2053)transcription regulating sequence from
gene At4g00360 65taaaaaggat ttaaaaagag agtcaacatt gcgttttaaa
tgcattttga ggtttggcaa 60accatctaca agctatctga catctacaga ctacgtacag
ttgtgttcca tttgttacaa 120ttacaacaat cagcttgttt gtttttggca
aattaagagt catgagtgtg tatacgaaat 180tactttttgg tggtctgaat
ttatgaatgc agttacacta acccttcttc aaaaataaaa 240atgaatgcgg
ttatactaaa ctatcaaata tatctactag caaatatgaa actacattca
300gtaacagtga tctctctttt tggatacaag atgaaacatt gttctcttct
catgctctaa 360atagaaaata ctctacgaaa tgaaaatgta tgtcatctta
taagaactat acatttttac 420tccgatggtt aattctttga aggattctcg
acagtttaga agtctgcaaa aaatataatc 480tcataagaaa aaaattctaa
gtctcttgat acaatgcata tagaaactga caaataatcg 540aagaaattgt
acttgagccc attacgacta ttgaaaattc tgattttgga tgaattcagc
600ggaaactaca aatttaagag tactttatgt gtataatatg aagcctatat
atatagaatt 660actaatgtaa taaaataaga accggtgaaa agggggacaa
gatcacaagg ttttcgcatt 720cagtgccttt acagagttat atatttgatg
atgttattgc ttacttgcct aatagtacta 780tactactata tatatctaag
gtaacacatg tatatatatg tcacatagac attactagta 840tatattatgt
acttctatca tatatttatg atattgcagt tgcagcgtac acaagtcagc
900tccttttgac ttttacatct catgaatgca ttgccatgac atctaatctt
actcgagatt 960tgtgcatgca cattattcac ttttgtcttt tgcaattttt
gttattgtaa aaaaaggaaa 1020aacaaatgta aaagagagag agaccagaaa
ggtctaacta aacctaaaga gtcaatgaaa 1080tgtgtttctt tcttgtggga
ttaatcaatt cactctttaa cacttcttta taccattgaa 1140gaaattagat
gaaagagtca cgagttgctt accaaatccc tcacaagaat tgagaactga
1200taaaccaaat tgagaagatt aaatatcacg tctccttttg atctctatta
taattaatcg 1260aaaataaaaa taagagtttt caacaaaacg tgatcattgg
tttacgatca cttgcaaagt 1320caaacctaaa acgtagcatt agtacactaa
ccttaaatac taattatatc atgcaaaccc 1380taatgtcatt acctaactat
acatgtgtaa tgtgttcaac agatcttctt aacccacatt 1440agatcaatat
taaacaataa aaagattctt atatattcta ctacttactt cttcttattc
1500ccatccatat ttttctgtgc ctttaggttc tcaactaatc tcatttaatt
tagctagcac 1560acagagaaac acacacgtat ataaataata tgataacaca
caaaaagact catatatata 1620aataattaga gtcattaaat gtggattcat
cattaaatga aacaactctt cttctctgta 1680caatttctct tcacaccttc
accaaattct ttgacttcaa aaatcttata aaatttatat 1740atctccaaaa
ccataaaacc aaaacgagtt ttcacaaata aattacttag ttgaaatttc
1800aaatctcatt caattagggt acactctctc aacaatccac attaatgagg
gttgctgctt 1860ctgatggcta gcagtaacag ttttatcgcc tccacttctt
aatgccatct ttttcctctt 1920ccctctcctt ctctatatat attttctgac
tctgcaaaac cttaattcat ccatctctca 1980aacaccattt ttggaaacac
catttcatat tccttaaact tttccatttt agtatcattt 2040catattcatt gat
2053661859DNAArabidopsis thalianapromoter(1)..(1859)transcription
regulating sequence from gene At4g00360 66aaaaagagag tcaacattgc
gttttaaatg cattttgagg tttggcaaac tatctacaag 60ctatctgaca tctacagact
acgtacagtt gtgttccatt tgttacaatt acaacaatca 120gcttgtttgt
ttttggcaaa ttaagagtca tgagtgtgta tacgaaatta ctttttggtg
180gtctgaattt atgaatgcag ttacactaac ccttcttcaa aaataaaaat
gaatgcggtt 240atactaaact atcaaatata tctactagca aatatgaaac
tacattcagt aacagtgatc 300tctctttttg gatacaagat gaaacattgt
tctcttctca tgctctaaat agaaaatact 360ctacgaaatg aaaatgtatg
tcatcttata agaactatac atttttactc cgatggttaa 420ttctttgaag
gattctcgac agtttagaag tctgcaaaaa atataatctc ataagaaaaa
480aattctaagt ctcttgatac aatgcatata gaaactgaca aataatcgaa
gaaattgtac 540ttgagcccat tacgactatt gaaaattctg attttggatg
aattcagcgg aaactacaaa 600tttaagagta ctttatgtgt ataatatgaa
gcctatatat atagaagtac taatgtaata 660aaataagaac cggtgaaaag
ggggacaaga tcacaaggtt ttcgcattca gtgcctttac 720agagttatat
atttgatgat gttattgctt acttgcctaa tagtactata ctactatata
780tatctaaggt aacacatgta tatatatgtc acatagacat tactagtata
tattatgtac 840ttctatcata tatttatgat attgcagttg cagcgtacac
aagtcagctc cttttgactt 900ttacatctca tgaatgcatt gccatgacat
ctaatcttac tcgagatttg tgcatgcaca 960ttattcactt ttgtcttttg
caatttttgt tattgtaaaa aaaggaaaaa caaatgtaaa 1020agagagagag
accagaaagg tctaactaaa cctaaagagt caatgaaatg tgtttctttc
1080ttgtgggatt aatcaattca ctctttaaca cttctttata ccattgaaga
aattagatga 1140aagagtcacg agttgcttac caaatccctc acaagaattg
agaactgata aaccaaattg 1200agaagattaa atatcacgtc tccttttgat
ctctattata attaatcgaa aataaaaata 1260agagttttca acaaaacgtg
atcattggtt tacgatcact tgcaaagtca gacctaaaac 1320gtagcattag
tacactaacc ttaaatatta attatatcat gcaaacccta gtgtcattac
1380ctaactatac atgtgtaatg tgttcaacag atcttcttaa cccacattag
atcaatatta 1440aacaataaaa aagattctta tatattctac tacttacttc
ttcttattcc catccatatt 1500tttctgtgcc ttaaggttct caactaatct
catttaattt agctagcaca cagagaaaca 1560cacacgtata taaataatat
gataacacac aaaaagactc atatatataa ataattagag 1620tcattaaatg
tggattcatc attaaatgaa acaactcttc ttctctgtac aatttctctt
1680cacaccttca ccaaattctt tgacttcaaa aatcttataa aatttatata
tctccaaaac 1740cataaaacca aaacgagttt tcacaaataa attacttagt
tgaaatttca aatctcattc 1800aattagggta cactctctca acaatccaca
ttaatgaggg ttgctgcttc tgatggcta 1859671870DNAArabidopsis
thalianapromoter(1)..(1870)transcription regulating sequence from
gene At4g00360 67taaaaaggat ttaaaaagag agtcaacatt gcgttttaaa
tgcattttga ggtttggcaa 60accatctaca agctatctga catctacaga ctacgtacag
ttgtgttcca tttgttacaa 120ttacaacaat cagcttgttt gtttttggca
aattaagagt catgagtgtg tatacgaaat 180tactttttgg tggtctgaat
ttatgaatgc agttacacta acccttcttc aaaaataaaa 240atgaatgcgg
ttatactaaa ctatcaaata tatctactag caaatatgaa actacattca
300gtaacagtga tctctctttt tggatacaag atgaaacatt gttctcttct
catgctctaa 360atagaaaata ctctacgaaa tgaaaatgta tgtcatctta
taagaactat acatttttac 420tccgatggtt aattctttga aggattctcg
acagtttaga agtctgcaaa aaatataatc 480tcataagaaa aaaattctaa
gtctcttgat acaatgcata tagaaactga caaataatcg 540aagaaattgt
acttgagccc attacgacta ttgaaaattc tgattttgga tgaattcagc
600ggaaactaca aatttaagag tactttatgt gtataatatg aagcctatat
atatagaatt 660actaatgtaa taaaataaga accggtgaaa agggggacaa
gatcacaagg ttttcgcatt 720cagtgccttt acagagttat atatttgatg
atgttattgc ttacttgcct aatagtacta 780tactactata tatatctaag
gtaacacatg tatatatatg tcacatagac attactagta 840tatattatgt
acttctatca tatatttatg atattgcagt tgcagcgtac acaagtcagc
900tccttttgac ttttacatct catgaatgca ttgccatgac atctaatctt
actcgagatt 960tgtgcatgca cattattcac ttttgtcttt tgcaattttt
gttattgtaa aaaaaggaaa 1020aacaaatgta aaagagagag agaccagaaa
ggtctaacta aacctaaaga gtcaatgaaa 1080tgtgtttctt tcttgtggga
ttaatcaatt cactctttaa cacttcttta taccattgaa 1140gaaattagat
gaaagagtca cgagttgctt accaaatccc tcacaagaat tgagaactga
1200taaaccaaat tgagaagatt aaatatcacg tctccttttg atctctatta
taattaatcg 1260aaaataaaaa taagagtttt caacaaaacg tgatcattgg
tttacgatca cttgcaaagt 1320caaacctaaa acgtagcatt agtacactaa
ccttaaatac taattatatc atgcaaaccc 1380taatgtcatt acctaactat
acatgtgtaa tgtgttcaac agatcttctt aacccacatt 1440agatcaatat
taaacaataa aaagattctt atatattcta ctacttactt cttcttattc
1500ccatccatat ttttctgtgc ctttaggttc tcaactaatc tcatttaatt
tagctagcac 1560acagagaaac acacacgtat ataaataata tgataacaca
caaaaagact catatatata 1620aataattaga gtcattaaat gtggattcat
cattaaatga aacaactctt cttctctgta 1680caatttctct tcacaccttc
accaaattct ttgacttcaa aaatcttata aaatttatat 1740atctccaaaa
ccataaaacc aaaacgagtt ttcacaaata aattacttag ttgaaatttc
1800aaatctcatt caattagggt acactctctc aacaatccac attaatgagg
gttgctgctt 1860ctgatggcta 1870682996DNAArabidopsis
thalianapromoter(1)..(2996)transcription regulating sequence from
gene At4g00360 68agaaaaagaa tcaaattcct ccatgtcgtt ctgtatcgcc
tccatgtacg taaagaaagt 60tggtcactct tacggaaaga caccattttt accaagtctg
accaatttat aaggaaagct 120atttttcact tgccttgtag tctattaaat
actagggatg gttcagcatc cgcaaacagt 180aaatgagaaa ccgcatgact
tgcatttacc acatttatac taattattaa cttatctatt 240tatgctttct
gaatattagc aatcagcact tttgtgcata gtacatataa atataaggag
300agaggatctt cttgtcgtag gccgcattgg ggagtaatta agcctcttga
ttgaccgtta 360agtagcgcca tatgttagac tgatgtgata caccacatca
tccattgtat ttatgtctcc 420caacaatcca tttttcataa taaactctcg
ataaaatttg attcaatctg atcaaaagcc 480tttcatgtta gtttttataa
ccataaactt tcttttgtat gacgaatttg ttcagagacc 540ataaaacatc
tcttgcgtta taagaatatt atctaaaatc aatggtcttt cgataaagac
600tgattgcgtt tctaaaacaa aatttatttt tctatttttt tatttattta
attttccctc 660ttatttttag ttgtttttat aatatactaa gcgaggtagt
ataaatctag aatcctgaac 720cagcagtcaa atatcactat ttaaagctaa
ctatttaggt ttaataatac cttaggatat 780tattaccatt tacgtaaaaa
agtttgaacc tggaaaacat atatgtctac gtagagtacc 840gaatattcta
aaccatttaa aatattttat aatgatcaac catgattgaa atcaagtaga
900aaaacttgaa caatgatctt ctagttcatt ttagttgttt cacttaaaaa
ggatttaaaa 960agagagtcaa cattgcgttt taaatgcatt ttgaggtttg
gcaaaccatc tacaagctat 1020ctgacatcta cagactacgt acagttgtgt
tccatttgtt acaattacaa caatcagctt 1080gtttgttttt ggcaaattaa
gagtcatgag tgtgtatacg aaattacttt ttggtggtct 1140gaatttatga
atgcagttac actaaccctt cttcaaaaat aaaaatgaat gcggttatac
1200taaactatca aatatatcta ctagcaaata tgaaactaca ttcagtaaca
gtgatctctc 1260tttttggata caagatgaaa cattgttctc ttctcatgct
ctaaatagaa aatactctac 1320gaaatgaaaa tgtatgtcat cttataagaa
ctatacattt ttactccgat ggttaattct 1380ttgaaggatt ctcgacagtt
tagaagtctg caaaaaatat aatctcataa gaaaaaaatt 1440ctaagtctct
tgatacaatg catatagaaa ctgacaaata atcgaagaaa ttgtacttga
1500gcccattacg actattgaaa attctgattt tggatgaatt cagcggaaac
tacaaattta 1560agagtacttt atgtgtataa tatgaagcct atatatatag
aagtactaat gtaataaaat 1620aagaaccggt gaaaaggggg acaagatcac
aaggttttcg cattcagtgc ctatacagag 1680ttatatattt gatgatgtta
ttgcttactt gcctgatagt actatactac tatatatatc 1740taaggtaaca
catgtatata tatgtcacat agacattact agtatatatt atgtacttct
1800atcatatatt tatgatattg cagttgcagc gtacacaagt cagctccctt
tgacttttac 1860atctcatgaa tgcattgcca tgacatctaa tcttactcga
gatttgtgca tgcgcattat 1920tcacttttgt cttttgcaat ttttgttatt
gtaaaagaag gaaaaacaaa tgtaaaagag 1980agagagacca gaaaggtcta
actaaaccta aagagtcaat gaaatgtgtt tctttcttgt 2040gggattaatc
aattcactct ttaacacttc tttataccat tgaagaaatt agatgaaaga
2100gtcacgagtt gcttaccaaa tccctcacaa gaattgagaa ctgataaacc
aaattgagaa 2160gattaaatat cacgtctcct tttgatctct attataatta
atcgaaaata aaaataagag 2220ttttcaacaa aacgtgatca ttggtttacg
atcacttgca aagtcagacc taaaacgtag 2280cattagtaca ctaaccttaa
atattaatta tatcatgcaa accctaatgt cattacctaa 2340ctatacatgt
gtaatgtgtt caacagatct tcttaaccca cattagatca atattaaaca
2400ataaaaaaga ttcttatata ttctactact tacttcttct tattcccatc
catatttttc 2460tgtgccttaa ggttctcaac taatctcatt taatttagct
agcacacaga gaaacacaca 2520cgtatataaa taatatgata acacacaaaa
agactcatat atataaataa ttagagtcat 2580taaatgtgga ttcatcatta
aatgaaacaa ctcttcttct ctgtacaatt tctcttcaca 2640ccttcaccaa
attctttgac ttcaaaaatc ttataaaatt tatatatctc caaaaccata
2700aaaccaaaac gagttttcac aaataaatta cttagttgaa atttcaaatc
tcattcaatt 2760agggtacact ctctcaacaa tccacattaa tgagggttgc
tgcttctgat ggctagcagt 2820aacagtttta tcgcctccac ttcttaatgc
catctttttc ctcttccctc tccttctcta 2880tatatatttt ctgactctgc
aaaaccttaa ttcatccatc tctcaaacac catttttgga 2940aacaccattt
cacattcctt aaacttttcc attttagtat catttcatat tcattg
2996693008DNAArabidopsis thalianapromoter(1)..(3008)transcription
regulating sequence from gene At4g00360 69aactaaggat aaagaaaaag
aatcaaattc ctccatatcg ttctgtatcg cctccatgta 60cgtaaagaaa gttggtcact
cttacggaaa gacaccattt ttaccaggtc tgagcaattt 120ataaggaaag
ctatttttca cttgccttgt actctattaa atactaggga tgattcagca
180tccgcaaaca gtaaatgaga aaccgcatga cttgcattta ccacctttat
actaattatt 240aacttatcta tttatgcttt ctgaatatta gcaatcagca
cttttgtgca tagtacatat 300aaatatgagg agagaggatc ttcttgtcgt
aggccgcatt ggggagtaat taagcctctt 360gattgaccgt taagtagcgc
catatgttag actgatgtga tacaccacat catccattgt 420atttatgtct
cccaacaatc catttttcat aataaactct cgataaaatt tgattcaatc
480tgatcataag cctttcatgt tagtttttat aaccataaac tttcttttgt
atgacgaatt 540tgttcagaga ccataaaaca tctcttgcgt tataagaata
ttatctaaaa tcaatggtct 600ttcgataaag actaattgtg tttctaaaac
aaaatttatt tttctatttt ctatttattt 660aattttcctt ctcattttta
gttgttttta taatatacta agcgaggtag tataaatcta 720gaatcctgaa
ccagcagtca aatatcacta tttaaagcta actatttagg tttaataata
780ccttaggata ttattaccat ttacgtaaaa aagtttgaac ctggaaaaca
tatatatcta 840cgtagagtac cgaatattct aaaccattta aaatatttta
taatgatcaa ccatgattga 900aatcaagtag aaaaacttga acaatgatct
tctagttcat tttagttgtt tcacttaaaa 960aggatttaaa aagagagtca
acattgcgtt ttaaatgcat tttgaggttt ggcaaaccat 1020ctacaagcta
tctgacatct acagactacg tacagttgtg ttccatttgt tacaattaca
1080acaatcagct tgtttgtttt tggcaaatta agagtcatga gtgtgtatac
gaaattactt 1140tttggtggtc tgaatttatg aatgcagtta cactaaccct
tcttcaaaaa taaaaatgaa 1200tgcggttata ctaaactatc aaatatatct
actagcaaat atgaaactac attcagtaac 1260agtgatctct ctttttggat
acaagatgaa acattgttct cttctcatgc tctaaataga 1320aaatactcta
cgaaatgaaa atgtatgtca tcttataaga actatacatt tttactccga
1380tggttaattc tttgaaggat tctcgacagt ttagaagtct gcaaaaaata
taatctcata 1440agaaaaaaat tctaagtctc ttgatacaat gcatatagaa
actgacaaat aatcgaagaa 1500attgtacttg agcccattac gactattgaa
aattctgatt ttggatgaat tcagcggaaa 1560ctacaaattt aagagtactt
tatgtgtata atatgaagcc tatatatata gaattactaa 1620tgtaataaaa
taagaaccgg tgaaaagggg gacaagatca caaggttttc gcattcagtg
1680cctttacaga gttatatatt tgatgatgtt attgcttact tgcctaatag
tactatacta 1740ctatatatat ctaaggtaac acatgtatat atatgtcaca
tagacattac tagtatatat 1800tatgtacttc tatcatatat ttatgatatt
gcagttgcag cgtacacaag tcagctcctt 1860ttgactttta catctcatga
atgcattgcc atgacatcta atcttactcg agatttgtgc 1920atgcacatta
ttcacttttg tcttttgcaa tttttgttat tgtaaaaaaa ggaaaaacaa
1980atgtaaaaga gagagagacc agaaaggtct aactaaacct aaagagtcaa
tgaaatgtgt 2040ttctttcttg tgggattaat caattcactc tttaacactt
ctttatacca ttgaagaaat 2100tagatgaaag agtcacgagt tgcttaccaa
atccctcaca agaattgaga actgataaac 2160caaattgaga agattaaata
tcacgtctcc ttttgatctc tattataatt aatcgaaaat 2220aaaaataaga
gttttcaaca aaacgtgatc attggtttac gatcacttgc aaagtcaaac
2280ctaaaacgta gcattagtac actaacctta aatactaatt atatcatgca
aaccctaatg 2340tcattaccta actatacatg tgtaatgtgt tcaacagatc
ttcttaaccc acattagatc 2400aatattaaac aataaaaaga ttcttatata
ttctactact tacttcttct tattcccatc 2460catatttttc tgtgccttta
ggttctcaac taatctcatt taatttagct agcacacaga 2520gaaacacaca
cgtatataaa taatatgata acacacaaaa agactcatat atataaataa
2580ttagagtcat taaatgtgga ttcatcatta aatgaaacaa ctcttcttct
ctgtacaatt 2640tctcttcaca ccttcaccaa attctttgac ttcaaaaatc
ttataaaatt tatatatctc 2700caaaaccata aaaccaaaac gagttttcac
aaataaatta cttagttgaa atttcaaatc 2760tcattcaatt agggtacact
ctctcaacaa tccacattaa tgagggttgc tgcttctgat 2820ggctagcagt
aacagtttta tcgcctccac ttcttaatgc catctttttc ctcttccctc
2880tccttctcta tatatatttt ctgactctgc aaaaccttaa ttcatccatc
tctcaaacac 2940catttttgga aacaccattt catattcctt aaacttttcc
attttagtat catttcatat 3000tcattgat 3008702815DNAArabidopsis
thalianapromoter(1)..(2815)transcription regulating sequence from
gene At4g00360 70agaaaaagaa tcaaattcct ccatgtcgtt ctgtatcgcc
tccatgtacg taaagaaagt 60tggtcactct tacggaaaga caccattttt accaagtctg
accaatttat aaggaaagct 120atttttcact tgccttgtag tctattaaat
actagggatg gttcagcatc cgcaaacagt 180aaatgagaaa ccgcatgact
tgcatttacc acatttatac taattattaa cttatctatt 240tatgctttct
gaatattagc aatcagcact tttgtgcata gtacatataa atataaggag
300agaggatctt cttgtcgtag gccgcattgg ggagtaatta agcctcttga
ttgaccgtta 360agtagcgcca tatgttagac tgatgtgata caccacatca
tccattgtat ttatgtctcc 420caacaatcca tttttcataa taaactctcg
ataaaatttg attcaatctg atcaaaagcc 480tttcatgtta gtttttataa
ccataaactt tcttttgtat gacgaatttg ttcagagacc 540ataaaacatc
tcttgcgtta taagaatatt atctaaaatc aatggtcttt cgataaagac
600tgattgcgtt tctaaaacaa aatttatttt tctatttttt tatttattta
attttccctc 660ttatttttag ttgtttttat aatatactaa gcgaggtagt
ataaatctag aatcctgaac 720cagcagtcaa atatcactat ttaaagctaa
ctatttaggt ttaataatac cttaggatat 780tattaccatt tacgtaaaaa
agtttgaacc tggaaaacat atatgtctac gtagagtacc 840gaatattcta
aaccatttaa aatattttat aatgatcaac catgattgaa atcaagtaga
900aaaacttgaa caatgatctt ctagttcatt ttagttgttt cacttaaaaa
ggatttaaaa 960agagagtcaa cattgcgttt taaatgcatt ttgaggtttg
gcaaaccatc tacaagctat 1020ctgacatcta cagactacgt acagttgtgt
tccatttgtt acaattacaa caatcagctt 1080gtttgttttt ggcaaattaa
gagtcatgag tgtgtatacg aaattacttt ttggtggtct 1140gaatttatga
atgcagttac actaaccctt cttcaaaaat aaaaatgaat gcggttatac
1200taaactatca aatatatcta ctagcaaata tgaaactaca ttcagtaaca
gtgatctctc 1260tttttggata caagatgaaa cattgttctc ttctcatgct
ctaaatagaa aatactctac 1320gaaatgaaaa tgtatgtcat cttataagaa
ctatacattt ttactccgat ggttaattct 1380ttgaaggatt ctcgacagtt
tagaagtctg caaaaaatat aatctcataa gaaaaaaatt 1440ctaagtctct
tgatacaatg catatagaaa ctgacaaata atcgaagaaa ttgtacttga
1500gcccattacg actattgaaa attctgattt tggatgaatt cagcggaaac
tacaaattta 1560agagtacttt atgtgtataa tatgaagcct atatatatag
aagtactaat gtaataaaat 1620aagaaccggt gaaaaggggg acaagatcac
aaggttttcg cattcagtgc ctatacagag 1680ttatatattt gatgatgtta
ttgcttactt gcctgatagt actatactac
tatatatatc 1740taaggtaaca catgtatata tatgtcacat agacattact
agtatatatt atgtacttct 1800atcatatatt tatgatattg cagttgcagc
gtacacaagt cagctccctt tgacttttac 1860atctcatgaa tgcattgcca
tgacatctaa tcttactcga gatttgtgca tgcgcattat 1920tcacttttgt
cttttgcaat ttttgttatt gtaaaagaag gaaaaacaaa tgtaaaagag
1980agagagacca gaaaggtcta actaaaccta aagagtcaat gaaatgtgtt
tctttcttgt 2040gggattaatc aattcactct ttaacacttc tttataccat
tgaagaaatt agatgaaaga 2100gtcacgagtt gcttaccaaa tccctcacaa
gaattgagaa ctgataaacc aaattgagaa 2160gattaaatat cacgtctcct
tttgatctct attataatta atcgaaaata aaaataagag 2220ttttcaacaa
aacgtgatca ttggtttacg atcacttgca aagtcagacc taaaacgtag
2280cattagtaca ctaaccttaa atattaatta tatcatgcaa accctaatgt
cattacctaa 2340ctatacatgt gtaatgtgtt caacagatct tcttaaccca
cattagatca atattaaaca 2400ataaaaaaga ttcttatata ttctactact
tacttcttct tattcccatc catatttttc 2460tgtgccttaa ggttctcaac
taatctcatt taatttagct agcacacaga gaaacacaca 2520cgtatataaa
taatatgata acacacaaaa agactcatat atataaataa ttagagtcat
2580taaatgtgga ttcatcatta aatgaaacaa ctcttcttct ctgtacaatt
tctcttcaca 2640ccttcaccaa attctttgac ttcaaaaatc ttataaaatt
tatatatctc caaaaccata 2700aaaccaaaac gagttttcac aaataaatta
cttagttgaa atttcaaatc tcattcaatt 2760agggtacact ctctcaacaa
tccacattaa tgagggttgc tgcttctgat ggcta 2815712825DNAArabidopsis
thalianapromoter(1)..(2825)transcription regulating sequence from
gene At4g00360 71aactaaggat aaagaaaaag aatcaaattc ctccatatcg
ttctgtatcg cctccatgta 60cgtaaagaaa gttggtcact cttacggaaa gacaccattt
ttaccaggtc tgagcaattt 120ataaggaaag ctatttttca cttgccttgt
actctattaa atactaggga tgattcagca 180tccgcaaaca gtaaatgaga
aaccgcatga cttgcattta ccacctttat actaattatt 240aacttatcta
tttatgcttt ctgaatatta gcaatcagca cttttgtgca tagtacatat
300aaatatgagg agagaggatc ttcttgtcgt aggccgcatt ggggagtaat
taagcctctt 360gattgaccgt taagtagcgc catatgttag actgatgtga
tacaccacat catccattgt 420atttatgtct cccaacaatc catttttcat
aataaactct cgataaaatt tgattcaatc 480tgatcataag cctttcatgt
tagtttttat aaccataaac tttcttttgt atgacgaatt 540tgttcagaga
ccataaaaca tctcttgcgt tataagaata ttatctaaaa tcaatggtct
600ttcgataaag actaattgtg tttctaaaac aaaatttatt tttctatttt
ctatttattt 660aattttcctt ctcattttta gttgttttta taatatacta
agcgaggtag tataaatcta 720gaatcctgaa ccagcagtca aatatcacta
tttaaagcta actatttagg tttaataata 780ccttaggata ttattaccat
ttacgtaaaa aagtttgaac ctggaaaaca tatatatcta 840cgtagagtac
cgaatattct aaaccattta aaatatttta taatgatcaa ccatgattga
900aatcaagtag aaaaacttga acaatgatct tctagttcat tttagttgtt
tcacttaaaa 960aggatttaaa aagagagtca acattgcgtt ttaaatgcat
tttgaggttt ggcaaaccat 1020ctacaagcta tctgacatct acagactacg
tacagttgtg ttccatttgt tacaattaca 1080acaatcagct tgtttgtttt
tggcaaatta agagtcatga gtgtgtatac gaaattactt 1140tttggtggtc
tgaatttatg aatgcagtta cactaaccct tcttcaaaaa taaaaatgaa
1200tgcggttata ctaaactatc aaatatatct actagcaaat atgaaactac
attcagtaac 1260agtgatctct ctttttggat acaagatgaa acattgttct
cttctcatgc tctaaataga 1320aaatactcta cgaaatgaaa atgtatgtca
tcttataaga actatacatt tttactccga 1380tggttaattc tttgaaggat
tctcgacagt ttagaagtct gcaaaaaata taatctcata 1440agaaaaaaat
tctaagtctc ttgatacaat gcatatagaa actgacaaat aatcgaagaa
1500attgtacttg agcccattac gactattgaa aattctgatt ttggatgaat
tcagcggaaa 1560ctacaaattt aagagtactt tatgtgtata atatgaagcc
tatatatata gaattactaa 1620tgtaataaaa taagaaccgg tgaaaagggg
gacaagatca caaggttttc gcattcagtg 1680cctttacaga gttatatatt
tgatgatgtt attgcttact tgcctaatag tactatacta 1740ctatatatat
ctaaggtaac acatgtatat atatgtcaca tagacattac tagtatatat
1800tatgtacttc tatcatatat ttatgatatt gcagttgcag cgtacacaag
tcagctcctt 1860ttgactttta catctcatga atgcattgcc atgacatcta
atcttactcg agatttgtgc 1920atgcacatta ttcacttttg tcttttgcaa
tttttgttat tgtaaaaaaa ggaaaaacaa 1980atgtaaaaga gagagagacc
agaaaggtct aactaaacct aaagagtcaa tgaaatgtgt 2040ttctttcttg
tgggattaat caattcactc tttaacactt ctttatacca ttgaagaaat
2100tagatgaaag agtcacgagt tgcttaccaa atccctcaca agaattgaga
actgataaac 2160caaattgaga agattaaata tcacgtctcc ttttgatctc
tattataatt aatcgaaaat 2220aaaaataaga gttttcaaca aaacgtgatc
attggtttac gatcacttgc aaagtcaaac 2280ctaaaacgta gcattagtac
actaacctta aatactaatt atatcatgca aaccctaatg 2340tcattaccta
actatacatg tgtaatgtgt tcaacagatc ttcttaaccc acattagatc
2400aatattaaac aataaaaaga ttcttatata ttctactact tacttcttct
tattcccatc 2460catatttttc tgtgccttta ggttctcaac taatctcatt
taatttagct agcacacaga 2520gaaacacaca cgtatataaa taatatgata
acacacaaaa agactcatat atataaataa 2580ttagagtcat taaatgtgga
ttcatcatta aatgaaacaa ctcttcttct ctgtacaatt 2640tctcttcaca
ccttcaccaa attctttgac ttcaaaaatc ttataaaatt tatatatctc
2700caaaaccata aaaccaaaac gagttttcac aaataaatta cttagttgaa
atttcaaatc 2760tcattcaatt agggtacact ctctcaacaa tccacattaa
tgagggttgc tgcttctgat 2820ggcta 2825722069DNAArabidopsis
thalianaCDS(184)..(1845)encoding putative cytochrome P450
72gcagtaacag ttttatcgcc tccacttctt aatgccatct ttttcctctt ccctctcctt
60ctctatatat attttctgac tctgcaaaac cttaattcat ccatctctca aacaccattt
120ttggaaacac catttcatat tccttaaact tttccatttt agtatcattt
catattcatt 180gat atg gat gtc tcc aac acg atg ctc ctt gta gct gtt
gtt gca gct 228 Met Asp Val Ser Asn Thr Met Leu Leu Val Ala Val Val
Ala Ala 1 5 10 15tac tgg cta tgg ttc cag cgg atc tca agg tgg cta
aag ggt cca cgt 276Tyr Trp Leu Trp Phe Gln Arg Ile Ser Arg Trp Leu
Lys Gly Pro Arg 20 25 30gtt tgg cca gtt ttg ggc agt ctt ccg ggt ctg
atc gag cag cgt gac 324Val Trp Pro Val Leu Gly Ser Leu Pro Gly Leu
Ile Glu Gln Arg Asp 35 40 45cgt atg cac gac tgg atc act gag aac ctc
cgt gcg tgt ggc ggc act 372Arg Met His Asp Trp Ile Thr Glu Asn Leu
Arg Ala Cys Gly Gly Thr 50 55 60tat cag aca tgt atc tgc gcc gta cct
ttc ttg gca aaa aag caa ggt 420Tyr Gln Thr Cys Ile Cys Ala Val Pro
Phe Leu Ala Lys Lys Gln Gly 65 70 75ctc gtg acc gtc acg tgc gat ccc
aag aac atc gaa cac atg ctc aag 468Leu Val Thr Val Thr Cys Asp Pro
Lys Asn Ile Glu His Met Leu Lys80 85 90 95acc agg ttc gac aac tac
cct aaa ggt cct acg tgg caa gcc gtg ttc 516Thr Arg Phe Asp Asn Tyr
Pro Lys Gly Pro Thr Trp Gln Ala Val Phe 100 105 110cat gac ttc ctc
ggc caa ggt atc ttc aac tcc gac ggt gac act tgg 564His Asp Phe Leu
Gly Gln Gly Ile Phe Asn Ser Asp Gly Asp Thr Trp 115 120 125ctc ttc
cag cgt aaa acc gcc gct ctt gaa ttc acc acc agg acg ttg 612Leu Phe
Gln Arg Lys Thr Ala Ala Leu Glu Phe Thr Thr Arg Thr Leu 130 135
140agg caa gcg atg ggt cgg tgg gtg aac cgg gga atc aag ctc cgg ttt
660Arg Gln Ala Met Gly Arg Trp Val Asn Arg Gly Ile Lys Leu Arg Phe
145 150 155tgt cca att ctc gaa acg gct cag aac aat tac gag ccg gtt
gat ctc 708Cys Pro Ile Leu Glu Thr Ala Gln Asn Asn Tyr Glu Pro Val
Asp Leu160 165 170 175caa gac tta ata cta cgg ctc aca ttc gac aac
att tgc ggt tta gca 756Gln Asp Leu Ile Leu Arg Leu Thr Phe Asp Asn
Ile Cys Gly Leu Ala 180 185 190ttc ggt aaa gac act cga acc tgc gca
ccg gga ctt ccc gag aac ggt 804Phe Gly Lys Asp Thr Arg Thr Cys Ala
Pro Gly Leu Pro Glu Asn Gly 195 200 205ttc gcc tcg gct ttc gac cga
gcc acc gaa gct tct ctc cag cgg ttt 852Phe Ala Ser Ala Phe Asp Arg
Ala Thr Glu Ala Ser Leu Gln Arg Phe 210 215 220att cta cca gag ttt
cta tgg agg ctg aag aaa tgg ctt gga ctc ggc 900Ile Leu Pro Glu Phe
Leu Trp Arg Leu Lys Lys Trp Leu Gly Leu Gly 225 230 235tta gaa gtc
agc ttg agc cgg agc cta gga gag atc gat ggg tat tta 948Leu Glu Val
Ser Leu Ser Arg Ser Leu Gly Glu Ile Asp Gly Tyr Leu240 245 250
255gat gct gtc att aat aca cgt aag caa gaa ttg ctg agt cag cga gag
996Asp Ala Val Ile Asn Thr Arg Lys Gln Glu Leu Leu Ser Gln Arg Glu
260 265 270agt ggg gtc cag cgt cac gac gat ctc ctc tct cgt ttc atg
aag aag 1044Ser Gly Val Gln Arg His Asp Asp Leu Leu Ser Arg Phe Met
Lys Lys 275 280 285aaa gac cag tcg tac agc gag acg ttc tta cga cac
gtg gcg ctt aac 1092Lys Asp Gln Ser Tyr Ser Glu Thr Phe Leu Arg His
Val Ala Leu Asn 290 295 300ttc atc cta gct gga cgt gac acg tca tca
gta gcg ttg agc tgg ttt 1140Phe Ile Leu Ala Gly Arg Asp Thr Ser Ser
Val Ala Leu Ser Trp Phe 305 310 315ttc tgg ctc atc acg acg cat ccg
acg gtt gag gat aag atc gtc cgc 1188Phe Trp Leu Ile Thr Thr His Pro
Thr Val Glu Asp Lys Ile Val Arg320 325 330 335gag ata tgc tcc gtt
ctg att gag aca cgt gga acc gat gta tcg tcg 1236Glu Ile Cys Ser Val
Leu Ile Glu Thr Arg Gly Thr Asp Val Ser Ser 340 345 350tgg acg gcg
gag ccg ttg gaa ttc gat gag gtc gac cgg ttg gtt tac 1284Trp Thr Ala
Glu Pro Leu Glu Phe Asp Glu Val Asp Arg Leu Val Tyr 355 360 365ctg
aag gcc gcg ctc tct gag acg ttg agg ctt tac ccg tcg gtg ccg 1332Leu
Lys Ala Ala Leu Ser Glu Thr Leu Arg Leu Tyr Pro Ser Val Pro 370 375
380gaa gat tca aag cac gtc gtg aac gac gat atc tta ccg gac gga act
1380Glu Asp Ser Lys His Val Val Asn Asp Asp Ile Leu Pro Asp Gly Thr
385 390 395ttc gta ccg gcg gga tcg tcg gtg act tat tcg atc tac gcg
gcg ggg 1428Phe Val Pro Ala Gly Ser Ser Val Thr Tyr Ser Ile Tyr Ala
Ala Gly400 405 410 415agg atg aag agc acg tgg gga gag gat tgc ttg
gaa ttc aaa ccg gag 1476Arg Met Lys Ser Thr Trp Gly Glu Asp Cys Leu
Glu Phe Lys Pro Glu 420 425 430agg tgg atc tcg ccg gac gat gga aaa
ttc gtg aat cac gat cag tac 1524Arg Trp Ile Ser Pro Asp Asp Gly Lys
Phe Val Asn His Asp Gln Tyr 435 440 445cga ttc gtg gcg ttt aac gcc
gga cct agg atc tgt cta gga aaa gat 1572Arg Phe Val Ala Phe Asn Ala
Gly Pro Arg Ile Cys Leu Gly Lys Asp 450 455 460cta gcg tat ctg cag
atg aag acg atc gcg gcg gcg gtt tta ctc agg 1620Leu Ala Tyr Leu Gln
Met Lys Thr Ile Ala Ala Ala Val Leu Leu Arg 465 470 475cat aga cta
acg gtg gcg ccg gga cac aag gtg gag cag aag atg tcg 1668His Arg Leu
Thr Val Ala Pro Gly His Lys Val Glu Gln Lys Met Ser480 485 490
495ttg act ttg ttc atg aag aac gga ctt ttg gtc aac gtg cac aag agg
1716Leu Thr Leu Phe Met Lys Asn Gly Leu Leu Val Asn Val His Lys Arg
500 505 510gat ttg gaa gtg atg atg aag agt ctc gta ccc aaa gag aga
aac gac 1764Asp Leu Glu Val Met Met Lys Ser Leu Val Pro Lys Glu Arg
Asn Asp 515 520 525gtc gtt gtt ctt aac gga aaa tgc aac ggc ggt atc
ggt gaa ggc gtc 1812Val Val Val Leu Asn Gly Lys Cys Asn Gly Gly Ile
Gly Glu Gly Val 530 535 540gcc gtt aat gcg gct gtg gcg gtt gcc gtg
taa tgcagcttct cgggttgata 1865Ala Val Asn Ala Ala Val Ala Val Ala
Val 545 550acatttttat agtaattaat acatacattt acctttgggt tttgtgattg
ttgtgtaaaa 1925cagtaaaaaa aatgatttcg ttgtgctatg taaactttgt
aagcaaatac tcttcttcct 1985aaagtaaaaa agtttgattg tttttgtttt
ggtttctttt tctccatccc catcaaattg 2045taagcttcaa aagagtcaag ttcc
206973553PRTArabidopsis thaliana 73Met Asp Val Ser Asn Thr Met Leu
Leu Val Ala Val Val Ala Ala Tyr1 5 10 15Trp Leu Trp Phe Gln Arg Ile
Ser Arg Trp Leu Lys Gly Pro Arg Val 20 25 30Trp Pro Val Leu Gly Ser
Leu Pro Gly Leu Ile Glu Gln Arg Asp Arg 35 40 45Met His Asp Trp Ile
Thr Glu Asn Leu Arg Ala Cys Gly Gly Thr Tyr 50 55 60Gln Thr Cys Ile
Cys Ala Val Pro Phe Leu Ala Lys Lys Gln Gly Leu65 70 75 80Val Thr
Val Thr Cys Asp Pro Lys Asn Ile Glu His Met Leu Lys Thr 85 90 95Arg
Phe Asp Asn Tyr Pro Lys Gly Pro Thr Trp Gln Ala Val Phe His 100 105
110Asp Phe Leu Gly Gln Gly Ile Phe Asn Ser Asp Gly Asp Thr Trp Leu
115 120 125Phe Gln Arg Lys Thr Ala Ala Leu Glu Phe Thr Thr Arg Thr
Leu Arg 130 135 140Gln Ala Met Gly Arg Trp Val Asn Arg Gly Ile Lys
Leu Arg Phe Cys145 150 155 160Pro Ile Leu Glu Thr Ala Gln Asn Asn
Tyr Glu Pro Val Asp Leu Gln 165 170 175Asp Leu Ile Leu Arg Leu Thr
Phe Asp Asn Ile Cys Gly Leu Ala Phe 180 185 190Gly Lys Asp Thr Arg
Thr Cys Ala Pro Gly Leu Pro Glu Asn Gly Phe 195 200 205Ala Ser Ala
Phe Asp Arg Ala Thr Glu Ala Ser Leu Gln Arg Phe Ile 210 215 220Leu
Pro Glu Phe Leu Trp Arg Leu Lys Lys Trp Leu Gly Leu Gly Leu225 230
235 240Glu Val Ser Leu Ser Arg Ser Leu Gly Glu Ile Asp Gly Tyr Leu
Asp 245 250 255Ala Val Ile Asn Thr Arg Lys Gln Glu Leu Leu Ser Gln
Arg Glu Ser 260 265 270Gly Val Gln Arg His Asp Asp Leu Leu Ser Arg
Phe Met Lys Lys Lys 275 280 285Asp Gln Ser Tyr Ser Glu Thr Phe Leu
Arg His Val Ala Leu Asn Phe 290 295 300Ile Leu Ala Gly Arg Asp Thr
Ser Ser Val Ala Leu Ser Trp Phe Phe305 310 315 320Trp Leu Ile Thr
Thr His Pro Thr Val Glu Asp Lys Ile Val Arg Glu 325 330 335Ile Cys
Ser Val Leu Ile Glu Thr Arg Gly Thr Asp Val Ser Ser Trp 340 345
350Thr Ala Glu Pro Leu Glu Phe Asp Glu Val Asp Arg Leu Val Tyr Leu
355 360 365Lys Ala Ala Leu Ser Glu Thr Leu Arg Leu Tyr Pro Ser Val
Pro Glu 370 375 380Asp Ser Lys His Val Val Asn Asp Asp Ile Leu Pro
Asp Gly Thr Phe385 390 395 400Val Pro Ala Gly Ser Ser Val Thr Tyr
Ser Ile Tyr Ala Ala Gly Arg 405 410 415Met Lys Ser Thr Trp Gly Glu
Asp Cys Leu Glu Phe Lys Pro Glu Arg 420 425 430Trp Ile Ser Pro Asp
Asp Gly Lys Phe Val Asn His Asp Gln Tyr Arg 435 440 445Phe Val Ala
Phe Asn Ala Gly Pro Arg Ile Cys Leu Gly Lys Asp Leu 450 455 460Ala
Tyr Leu Gln Met Lys Thr Ile Ala Ala Ala Val Leu Leu Arg His465 470
475 480Arg Leu Thr Val Ala Pro Gly His Lys Val Glu Gln Lys Met Ser
Leu 485 490 495Thr Leu Phe Met Lys Asn Gly Leu Leu Val Asn Val His
Lys Arg Asp 500 505 510Leu Glu Val Met Met Lys Ser Leu Val Pro Lys
Glu Arg Asn Asp Val 515 520 525Val Val Leu Asn Gly Lys Cys Asn Gly
Gly Ile Gly Glu Gly Val Ala 530 535 540Val Asn Ala Ala Val Ala Val
Ala Val545 550741564DNAArabidopsis
thalianapromoter(1)..(1564)transcription regulating sequence from
gene At2g48030 74aaaccagtct ctcacactaa agaaacactt tgtcatattt
gtacatactg agcggaatat 60tctgagggat ttgttttgtt ttcaatcagc ttgtagttga
ttattgtagt tgcgacataa 120tgaaaagagg agaaatgaga gatgggttca
aagaagcatt ccacattcag aagtccaatc 180tcctaaaaga ctaaaccagc
caaaatgaaa agaggagaca gagccacaca tccgagacga 240tgggccgaaa
cacacatagt ataattgaat agtgggccgt ggcccaatat taaataatca
300tcccttgatt attgattatc tatgacctca taaacgataa taattactaa
ttagtagtct 360acgaatgaga aatataataa aatagtcgtt ttctagtgtg
tttgttaagt tgttatagat 420caatatacaa gtatcattca ttggaagaca
acaacatgta tatatctgag tgggaatgtg 480gcagaaaaag gatttggata
aaataagtac acgaagaaaa aagacagagt atgtaatgcc 540gttttggtag
gatggaaaca gcatgcagtt gttttggcaa ctgtattgta gtattagtat
600tattagaaaa tccagctaag agattattag actttaccca tatctaaata
agtcatcctt 660ccttaccttt ttcatataca aaacaattaa cacttgacct
aacttgcaac gctctctctc 720gattccatcc ttttttgttc ttttcttttt
gacacttttt ttttctttct ttcttttctt 780aaatagatta ttattcaatc
tttcctcctc catataataa taaaagtgat tgtgactgat 840atcaaattcc
ttatttaaat accacaatca cacagataca acatattact aattttaaat
900tacagagcag ttggtccaca tatgcttgaa attgatagtc taattagttt
taatgaggag 960gtccatgtca aataggcctg acccaccagc tatatatata
ccatgcatta aacctatata 1020taaaaataaa atctctataa ctttttttgg
gagttgttaa tttccttttc gttaattata 1080gaatatgctc cctttctctt
ctctgacgat ggttcataaa cctattcctt cgtagttgtt 1140taacgtccat
aataattgta tacttttagt tcgagattta cctaaacaaa acaaaaacag
1200aataccatac tgtatttcat gatttctgta ttggaaaata aaagactgaa
accaaatgat 1260tggtgggctt ttctaaaata atagtaataa aatacaatta
agacacacaa aaaagaaaag 1320gatttggtac atttattaac atcgatgatg
agttgataat gccacgtaag ccaaatacca 1380ttttcccaag aagctcatat
gaccttcttt tttgtgcagc aaactccaca ttccacaact 1440ctctctctct
ctcatttctt cgtataaaag gaaatctata tcatctctct ctctcattgt
1500caaaactcaa aagtatatac ttttactagt tttagactta ataaatgcga
tcgccctttt 1560ttta 1564751578DNAArabidopsis
thalianapromoter(1)..(1578)transcription regulating sequence from
gene At2g48030 75tgcttaggtc caaaaccagt ctctcacact aaagaaacac
tttgtcatat ttgtacatac 60tgagcggaat attctgaggg atttgttttg ttttcaatca
gcttgtagtt gattattgta 120gttgcgacat aatgaaaaga ggagaaatga
gagatgggtt caaagaagca ttccacattc 180agaagtccaa tctcctaaaa
gactaaacca gccaaaatga aaagaggaga cagagccaca 240catccgagac
gatgggccga aacacacata gtataattga atgtgggccg tggcccaata
300ttaaataatc atcccttgat tattgattat ctatgacctc ataaacgata
ataattacta 360attagtactc tacgaatgag aaatataata aaatagtcgt
tttctagtgt gtttgttaag 420ttgttataga tcaatataca agtatcattc
attggaagac aacaacatgt atatatctga 480gtgggaatgt ggcagaaaaa
ggatttggat aaaataagta cacgaagaaa aaagacagag 540tatgtaatgc
cgttttggta ggatggaaac agcatgcagt tgttttggca actgtattgt
600agtattagta ttattagaaa atccagctaa gagattatat tagactttac
ccatatctaa 660ataagtcatc cttccttacc tttttcatat acaaaacaat
taacacttga cctaacttgc 720aacgctctct ctcgattcca tccttttttg
ttcttttctt tttgacactt ttttttttct 780ttctttcttt tcttaaatag
attattattc aatctttcct cctccatata ataataaaag 840tgattgtgac
tgatatcaaa ttccttattt aaataccaca atcacacaga tacaacatat
900tactaatttt aaattacaga gcagttggtc cacatatgct tgaaattgat
agtctaatta 960gttttaatga ggaggtccat gtcaaatagg cctgacccac
tagctatata tataccatgc 1020attaaaccta tatataaaaa taaaatctct
ataacttttt ttgggagttg ttaatttcct 1080tttcgttaat tatagaatat
gctccctttc tcttctctga cgatggttca taaacctatt 1140ccttcgtagt
tgtttaacgt ccataataat tgtatacttt tagttcgaga tttacctaaa
1200caaaacaaaa acagaatacc atactgtatt tcatgatttc tgtattggaa
aataaaagac 1260tgaaaccaaa tgattggtgg gcttttctaa aataatagta
ataaaataca attaagacac 1320acaaaaaaga aaaggatttg gtacatttat
taacatcgat gatgagttga taatgccacg 1380taagccaaat accattttcc
caagaagctc atatgacctt cttttttgtg cagcaaactc 1440cacattccac
aactctctct ctctcatttc ttcgtataaa aggaaatcta tatcatctct
1500ctctctcatt gtcaaaactc aaaagtatat acttttacta gttttagact
taataaatgc 1560gatcgccctt tttttagt 1578761404DNAArabidopsis
thalianapromoter(1)..(1404)transcription regulating sequence from
gene At2g48030 76aaaccagtct ctcacactaa agaaacactt tgtcatattt
gtacatactg agcggaatat 60tctgagggat ttgttttgtt ttcaatcagc ttgtagttga
ttattgtagt tgcgacataa 120tgaaaagagg agaaatgaga gatgggttca
aagaagcatt ccacattcag aagtccaatc 180tcctaaaaga ctaaaccagc
caaaatgaaa agaggagaca gagccacaca tccgagacga 240tgggccgaaa
cacacatagt ataattgaat agtgggccgt ggcccaatat taaataatca
300tcccttgatt attgattatc tatgacctca taaacgataa taattactaa
ttagtagtct 360acgaatgaga aatataataa aatagtcgtt ttctagtgtg
tttgttaagt tgttatagat 420caatatacaa gtatcattca ttggaagaca
acaacatgta tatatctgag tgggaatgtg 480gcagaaaaag gatttggata
aaataagtac acgaagaaaa aagacagagt atgtaatgcc 540gttttggtag
gatggaaaca gcatgcagtt gttttggcaa ctgtattgta gtattagtat
600tattagaaaa tccagctaag agattattag actttaccca tatctaaata
agtcatcctt 660ccttaccttt ttcatataca aaacaattaa cacttgacct
aacttgcaac gctctctctc 720gattccatcc ttttttgttc ttttcttttt
gacacttttt ttttctttct ttcttttctt 780aaatagatta ttattcaatc
tttcctcctc catataataa taaaagtgat tgtgactgat 840atcaaattcc
ttatttaaat accacaatca cacagataca acatattact aattttaaat
900tacagagcag ttggtccaca tatgcttgaa attgatagtc taattagttt
taatgaggag 960gtccatgtca aataggcctg acccaccagc tatatatata
ccatgcatta aacctatata 1020taaaaataaa atctctataa ctttttttgg
gagttgttaa tttccttttc gttaattata 1080gaatatgctc cctttctctt
ctctgacgat ggttcataaa cctattcctt cgtagttgtt 1140taacgtccat
aataattgta tacttttagt tcgagattta cctaaacaaa acaaaaacag
1200aataccatac tgtatttcat gatttctgta ttggaaaata aaagactgaa
accaaatgat 1260tggtgggctt ttctaaaata atagtaataa aatacaatta
agacacacaa aaaagaaaag 1320gatttggtac atttattaac atcgatgatg
agttgataat gccacgtaag ccaaatacca 1380ttttcccaag aagctcatat gacc
1404771418DNAArabidopsis thalianapromoter(1)..(1418)transcription
regulating sequence from gene At2g48030 77tgcttaggtc caaaaccagt
ctctcacact aaagaaacac tttgtcatat ttgtacatac 60tgagcggaat attctgaggg
atttgttttg ttttcaatca gcttgtagtt gattattgta 120gttgcgacat
aatgaaaaga ggagaaatga gagatgggtt caaagaagca ttccacattc
180agaagtccaa tctcctaaaa gactaaacca gccaaaatga aaagaggaga
cagagccaca 240catccgagac gatgggccga aacacacata gtataattga
atgtgggccg tggcccaata 300ttaaataatc atcccttgat tattgattat
ctatgacctc ataaacgata ataattacta 360attagtactc tacgaatgag
aaatataata aaatagtcgt tttctagtgt gtttgttaag 420ttgttataga
tcaatataca agtatcattc attggaagac aacaacatgt atatatctga
480gtgggaatgt ggcagaaaaa ggatttggat aaaataagta cacgaagaaa
aaagacagag 540tatgtaatgc cgttttggta ggatggaaac agcatgcagt
tgttttggca actgtattgt 600agtattagta ttattagaaa atccagctaa
gagattatat tagactttac ccatatctaa 660ataagtcatc cttccttacc
tttttcatat acaaaacaat taacacttga cctaacttgc 720aacgctctct
ctcgattcca tccttttttg ttcttttctt tttgacactt ttttttttct
780ttctttcttt tcttaaatag attattattc aatctttcct cctccatata
ataataaaag 840tgattgtgac tgatatcaaa ttccttattt aaataccaca
atcacacaga tacaacatat 900tactaatttt aaattacaga gcagttggtc
cacatatgct tgaaattgat agtctaatta 960gttttaatga ggaggtccat
gtcaaatagg cctgacccac tagctatata tataccatgc 1020attaaaccta
tatataaaaa taaaatctct ataacttttt ttgggagttg ttaatttcct
1080tttcgttaat tatagaatat gctccctttc tcttctctga cgatggttca
taaacctatt 1140ccttcgtagt tgtttaacgt ccataataat tgtatacttt
tagttcgaga tttacctaaa 1200caaaacaaaa acagaatacc atactgtatt
tcatgatttc tgtattggaa aataaaagac 1260tgaaaccaaa tgattggtgg
gcttttctaa aataatagta ataaaataca attaagacac 1320acaaaaaaga
aaaggatttg gtacatttat taacatcgat gatgagttga taatgccacg
1380taagccaaat accattttcc caagaagctc atatgacc
1418781784DNAArabidopsis thalianaCDS(161)..(1477)encoding
endonuclease/exonuclease/phosphatase family protein 78ttcttttttg
tgcagcaaac tccacattcc acaactctct ctctctcatt tcttcgtata 60aaaggaaatc
tatatcatct ctctctctca ttgtcaaaac tcaaaagtat atacttttac
120tagttttaga cttaataaat gcgatcgccc tttttttagt atg ttg aac ctc att
175 Met Leu Asn Leu Ile 1 5gct ttc ctc cgc cgt cgt ctc cgc cgt ccc
cgg aaa gcc aga atc tcc 223Ala Phe Leu Arg Arg Arg Leu Arg Arg Pro
Arg Lys Ala Arg Ile Ser 10 15 20gtc aac cac cac cac ctc tca gtt gat
tct tct cct gag act cat cat 271Val Asn His His His Leu Ser Val Asp
Ser Ser Pro Glu Thr His His 25 30 35cat caa aat ggc ttt tca tcg gca
gcc gcc atc cac cca aac ccg gac 319His Gln Asn Gly Phe Ser Ser Ala
Ala Ala Ile His Pro Asn Pro Asp 40 45 50aaa acc atc aca gtg gcc acc
ttt aac gct gct atg ttc tcc atg gct 367Lys Thr Ile Thr Val Ala Thr
Phe Asn Ala Ala Met Phe Ser Met Ala 55 60 65ccc gcc gtc cct agc aac
aag ggc ttg cca ttc cgg tcc aag tct acg 415Pro Ala Val Pro Ser Asn
Lys Gly Leu Pro Phe Arg Ser Lys Ser Thr70 75 80 85gtt gac cgc ccc
aag agc att ctc aag ccc atg aac gcc gcc gca tct 463Val Asp Arg Pro
Lys Ser Ile Leu Lys Pro Met Asn Ala Ala Ala Ser 90 95 100cct act
cac gac tct aga aag caa cag agg ttt gcc aaa tcc agg cct 511Pro Thr
His Asp Ser Arg Lys Gln Gln Arg Phe Ala Lys Ser Arg Pro 105 110
115agg aga gtc tcc atc aat ctc ccc gac aac gag atc agc cgc cag ctc
559Arg Arg Val Ser Ile Asn Leu Pro Asp Asn Glu Ile Ser Arg Gln Leu
120 125 130agc ttc cgg gag gat cca cag cac tct ccc ctc agg ccc ggc
gag atc 607Ser Phe Arg Glu Asp Pro Gln His Ser Pro Leu Arg Pro Gly
Glu Ile 135 140 145ggt ctc cgg agc acc aga acg gct ctg gaa gtg ctg
agt gag cta gac 655Gly Leu Arg Ser Thr Arg Thr Ala Leu Glu Val Leu
Ser Glu Leu Asp150 155 160 165gca gac gtg ctg gct ctt caa gac gtc
aag gcg gac gag gct gac caa 703Ala Asp Val Leu Ala Leu Gln Asp Val
Lys Ala Asp Glu Ala Asp Gln 170 175 180atg aga ccg ctc tcc gat ctc
gct gcg gcg ctg ggg atg aat tac gtc 751Met Arg Pro Leu Ser Asp Leu
Ala Ala Ala Leu Gly Met Asn Tyr Val 185 190 195ttc gcc gag agc tgg
gcg ccg gag tac ggc aac gcc att ctc tct aag 799Phe Ala Glu Ser Trp
Ala Pro Glu Tyr Gly Asn Ala Ile Leu Ser Lys 200 205 210tgg ccc atc
aaa agc tcc aat gtt cta aga atc ttc gac cac act gat 847Trp Pro Ile
Lys Ser Ser Asn Val Leu Arg Ile Phe Asp His Thr Asp 215 220 225ttc
agg aac gtg ttg aag gcg agc ata gag gtt ccc ggg agc ggg gaa 895Phe
Arg Asn Val Leu Lys Ala Ser Ile Glu Val Pro Gly Ser Gly Glu230 235
240 245gtg gag ttt cac tgc act cat ctg gat cac ttg gac gag aag tgg
aga 943Val Glu Phe His Cys Thr His Leu Asp His Leu Asp Glu Lys Trp
Arg 250 255 260atg aag caa gtc gat gcc att atc caa tcc acc aac gta
ccg cac ata 991Met Lys Gln Val Asp Ala Ile Ile Gln Ser Thr Asn Val
Pro His Ile 265 270 275ctc gct ggt gct ctc aat tct ctc gac gaa tcc
gat tat tct cct gag 1039Leu Ala Gly Ala Leu Asn Ser Leu Asp Glu Ser
Asp Tyr Ser Pro Glu 280 285 290aga tgg acc gac atc gtc aag tat tac
gaa gag atg ggt aag cca ata 1087Arg Trp Thr Asp Ile Val Lys Tyr Tyr
Glu Glu Met Gly Lys Pro Ile 295 300 305cca aaa gca caa gtg atg aga
ttc tta aag agc aaa gaa tac act gac 1135Pro Lys Ala Gln Val Met Arg
Phe Leu Lys Ser Lys Glu Tyr Thr Asp310 315 320 325gct aag gac ttt
gct gga gaa tgc gaa tct gtg gtt gtt gtc gcc aaa 1183Ala Lys Asp Phe
Ala Gly Glu Cys Glu Ser Val Val Val Val Ala Lys 330 335 340ggc caa
agt gtg cag ggg aca tgc aag tac ggg aca cgc gtg gac tac 1231Gly Gln
Ser Val Gln Gly Thr Cys Lys Tyr Gly Thr Arg Val Asp Tyr 345 350
355ata ctt gct tcc tcc gat tca ccg tac cgg ttc gtg cca ggg tcg tat
1279Ile Leu Ala Ser Ser Asp Ser Pro Tyr Arg Phe Val Pro Gly Ser Tyr
360 365 370tcc gtg ttg tct tcc aaa gga acc tcg gac cat cac ata gtc
aaa gtc 1327Ser Val Leu Ser Ser Lys Gly Thr Ser Asp His His Ile Val
Lys Val 375 380 385gat gtt gta aaa gct aca tcg atc aac gtc aac gag
cag gag cag cga 1375Asp Val Val Lys Ala Thr Ser Ile Asn Val Asn Glu
Gln Glu Gln Arg390 395 400 405ccg ata aga tca cat aag ctg cag aga
att aca gcg act acg tat aat 1423Pro Ile Arg Ser His Lys Leu Gln Arg
Ile Thr Ala Thr Thr Tyr Asn 410 415 420aat aac tcg tct ctc aca aag
gcc tcg tgg agg aca cac tac tat aaa 1471Asn Asn Ser Ser Leu Thr Lys
Ala Ser Trp Arg Thr His Tyr Tyr Lys 425 430 435gca tga acaatcaaca
tatatattac aaaatgttac aggttttttt tcgactgttg 1527Alagttgattaac
acaatttgag atctgtgtat agttaataat cgtgctagtc tctgtgttga
1587acgtgaagtc tgatatactt ttgcctagat ttccattttt tctacatcct
aattttggtc 1647tgcttttaat gttaattaga caaggtggct tctttaaccg
aaacctgtag tattcatgag 1707tgagtaagta ctactgtact cttgtttttt
tttctgtttg tagtttctca aaatgagatt 1767aattagcttt cttgaaa
178479438PRTArabidopsis thaliana 79Met Leu Asn Leu Ile Ala Phe Leu
Arg Arg Arg Leu Arg Arg Pro Arg1 5 10 15Lys Ala Arg Ile Ser Val Asn
His His His Leu Ser Val Asp Ser Ser 20 25 30Pro Glu Thr His His His
Gln Asn Gly Phe Ser Ser Ala Ala Ala Ile 35 40 45His Pro Asn Pro Asp
Lys Thr Ile Thr Val Ala Thr Phe Asn Ala Ala 50 55 60Met Phe Ser Met
Ala Pro Ala Val Pro Ser Asn Lys Gly Leu Pro Phe65 70 75 80Arg Ser
Lys Ser Thr Val Asp Arg Pro Lys Ser Ile Leu Lys Pro Met 85 90 95Asn
Ala Ala Ala Ser Pro Thr His Asp Ser Arg Lys Gln Gln Arg Phe 100 105
110Ala Lys Ser Arg Pro Arg Arg Val Ser Ile Asn Leu Pro Asp Asn Glu
115 120 125Ile Ser Arg Gln Leu Ser Phe Arg Glu Asp Pro Gln His Ser
Pro Leu 130 135 140Arg Pro Gly Glu Ile Gly Leu Arg Ser Thr Arg Thr
Ala Leu Glu Val145 150 155 160Leu Ser Glu Leu Asp Ala Asp Val Leu
Ala Leu Gln Asp Val Lys Ala 165 170 175Asp Glu Ala Asp Gln Met Arg
Pro Leu Ser Asp Leu Ala Ala Ala Leu 180 185 190Gly Met Asn Tyr Val
Phe Ala Glu Ser Trp Ala Pro Glu Tyr Gly Asn 195 200 205Ala Ile Leu
Ser Lys Trp Pro Ile Lys Ser Ser Asn Val Leu Arg Ile 210 215 220Phe
Asp His Thr Asp Phe Arg Asn Val Leu Lys Ala Ser Ile Glu Val225 230
235 240Pro Gly Ser Gly Glu Val Glu Phe His Cys Thr His Leu Asp His
Leu 245 250 255Asp Glu Lys Trp Arg Met Lys Gln Val Asp Ala Ile Ile
Gln Ser Thr 260 265 270Asn Val Pro His Ile Leu Ala Gly Ala Leu Asn
Ser Leu Asp Glu Ser 275 280 285Asp Tyr Ser Pro Glu Arg Trp Thr Asp
Ile Val Lys Tyr Tyr Glu Glu 290 295 300Met Gly Lys Pro Ile Pro Lys
Ala Gln Val Met Arg Phe Leu Lys Ser305 310 315 320Lys Glu Tyr Thr
Asp Ala Lys Asp Phe Ala Gly Glu Cys Glu Ser Val 325 330 335Val Val
Val Ala Lys Gly Gln Ser Val Gln Gly Thr Cys Lys Tyr Gly 340 345
350Thr Arg Val Asp Tyr Ile Leu Ala Ser Ser Asp Ser Pro Tyr Arg Phe
355 360 365Val Pro Gly Ser Tyr Ser Val Leu Ser Ser Lys Gly Thr Ser
Asp His 370 375 380His Ile Val Lys Val Asp Val Val Lys Ala Thr Ser
Ile Asn Val Asn385 390 395 400Glu Gln Glu Gln Arg Pro Ile Arg Ser
His Lys Leu Gln Arg Ile Thr 405 410 415Ala Thr Thr Tyr Asn Asn Asn
Ser Ser Leu Thr Lys Ala Ser Trp Arg 420 425 430Thr His Tyr Tyr Lys
Ala 43580524DNAArabidopsis thalianapromoter(1)..(524)transcription
regulating sequence from gene At2g38590 80agtttttgta aaactcaaca
atacattgga ggcagagaag atgcatcatc aaatctatat 60gtctccctct ctcttttata
atttgaaggg acctgagaat cattgattag ctttgcacaa 120tatttagagt
acacaaatgt ttattgtaat tttttgatag gataacttgg tgatgacaat
180gatgttgatt actgttgttt tgttctccat ctttttcaga aaatcttgaa
agttgaaaca 240taaatcttaa gcatctgagc aaagacaaga ctgatgtatg
caacaaactc aattatgtct 300tacataagct ggtccagttt attttttctt
caaagtagga ccgtaaaagt gaagccataa 360aacaaacata acatcattaa
aagcctaact agattctaaa gcccacgatt agcccagctt 420tgtttagggt
ttttgtgtat cttcaattgg tgtgttggtg tatactctca tatttccctt
480tgccgcagag aaagaaaaaa aaaaatcatt gttgcaattt gaaa
52481539DNAArabidopsis thalianapromoter(1)..(539)transcription
regulating sequence from gene At2g38590 81cggcaaagaa atagtttttg
taaaactcaa caatacattg gaggcagaga agatgcatca 60tcaaatctat atgtctccct
ctctctttta taatttgaag ggacctgaga atcattgatt 120agctttgcac
aatatttaga gtacacaaat gtttattgta attttttgat aggataactt
180ggtgatgaca atgatgttga ttactgttgt tttgttctcc atctttttca
gaaaatcttg 240aaagttgaaa cataaatctt aagcatctga gcaaagacaa
gactgatgta tgcaacaaac 300tcaattatgt cttacataag ctggtccagt
ttattttttc ttcaaagtag gaccgtaaaa 360gtgaagccat aaaacaaaca
taacatcatt aaaagcctaa ctagattcta aagcccacga 420ttagcccagc
tttgtttagg gtttttgtgt atcttcaatt ggtgtgttgg tgtatactct
480cataatttcc ctttgccgca gagaaagaaa aaaaaaaatc attgttgcaa tttgaaagt
539821288DNAArabidopsis thalianaCDS(1)..(1275)encoding F-box family
protein 82atg acg acg atg atc tct aat ctt cca agg gtt ttg ata gag
gag att 48Met Thr Thr Met Ile Ser Asn Leu Pro Arg Val Leu Ile Glu
Glu Ile1 5 10 15ttt ttt agg gtt cct ttg aaa tct ttg aga gca gtg aga
tta act tgc 96Phe Phe Arg Val Pro Leu Lys Ser Leu Arg Ala Val Arg
Leu Thr Cys 20 25 30aaa agt tgg aac act cta tcc aaa agt agg agc ttt
agg aag ttg tac 144Lys Ser Trp Asn Thr Leu Ser Lys Ser Arg Ser Phe
Arg Lys Leu Tyr 35 40 45att agt aaa aga gca aca aga gaa gag gag tcc
atg atg atc gct atg 192Ile Ser Lys Arg Ala Thr Arg Glu Glu Glu Ser
Met Met Ile Ala Met 50 55 60atg aat ttc gat ctt tat tca atg agg gtc
gta gtt gac gac gac gtt 240Met Asn Phe Asp Leu Tyr Ser Met Arg Val
Val Val Asp Asp Asp Val65 70 75 80gat cca tct aaa gcg ttt aaa aaa
aaa agg aat aaa aaa tct ata gcg 288Asp Pro Ser Lys Ala Phe Lys Lys
Lys Arg Asn Lys Lys Ser Ile Ala 85 90 95ttt aaa cgt caa cct att ttc
ctt gat gaa caa gtc aag ata tct caa 336Phe Lys Arg Gln Pro Ile Phe
Leu Asp Glu Gln Val Lys Ile Ser Gln 100 105 110gtt ttt cac tgt gaa
ggt
tta ttg tta tgc ttc tta aaa gaa gac gat 384Val Phe His Cys Glu Gly
Leu Leu Leu Cys Phe Leu Lys Glu Asp Asp 115 120 125aca agg gtt gtc
gtt tgg aat ccg tat tgc gga caa aca agg tgg atc 432Thr Arg Val Val
Val Trp Asn Pro Tyr Cys Gly Gln Thr Arg Trp Ile 130 135 140caa ctc
aga tat tct cac cgt cca cac aaa gac agg ttc att tac gct 480Gln Leu
Arg Tyr Ser His Arg Pro His Lys Asp Arg Phe Ile Tyr Ala145 150 155
160cta gga tac aag gat aag gaa tct cgt ggt agc ttc caa tta ttg agg
528Leu Gly Tyr Lys Asp Lys Glu Ser Arg Gly Ser Phe Gln Leu Leu Arg
165 170 175ttt gta gat tat ttc cta gga gca ccc aaa aat caa tat ttt
tgg tat 576Phe Val Asp Tyr Phe Leu Gly Ala Pro Lys Asn Gln Tyr Phe
Trp Tyr 180 185 190gaa att tac gat ttt aac tct gat tca tgg aca act
ctt gat gtc act 624Glu Ile Tyr Asp Phe Asn Ser Asp Ser Trp Thr Thr
Leu Asp Val Thr 195 200 205cca cac tgg tgt ata tac tgt tgt gac cgt
ggt gtt tct ctc aac gga 672Pro His Trp Cys Ile Tyr Cys Cys Asp Arg
Gly Val Ser Leu Asn Gly 210 215 220aac act tac tgg tgt gct aaa gaa
agg aaa gca gaa gac gat att gtc 720Asn Thr Tyr Trp Cys Ala Lys Glu
Arg Lys Ala Glu Asp Asp Ile Val225 230 235 240gat cac atc ata tct
ttt gat ttt aca aac gag aga ttt ggg ccg ctt 768Asp His Ile Ile Ser
Phe Asp Phe Thr Asn Glu Arg Phe Gly Pro Leu 245 250 255ttg cct ttg
ccg tct aag gtt atg gaa cat gaa tat gaa att gta act 816Leu Pro Leu
Pro Ser Lys Val Met Glu His Glu Tyr Glu Ile Val Thr 260 265 270tta
tct tat gtt aaa gaa gag aag ctt gca gct tta ttt cag cat tac 864Leu
Ser Tyr Val Lys Glu Glu Lys Leu Ala Ala Leu Phe Gln His Tyr 275 280
285gaa gca gat tgg aat gag ttt gat ata tgg att acg act aag att gat
912Glu Ala Asp Trp Asn Glu Phe Asp Ile Trp Ile Thr Thr Lys Ile Asp
290 295 300gct gaa gtg gtg tcg tgg agc atg ttc ttg aga atg gat acg
gga cct 960Ala Glu Val Val Ser Trp Ser Met Phe Leu Arg Met Asp Thr
Gly Pro305 310 315 320agg ata gag gtt cca cat ata tgt gaa ggt ttc
ttc att gat gag gag 1008Arg Ile Glu Val Pro His Ile Cys Glu Gly Phe
Phe Ile Asp Glu Glu 325 330 335aag aaa gtc gcc atg ggt ttt gaa gaa
gac ttc gat cgc aaa aca ttt 1056Lys Lys Val Ala Met Gly Phe Glu Glu
Asp Phe Asp Arg Lys Thr Phe 340 345 350atc atc att gga gaa gct gga
tac gta aga aaa ttg gat atc aaa gca 1104Ile Ile Ile Gly Glu Ala Gly
Tyr Val Arg Lys Leu Asp Ile Lys Ala 355 360 365cat gta gac aga aaa
tgt cgg cca act gtg tgt tct tat gtt cca agt 1152His Val Asp Arg Lys
Cys Arg Pro Thr Val Cys Ser Tyr Val Pro Ser 370 375 380ttg gtc caa
atc aag aaa cct gca cga ggc aaa agg aaa aga caa agc 1200Leu Val Gln
Ile Lys Lys Pro Ala Arg Gly Lys Arg Lys Arg Gln Ser385 390 395
400agc tta gaa aag cgt cta ttt gat caa aac atg ttg aga ctt gaa gca
1248Ser Leu Glu Lys Arg Leu Phe Asp Gln Asn Met Leu Arg Leu Glu Ala
405 410 415ttt aaa aag cta ggc ggc tac ttc tag aagcaatatg cag
1288Phe Lys Lys Leu Gly Gly Tyr Phe 42083424PRTArabidopsis thaliana
83Met Thr Thr Met Ile Ser Asn Leu Pro Arg Val Leu Ile Glu Glu Ile1
5 10 15Phe Phe Arg Val Pro Leu Lys Ser Leu Arg Ala Val Arg Leu Thr
Cys 20 25 30Lys Ser Trp Asn Thr Leu Ser Lys Ser Arg Ser Phe Arg Lys
Leu Tyr 35 40 45Ile Ser Lys Arg Ala Thr Arg Glu Glu Glu Ser Met Met
Ile Ala Met 50 55 60Met Asn Phe Asp Leu Tyr Ser Met Arg Val Val Val
Asp Asp Asp Val65 70 75 80Asp Pro Ser Lys Ala Phe Lys Lys Lys Arg
Asn Lys Lys Ser Ile Ala 85 90 95Phe Lys Arg Gln Pro Ile Phe Leu Asp
Glu Gln Val Lys Ile Ser Gln 100 105 110Val Phe His Cys Glu Gly Leu
Leu Leu Cys Phe Leu Lys Glu Asp Asp 115 120 125Thr Arg Val Val Val
Trp Asn Pro Tyr Cys Gly Gln Thr Arg Trp Ile 130 135 140Gln Leu Arg
Tyr Ser His Arg Pro His Lys Asp Arg Phe Ile Tyr Ala145 150 155
160Leu Gly Tyr Lys Asp Lys Glu Ser Arg Gly Ser Phe Gln Leu Leu Arg
165 170 175Phe Val Asp Tyr Phe Leu Gly Ala Pro Lys Asn Gln Tyr Phe
Trp Tyr 180 185 190Glu Ile Tyr Asp Phe Asn Ser Asp Ser Trp Thr Thr
Leu Asp Val Thr 195 200 205Pro His Trp Cys Ile Tyr Cys Cys Asp Arg
Gly Val Ser Leu Asn Gly 210 215 220Asn Thr Tyr Trp Cys Ala Lys Glu
Arg Lys Ala Glu Asp Asp Ile Val225 230 235 240Asp His Ile Ile Ser
Phe Asp Phe Thr Asn Glu Arg Phe Gly Pro Leu 245 250 255Leu Pro Leu
Pro Ser Lys Val Met Glu His Glu Tyr Glu Ile Val Thr 260 265 270Leu
Ser Tyr Val Lys Glu Glu Lys Leu Ala Ala Leu Phe Gln His Tyr 275 280
285Glu Ala Asp Trp Asn Glu Phe Asp Ile Trp Ile Thr Thr Lys Ile Asp
290 295 300Ala Glu Val Val Ser Trp Ser Met Phe Leu Arg Met Asp Thr
Gly Pro305 310 315 320Arg Ile Glu Val Pro His Ile Cys Glu Gly Phe
Phe Ile Asp Glu Glu 325 330 335Lys Lys Val Ala Met Gly Phe Glu Glu
Asp Phe Asp Arg Lys Thr Phe 340 345 350Ile Ile Ile Gly Glu Ala Gly
Tyr Val Arg Lys Leu Asp Ile Lys Ala 355 360 365His Val Asp Arg Lys
Cys Arg Pro Thr Val Cys Ser Tyr Val Pro Ser 370 375 380Leu Val Gln
Ile Lys Lys Pro Ala Arg Gly Lys Arg Lys Arg Gln Ser385 390 395
400Ser Leu Glu Lys Arg Leu Phe Asp Gln Asn Met Leu Arg Leu Glu Ala
405 410 415Phe Lys Lys Leu Gly Gly Tyr Phe 420841988DNAArabidopsis
thalianapromoter(1)..(1988)transcription regulating sequence from
gene At1g23000 84atttaattat agtgatgaga aaaggactaa atacattttt
tttttggttg tcaacaagaa 60catatgatat aaagctctaa caaaaaaaga agaactaaat
atgaaaatat gatcagagga 120caggtaagta cattgtcggg tgatgacatg
actaattatt aatttcgacc ccaattttaa 180aacatacata ttctaaaatt
ctattatggc atgtgaaatt gtgaaaagac acaaaactaa 240tggaccgatg
tggagtaaca cagaaaacgc gtggcgtaga gaggacttga taattaggag
300tcaaggttgc gttgatgctg tgaggtccag ggttttgtag cttttcttct
attgtcacgt 360gtacggtgtt tgggaaatga acggctaaat cattcaactt
gaaacactaa tcacggcatt 420tcacgaaata agaggataat attggtatat
gcagtttaca tcaagagtta aataccataa 480tgtaatgttg gctacggatc
taacaaatca tgtgagcaat acgagttttg gtaactataa 540ttttcgtgaa
aggaacatat attggactat tggaggtagc tggcaactct caaatcttaa
600agtgaagtat attttttgta tgcaaaatca agatgatagt atgtatgatg
atcatgtata 660tatttattgt ggacagttaa attcgtttga tgtagataaa
tgcactgatt attgatttgt 720atgcggtcaa aactatatag taatcgagcc
atcgaggtgg tgtggtttat tggtgttatt 780cgagtggtag aaatttttct
atcaaggggg aaagtcctac tatctacgac cattattgct 840cttttatttt
gaccagcata ctcattttaa ctgacaagac tagactgcat gacatttcat
900agactttggg attagtatgt gttttagctc atgatctagg ttaaaaaaga
gaatgtgatt 960attagtaaat atattttaat agctaaccaa atataatgta
atcacgtaat ctttgatcat 1020atgtatgatt tgtaaactca cattagatga
agttgaaaat ccatcgaggg tttgcgattt 1080ttttaatgtt tttttattta
tgtgttatat agatttggaa agaaattaag tttaagtggg 1140ggaatcaaag
ggccaacatt gaacagccag actaaaaaac attaaacaga ttaatgggct
1200taatgcgtaa tgcgcctctt tttttgttta aggcccataa tagttttatt
taaaaggccg 1260aaatgtaagg caacagtagc actacactaa caaggtcagt
cagaatgtgt cactggccac 1320tgctcataaa tcaagaaatc aaaatattat
atgtaatttt ttttttctta aaaccaaaaa 1380cgtagtatag tgaaaatatt
cttaaaaaca catttttatt ttttctaaaa gaaattagta 1440ttattctaaa
aacaaaaaga agaaaaaagt tgggggaaag agaggggaga agctcgagaa
1500gagtgtggga gaaggcaaac cccaaacact gactagacac agaccgttgg
gcccgactct 1560tcatgcatgc actcttctga caacattttc aatttctcca
tatctctctc tattgttttt 1620tcatttcttg gtttcattaa tatcatagtt
tatgaataat actacaagta ctatgtaaaa 1680tatgccttaa ataaagatat
cgtacaattc aaaatgcttt tgttggaatg caatttaaac 1740tccaaaattc
agaaatttaa tgaggaagtg tggtcaggtc gctgttactt ttaagtgtca
1800gtcttcactc actcactcac tcaacatcgt ttcaaatttc agagcaaaag
tctctaaacc 1860caaagcctct ccacttaatt gcctattctt caaaaggcga
gtttccttta ccaagcttgc 1920aaagttatct aaactccaaa caatatctaa
aacgcgtgta ctcatttctc ttattctttc 1980ttcttaag
1988852010DNAArabidopsis thalianapromoter(1)..(2010)transcription
regulating sequence from gene At1g23000 85acattaggaa caatttaatt
atagtgatga gaaaaggact aaatacatta tttttttggt 60tgtcaacaag aacatatgat
ataaagctct aacaaaaaaa gaagaactaa atatgaaaat 120atgatcagag
gacaggtaag tacattgtcg ggtgatgaca tgactaatta ttaatttcga
180ccccaattta aaaacataca tattctaaaa ttctattatg gcatgtgaaa
ttgtgaaaag 240acacaaaact aatggaccga tgtggagtaa cacagaaaac
gcgtggcgta gagaggactt 300gataattagg agtcaaggtt gcgttgatgc
tgtgaggtcc agggttttgt agcttttctt 360ctattgtcac gtgtacggtg
tttgggaaat gaacggctaa atcattcaac ttgaaacact 420aatcacggca
tttcacgaaa taagaggata atattggtat atgcagttta catcaagagt
480taaataccat aatgtaatgt tggctacgga tctaacaaat catgtgagca
atacgagttt 540tggtaactat aattttcgtg aaaggaacat atattggact
attggaggta gctggcaact 600ctcaaatctt aaagtgaagt atattttttg
tatgcaaaat caagatgata gtatgtatga 660tgatcatgta tatatttatt
gtggacagtt aaattcgttt gatgtagata aatgcactga 720ttattgattt
gtatgcggtc aaaactatat agtaatcgag ccatcgaggt ggtgtggttt
780attggtgtta ttcgagtggt agaaattttt ctatcaaggg ggaaagtcct
actatctacg 840accattattg ctcttttctt ttgaccagca tactcatttt
aactgacaag actagactgc 900atgacatttc atagactttg ggattagtat
gtgttttagc tcatgatcta ggttaaaaaa 960gagaatgtga ttattagtaa
acatatttta aaagctaacc aaatataatg taatcacgta 1020atctttgatc
atatgtatga tttgtaaact cccattagat caagttgaaa atccatcgag
1080ggtttgcgat ttttttaatg tttttttatt tatgtgttat atagatttgg
aaagaaatta 1140agtttaagtg ggggaatcaa agggccaaca ttgaacagcc
agactaaaaa aacattaaac 1200agattaatgg gcttaatgcg taatgcgtct
ctttttttgt ttaaggccca taatagtttt 1260attaaaaagg ccgaaatgta
aggcaacact agcactacac taacaaggtc agtcagaatg 1320tgctcactgg
ccactgctca taaatcaaga aatcaaaata ttgcatgcaa tttttttctt
1380aaaaccaaaa acgtagtata gtgaaaatat tctaaaaata atatgtatgt
cagctcattt 1440ttactttttc taaaagaaat tagtattatt ctataaacaa
aaagaagaaa aaagttgggg 1500gaaagagagg ggagaagctc gagaagagtg
tgggagaagg caaaccccaa acactgacta 1560gacacagacc gttgggcccg
actcttcatg catgcactct tctgacaaca ttttcaattt 1620ctccatatct
ctctctattg ttttttcatt tcttagtttc attaatatca tagtttatga
1680ataatactac aagtactatg taaaatatgc cttaaataaa gatatcgtac
aattcaaaat 1740gcttttgttg gaatgcaatt taaactccaa aattcagaat
ttaatgagga agtgtggtca 1800ggtcgctgtt acttttaagt gtcagtcttc
actcactcac tcaacatcgt ttcaaatttc 1860agagcaaaag tctctaaacc
caaagcctct ccacttaatt gcctattctt caaaaggcga 1920gtttccttta
ccaagcttgc aaagttatct aaactccaaa cgatatctaa aacgcgtgta
1980ctcatttctc ttattctttc ttcttaagcc 2010861776DNAArabidopsis
thalianapromoter(1)..(1776)transcription regulating sequence from
gene At1g23000 86atttaattat agtgatgaga aaaggactaa atacattttt
tttttggttg tcaacaagaa 60catatgatat aaagctctaa caaaaaaaga agaactaaat
atgaaaatat gatcagagga 120caggtaagta cattgtcggg tgatgacatg
actaattatt aatttcgacc ccaattttaa 180aacatacata ttctaaaatt
ctattatggc atgtgaaatt gtgaaaagac acaaaactaa 240tggaccgatg
tggagtaaca cagaaaacgc gtggcgtaga gaggacttga taattaggag
300tcaaggttgc gttgatgctg tgaggtccag ggttttgtag cttttcttct
attgtcacgt 360gtacggtgtt tgggaaatga acggctaaat cattcaactt
gaaacactaa tcacggcatt 420tcacgaaata agaggataat attggtatat
gcagtttaca tcaagagtta aataccataa 480tgtaatgttg gctacggatc
taacaaatca tgtgagcaat acgagttttg gtaactataa 540ttttcgtgaa
aggaacatat attggactat tggaggtagc tggcaactct caaatcttaa
600agtgaagtat attttttgta tgcaaaatca agatgatagt atgtatgatg
atcatgtata 660tatttattgt ggacagttaa attcgtttga tgtagataaa
tgcactgatt attgatttgt 720atgcggtcaa aactatatag taatcgagcc
atcgaggtgg tgtggtttat tggtgttatt 780cgagtggtag aaatttttct
atcaaggggg aaagtcctac tatctacgac cattattgct 840cttttatttt
gaccagcata ctcattttaa ctgacaagac tagactgcat gacatttcat
900agactttggg attagtatgt gttttagctc atgatctagg ttaaaaaaga
gaatgtgatt 960attagtaaat atattttaat agctaaccaa atataatgta
atcacgtaat ctttgatcat 1020atgtatgatt tgtaaactca cattagatga
agttgaaaat ccatcgaggg tttgcgattt 1080ttttaatgtt tttttattta
tgtgttatat agatttggaa agaaattaag tttaagtggg 1140ggaatcaaag
ggccaacatt gaacagccag actaaaaaac attaaacaga ttaatgggct
1200taatgcgtaa tgcgcctctt tttttgttta aggcccataa tagttttatt
taaaaggccg 1260aaatgtaagg caacagtagc actacactaa caaggtcagt
cagaatgtgt cactggccac 1320tgctcataaa tcaagaaatc aaaatattat
atgtaatttt ttttttctta aaaccaaaaa 1380cgtagtatag tgaaaatatt
cttaaaaaca catttttatt ttttctaaaa gaaattagta 1440ttattctaaa
aacaaaaaga agaaaaaagt tgggggaaag agaggggaga agctcgagaa
1500gagtgtggga gaaggcaaac cccaaacact gactagacac agaccgttgg
gcccgactct 1560tcatgcatgc actcttctga caacattttc aatttctcca
tatctctctc tattgttttt 1620tcatttcttg gtttcattaa tatcatagtt
tatgaataat actacaagta ctatgtaaaa 1680tatgccttaa ataaagatat
cgtacaattc aaaatgcttt tgttggaatg caatttaaac 1740tccaaaattc
agaaatttaa tgaggaagtg tggtca 1776871800DNAArabidopsis
thalianapromoter(1)..(1800)transcription regulating sequence from
gene At1g23000 87acattaggaa caatttaatt atagtgatga gaaaaggact
aaatacatta tttttttggt 60tgtcaacaag aacatatgat ataaagctct aacaaaaaaa
gaagaactaa atatgaaaat 120atgatcagag gacaggtaag tacattgtcg
ggtgatgaca tgactaatta ttaatttcga 180ccccaattta aaaacataca
tattctaaaa ttctattatg gcatgtgaaa ttgtgaaaag 240acacaaaact
aatggaccga tgtggagtaa cacagaaaac gcgtggcgta gagaggactt
300gataattagg agtcaaggtt gcgttgatgc tgtgaggtcc agggttttgt
agcttttctt 360ctattgtcac gtgtacggtg tttgggaaat gaacggctaa
atcattcaac ttgaaacact 420aatcacggca tttcacgaaa taagaggata
atattggtat atgcagttta catcaagagt 480taaataccat aatgtaatgt
tggctacgga tctaacaaat catgtgagca atacgagttt 540tggtaactat
aattttcgtg aaaggaacat atattggact attggaggta gctggcaact
600ctcaaatctt aaagtgaagt atattttttg tatgcaaaat caagatgata
gtatgtatga 660tgatcatgta tatatttatt gtggacagtt aaattcgttt
gatgtagata aatgcactga 720ttattgattt gtatgcggtc aaaactatat
agtaatcgag ccatcgaggt ggtgtggttt 780attggtgtta ttcgagtggt
agaaattttt ctatcaaggg ggaaagtcct actatctacg 840accattattg
ctcttttctt ttgaccagca tactcatttt aactgacaag actagactgc
900atgacatttc atagactttg ggattagtat gtgttttagc tcatgatcta
ggttaaaaaa 960gagaatgtga ttattagtaa acatatttta aaagctaacc
aaatataatg taatcacgta 1020atctttgatc atatgtatga tttgtaaact
cccattagat caagttgaaa atccatcgag 1080ggtttgcgat ttttttaatg
tttttttatt tatgtgttat atagatttgg aaagaaatta 1140agtttaagtg
ggggaatcaa agggccaaca ttgaacagcc agactaaaaa aacattaaac
1200agattaatgg gcttaatgcg taatgcgtct ctttttttgt ttaaggccca
taatagtttt 1260attaaaaagg ccgaaatgta aggcaacact agcactacac
taacaaggtc agtcagaatg 1320tgctcactgg ccactgctca taaatcaaga
aatcaaaata ttgcatgcaa tttttttctt 1380aaaaccaaaa acgtagtata
gtgaaaatat tctaaaaata atatgtatgt cagctcattt 1440ttactttttc
taaaagaaat tagtattatt ctataaacaa aaagaagaaa aaagttgggg
1500gaaagagagg ggagaagctc gagaagagtg tgggagaagg caaaccccaa
acactgacta 1560gacacagacc gttgggcccg actcttcatg catgcactct
tctgacaaca ttttcaattt 1620ctccatatct ctctctattg ttttttcatt
tcttagtttc attaatatca tagtttatga 1680ataatactac aagtactatg
taaaatatgc cttaaataaa gatatcgtac aattcaaaat 1740gcttttgttg
gaatgcaatt taaactccaa aattcagaat ttaatgagga agtgtggtca
1800881492DNAArabidopsis thalianaCDS(211)..(1287)encoding
heavy-metal-associated domain- containing protein 88ggtcgctgtt
acttttaagt gtcagtcttc actcactcac tcaacatcgt ttcaaatttc 60agagcaaaag
tctctaaacc caaagcctct ccacttaatt gcctattctt caaaaggcga
120gtttccttta ccaagcttgc aaagttatct aaactccaaa cgatatctaa
aacgcgtgta 180ctcatttctc ttattctttc ttcttaagcc atg act aaa gat gaa
gac ttt aag 234 Met Thr Lys Asp Glu Asp Phe Lys 1 5ctc cta aag atc
cag acg ttt tca ctc aga gtc aac att cac tgc gag 282Leu Leu Lys Ile
Gln Thr Phe Ser Leu Arg Val Asn Ile His Cys Glu 10 15 20ggc tgt aac
aag aaa gtc aag aaa ctt ctt cag agg atc gaa gga gtt 330Gly Cys Asn
Lys Lys Val Lys Lys Leu Leu Gln Arg Ile Glu Gly Val25 30 35 40tgc
cac gtg aag ata gaa gca gaa cat caa aag gtg act gtt tca ggg 378Cys
His Val Lys Ile Glu Ala Glu His Gln Lys Val Thr Val Ser Gly 45 50
55tca gtt gac tca gcc aca ctc atc aac aag ctt gtt aaa gcc ggg aaa
426Ser Val Asp Ser Ala Thr Leu Ile Asn Lys Leu Val Lys Ala Gly Lys
60 65 70cac gcc gag ctt tgg tct cct aac cca aac caa aac cag cct caa
aag 474His Ala Glu Leu Trp Ser Pro Asn Pro Asn Gln Asn Gln Pro Gln
Lys 75 80 85ccc aag act aac gac ttc atc aag aac gtt aat caa aag ggt
cag aag 522Pro Lys Thr Asn Asp Phe Ile Lys Asn Val
Asn Gln Lys Gly Gln Lys 90 95 100cag ggg tct gcc aaa agt ggt att
gaa gcc tgt aaa ccc aag aac ggc 570Gln Gly Ser Ala Lys Ser Gly Ile
Glu Ala Cys Lys Pro Lys Asn Gly105 110 115 120cct aaa ggt gca gcc
ttt gta gct gag gaa gat gga gat ggc agt gaa 618Pro Lys Gly Ala Ala
Phe Val Ala Glu Glu Asp Gly Asp Gly Ser Glu 125 130 135gag gaa gac
gga gat gtc cag ttt cct aaa ccg gcg aat cag cag cag 666Glu Glu Asp
Gly Asp Val Gln Phe Pro Lys Pro Ala Asn Gln Gln Gln 140 145 150caa
caa aac gtc gtc aac gcc aag aaa aac agt gga gga gct gcg atg 714Gln
Gln Asn Val Val Asn Ala Lys Lys Asn Ser Gly Gly Ala Ala Met 155 160
165aac aat gga aac aac gga gtc aac gca gca tca aag aaa gtc aac caa
762Asn Asn Gly Asn Asn Gly Val Asn Ala Ala Ser Lys Lys Val Asn Gln
170 175 180aaa cag agc aac cat aac cag aac act caa caa gta atg gcg
gct atg 810Lys Gln Ser Asn His Asn Gln Asn Thr Gln Gln Val Met Ala
Ala Met185 190 195 200aga atg aga acc gcc ggg aaa atg agc act ggt
gtt gaa gcc aac gag 858Arg Met Arg Thr Ala Gly Lys Met Ser Thr Gly
Val Glu Ala Asn Glu 205 210 215att gga gct ctc atg ggt ctt gct ggc
ttc aac ggc gca acc aac gca 906Ile Gly Ala Leu Met Gly Leu Ala Gly
Phe Asn Gly Ala Thr Asn Ala 220 225 230gtg aat cac cct cca aat ggg
att caa caa cag ctt caa gct cca ccg 954Val Asn His Pro Pro Asn Gly
Ile Gln Gln Gln Leu Gln Ala Pro Pro 235 240 245tta aac aac gtc aac
ggc gtc act aat cac aac ctc acc aat agt aat 1002Leu Asn Asn Val Asn
Gly Val Thr Asn His Asn Leu Thr Asn Ser Asn 250 255 260gga ggc atg
atg atg aac atg aat ggg tac aat aat cat cat cca atg 1050Gly Gly Met
Met Met Asn Met Asn Gly Tyr Asn Asn His His Pro Met265 270 275
280aac atg cag agt agg caa atg atg cat cag cct cag caa atg atg tac
1098Asn Met Gln Ser Arg Gln Met Met His Gln Pro Gln Gln Met Met Tyr
285 290 295caa aga tca tct ttc gtt cca gca tca agc aat ggg tat tat
tac aat 1146Gln Arg Ser Ser Phe Val Pro Ala Ser Ser Asn Gly Tyr Tyr
Tyr Asn 300 305 310tat acc cct agt ccc tac agt tat tat cct tat tac
cct tac gca agt 1194Tyr Thr Pro Ser Pro Tyr Ser Tyr Tyr Pro Tyr Tyr
Pro Tyr Ala Ser 315 320 325gat cag tac caa caa caa agt agt cat tca
cat gca aca aac atg tct 1242Asp Gln Tyr Gln Gln Gln Ser Ser His Ser
His Ala Thr Asn Met Ser 330 335 340agt gaa gaa gac gct ggt aat aac
aat agc tgc aac atc atg taa 1287Ser Glu Glu Asp Ala Gly Asn Asn Asn
Ser Cys Asn Ile Met345 350 355agtgtggatg cttgctaagg aagatatcta
tcttttgctt ttggaaacaa gacttaaaag 1347tttaatcttt tctggagtgt
gatatattag tgtttggttt ttctttctct ttttttttgg 1407tgtgtgtcat
gtagctgttt tggactgaca tgaagttgtg ttggttgata tataaaatcc
1467tagaatgtta ttgatgtttc ttgtg 149289358PRTArabidopsis thaliana
89Met Thr Lys Asp Glu Asp Phe Lys Leu Leu Lys Ile Gln Thr Phe Ser1
5 10 15Leu Arg Val Asn Ile His Cys Glu Gly Cys Asn Lys Lys Val Lys
Lys 20 25 30Leu Leu Gln Arg Ile Glu Gly Val Cys His Val Lys Ile Glu
Ala Glu 35 40 45His Gln Lys Val Thr Val Ser Gly Ser Val Asp Ser Ala
Thr Leu Ile 50 55 60Asn Lys Leu Val Lys Ala Gly Lys His Ala Glu Leu
Trp Ser Pro Asn65 70 75 80Pro Asn Gln Asn Gln Pro Gln Lys Pro Lys
Thr Asn Asp Phe Ile Lys 85 90 95Asn Val Asn Gln Lys Gly Gln Lys Gln
Gly Ser Ala Lys Ser Gly Ile 100 105 110Glu Ala Cys Lys Pro Lys Asn
Gly Pro Lys Gly Ala Ala Phe Val Ala 115 120 125Glu Glu Asp Gly Asp
Gly Ser Glu Glu Glu Asp Gly Asp Val Gln Phe 130 135 140Pro Lys Pro
Ala Asn Gln Gln Gln Gln Gln Asn Val Val Asn Ala Lys145 150 155
160Lys Asn Ser Gly Gly Ala Ala Met Asn Asn Gly Asn Asn Gly Val Asn
165 170 175Ala Ala Ser Lys Lys Val Asn Gln Lys Gln Ser Asn His Asn
Gln Asn 180 185 190Thr Gln Gln Val Met Ala Ala Met Arg Met Arg Thr
Ala Gly Lys Met 195 200 205Ser Thr Gly Val Glu Ala Asn Glu Ile Gly
Ala Leu Met Gly Leu Ala 210 215 220Gly Phe Asn Gly Ala Thr Asn Ala
Val Asn His Pro Pro Asn Gly Ile225 230 235 240Gln Gln Gln Leu Gln
Ala Pro Pro Leu Asn Asn Val Asn Gly Val Thr 245 250 255Asn His Asn
Leu Thr Asn Ser Asn Gly Gly Met Met Met Asn Met Asn 260 265 270Gly
Tyr Asn Asn His His Pro Met Asn Met Gln Ser Arg Gln Met Met 275 280
285His Gln Pro Gln Gln Met Met Tyr Gln Arg Ser Ser Phe Val Pro Ala
290 295 300Ser Ser Asn Gly Tyr Tyr Tyr Asn Tyr Thr Pro Ser Pro Tyr
Ser Tyr305 310 315 320Tyr Pro Tyr Tyr Pro Tyr Ala Ser Asp Gln Tyr
Gln Gln Gln Ser Ser 325 330 335His Ser His Ala Thr Asn Met Ser Ser
Glu Glu Asp Ala Gly Asn Asn 340 345 350Asn Ser Cys Asn Ile Met
3559027DNAArtificial sequenceOligonucleotide primer 90aacttgggat
ccttttatct gtataat 279123DNAArtificial sequenceOligonucleotide
primer 91accaaaccat ggtgattatt gac 239228DNAArtificial
sequenceOligonucleotide primer 92gattttccat ggttctataa agaaaatg
289327DNAArtificial sequenceOligonucleotide primer 93tactttggat
ccctcccttt ttatgtc 279423DNAArtificial sequenceOligonucleotide
primer 94accaaaccat ggtgattatt gac 239528DNAArtificial
sequenceOligonucleotide primer 95gattttccat ggttctataa agaaaatg
289628DNAArtificial sequenceOligonucleotide primer 96ataattggat
ccactagtca tctatttc 289723DNAArtificial sequenceOligonucleotide
primer 97accaaaccat ggtgattatt gac 239828DNAArtificial
sequenceOligonucleotide primer 98gattttccat ggttctataa agaaaatg
289924DNAArtificial sequenceOligonucleotide primer 99taaaagctcg
agctcgtcat ggac 2410027DNAArtificial sequenceOligonucleotide primer
100gatgtgggat cccagaaaga tcagtcc 2710130DNAArtificial
sequenceOligonucleotide primer 101agggtaaagc ttctgattga gaaaggacac
3010223DNAArtificial sequenceOligonucleotide primer 102caataagaat
tcggttagtt ttc 2310327DNAArtificial sequenceOligonucleotide primer
103gatgtggaat tccagaaaga tcagtcc 2710430DNAArtificial
sequenceoligonucleotide primer 104agggtaaagc ttctgattga gaaaggacac
3010524DNAArtificial sequenceOligonucleotide primer 105taaaagctcg
agctcgtcat ggac 2410625DNAArtificial sequenceOligonucleotide primer
106tcttacccat gggaggttag gtagg 2510728DNAArtificial
sequenceOligonucleotide primer 107ggaaggccat ggaagggaga cttctgac
2810830DNAArtificial sequenceOligonucleotide primer 108agggtaaagc
ttctgattga gaaaggacac 3010925DNAArtificial sequenceoligonucleotide
primer 109tcttacccat gggaggttag gtagg 2511028DNAArtificial
sequenceOligonucleotide primer 110ggaaggccat ggaagggaga cttctgac
2811127DNAArtificial sequenceOligonucleotide primer 111taagctttct
ttttgttgtg gaggata 2711228DNAArtificial sequenceOligonucleotide
primer 112atctagatag aattctttaa cttgtgct 2811330DNAArtificial
sequenceOligonucleotide primer 113taatctggat ccttgctttc aactccaaaa
3011430DNAArtificial sequenceOligonucleotide primer 114gcttttccat
ggattgcttg aaaattgatg 3011528DNAArtificial sequenceOligonucleotide
primer 115tgtcgacttt atctaatgga taaccacc 2811625DNAArtificial
sequenceOligonucleotide primer 116tcccgggtta attcatatat acaac
2511730DNAArtificial sequenceOligonucleotide primer 117tttaactctc
gagtgccgtt tcagttttgt 3011828DNAArtificial sequenceOligonucleotide
primer 118tgtcgacttt atctaatgga taaccacc 2811928DNAArtificial
sequenceOligonucleotide primer 119tcccgggaaa aatctcagct ggctctcg
2812030DNAArtificial sequenceOligonucleotide primer 120tttaactctc
gagtgccgtt tcagttttgt 3012124DNAArtificial sequenceOligonucleotide
primer 121aggatcctgt tattttagct aaag 2412225DNAArtificial
sequenceOligonucleotide primer 122tccatggtta attcatatat acaac
2512329DNAArtificial sequenceOligonucleotide primer 123tttaactctc
gagtgccgtt tcagttttg 2912425DNAArtificial sequenceOligonucleotide
primer 124agagatggat ccggtgagtg ctacg 2512527DNAArtificial
sequenceOligonucleotide primer 125ggttgtccat ggtgtctctt atatggc
2712628DNAArtificial sequenceOligonucleotide primer 126tttttcccat
ggttcatgag aagttgtg 2812728DNAArtificial sequenceOligonucleotide
primer 127gagttaggat ccctgaatac gatcgatc 2812827DNAArtificial
sequenceOligonucleotide primer 128ggttgtccat ggtgtctctt atatggc
2712928DNAArtificial sequenceOligonucleotide primer 129tttttcccat
ggttcatgag aagttgtg 2813024DNAArtificial sequenceOligonucleotide
primer 130cttgtgggat ccatcaattc actc 2413128DNAArtificial
sequenceOligonucleotide primer 131agacatccat ggcaatgaat atgaaatg
2813231DNAArtificial sequenceOligonucleotide primer 132tagccatggg
aagcagcaac cctcattaat g 3113327DNAArtificial
sequenceOligonucleotide primer 133taaaaaggat ccaaaaagag agtcaac
2713428DNAArtificial sequenceOligonucleotide primer 134agacatccat
ggcaatgaat atgaaatg 2813531DNAArtificial sequenceOligonucleotide
primer 135tagccatggg aagcagcaac cctcattaat g 3113624DNAArtificial
sequenceOligonucleotide primer 136aactaaggat ccagaaaaag aatc
2413728DNAArtificial sequenceOligonucleotide primer 137agacatccat
ggcaatgaat atgaaatg 2813831DNAArtificial sequenceOligonucleotide
primer 138tagccatggg aagcagcaac cctcattaat g 3113925DNAArtificial
sequenceOligonucleotide primer 139tgcttaggat ccaaaccagt ctctc
2514025DNAArtificial sequenceOligonucleotide primer 140ggttcaccat
ggtaaaaaaa gggcg 2514127DNAArtificial sequenceOligonucleotide
primer 141ggtcatccat ggttcttggg aaaatgg 2714228DNAArtificial
sequenceOligonucleotide primer 142cggcaaggat ccagtttttg taaaactc
2814326DNAArtificial sequenceOligonucleotide primer 143tcgtcgccat
ggtttcaaat tgcaac 2614426DNAArtificial sequenceOligonucleotide
primer 144acattaggat ccatttaatt atagtg 2614525DNAArtificial
sequenceOligonucleotide primer 145ctttagccat ggcttaagaa gaaag
2514630DNAArtificial sequenceOligonucleotide primer 146tgaccatggt
tcctcattaa attctgaatt 301478986DNAArtificial sequencebinary vector
pSUN0301 147cgttgtaaaa cgacggccag tgaattcgag ctcggtacct cgagcccggg
cgatatcgga 60tccactagtc tagagtcgat cgaccatggt acgtcctgta gaaaccccaa
cccgtgaaat 120caaaaaactc gacggcctgt gggcattcag tctggatcgc
gaaaactgtg gaattggtca 180gcgttggtgg gaaagcgcgt tacaagaaag
ccgggcaatt gctgtgccag gcagttttaa 240cgatcagttc gccgatgcag
atattcgtaa ttatgcgggc aacgtctggt atcagcgcga 300agtctttata
ccgaaaggtt gggcaggcca gcgtatcgtg ctgcgtttcg atgcggtcac
360tcattacggc aaagtgtggg tcaataatca ggaagtgatg gagcatcagg
gcggctatac 420gccatttgaa gccgatgtca cgccgtatgt tattgccggg
aaaagtgtac gtaagtttct 480gcttctacct ttgatatata tataataatt
atcattaatt agtagtaata taatatttca 540aatatttttt tcaaaataaa
agaatgtagt atatagcaat tgcttttctg tagtttataa 600gtgtgtatat
tttaatttat aacttttcta atatatgacc aaaatttgtt gatgtgcagg
660tatcaccgtt tgtgtgaaca acgaactgaa ctggcagact atcccgccgg
gaatggtgat 720taccgacgaa aacggcaaga aaaagcagtc ttacttccat
gatttcttta actatgccgg 780aatccatcgc agcgtaatgc tctacaccac
gccgaacacc tgggtggacg atatcaccgt 840ggtgacgcat gtcgcgcaag
actgtaacca cgcgtctgtt gactggcagg tggtggccaa 900tggtgatgtc
agcgttgaac tgcgtgatgc ggatcaacag gtggttgcaa ctggacaagg
960cactagcggg actttgcaag tggtgaatcc gcacctctgg caaccgggtg
aaggttatct 1020ctatgaactg tgcgtcacag ccaaaagcca gacagagtgt
gatatctacc cgcttcgcgt 1080cggcatccgg tcagtggcag tgaagggcga
acagttcctg attaaccaca aaccgttcta 1140ctttactggc tttggtcgtc
atgaagatgc ggacttacgt ggcaaaggat tcgataacgt 1200gctgatggtg
cacgaccacg cattaatgga ctggattggg gccaactcct accgtacctc
1260gcattaccct tacgctgaag agatgctcga ctgggcagat gaacatggca
tcgtggtgat 1320tgatgaaact gctgctgtcg gctttaacct ctctttaggc
attggtttcg aagcgggcaa 1380caagccgaaa gaactgtaca gcgaagaggc
agtcaacggg gaaactcagc aagcgcactt 1440acaggcgatt aaagagctga
tagcgcgtga caaaaaccac ccaagcgtgg tgatgtggag 1500tattgccaac
gaaccggata cccgtccgca agtgcacggg aatatttcgc cactggcgga
1560agcaacgcgt aaactcgacc cgacgcgtcc gatcacctgc gtcaatgtaa
tgttctgcga 1620cgctcacacc gataccatca gcgatctctt tgatgtgctg
tgcctgaacc gttattacgg 1680atggtatgtc caaagcggcg atttggaaac
ggcagagaag gtactggaaa aagaacttct 1740ggcctggcag gagaaactgc
atcagccgat tatcatcacc gaatacggcg tggatacgtt 1800agccgggctg
cactcaatgt acaccgacat gtggagtgaa gagtatcagt gtgcatggct
1860ggatatgtat caccgcgtct ttgatcgcgt cagcgccgtc gtcggtgaac
aggtatggaa 1920tttcgccgat tttgcgacct cgcaaggcat attgcgcgtt
ggcggtaaca agaaagggat 1980cttcactcgc gaccgcaaac cgaagtcggc
ggcttttctg ctgcaaaaac gctggactgg 2040catgaacttc ggtgaaaaac
cgcagcaggg aggcaaacaa tgaatcaaca actctcctgg 2100cgcaccatcg
tcggctacag cctcgggaat tgctaccgag ctcggtaccc ggcgcaaaaa
2160tcaccagtct ctctctacaa atctatctct ctctattttt ctccagaata
atgtgtgagt 2220agttcccaga taagggaatt agggttctta tagggtttcg
ctcatgtgtt gagcatataa 2280gaaaccctta gtatgtattt gtatttgtaa
aatacttcta tcaataaaat ttctaattcc 2340taaaaccaaa atccagtgac
cgggtaccga gctcgaattt cgacctgcag gcatgcaagc 2400ttggcgtaat
catggtcata gctgtttcct actagatctg attgtcgttt cccgccttca
2460gtttaaacta tcagtgtttg acaggatata ttggcgggta aacctaagag
aaaagagcgt 2520ttattagaat aatcggatat ttaaaagggc gtgaaaaggt
ttatccgttc gtccatttgt 2580atgtccatga taagtcgcgc tgtatgtgtt
tgtttgaata ttcatggaac gcagtggcgg 2640ttttcatggc ttgttatgac
tgtttttttg gggtacagtc tatgcctcgg gcatccaagc 2700agcaagcgcg
ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc
2760agcagggcag tcgccctaaa acaaagttaa acatcatggg ggaagcggtg
atcgccgaag 2820tatcgactca actatcagag gtagttggcg tcatcgagcg
ccatctcgaa ccgacgttgc 2880tggccgtaca tttgtacggc tccgcagtgg
atggcggcct gaagccacac agtgatattg 2940atttgctggt tacggtgacc
gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg 3000accttttgga
aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca
3060ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc
gaactgcaat 3120ttggagaatg gcagcgcaat gacattcttg caggtatctt
cgagccagcc acgatcgaca 3180ttgatctggc
tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag
3240cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg
ctaaatgaaa 3300ccttaacgct atggaactcg ccgcccgact gggctggcga
tgagcgaaat gtagtgctta 3360cgttgtcccg catttggtac agcgcagtaa
ccggcaaaat cgcgccgaag gatgtcgctg 3420ccgactgggc aatggagcgc
ctgccggccc agtatcagcc cgtcatactt gaagctagac 3480aggcttatct
tggacaagaa gaagatcgct tggcctcgcg cgcagatcag ttggaagaat
3540ttgtccacta cgtgaaaggc gagatcacca aggtagtcgg caaataatgt
ctagctagaa 3600attcgttcaa gccgacgccg cttcgcggcg cggcttaact
caagcgttag atgcactaag 3660cacataattg ctcacagcca aactatcagg
tcaagtctgc ttttattatt tttaagcgtg 3720cataataagc cctacacaaa
ttgggagata tatcatgcat gaccaaaatc ccttaacgtg 3780agttttcgtt
ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
3840ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
ccagcggtgg 3900tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
ggtaactggc ttcagcagag 3960cgcagatacc aaatactgtc cttctagtgt
agccgtagtt aggccaccac ttcaagaact 4020ctgtagcacc gcctacatac
ctcgctctgc taatcctgtt accagtggct gctgccagtg 4080gcgataagtc
gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
4140ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
acctacaccg 4200aactgagata cctacagcgt gagctatgag aaagcgccac
gcttcccgaa gggagaaagg 4260cggacaggta tccggtaagc ggcagggtcg
gaacaggaga gcgcacgagg gagcttccag 4320ggggaaacgc ctggtatctt
tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 4380gatttttgtg
atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
4440ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
gcgttatccc 4500ctgattctgt ggataaccgt attaccgcct ttgagtgagc
tgataccgct cgccgcagcc 4560gaacgaccga gcgcagcgag tcagtgagcg
aggaagcgga agagcgcctg atgcggtatt 4620ttctccttac gcatctgtgc
ggtatttcac accgcatagg ccgcgatagg ccgacgcgaa 4680gcggcggggc
gtagggagcg cagcgaccga agggtaggcg ctttttgcag ctcttcggct
4740gtgcgctggc cagacagtta tgcacaggcc aggcgggttt taagagtttt
aataagtttt 4800aaagagtttt aggcggaaaa atcgcctttt ttctctttta
tatcagtcac ttacatgtgt 4860gaccggttcc caatgtacgg ctttgggttc
ccaatgtacg ggttccggtt cccaatgtac 4920ggctttgggt tcccaatgta
cgtgctatcc acaggaaaga gaccttttcg acctttttcc 4980cctgctaggg
caatttgccc tagcatctgc tccgtacatt aggaaccggc ggatgcttcg
5040ccctcgatca ggttgcggta gcgcatgact aggatcgggc cagcctgccc
cgcctcctcc 5100ttcaaatcgt actccggcag gtcatttgac ccgatcagct
tgcgcacggt gaaacagaac 5160ttcttgaact ctccggcgct gccactgcgt
tcgtagatcg tcttgaacaa ccatctggct 5220tctgccttgc ctgcggcgcg
gcgtgccagg cggtagagaa aacggccgat gccgggatcg 5280atcaaaaagt
aatcggggtg aaccgtcagc acgtccgggt tcttgccttc tgtgatctcg
5340cggtacatcc aatcagctag ctcgatctcg atgtactccg gccgcccggt
ttcgctcttt 5400acgatcttgt agcggctaat caaggcttca ccctcggata
ccgtcaccag gcggccgttc 5460ttggccttct tcgtacgctg catggcaacg
tgcgtggtgt ttaaccgaat gcaggtttct 5520accaggtcgt ctttctgctt
tccgccatcg gctcgccggc agaacttgag tacgtccgca 5580acgtgtggac
ggaacacgcg gccgggcttg tctcccttcc cttcccggta tcggttcatg
5640gattcggtta gatgggaaac cgccatcagt accaggtcgt aatcccacac
actggccatg 5700ccggccggcc ctgcggaaac ctctacgtgc ccgtctggaa
gctcgtagcg gatcacctcg 5760ccagctcgtc ggtcacgctt cgacagacgg
aaaacggcca cgtccatgat gctgcgacta 5820tcgcgggtgc ccacgtcata
gagcatcgga acgaaaaaat ctggttgctc gtcgcccttg 5880ggcggcttcc
taatcgacgg cgcaccggct gccggcggtt gccgggattc tttgcggatt
5940cgatcagcgg ccccttgcca cgattcaccg gggcgtgctt ctgcctcgat
gcgttgccgc 6000tgggcggcct gcgcggcctt caacttctcc accaggtcat
cacccagcgc cgcgccgatt 6060tgtaccgggc cggatggttt gcgaccgctc
acgccgattc ctcgggcttg ggggttccag 6120tgccattgca gggccggcag
acaacccagc cgcttacgcc tggccaaccg cccgttcctc 6180cacacatggg
gcattccacg gcgtcggtgc ctggttgttc ttgattttcc atgccgcctc
6240ctttagccgc taaaattcat ctactcattt attcatttgc tcatttactc
tggtagctgc 6300gcgatgtatt cagatagcag ctcggtaatg gtcttgcctt
ggcgtaccgc gtacatcttc 6360agcttggtgt gatcctccgc cggcaactga
aagttgaccc gcttcatggc tggcgtgtct 6420gccaggctgg ccaacgttgc
agccttgctg ctgcgtgcgc tcggacggcc ggcacttagc 6480gtgtttgtgc
ttttgctcat tttctcttta cctcattaac tcaaatgagt tttgatttaa
6540tttcagcggc cagcgcctgg acctcgcggg cagcgtcgcc ctcgggttct
gattcaagaa 6600cggttgtgcc ggcggcggca gtgcctgggt agctcacgcg
ctgcgtgata cgggactcaa 6660gaatgggcag ctcgtacccg gccagcgcct
cggcaacctc accgccgatg cgcgtgcctt 6720tgatcgcccg cgacacgaca
aaggccgctt gtagccttcc atccgtgacc tcaatgcgct 6780gcttaaccag
ctccaccagg tcggcggtgg cccatatgtc gtaagggctt ggctgcaccg
6840gaatcagcac gaagtcggct gccttgatcg cggacacagc caagtccgcc
gcctggggcg 6900ctccgtcgat cactacgaag tcgcgccggc cgatggcctt
cacgtcgcgg tcaatcgtcg 6960ggcggtcgat gccgacaacg gttagcggtt
gatcttcccg cacggccgcc caatcgcggg 7020cactgccctg gggatcggaa
tcgactaaca gaacatcggc cccggcgagt tgcagggcgc 7080gggctagatg
ggttgcgatg gtcgtcttgc ctgacccgcc tttctggtta agtacagcga
7140taaccttcat gcgttcccct tgcgtatttg tttatttact catcgcatca
tatacgcagc 7200gaccgcatga cgcaagctgt tttactcaaa tacacatcac
ctttttagac gcgtggtgat 7260tttgtgccga gctgccggtc ggggagctgt
tggctggctg gtggcaggat atattgtggt 7320gtaaacaaat tgacgcttag
acaacttaat aacacattgc ggacgtcttt aatgtactga 7380attaacatcc
gtttgatact tgtctaaaat tggctgattt cgagtgcatc tatgcataaa
7440aacaatctaa tgacaattat taccaagcag tgatcctgtc aaacactgat
agtttaaact 7500gaaggcggga aacgacaatc tgatcatgag cggagaatta
agggagtcac gttatgaccc 7560ccgccgatga cgcgggacaa gccgttttac
gtttggaact gacagaaccg caacgttgaa 7620ggagccactc agccgcgggt
ttctggagtt taatgagcta agcacatacg tcagaaacca 7680ttattgcgcg
ttcaaaagtc gcctaaggtc actatcagct agcaaatatt tcttgtcaaa
7740aatgctccac tgacgttcca taaattcccc tcggtatcca attagagtct
catattcact 7800ctcaatccaa ataatctgca ccggatctgg atcgtttcgc
atgattgaac aagatggatt 7860gcacgcaggt tctccggccg cttgggtgga
gaggctattc ggctatgact gggcacaaca 7920gacaatcggc tgctctgatg
ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct 7980ttttgtcaag
accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
8040atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
tcactgaagc 8100gggaagggac tggctgctat tgggcgaagt gccggggcag
gatctcctgt catctcacct 8160tgctcctgcc gagaaagtat ccatcatggc
tgatgcaatg cggcggctgc atacgcttga 8220tccggctacc tgcccattcg
accaccaagc gaaacatcgc atcgagcgag cacgtactcg 8280gatggaagcc
ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
8340agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
tcgtcgtgac 8400acatggcgat gcctgcttgc cgaatatcat ggtggaaaat
ggccgctttt ctggattcat 8460cgactgtggc cggctgggtg tggcggaccg
ctatcaggac atagcgttgg ctacccgtga 8520tattgctgaa gagcttggcg
gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc 8580cgctcccgat
tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
8640acccaagctc tagatcttgc tgcgttcgga tattttcgtg gagttcccgc
cacagacccg 8700gatgatcccc gatcgttcaa acatttggca ataaagtttc
ttaagattga atcctgttgc 8760cggtcttgcg atgattatca tataatttct
gttgaattac gttaagcatg taataattaa 8820catgtaatgc atgacgttat
ttatgagatg ggtttttatg attagagtcc cgcaattata 8880catttaatac
gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc
8940ggtgtcatct atgttactag atcgggcctc ctgtcaagct ctgagt
89861481106DNAArabidopsis thalianapromoter(1)..(1106)transcription
regulating sequence from Arabidopsis thaliana gene At3g15510
148agcgagtatt tgattttatg tgttagtgta aatggcgaaa gcgatatata
tcttgaaatt 60atgtacaaat tttttgtttt gttaagatat atcgttttcg acgttaattc
cagcctcctc 120tccattgtcg tagttcgtcc gtgtcgcgag gctatacata
aaagtcataa acgcagagaa 180agaaaattga tatttaattc aatggttaat
tgtagctatt atgtaattag caatgactaa 240tctaacctta tgcagtttgg
ttaatttagt aattatacac agactcgaca tgattagtca 300ggctttgtat
atgtgtcgca ttgtcgccac attaagatcc tttagtgcaa ccaaaacaca
360ctaaactcgg ctgaaatatg gggcatcttg taccttttaa ttatcgctga
ttactgattc 420acatcaaata tatggttcca aattatttta ttaactcaag
tgtatagtaa tgtttgactc 480ttttttctgc aaatgtttcg aagacaaagt
gctattaagt tttatcaaaa ctatgatgtt 540ttgtagtttt agatgattat
ataacgtttg tcaatctgtc ttttaattta cttaaatcat 600tagggcaggt
aactatattt aaataaaaaa cagagattgt gtatgagttt gaaataaaaa
660cgtgttaaaa tggaaaaaat aaaccatgcc aagtatttac tttactacaa
aggaaaaagt 720atacagaatt tttacatgat gtcgtatgaa ttacatcatg
taataatagt aacttagata 780ttctgtaaga aaattagatt tgcataatta
cgttaatatc cgagggtgta tataaataag 840aaagaaagaa cagaggatta
aaacccacaa attggcgttt tatacatccc catttaaaaa 900gaatggtaac
gccgactctg tgaagcattt ttcagcactc ctttcctttc ccccacaaac
960taaacaaaaa accattaaca aaaaaaccat tcacaaaaaa aatagagaga
atcatctgag 1020agagtagtag tgatgatatg atcgcttctt ctcctacaat
ctcagaaacc tccgatcacg 1080gttttagata tcttctacaa cggata
11061491115DNAArabidopsis thalianapromoter(1)..(1115)transcription
regulating sequence from Arabidopsis thaliana gene At3g15510
149aagatatagc gagtatttga ttttatgtgt tagtgtaaat ggcgaaagcg
atatatatct 60tgaaattatg tacaaatttt ttgttttgtt aagatatatc gttttcgacg
ttaattccag 120cctcctctcc attgtcgtag ttcgtccgtg tcgcgaggct
atacataaaa gtcataaacg 180cagagaaaga aaattgatat ttaattcaat
ggttaattgt agctattatg taattagcaa 240tgactaatct aaccttatgc
agtttggtta atttagtaat tatacacaga ctcgacatga 300ttagtcaggc
tttgtatatg tgtcgcattg tcgccacatt aagatccttt agtgcaacca
360aaacacacta aactcggctg aaatatgggg catcttgtac cttttaatta
tcgctgatta 420ctgattcaca tcaaatatat ggttccaaat tattttatta
actcaagtgt atagtaatgt 480ttgactcttt tttctgcaaa tgtttcgaag
acaaagtgct attaagtttt atcaaaacta 540tgatgttttg tagttttaga
tgattatata acgtttgtca atctgtcttt taatttactt 600aaatcattag
ggcaggtaac tatatttaaa taaaaaacag agattgtgta tgagtttgaa
660ataaaaacgt gttaaaatgg aaaaaataaa ccatgccaag tatttacttt
actacaaagg 720aaaaagtata cagaattttt acatgatgtc gtatgaatta
catcatgtaa taatagtaac 780ttagatattc tgtaagaaaa ttagatttgc
ataattacgt taatatccga gggtgtatat 840aaataagaaa gaaagaacag
aggattaaaa cccacaaatt ggcgttttat acatccccat 900ttaaaaagaa
tggtaacgcc gactctgtga agcatttttc agcactcctt tcctttcccc
960cacaaactaa acaaaaaacc attaacaaaa aaaccattca caaaaaaaat
agagagaatc 1020atctgagaga gtagtagtga tgatatgatc gcttcttctc
ctacaatctc agaaacctcc 1080gatcacggtt ttagatatct tctacaacgg ataca
1115150923DNAArabidopsis thalianapromoter(1)..(923)transcription
regulating sequence from Arabidopsis thaliana gene At3g15510
150agcgagtatt tgattttatg tgttagtgta aatggcgaaa gcgatatata
tcttgaaatt 60atgtacaaat tttttgtttt gttaagatat atcgttttcg acgttaattc
cagcctcctc 120tccattgtcg tagttcgtcc gtgtcgcgag gctatacata
aaagtcataa acgcagagaa 180agaaaattga tatttaattc aatggttaat
tgtagctatt atgtaattag caatgactaa 240tctaacctta tgcagtttgg
ttaatttagt aattatacac agactcgaca tgattagtca 300ggctttgtat
atgtgtcgca ttgtcgccac attaagatcc tttagtgcaa ccaaaacaca
360ctaaactcgg ctgaaatatg gggcatcttg taccttttaa ttatcgctga
ttactgattc 420acatcaaata tatggttcca aattatttta ttaactcaag
tgtatagtaa tgtttgactc 480ttttttctgc aaatgtttcg aagacaaagt
gctattaagt tttatcaaaa ctatgatgtt 540ttgtagtttt agatgattat
ataacgtttg tcaatctgtc ttttaattta cttaaatcat 600tagggcaggt
aactatattt aaataaaaaa cagagattgt gtatgagttt gaaataaaaa
660cgtgttaaaa tggaaaaaat aaaccatgcc aagtatttac tttactacaa
aggaaaaagt 720atacagaatt tttacatgat gtcgtatgaa ttacatcatg
taataatagt aacttagata 780ttctgtaaga aaattagatt tgcataatta
cgttaatatc cgagggtgta tataaataag 840aaagaaagaa cagaggatta
aaacccacaa attggcgttt tatacatccc catttaaaaa 900gaatggtaac
gccgactctg tga 923151930DNAArabidopsis
thalianapromoter(1)..(930)transcription regulating sequence from
Arabidopsis thaliana gene At3g15510 151aagatatagc gagtatttga
ttttatgtgt tagtgtaaat ggcgaaagcg atatatatct 60tgaaattatg tacaaatttt
ttgttttgtt aagatatatc gttttcgacg ttaattccag 120cctcctctcc
attgtcgtag ttcgtccgtg tcgcgaggct atacataaaa gtcataaacg
180cagagaaaga aaattgatat ttaattcaat ggttaattgt agctattatg
taattagcaa 240tgactaatct aaccttatgc agtttggtta atttagtaat
tatacacaga ctcgacatga 300ttagtcaggc tttgtatatg tgtcgcattg
tcgccacatt aagatccttt agtgcaacca 360aaacacacta aactcggctg
aaatatgggg catcttgtac cttttaatta tcgctgatta 420ctgattcaca
tcaaatatat ggttccaaat tattttatta actcaagtgt atagtaatgt
480ttgactcttt tttctgcaaa tgtttcgaag acaaagtgct attaagtttt
atcaaaacta 540tgatgttttg tagttttaga tgattatata acgtttgtca
atctgtcttt taatttactt 600aaatcattag ggcaggtaac tatatttaaa
taaaaaacag agattgtgta tgagtttgaa 660ataaaaacgt gttaaaatgg
aaaaaataaa ccatgccaag tatttacttt actacaaagg 720aaaaagtata
cagaattttt acatgatgtc gtatgaatta catcatgtaa taatagtaac
780ttagatattc tgtaagaaaa ttagatttgc ataattacgt taatatccga
gggtgtatat 840aaataagaaa gaaagaacag aggattaaaa cccacaaatt
ggcgttttat acatccccat 900ttaaaaagaa tggtaacgcc gactctgtga
9301521845DNAArabidopsis thalianapromoter(1)..(1845)transcription
regulating sequence from Arabidopsis thaliana gene At3g15510
152aagtgctcta aactgacaaa actggagaaa tagtaggctg tatttgattt
tgtgaaccaa 60ccaaaaaacc tttgtatttg atttataata ttgtgtgttt taaacataaa
gaaaaagata 120tgagataagg aatctcatgg tcagaagtca aaactagaaa
gtcataaaat aaaccactcc 180taagaatctg ataagtaaac aaactatata
tttgcacact aaacgtaatt tagtggtcga 240aaaatatgca tgggttataa
tcatcttatg acgttggaaa aaaaacaaga ataaatcata 300taataatgaa
aatataaaag taatcgaaac ttaaaaatga catgaaattg gtcgatttgt
360ttgtttccat aatatttatt atctaaaatt caaaaaatat tctggtggtg
cgattatctc 420aataacaaaa gcgtgacatg tgacatctga aacgtaaaac
tttttttttt ttcccacacc 480cacccggcca tctttttcca cgtcatcaat
ttgttacctt ctctggcgat ttctgtgggc 540cctactgccc cgattgccac
gtatgaatat atcgtccata ggtatcacac gtgtcgatta 600ttcattggat
atttaacaag aacagtaacc acacaaaatt aataaaatcg tattacagca
660tacaagatga aattagaaaa tttcttcaac ttttttttgt tttgcgatat
tgtcttgata 720atgtattata gtaagatata gcgagtattt gattttatgt
gttagtgtaa atggcgaaag 780cgatatatat cttgaaatta tgtacaaatt
ttttgttttg ttaagatata tcgttttcga 840cgttaattcc agcctcctct
ccattgtcgt agttcgtccg tgtcgcgagg ctatacataa 900aagtcataaa
cgcagagaaa gaaaattgat atttaattca atggttaatt gtagctatta
960tgtaattagc aatgactaat ctaaccttat gcagtttggt taatttagta
attatacaca 1020gactcgacat gattagtcag gctttgtata tgtgtcgcat
tgtcgccaca ttaagatcct 1080ttagtgcaac caaaacacac taaactcggc
tgaaatatgg ggcatcttgt accttttaat 1140tatcgctgat tactgattca
catcaaatat atggttccaa attattttat taactcaagt 1200gtatagtaat
gtttgactct tttttctgca aatgtttcga agacaaagtg ctattaagtt
1260ttatcaaaac tatgatgttt tgtagtttta gatgattata taacgtttgt
caatctgtct 1320tttaatttac ttaaatcatt agggcaggta actatattta
aataaaaaac agagattgtg 1380tatgagtttg aaataaaaac gtgttaaaat
ggaaaaaata aaccatgcca agtatttact 1440ttactacaaa ggaaaaagta
tacagaattt ttacatgatg tcgtatgaat tacatcatgt 1500aataatagta
acttagatat tctgtaagaa aattagattt gcataattac gttaatatcc
1560gagggtgtat ataaataaga aagaaagaac agaggattaa aacccacaaa
ttggcgtttt 1620atacatcccc atttaaaaag aatggtaacg ccgactctgt
gaagcatttt tcagcactcc 1680tttcctttcc cccacaaact aaacaaaaaa
ccattaacaa aaaaaccatt cacaaaaaaa 1740atagagagaa tcatctgaga
gagtagtagt gatgatatga tcgcttcttc tcctacaatc 1800tcagaaacct
ccgatcacgg ttttagatat cttctacaac ggata 18451531854DNAArabidopsis
thalianapromoter(1)..(1854)transcription regulating sequence from
Arabidopsis thaliana gene At3g15510 153tcaaacaaag tgctctaaac
tgacaaaact ggagaaatag taggctgtat ttgattttgt 60gaaccaacca aaaaaccttt
gtatttgatt tataatattg tgtgttttaa acataaagaa 120aaagatatga
gataaggaat ctcatggtca gaagtcaaaa ctagaaagtc ataaaataaa
180ccactcctaa gaatctgata agtaaacaaa ctatatattt gcacactaaa
cgtaatttag 240tggtcgaaaa atatgcatgg gttataatca tcttatgacg
ttggaaaaaa aacaagaata 300aatcatataa taatgaaaat ataaaagtaa
tcgaaactta aaaatgacat gaaattggtc 360gatttgtttg tttccataat
atttattatc taaaattcaa aaaatattct ggtggtgcga 420ttatctcaat
aacaaaagcg tgacatgtga catctgaaac gtaaaacttt tttttttttc
480ccacacccac ccggccatct ttttccacgt catcaatttg ttaccttctc
tggcgatttc 540tgtgggccct actgccccga ttgccacgta tgaatatatc
gtccataggt atcacacgtg 600tcgattattc attggatatt taacaagaac
agtaaccaca caaaattaat aaaatcgtat 660tacagcatac aagatgaaat
tagaaaattt cttcaacttt tttttgtttt gcgatattgt 720cttgataatg
tattatagta agatatagcg agtatttgat tttatgtgtt agtgtaaatg
780gcgaaagcga tatatatctt gaaattatgt acaaattttt tgttttgtta
agatatatcg 840ttttcgacgt taattccagc ctcctctcca ttgtcgtagt
tcgtccgtgt cgcgaggcta 900tacataaaag tcataaacgc agagaaagaa
aattgatatt taattcaatg gttaattgta 960gctattatgt aattagcaat
gactaatcta accttatgca gtttggttaa tttagtaatt 1020atacacagac
tcgacatgat tagtcaggct ttgtatatgt gtcgcattgt cgccacatta
1080agatccttta gtgcaaccaa aacacactaa actcggctga aatatggggc
atcttgtacc 1140ttttaattat cgctgattac tgattcacat caaatatatg
gttccaaatt attttattaa 1200ctcaagtgta tagtaatgtt tgactctttt
ttctgcaaat gtttcgaaga caaagtgcta 1260ttaagtttta tcaaaactat
gatgttttgt agttttagat gattatataa cgtttgtcaa 1320tctgtctttt
aatttactta aatcattagg gcaggtaact atatttaaat aaaaaacaga
1380gattgtgtat gagtttgaaa taaaaacgtg ttaaaatgga aaaaataaac
catgccaagt 1440atttacttta ctacaaagga aaaagtatac agaattttta
catgatgtcg tatgaattac 1500atcatgtaat aatagtaact tagatattct
gtaagaaaat tagatttgca taattacgtt 1560aatatccgag ggtgtatata
aataagaaag aaagaacaga ggattaaaac ccacaaattg 1620gcgttttata
catccccatt taaaaagaat ggtaacgccg actctgtgaa gcatttttca
1680gcactccttt cctttccccc acaaactaaa caaaaaacca ttaacaaaaa
aaccattcac 1740aaaaaaaata gagagaatca tctgagagag tagtagtgat
gatatgatcg cttcttctcc 1800tacaatctca gaaacctccg atcacggttt
tagatatctt ctacaacgga taca 18541541662DNAArabidopsis
thalianapromoter(1)..(1662)transcription regulating sequence from
Arabidopsis thaliana gene At3g15510 154aagtgctcta aactgacaaa
actggagaaa tagtaggctg tatttgattt tgtgaaccaa 60ccaaaaaacc tttgtatttg
atttataata ttgtgtgttt taaacataaa gaaaaagata 120tgagataagg
aatctcatgg tcagaagtca aaactagaaa gtcataaaat aaaccactcc
180taagaatctg ataagtaaac aaactatata tttgcacact aaacgtaatt
tagtggtcga 240aaaatatgca tgggttataa tcatcttatg acgttggaaa
aaaaacaaga ataaatcata 300taataatgaa aatataaaag taatcgaaac
ttaaaaatga catgaaattg gtcgatttgt 360ttgtttccat aatatttatt
atctaaaatt caaaaaatat tctggtggtg cgattatctc 420aataacaaaa
gcgtgacatg tgacatctga aacgtaaaac tttttttttt ttcccacacc
480cacccggcca tctttttcca cgtcatcaat ttgttacctt ctctggcgat
ttctgtgggc 540cctactgccc cgattgccac gtatgaatat atcgtccata
ggtatcacac gtgtcgatta 600ttcattggat atttaacaag aacagtaacc
acacaaaatt aataaaatcg tattacagca 660tacaagatga aattagaaaa
tttcttcaac ttttttttgt tttgcgatat tgtcttgata 720atgtattata
gtaagatata gcgagtattt gattttatgt gttagtgtaa atggcgaaag
780cgatatatat cttgaaatta tgtacaaatt ttttgttttg ttaagatata
tcgttttcga 840cgttaattcc agcctcctct ccattgtcgt agttcgtccg
tgtcgcgagg ctatacataa 900aagtcataaa cgcagagaaa gaaaattgat
atttaattca atggttaatt gtagctatta 960tgtaattagc aatgactaat
ctaaccttat gcagtttggt taatttagta attatacaca 1020gactcgacat
gattagtcag gctttgtata tgtgtcgcat tgtcgccaca ttaagatcct
1080ttagtgcaac caaaacacac taaactcggc tgaaatatgg ggcatcttgt
accttttaat 1140tatcgctgat tactgattca catcaaatat atggttccaa
attattttat taactcaagt 1200gtatagtaat gtttgactct tttttctgca
aatgtttcga agacaaagtg ctattaagtt 1260ttatcaaaac tatgatgttt
tgtagtttta gatgattata taacgtttgt caatctgtct 1320tttaatttac
ttaaatcatt agggcaggta actatattta aataaaaaac agagattgtg
1380tatgagtttg aaataaaaac gtgttaaaat ggaaaaaata aaccatgcca
agtatttact 1440ttactacaaa ggaaaaagta tacagaattt ttacatgatg
tcgtatgaat tacatcatgt 1500aataatagta acttagatat tctgtaagaa
aattagattt gcataattac gttaatatcc 1560gagggtgtat ataaataaga
aagaaagaac agaggattaa aacccacaaa ttggcgtttt 1620atacatcccc
atttaaaaag aatggtaacg ccgactctgt ga 16621551669DNAArabidopsis
thalianapromoter(1)..(1669)transcription regulating sequence from
Arabidopsis thaliana gene At3g15510 155tcaaacaaag tgctctaaac
tgacaaaact ggagaaatag taggctgtat ttgattttgt 60gaaccaacca aaaaaccttt
gtatttgatt tataatattg tgtgttttaa acataaagaa 120aaagatatga
gataaggaat ctcatggtca gaagtcaaaa ctagaaagtc ataaaataaa
180ccactcctaa gaatctgata agtaaacaaa ctatatattt gcacactaaa
cgtaatttag 240tggtcgaaaa atatgcatgg gttataatca tcttatgacg
ttggaaaaaa aacaagaata 300aatcatataa taatgaaaat ataaaagtaa
tcgaaactta aaaatgacat gaaattggtc 360gatttgtttg tttccataat
atttattatc taaaattcaa aaaatattct ggtggtgcga 420ttatctcaat
aacaaaagcg tgacatgtga catctgaaac gtaaaacttt tttttttttc
480ccacacccac ccggccatct ttttccacgt catcaatttg ttaccttctc
tggcgatttc 540tgtgggccct actgccccga ttgccacgta tgaatatatc
gtccataggt atcacacgtg 600tcgattattc attggatatt taacaagaac
agtaaccaca caaaattaat aaaatcgtat 660tacagcatac aagatgaaat
tagaaaattt cttcaacttt tttttgtttt gcgatattgt 720cttgataatg
tattatagta agatatagcg agtatttgat tttatgtgtt agtgtaaatg
780gcgaaagcga tatatatctt gaaattatgt acaaattttt tgttttgtta
agatatatcg 840ttttcgacgt taattccagc ctcctctcca ttgtcgtagt
tcgtccgtgt cgcgaggcta 900tacataaaag tcataaacgc agagaaagaa
aattgatatt taattcaatg gttaattgta 960gctattatgt aattagcaat
gactaatcta accttatgca gtttggttaa tttagtaatt 1020atacacagac
tcgacatgat tagtcaggct ttgtatatgt gtcgcattgt cgccacatta
1080agatccttta gtgcaaccaa aacacactaa actcggctga aatatggggc
atcttgtacc 1140ttttaattat cgctgattac tgattcacat caaatatatg
gttccaaatt attttattaa 1200ctcaagtgta tagtaatgtt tgactctttt
ttctgcaaat gtttcgaaga caaagtgcta 1260ttaagtttta tcaaaactat
gatgttttgt agttttagat gattatataa cgtttgtcaa 1320tctgtctttt
aatttactta aatcattagg gcaggtaact atatttaaat aaaaaacaga
1380gattgtgtat gagtttgaaa taaaaacgtg ttaaaatgga aaaaataaac
catgccaagt 1440atttacttta ctacaaagga aaaagtatac agaattttta
catgatgtcg tatgaattac 1500atcatgtaat aatagtaact tagatattct
gtaagaaaat tagatttgca taattacgtt 1560aatatccgag ggtgtatata
aataagaaag aaagaacaga ggattaaaac ccacaaattg 1620gcgttttata
catccccatt taaaaagaat ggtaacgccg actctgtga
16691561625DNAArabidopsis thalianaCDS(186)..(1280)encoding
Arabidopsis thaliana no apical meristem (NAM) family protein (NAC2)
156agcatttttc agcactcctt tcctttcccc cacaaactaa acaaaaaacc
attaacaaaa 60aaaccattca caaaaaaaat agagagaatc atctgagaga gtagtagtga
tgatatgatc 120gcttcttctc ctacaatctc agaaacctcc gatcacggtt
ttagatatct tctacaacgg 180ataca atg gag agc acc gat tct tcc ggt ggt
cca cca ccg cca caa cct 230 Met Glu Ser Thr Asp Ser Ser Gly Gly Pro
Pro Pro Pro Gln Pro 1 5 10 15aac ctt cct cca ggc ttc cgg ttt cac
cct acc gac gaa gag ctt gtt 278Asn Leu Pro Pro Gly Phe Arg Phe His
Pro Thr Asp Glu Glu Leu Val 20 25 30gtt cac tac ctc aaa cgc aaa gca
gcc tct gct cct tta cct gtc gcc 326Val His Tyr Leu Lys Arg Lys Ala
Ala Ser Ala Pro Leu Pro Val Ala 35 40 45atc atc gcc gaa gtc gat ctc
tat aaa ttt gat cca tgg gaa ctt ccc 374Ile Ile Ala Glu Val Asp Leu
Tyr Lys Phe Asp Pro Trp Glu Leu Pro 50 55 60gct aaa gca tcg ttt gga
gaa caa gaa tgg tac ttc ttt agt cca cga 422Ala Lys Ala Ser Phe Gly
Glu Gln Glu Trp Tyr Phe Phe Ser Pro Arg 65 70 75gat cgg aag tat cca
aac gga gca aga cca aac aga gcg gcg act tca 470Asp Arg Lys Tyr Pro
Asn Gly Ala Arg Pro Asn Arg Ala Ala Thr Ser80 85 90 95ggt tat tgg
aaa gcg acc ggt aca gat aaa ccg gta ctt gct tcc gac 518Gly Tyr Trp
Lys Ala Thr Gly Thr Asp Lys Pro Val Leu Ala Ser Asp 100 105 110ggt
aac caa aag gtg ggc gtg aag aag gca cta gtc ttc tac agt ggt 566Gly
Asn Gln Lys Val Gly Val Lys Lys Ala Leu Val Phe Tyr Ser Gly 115 120
125aaa cca cca aaa ggc gtt aaa agt gat tgg atc atg cat gag tat cgt
614Lys Pro Pro Lys Gly Val Lys Ser Asp Trp Ile Met His Glu Tyr Arg
130 135 140ctc atc gaa aac aaa cca aac aat cga cct cct ggc tgt gat
ttc ggc 662Leu Ile Glu Asn Lys Pro Asn Asn Arg Pro Pro Gly Cys Asp
Phe Gly 145 150 155aac aaa aaa aac tca ctc aga ctt gat gat tgg gtg
tta tgt aga atc 710Asn Lys Lys Asn Ser Leu Arg Leu Asp Asp Trp Val
Leu Cys Arg Ile160 165 170 175tac aag aag aac aac gca agt cga cat
gtt gat aac gat aag gat cat 758Tyr Lys Lys Asn Asn Ala Ser Arg His
Val Asp Asn Asp Lys Asp His 180 185 190gat atg atc gat tac att ttc
agg aag att cct ccg tct tta tca atg 806Asp Met Ile Asp Tyr Ile Phe
Arg Lys Ile Pro Pro Ser Leu Ser Met 195 200 205gcg gct gct tct aca
gga ctt cac caa cat cat cat aat gtc tca aga 854Ala Ala Ala Ser Thr
Gly Leu His Gln His His His Asn Val Ser Arg 210 215 220tca atg aat
ttc ttc cct ggc aaa ttc tcc ggt ggt ggt tac ggg att 902Ser Met Asn
Phe Phe Pro Gly Lys Phe Ser Gly Gly Gly Tyr Gly Ile 225 230 235ttc
tct gac ggt ggt aac acg agt ata tac gac ggc ggt ggc atg atc 950Phe
Ser Asp Gly Gly Asn Thr Ser Ile Tyr Asp Gly Gly Gly Met Ile240 245
250 255aac aat att ggt act gac tca gta gat cac gac aat aac gct gac
gtc 998Asn Asn Ile Gly Thr Asp Ser Val Asp His Asp Asn Asn Ala Asp
Val 260 265 270gtt ggt tta aat cat gct tcg tcg tca ggt cct atg atg
atg gcg aat 1046Val Gly Leu Asn His Ala Ser Ser Ser Gly Pro Met Met
Met Ala Asn 275 280 285ttg aaa cga act ctc ccg gtg ccg tat tgg cct
gta gca gat gag gag 1094Leu Lys Arg Thr Leu Pro Val Pro Tyr Trp Pro
Val Ala Asp Glu Glu 290 295 300caa gat gca tct ccg agc aaa cgg ttt
cac ggt gta gga gga gga gga 1142Gln Asp Ala Ser Pro Ser Lys Arg Phe
His Gly Val Gly Gly Gly Gly 305 310 315gga gat tgt tcg aac atg tct
tcc tcc atg atg gaa gag act cca cca 1190Gly Asp Cys Ser Asn Met Ser
Ser Ser Met Met Glu Glu Thr Pro Pro320 325 330 335ttg atg caa caa
caa ggt ggt gtg tta gga gat gga tta ttc aga acg 1238Leu Met Gln Gln
Gln Gly Gly Val Leu Gly Asp Gly Leu Phe Arg Thr 340 345 350aca tcg
tac caa tta ccc ggt tta aat tgg tac tct tct taa 1280Thr Ser Tyr Gln
Leu Pro Gly Leu Asn Trp Tyr Ser Ser 355 360tcaaatgtgt ttcgccgccg
gtgtgaagaa ttttccggtg acagtgaaga tttttttccg 1340attggtgggg
tcatttgcat gcattatata atttgagatt tgtgtatatg ttttgggtta
1400attaattggt cacaggggtt agaggaagaa gattatttag agagcggaga
agaagtgagg 1460caacatatag aaaacatagg tttaaaaaat attactgttt
gttaagttta atgattaaat 1520gtataaatct tatacttaga tgtttatata
tagtttgtat agatgcatat agtttctctc 1580aatgctattt tgagattgta
ttttcttgta aaggaaatgt ttttt 1625157364PRTArabidopsis thaliana
157Met Glu Ser Thr Asp Ser Ser Gly Gly Pro Pro Pro Pro Gln Pro Asn1
5 10 15Leu Pro Pro Gly Phe Arg Phe His Pro Thr Asp Glu Glu Leu Val
Val 20 25 30His Tyr Leu Lys Arg Lys Ala Ala Ser Ala Pro Leu Pro Val
Ala Ile 35 40 45Ile Ala Glu Val Asp Leu Tyr Lys Phe Asp Pro Trp Glu
Leu Pro Ala 50 55 60Lys Ala Ser Phe Gly Glu Gln Glu Trp Tyr Phe Phe
Ser Pro Arg Asp65 70 75 80Arg Lys Tyr Pro Asn Gly Ala Arg Pro Asn
Arg Ala Ala Thr Ser Gly 85 90 95Tyr Trp Lys Ala Thr Gly Thr Asp Lys
Pro Val Leu Ala Ser Asp Gly 100 105 110Asn Gln Lys Val Gly Val Lys
Lys Ala Leu Val Phe Tyr Ser Gly Lys 115 120 125Pro Pro Lys Gly Val
Lys Ser Asp Trp Ile Met His Glu Tyr Arg Leu 130 135 140Ile Glu Asn
Lys Pro Asn Asn Arg Pro Pro Gly Cys Asp Phe Gly Asn145 150 155
160Lys Lys Asn Ser Leu Arg Leu Asp Asp Trp Val Leu Cys Arg Ile Tyr
165 170 175Lys Lys Asn Asn Ala Ser Arg His Val Asp Asn Asp Lys Asp
His Asp 180 185 190Met Ile Asp Tyr Ile Phe Arg Lys Ile Pro Pro Ser
Leu Ser Met Ala 195 200 205Ala Ala Ser Thr Gly Leu His Gln His His
His Asn Val Ser Arg Ser 210 215 220Met Asn Phe Phe Pro Gly Lys Phe
Ser Gly Gly Gly Tyr Gly Ile Phe225 230 235 240Ser Asp Gly Gly Asn
Thr Ser Ile Tyr Asp Gly Gly Gly Met Ile Asn 245 250 255Asn Ile Gly
Thr Asp Ser Val Asp His Asp Asn Asn Ala Asp Val Val 260 265 270Gly
Leu Asn His Ala Ser Ser Ser Gly Pro Met Met Met Ala Asn Leu 275 280
285Lys Arg Thr Leu Pro Val Pro Tyr Trp Pro Val Ala Asp Glu Glu Gln
290 295 300Asp Ala Ser Pro Ser Lys Arg Phe His Gly Val Gly Gly Gly
Gly Gly305 310 315 320Asp Cys Ser Asn Met Ser Ser Ser Met Met Glu
Glu Thr Pro Pro Leu 325 330 335Met Gln Gln Gln Gly Gly Val Leu Gly
Asp Gly Leu Phe Arg Thr Thr 340 345 350Ser Tyr Gln Leu Pro Gly Leu
Asn Trp Tyr Ser Ser 355 3601581017DNAArabidopsis
thalianapromoter(1)..(1017)transcription regulating sequence from
Arabidopsis thaliana gene At3g50870 158ttcttcttct tctacgtttt
ttattttctg ttgaattttc ctccacaaaa tcgataattt 60caattttttc atacaatatc
atagttctag taaagtaaac attcgtgtgt gagtgacata 120ataattgctt
caaaaagaaa taacgaatta ataagagttg tgtgcttgat ggtccaaaag
180tcgtatgttt ttcaccatca caataagatt taccatatct acttaaacca
aacttcaatc 240tatgatattt ggaagctagc aatgaatgat tttgttttag
catatgttac cgagcctatt 300catctttaga ttgacattcg cattttaaag
ttttgtgtcc aacaactagc aaacccttcg 360ctttttaaac caagatcagt
ttttctacat agtttcgact ttttgttatt ttacttccca 420tcatattttg
ttttatttac tactgacatt ttcataatgt acggtacatt ttcatatatt
480agcatcctta tctaaaagct aatcaactcc ataacgtact tgaaataggg
atattttagt 540tgagttaatt attcaaaaca atcacacagt gacacagact
ctatgtagac caagcatgtg 600ttatgtaatt agaaaaaata aactgtgatt
aaaatatctt ttaacttgtt aacttggctg 660cctaaagaaa cttgatactg
tatcatcgtt catcgtattt tgtaattaat aaaacataaa 720aaagcaagga
aaacccattt gagtaaatga tatattaata ttacttcgta aaaagctgcg
780tccccacata ttctattgat tttaaatact attcgtatta atataaacct
gcatataagc 840gtacatatac atttctgatt ctatatatag gtgtgagacc
ttcttctcca ttctacactt 900cactccaacg tgaaacctgc aaactgaact
tatctcttca tattttcttt agcttctctt 960aattttacac ttagggtttt
caaaccagta gagagagaga gaagtgtgta agagcga 10171591008DNAArabidopsis
thalianapromoter(1)..(1008)transcription regulating sequence from
Arabidopsis thaliana gene At3g50870 159cactctctcc ttcttcttct
tctacgtttt ttattttctg ttaaatttcc tccacaaaat 60cgataatttc aattttttca
tacaatatca tagttctagt aaagtaaaca ttcgtgtgtg 120agtgacataa
taattgcttc aaaaagaaat aacgaattaa taagagttgt gtgcttgatg
180gtccaaaagt cgtatgtttt tcaccatcac aataagattt accatatcta
cttaaaccaa 240acttcaatct atgatatttg gaagctagca atgaatgatt
ttgttttagc atatgttacc 300gagcctattc atctttagat tgaaagtttt
gtgtccaaca actagcaaac ccttcgcttt 360ttaaaccaag atcagttttt
ctacatagtt tcgacttttt gttattttac ttcccatcat 420attttgtttt
atttactact gacattttca taatgtacgg tacattttca tatattagca
480tccttatcta aaagctaatc aactccataa cgtacttgaa atagggatat
tttagttgaa 540ttattcaaaa caatcacaca gtgacacaga ctctatgtag
accaagcatg tgttatgtaa 600ttagaaaaaa taaactgtga ttaaaatatc
ttttaacttg ttaacttggc tgcctaaaga 660aacttgatac tgtatcatcg
ttcatcgtat tttgtaatta ataaaacata aaaaagcaag 720gaaaacccat
ttgagtaaat gatatattaa tattacttcg taaaaagctg cgtccccaca
780tattctattg attttaaata ctattcgtat taatataaac ctgcatataa
gcgtacatat 840acatttctga ttctatatat aggtgtgaga ccttcttctc
cattctacac ttcactccaa 900cgtgaaacct gcaaactgaa cttatctctt
catattttct ttagcttctc ttaattttac 960acttagggtt ttcaaaccag
tagagagaga gagaagtgtg taagagcg 1008160894DNAArabidopsis
thalianapromoter(1)..(894)transcription regulating sequence from
Arabidopsis thaliana gene At3g50870 160ttcttcttct tctacgtttt
ttattttctg ttgaattttc ctccacaaaa tcgataattt 60caattttttc atacaatatc
atagttctag taaagtaaac attcgtgtgt gagtgacata 120ataattgctt
caaaaagaaa taacgaatta ataagagttg tgtgcttgat ggtccaaaag
180tcgtatgttt ttcaccatca caataagatt taccatatct acttaaacca
aacttcaatc 240tatgatattt ggaagctagc aatgaatgat tttgttttag
catatgttac cgagcctatt 300catctttaga ttgacattcg cattttaaag
ttttgtgtcc aacaactagc aaacccttcg 360ctttttaaac caagatcagt
ttttctacat agtttcgact ttttgttatt ttacttccca 420tcatattttg
ttttatttac tactgacatt ttcataatgt acggtacatt ttcatatatt
480agcatcctta tctaaaagct aatcaactcc ataacgtact tgaaataggg
atattttagt 540tgagttaatt attcaaaaca atcacacagt gacacagact
ctatgtagac caagcatgtg 600ttatgtaatt agaaaaaata aactgtgatt
aaaatatctt ttaacttgtt aacttggctg 660cctaaagaaa cttgatactg
tatcatcgtt catcgtattt tgtaattaat aaaacataaa 720aaagcaagga
aaacccattt gagtaaatga tatattaata ttacttcgta aaaagctgcg
780tccccacata ttctattgat tttaaatact attcgtatta atataaacct
gcatataagc 840gtacatatac atttctgatt ctatatatag gtgtgagacc
ttcttctcca ttct 894161886DNAArabidopsis
thalianapromoter(1)..(886)transcription regulating sequence from
Arabidopsis thaliana gene At3g50870 161cactctctcc ttcttcttct
tctacgtttt ttattttctg ttaaatttcc tccacaaaat 60cgataatttc aattttttca
tacaatatca tagttctagt aaagtaaaca ttcgtgtgtg 120agtgacataa
taattgcttc aaaaagaaat aacgaattaa taagagttgt gtgcttgatg
180gtccaaaagt cgtatgtttt tcaccatcac aataagattt accatatcta
cttaaaccaa 240acttcaatct atgatatttg gaagctagca atgaatgatt
ttgttttagc atatgttacc 300gagcctattc atctttagat tgaaagtttt
gtgtccaaca actagcaaac ccttcgcttt 360ttaaaccaag atcagttttt
ctacatagtt tcgacttttt gttattttac ttcccatcat 420attttgtttt
atttactact gacattttca taatgtacgg tacattttca tatattagca
480tccttatcta aaagctaatc aactccataa cgtacttgaa atagggatat
tttagttgaa 540ttattcaaaa caatcacaca gtgacacaga ctctatgtag
accaagcatg tgttatgtaa 600ttagaaaaaa taaactgtga ttaaaatatc
ttttaacttg ttaacttggc tgcctaaaga 660aacttgatac tgtatcatcg
ttcatcgtat tttgtaatta ataaaacata aaaaagcaag 720gaaaacccat
ttgagtaaat gatatattaa tattacttcg taaaaagctg cgtccccaca
780tattctattg attttaaata ctattcgtat taatataaac ctgcatataa
gcgtacatat 840acatttctga ttctatatat aggtgtgaga ccttcttctc cattct
8861622017DNAArabidopsis thalianapromoter(1)..(2017)transcription
regulating sequence from Arabidopsis thaliana gene At3g50870
162atcaatagaa gagtaattaa tcgttccatc ggaacatccg atgaaattgg
aacgagacct 60atacagaaga aaacaaaaga gcaactaatc atcttgatca catgcccaat
atgcatttaa 120gtaaattgga gagaatgata tagaaaaaca agacattatc
tacaactaca agatatcaaa 180gcactcggtt aaccgcgtcc ctgtgaagtg
ttagttatca gtacatgaat tgtgatttca 240gcatcttggt cctttttttt
ttggacaacc gtcatcttta ttcttgatcg gtttttataa 300cgtacaaaca
tatactattt caacagtttt cttttaattt ttcttttttt ttgaatatac
360actttctttc ttttattttt ttcattacaa aattttcaaa aaatagtgtt
cggcaaaaaa 420aaaaattaga tcggccaatg cattatatcc cctagcgtaa
gctctttttc aaccaatcga 480gagtccatgt tttcaataaa aatgggtttt
attagtaaca gtagttaaag catttaacat 540acgtaatccc aaaaaaataa
aataattctg tatagggacg aagactagta ttcgttgacg 600acaagagtag
agtagactct agctagctgc aagttttgaa gaaatctaat aaccaaatct
660aagtcatttt acttacatca tcatggatca tcattaacca catgaggctg
agcagatatc 720gaacgtgtga ttgcgatgtg gagaagtaaa ggacgaggcc
acgcgcaact gagcaggagc 780gcgtgagatt cagggtttta gggcagagat
acggaaagag acagaggttt tatagcatta 840tattgaaaga gaacaaataa
aaataaaaat atgagcctcg tgatcggaga tttttgcagt 900gatgactgta
ctcttcttcc tcttctctcc cccttgtcac gtgggctaaa ttttgaccac
960ccttcttcaa tgaccatcaa catcacatca cactctctcc ttcttcttct
tctacgtttt 1020ttattttctg ttgaattttc ctccacaaaa tcgataattt
caattttttc atacaatatc 1080atagttctag taaagtaaac attcgtgtgt
gagtgacata ataattgctt caaaaagaaa
1140taacgaatta ataagagttg tgtgcttgat ggtccaaaag tcgtatgttt
ttcaccatca 1200caataagatt taccatatct acttaaacca aacttcaatc
tatgatattt ggaagctagc 1260aatgaatgat tttgttttag catatgttac
cgagcctatt catctttaga ttgacattcg 1320cattttaaag ttttgtgtcc
aacaactagc aaacccttcg ctttttaaac caagatcagt 1380ttttctacat
agtttcgact ttttgttatt ttacttccca tcatattttg ttttatttac
1440tactgacatt ttcataatgt acggtacatt ttcatatatt agcatcctta
tctaaaagct 1500aatcaactcc ataacgtact tgaaataggg atattttagt
tgagttaatt attcaaaaca 1560atcacacagt gacacagact ctatgtagac
caagcatgtg ttatgtaatt agaaaaaata 1620aactgtgatt aaaatatctt
ttaacttgtt aacttggctg cctaaagaaa cttgatactg 1680tatcatcgtt
catcgtattt tgtaattaat aaaacataaa aaagcaagga aaacccattt
1740gagtaaatga tatattaata ttacttcgta aaaagctgcg tccccacata
ttctattgat 1800tttaaatact attcgtatta atataaacct gcatataagc
gtacatatac atttctgatt 1860ctatatatag gtgtgagacc ttcttctcca
ttctacactt cactccaacg tgaaacctgc 1920aaactgaact tatctcttca
tattttcttt agcttctctt aattttacac ttagggtttt 1980caaaccagta
gagagagaga gaagtgtgta agagcga 20171632004DNAArabidopsis
thalianapromoter(1)..(2004)transcription regulating sequence from
Arabidopsis thaliana gene At3g50870 163tataaacatc aatagaagag
taattaatcg ttccatcgga acatccgatg aaattggaac 60gagacctata cagaagaaaa
caaaagagca actaatcatc ttgatcacat gcccaatatg 120catttaagta
aattggagag aatgatatag aaaaacaaga cattatctac aactacaaga
180tatcaaagca ctcggttaac cgcgtccctg tgaagtgtta gttatcagta
catgaattgt 240gatttcagca tcttggtcct tttttttttg gacaaccgtc
atctttttct tgatcggttt 300ttataacgta caaacatata ctatttcaac
agttttcttt taattttttt tttttttgaa 360tatacacttt ctttctttta
tttgtttgat tacaaaattt tcaaaaaata gtgttcggca 420aaaaaaaaaa
ttagatcggc caatgcatta tatcccctag cgtaagctct ttttcaacca
480atcgagagtc catgttttca ataaaaatag ggttttatta gtaacagtag
ttaaagcatt 540taacatacgt aatcccaaaa aaataaaata attctgtata
gggacgaaga ctagtattcg 600ttgacgacaa gagtagagta gactctagct
agctgcaagt tttgaagaaa tctaataacc 660aaatctaagt cattttactt
acatcatcat ggatcatcat taaccacatg aggctgagca 720gatatcgaac
gtgtgattgc gatgtggaga agtaaaggac gaggccacgc gcaactgagc
780aggagcgcgt gagattcagg gttttagggc agagatacgg aaagagacag
aggttttata 840gcattatatt gaaagagaac aaataaaaat aaaaatatga
gcctcgtgat cggagatttt 900tgcagtgatg actgtactct tcttcctctt
ctctccccct tgtcacgtgg gctaaatttt 960gaccaccctt cttcaatgac
catcaacatc gcatcacact ctctccttct tcttcttcta 1020cgttttttat
tttctgttaa atttcctcca caaaatcgat aatttcaatt ttttcataca
1080atatcatagt tctagtaaag taaacattcg tgtgtgagtg acataataat
tgcttcaaaa 1140agaaataacg aattaataag agttgtgtgc ttgatggtcc
aaaagtcgta tgtttttcac 1200catcacaata agatttacca tatctactta
aaccaaactt caatctatga tatttggaag 1260ctagcaatga atgattttgt
tttagcatat gttaccgagc ctattcatct ttagattgaa 1320agttttgtgt
ccaacaacta gcaaaccctt cgctttttaa accaagatca gtttttctac
1380atagtttcga ctttttgtta ttttacttcc catcatattt tgttttattt
actactgaca 1440ttttcataat gtacggtaca ttttcatata ttagcatcct
tatctaaaag ctaatcaact 1500ccataacgta cttgaaatag ggatatttta
gttgaattat tcaaaacaat cacacagtga 1560cacagactct atgtagacca
agcatgtgtt atgtaattag aaaaaataaa ctgtgattaa 1620aatatctttt
aacttgttaa cttggctgcc taaagaaact tgatactgta tcatcgttca
1680tcgtattttg taattaataa aacataaaaa agcaaggaaa acccatttga
gtaaatgata 1740tattaatatt acttcgtaaa aagctgcgtc cccacatatt
ctattgattt taaatactat 1800tcgtattaat ataaacctgc atataagcgt
acatatacat ttctgattct atatataggt 1860gtgagacctt cttctccatt
ctacacttca ctccaacgtg aaacctgcaa actgaactta 1920tctcttcata
ttttctttag cttctcttaa ttttacactt agggttttca aaccagtaga
1980gagagagaga agtgtgtaag agcg 20041641894DNAArabidopsis
thalianapromoter(1)..(1894)transcription regulating sequence from
Arabidopsis thaliana gene At3g50870 164atcaatagaa gagtaattaa
tcgttccatc ggaacatccg atgaaattgg aacgagacct 60atacagaaga aaacaaaaga
gcaactaatc atcttgatca catgcccaat atgcatttaa 120gtaaattgga
gagaatgata tagaaaaaca agacattatc tacaactaca agatatcaaa
180gcactcggtt aaccgcgtcc ctgtgaagtg ttagttatca gtacatgaat
tgtgatttca 240gcatcttggt cctttttttt ttggacaacc gtcatcttta
ttcttgatcg gtttttataa 300cgtacaaaca tatactattt caacagtttt
cttttaattt ttcttttttt ttgaatatac 360actttctttc ttttattttt
ttcattacaa aattttcaaa aaatagtgtt cggcaaaaaa 420aaaaattaga
tcggccaatg cattatatcc cctagcgtaa gctctttttc aaccaatcga
480gagtccatgt tttcaataaa aatgggtttt attagtaaca gtagttaaag
catttaacat 540acgtaatccc aaaaaaataa aataattctg tatagggacg
aagactagta ttcgttgacg 600acaagagtag agtagactct agctagctgc
aagttttgaa gaaatctaat aaccaaatct 660aagtcatttt acttacatca
tcatggatca tcattaacca catgaggctg agcagatatc 720gaacgtgtga
ttgcgatgtg gagaagtaaa ggacgaggcc acgcgcaact gagcaggagc
780gcgtgagatt cagggtttta gggcagagat acggaaagag acagaggttt
tatagcatta 840tattgaaaga gaacaaataa aaataaaaat atgagcctcg
tgatcggaga tttttgcagt 900gatgactgta ctcttcttcc tcttctctcc
cccttgtcac gtgggctaaa ttttgaccac 960ccttcttcaa tgaccatcaa
catcacatca cactctctcc ttcttcttct tctacgtttt 1020ttattttctg
ttgaattttc ctccacaaaa tcgataattt caattttttc atacaatatc
1080atagttctag taaagtaaac attcgtgtgt gagtgacata ataattgctt
caaaaagaaa 1140taacgaatta ataagagttg tgtgcttgat ggtccaaaag
tcgtatgttt ttcaccatca 1200caataagatt taccatatct acttaaacca
aacttcaatc tatgatattt ggaagctagc 1260aatgaatgat tttgttttag
catatgttac cgagcctatt catctttaga ttgacattcg 1320cattttaaag
ttttgtgtcc aacaactagc aaacccttcg ctttttaaac caagatcagt
1380ttttctacat agtttcgact ttttgttatt ttacttccca tcatattttg
ttttatttac 1440tactgacatt ttcataatgt acggtacatt ttcatatatt
agcatcctta tctaaaagct 1500aatcaactcc ataacgtact tgaaataggg
atattttagt tgagttaatt attcaaaaca 1560atcacacagt gacacagact
ctatgtagac caagcatgtg ttatgtaatt agaaaaaata 1620aactgtgatt
aaaatatctt ttaacttgtt aacttggctg cctaaagaaa cttgatactg
1680tatcatcgtt catcgtattt tgtaattaat aaaacataaa aaagcaagga
aaacccattt 1740gagtaaatga tatattaata ttacttcgta aaaagctgcg
tccccacata ttctattgat 1800tttaaatact attcgtatta atataaacct
gcatataagc gtacatatac atttctgatt 1860ctatatatag gtgtgagacc
ttcttctcca ttct 18941651882DNAArabidopsis
thalianapromoter(1)..(1882)transcription regulating sequence from
Arabidopsis thaliana gene At3g50870 165tataaacatc aatagaagag
taattaatcg ttccatcgga acatccgatg aaattggaac 60gagacctata cagaagaaaa
caaaagagca actaatcatc ttgatcacat gcccaatatg 120catttaagta
aattggagag aatgatatag aaaaacaaga cattatctac aactacaaga
180tatcaaagca ctcggttaac cgcgtccctg tgaagtgtta gttatcagta
catgaattgt 240gatttcagca tcttggtcct tttttttttg gacaaccgtc
atctttttct tgatcggttt 300ttataacgta caaacatata ctatttcaac
agttttcttt taattttttt tttttttgaa 360tatacacttt ctttctttta
tttgtttgat tacaaaattt tcaaaaaata gtgttcggca 420aaaaaaaaaa
ttagatcggc caatgcatta tatcccctag cgtaagctct ttttcaacca
480atcgagagtc catgttttca ataaaaatag ggttttatta gtaacagtag
ttaaagcatt 540taacatacgt aatcccaaaa aaataaaata attctgtata
gggacgaaga ctagtattcg 600ttgacgacaa gagtagagta gactctagct
agctgcaagt tttgaagaaa tctaataacc 660aaatctaagt cattttactt
acatcatcat ggatcatcat taaccacatg aggctgagca 720gatatcgaac
gtgtgattgc gatgtggaga agtaaaggac gaggccacgc gcaactgagc
780aggagcgcgt gagattcagg gttttagggc agagatacgg aaagagacag
aggttttata 840gcattatatt gaaagagaac aaataaaaat aaaaatatga
gcctcgtgat cggagatttt 900tgcagtgatg actgtactct tcttcctctt
ctctccccct tgtcacgtgg gctaaatttt 960gaccaccctt cttcaatgac
catcaacatc gcatcacact ctctccttct tcttcttcta 1020cgttttttat
tttctgttaa atttcctcca caaaatcgat aatttcaatt ttttcataca
1080atatcatagt tctagtaaag taaacattcg tgtgtgagtg acataataat
tgcttcaaaa 1140agaaataacg aattaataag agttgtgtgc ttgatggtcc
aaaagtcgta tgtttttcac 1200catcacaata agatttacca tatctactta
aaccaaactt caatctatga tatttggaag 1260ctagcaatga atgattttgt
tttagcatat gttaccgagc ctattcatct ttagattgaa 1320agttttgtgt
ccaacaacta gcaaaccctt cgctttttaa accaagatca gtttttctac
1380atagtttcga ctttttgtta ttttacttcc catcatattt tgttttattt
actactgaca 1440ttttcataat gtacggtaca ttttcatata ttagcatcct
tatctaaaag ctaatcaact 1500ccataacgta cttgaaatag ggatatttta
gttgaattat tcaaaacaat cacacagtga 1560cacagactct atgtagacca
agcatgtgtt atgtaattag aaaaaataaa ctgtgattaa 1620aatatctttt
aacttgttaa cttggctgcc taaagaaact tgatactgta tcatcgttca
1680tcgtattttg taattaataa aacataaaaa agcaaggaaa acccatttga
gtaaatgata 1740tattaatatt acttcgtaaa aagctgcgtc cccacatatt
ctattgattt taaatactat 1800tcgtattaat ataaacctgc atataagcgt
acatatacat ttctgattct atatataggt 1860gtgagacctt cttctccatt ct
18821661249DNAArabidopsis thalianaCDS(123)..(1010)encoding
Arabidopsis thaliana zinc finger (GATA type) family protein
166acacttcact ccaacgtgaa acctgcaaac tgaacttatc tcttcatatt
ttctttagct 60tctcttaatt ttacacttag ggttttcaaa ccagtagaga gagagagaag
tgtgtaagag 120cg atg atg cag act ccg tac act act tca acg cag ggg
caa tat tgt 167 Met Met Gln Thr Pro Tyr Thr Thr Ser Thr Gln Gly Gln
Tyr Cys 1 5 10 15cat tct tgt gga atg ttc cac cac cat agc caa agc
tgc tgc tac aac 215His Ser Cys Gly Met Phe His His His Ser Gln Ser
Cys Cys Tyr Asn 20 25 30aac aac aac aac tcc aac gcc ggt tct tac tcg
atg gtc ttc tcc atg 263Asn Asn Asn Asn Ser Asn Ala Gly Ser Tyr Ser
Met Val Phe Ser Met 35 40 45caa aac ggt ggc gtt ttc gag cag aac ggt
gag gac tat cat cac tct 311Gln Asn Gly Gly Val Phe Glu Gln Asn Gly
Glu Asp Tyr His His Ser 50 55 60tcc tcc ctc gtt gac tgc act ctc tct
ctt gga act cct tct acg agg 359Ser Ser Leu Val Asp Cys Thr Leu Ser
Leu Gly Thr Pro Ser Thr Arg 65 70 75ctt tgt gag gaa gat gag aaa cgt
aga cgc tct act tca tct ggt gct 407Leu Cys Glu Glu Asp Glu Lys Arg
Arg Arg Ser Thr Ser Ser Gly Ala80 85 90 95tct tct tgc atc tcc aac
ttt tgg gac ttg att cac acc aaa aac aac 455Ser Ser Cys Ile Ser Asn
Phe Trp Asp Leu Ile His Thr Lys Asn Asn 100 105 110aac tcc aaa acg
gca ccg tac aat aac gtt cct tct ttc tcc gct aac 503Asn Ser Lys Thr
Ala Pro Tyr Asn Asn Val Pro Ser Phe Ser Ala Asn 115 120 125aag cca
agt cgc ggt tgt tcc ggt ggt ggt ggt ggc gga gga ggc ggt 551Lys Pro
Ser Arg Gly Cys Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly 130 135
140ggc gga ggt gac tct ctt ctc gct aga cgc tgt gcc aac tgt gac act
599Gly Gly Gly Asp Ser Leu Leu Ala Arg Arg Cys Ala Asn Cys Asp Thr
145 150 155act tct act cca cta tgg agg aat ggt cct aga ggc cct aag
tcc cta 647Thr Ser Thr Pro Leu Trp Arg Asn Gly Pro Arg Gly Pro Lys
Ser Leu160 165 170 175tgc aac gca tgc ggc att cgt ttc aag aag gaa
gag aga aga act act 695Cys Asn Ala Cys Gly Ile Arg Phe Lys Lys Glu
Glu Arg Arg Thr Thr 180 185 190gcg gct aca gga aac acc gtc gtc gga
gct gca ccg gtt caa acc gac 743Ala Ala Thr Gly Asn Thr Val Val Gly
Ala Ala Pro Val Gln Thr Asp 195 200 205cag tac ggg cat cac aac tct
ggc tac aat aat tac cat gct gcc act 791Gln Tyr Gly His His Asn Ser
Gly Tyr Asn Asn Tyr His Ala Ala Thr 210 215 220aat aac aac aat aat
aat ggt act ccg tgg gct cat cac cac tcg acg 839Asn Asn Asn Asn Asn
Asn Gly Thr Pro Trp Ala His His His Ser Thr 225 230 235cag agg gtt
ccg tgt aat tat ccg gca aat gag atc agg ttc atg gat 887Gln Arg Val
Pro Cys Asn Tyr Pro Ala Asn Glu Ile Arg Phe Met Asp240 245 250
255gat tac ggc agt gga gta gca aac aac gtt gaa tcc gac ggt gct cac
935Asp Tyr Gly Ser Gly Val Ala Asn Asn Val Glu Ser Asp Gly Ala His
260 265 270ggc ggt gtt ccg ttc ctt tct tgg agg ctt aat gta gcg gat
agg gca 983Gly Gly Val Pro Phe Leu Ser Trp Arg Leu Asn Val Ala Asp
Arg Ala 275 280 285agt ctt gtc cat gac ttt acc aga tga aaggagaata
gaaaacgacg 1030Ser Leu Val His Asp Phe Thr Arg 290 295ttagattctc
ttaaaatttc catgtggttt aattatgtct aattaaaaaa aaaccaagaa
1090aatatttctg aaaaaaatag aagtgtatat cgagagacga tgttgatgtt
tattgatagt 1150gacggtctaa cttatggttt atctataaag taccgttctg
gtcaattttt aaaatcattt 1210tctaatattt attttaaaaa tataaccttc
tttttatgg 1249167295PRTArabidopsis thaliana 167Met Met Gln Thr Pro
Tyr Thr Thr Ser Thr Gln Gly Gln Tyr Cys His1 5 10 15Ser Cys Gly Met
Phe His His His Ser Gln Ser Cys Cys Tyr Asn Asn 20 25 30Asn Asn Asn
Ser Asn Ala Gly Ser Tyr Ser Met Val Phe Ser Met Gln 35 40 45Asn Gly
Gly Val Phe Glu Gln Asn Gly Glu Asp Tyr His His Ser Ser 50 55 60Ser
Leu Val Asp Cys Thr Leu Ser Leu Gly Thr Pro Ser Thr Arg Leu65 70 75
80Cys Glu Glu Asp Glu Lys Arg Arg Arg Ser Thr Ser Ser Gly Ala Ser
85 90 95Ser Cys Ile Ser Asn Phe Trp Asp Leu Ile His Thr Lys Asn Asn
Asn 100 105 110Ser Lys Thr Ala Pro Tyr Asn Asn Val Pro Ser Phe Ser
Ala Asn Lys 115 120 125Pro Ser Arg Gly Cys Ser Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly 130 135 140Gly Gly Asp Ser Leu Leu Ala Arg Arg
Cys Ala Asn Cys Asp Thr Thr145 150 155 160Ser Thr Pro Leu Trp Arg
Asn Gly Pro Arg Gly Pro Lys Ser Leu Cys 165 170 175Asn Ala Cys Gly
Ile Arg Phe Lys Lys Glu Glu Arg Arg Thr Thr Ala 180 185 190Ala Thr
Gly Asn Thr Val Val Gly Ala Ala Pro Val Gln Thr Asp Gln 195 200
205Tyr Gly His His Asn Ser Gly Tyr Asn Asn Tyr His Ala Ala Thr Asn
210 215 220Asn Asn Asn Asn Asn Gly Thr Pro Trp Ala His His His Ser
Thr Gln225 230 235 240Arg Val Pro Cys Asn Tyr Pro Ala Asn Glu Ile
Arg Phe Met Asp Asp 245 250 255Tyr Gly Ser Gly Val Ala Asn Asn Val
Glu Ser Asp Gly Ala His Gly 260 265 270Gly Val Pro Phe Leu Ser Trp
Arg Leu Asn Val Ala Asp Arg Ala Ser 275 280 285Leu Val His Asp Phe
Thr Arg 290 2951681300DNAArabidopsis
thalianapromoter(1)..(1300)transcription regulating sequence from
Arabidopsis thaliana gene At4g00220 168atccgcactg actagactat
acattacata tatcttgtaa tttcggaaag ttcaaatatt 60tgtgaaatat atgcactagt
tggagtttcc ttatttgtac cttaatcgaa tattgagtaa 120acatgattcc
ctgacccaaa tatagattgc gtacatcaac ttattaatac cgtcaataaa
180tacgagtttt tgcatataat cattacatgg tcatatgtaa taaatctagt
tgttttggaa 240attgcatatt ctctccctcc ctctttatca attttacata
tatgtagcat cagcaatata 300tgtgaatttg ttacattagt agtttagttt
gtattttaat ttttttggat aaattatgaa 360ttctattacc aaaattgaac
attgtttttt acaaatactt tggggcagtg aatttctaaa 420accaaaaaaa
aaacagttag taatttgtat aggtattatc gccgtggtat acacttaatt
480caaatatcat aatatacttc gtgtcatata cgacaaaagt aaaattaaag
tttgcaaaaa 540aaattttatt caaagaatct aacggaaaaa aaccgcttaa
attttctaga agatattgcc 600aaatgttgat aaaatatctg attaaccaaa
aagaaaataa cgcatagcgg tgagccgtta 660catttcgact acaaagttat
cacattaaaa taccgccaaa aattagtaat taattagatt 720atatatatat
atttaagctc attttataca tttatttgtt tgtaaaatta atttctcata
780gtattagatt gaatgttcat atattacaat tggtatatac cataagatag
ttgtgtggtt 840cgatgtaaca aaaataaaat taaaccagaa gtagaaaaag
tgaaggaaag ggaataacaa 900tgaagaagtt gaaagatatg gaatgttttg
cttcacgcaa agagcttaac acgagacaag 960tacgctccat cgcgttgctt
tgaagtcttt tttttcttct tcttcttctt gtcttaacct 1020cttttttttt
ttttagtctt ggtaataaca agactaatta aattattatt ttactttttt
1080tattatctac caacaaccaa accctagttt tgcttacgtc cgatctcctc
cttctctctt 1140ttcttctctc ccaaacgttg cctttctttt ttttctctaa
tcacgaacca aaaagcatca 1200taatctcgat gagttaaatc catgtatctc
aaaaccctaa tcaccactta gctagtaaaa 1260ggaaaggaaa ataaaaagga
cttgtgtggt agaaaaggac 13001691337DNAArabidopsis
thalianapromoter(1)..(1337)transcription regulating sequence from
Arabidopsis thaliana gene At4g00220 169aatatattgg agatccgcac
tgactagact atacattaca tagatcttgt aatttcggaa 60agttcaaata tttgtgaaat
atatgcacta gttggagttt ccttatttgt accttaatcg 120aatattgagt
aaacatgatt ccctgaccca aatatagatt gcgtacatca actcattaat
180accgtcaata aatacgagtt tttgcatata atcattacat ggtcatatgt
aataaatcta 240gttgttttgg aaattgcata ttctctccct ccctctttat
caattttaca tatatgtagc 300atcagcaata tatgtgaatt tgttacatta
gtagtttagt ttgtatttta attttttgga 360taaattatga attctattac
caaaattgaa cattgttttt tacaaatact ttggggcagt 420gaatttctaa
aaccaaaaaa aaaaagttag taatttgtat aggtattatc gccgtggtat
480acacttaatt caaatatcat aatatacatg ttcgtgtcat atacgacaaa
agtaaaatta 540aagtttgcaa aaaaaatttt atttaaagaa tctaacggaa
aaaaaccgct taaattttct 600agaaaatgtt gccaaatgtt gataaaatat
ccgattaacc aaaaagaaaa taacgcatag 660cggtgaaccg ttacatttcg
actacaaagt tatcacatta aaatcaccgc caaaaattag 720taattaatta
gattatatat atatatatat atatatatat atatatattt aagctcattt
780tatacattta tttgtttgta aaattaattt ctcatagtat tagattgaat
gttcatatat 840tacaattggt atataccata agatagttgt gtggttcgat
gtaacaaaaa taaaattaaa 900ccagaagtag aaaaagtgaa ggaaagggaa
taacaatgaa gaagttgaaa gatatggaat 960gttttgcttc acgcaaagag
cttaacacga gacaagtacg ctccatcgcg ttgctttgaa 1020gtcttttttt
tcttcttctt cttcttgtct taacctcttt tttatttttt agtcttggta
1080ataacaagac taattaaatt attattttac
tttttttatt atctaccaac aaccaaaccc 1140tagttttgct tacgtccgat
ctcctccttc tctcttttct tctctcccaa acgttccctt 1200tctttttttt
ctctaatcac gaaccaaaaa gcatcataat ctcgatgagt taaatccatg
1260tatctcaaaa ccctaatcac cacttagcta gtaaaaggaa aggaaaataa
aaaggacttg 1320tgtggtagaa aaggaag 13371701190DNAArabidopsis
thalianapromoter(1)..(1190)transcription regulating sequence from
Arabidopsis thaliana gene At4g00220 170atccgcactg actagactat
acattacata tatcttgtaa tttcggaaag ttcaaatatt 60tgtgaaatat atgcactagt
tggagtttcc ttatttgtac cttaatcgaa tattgagtaa 120acatgattcc
ctgacccaaa tatagattgc gtacatcaac ttattaatac cgtcaataaa
180tacgagtttt tgcatataat cattacatgg tcatatgtaa taaatctagt
tgttttggaa 240attgcatatt ctctccctcc ctctttatca attttacata
tatgtagcat cagcaatata 300tgtgaatttg ttacattagt agtttagttt
gtattttaat ttttttggat aaattatgaa 360ttctattacc aaaattgaac
attgtttttt acaaatactt tggggcagtg aatttctaaa 420accaaaaaaa
aaacagttag taatttgtat aggtattatc gccgtggtat acacttaatt
480caaatatcat aatatacttc gtgtcatata cgacaaaagt aaaattaaag
tttgcaaaaa 540aaattttatt caaagaatct aacggaaaaa aaccgcttaa
attttctaga agatattgcc 600aaatgttgat aaaatatctg attaaccaaa
aagaaaataa cgcatagcgg tgagccgtta 660catttcgact acaaagttat
cacattaaaa taccgccaaa aattagtaat taattagatt 720atatatatat
atttaagctc attttataca tttatttgtt tgtaaaatta atttctcata
780gtattagatt gaatgttcat atattacaat tggtatatac cataagatag
ttgtgtggtt 840cgatgtaaca aaaataaaat taaaccagaa gtagaaaaag
tgaaggaaag ggaataacaa 900tgaagaagtt gaaagatatg gaatgttttg
cttcacgcaa agagcttaac acgagacaag 960tacgctccat cgcgttgctt
tgaagtcttt tttttcttct tcttcttctt gtcttaacct 1020cttttttttt
ttttagtctt ggtaataaca agactaatta aattattatt ttactttttt
1080tattatctac caacaaccaa accctagttt tgcttacgtc cgatctcctc
cttctctctt 1140ttcttctctc ccaaacgttg cctttctttt ttttctctaa
tcacgaacca 11901711226DNAArabidopsis
thalianapromoter(1)..(1226)transcription regulating sequence from
Arabidopsis thaliana gene At4g00220 171aatatattgg agatccgcac
tgactagact atacattaca tagatcttgt aatttcggaa 60agttcaaata tttgtgaaat
atatgcacta gttggagttt ccttatttgt accttaatcg 120aatattgagt
aaacatgatt ccctgaccca aatatagatt gcgtacatca actcattaat
180accgtcaata aatacgagtt tttgcatata atcattacat ggtcatatgt
aataaatcta 240gttgttttgg aaattgcata ttctctccct ccctctttat
caattttaca tatatgtagc 300atcagcaata tatgtgaatt tgttacatta
gtagtttagt ttgtatttta attttttgga 360taaattatga attctattac
caaaattgaa cattgttttt tacaaatact ttggggcagt 420gaatttctaa
aaccaaaaaa aaaaagttag taatttgtat aggtattatc gccgtggtat
480acacttaatt caaatatcat aatatacatg ttcgtgtcat atacgacaaa
agtaaaatta 540aagtttgcaa aaaaaatttt atttaaagaa tctaacggaa
aaaaaccgct taaattttct 600agaaaatgtt gccaaatgtt gataaaatat
ccgattaacc aaaaagaaaa taacgcatag 660cggtgaaccg ttacatttcg
actacaaagt tatcacatta aaatcaccgc caaaaattag 720taattaatta
gattatatat atatatatat atatatatat atatatattt aagctcattt
780tatacattta tttgtttgta aaattaattt ctcatagtat tagattgaat
gttcatatat 840tacaattggt atataccata agatagttgt gtggttcgat
gtaacaaaaa taaaattaaa 900ccagaagtag aaaaagtgaa ggaaagggaa
taacaatgaa gaagttgaaa gatatggaat 960gttttgcttc acgcaaagag
cttaacacga gacaagtacg ctccatcgcg ttgctttgaa 1020gtcttttttt
tcttcttctt cttcttgtct taacctcttt tttatttttt agtcttggta
1080ataacaagac taattaaatt attattttac tttttttatt atctaccaac
aaccaaaccc 1140tagttttgct tacgtccgat ctcctccttc tctcttttct
tctctcccaa acgttccctt 1200tctttttttt ctctaatcac gaacca
12261723232DNAArabidopsis thalianapromoter(1)..(3232)transcription
regulating sequence from Arabidopsis thaliana gene At4g00220
172ttttatgtca aagacgtaac gttttgtttg ggggaagttg tgtcgtcgtc
gaccacagtt 60attatatatc caatccaaag agatgtttaa gagagagaga tggatgggtt
tgtttatgaa 120taaatgtttt taagttgatg tggagcatag aggatcgtgt
caggcaagaa aatcatgtct 180tgcttttggt ggtgggtcat ttataatcat
tacatttttc tataacttga tcatcttttc 240ttttgtatgt ccatattttt
gttttttgtt ttccttggtg tgtttcactt ttctgtacat 300agttgaatca
cacactcaag aaacaagcta ttttcaaaca agtgaaagaa ttatctattt
360atggaatcct aaaatcaatg aaagcatata acacaaggag ggataacata
tacttgtaaa 420ttgaaaagta gacaaatcaa caatatatat catctaaatt
agcaatcata ggtttaagac 480ataaaaagat tttgtatatg ccactgagaa
tctagaacta actatcagtc tatcacacaa 540gcaaggccta gaaagtagtg
aacaagtaag gatttagaat tttagatagt gagtaatgaa 600gatatatata
ggctggtggg ttcagttaac aaggtactaa aatcggatta taacgaggga
660tcaagtggtg gggattatga attattatag taatgagaat gaatgtacta
gtactaatta 720gaatgagtaa agcttggaag caacggcaga gcagatttgt
gatatataca aaggacaaac 780aaacacaaat gctttgcgtg aagcaaaaca
caattagctg cttgctgacg tatttctctc 840tcaccactca gccgcaggtt
tccctgtcac acacacgtgc gatccctcac cacacgtgtg 900taattctttt
atatatgtta tatactgtat ataatttttt gtcaaactag ttctctatgg
960acagctttcg tgatagaatt atagagagaa aatcgagatg tatgttgctt
cttgtttcta 1020caaatcgtat agtcagaaac agacatgaca ctgatcactg
tttgcttccc ccgcaagtac 1080aacctcagtc agtcagttac tgtcatgtct
ctctttctct ctgtttgaca agatccctga 1140cccttgttca cttccctaat
ttaaagcctt ctccttaatg ggccgggcct tgtatctgtt 1200tcggatcctt
ctttatagcg aaatgattaa tagtagtgat tacacctaaa atgtacgtat
1260atgacaaaaa gaatagtttt tttcttctgt ttctcttgta ctattaggcc
tagtgaatgt 1320attaaataca aacagtgtac attagcgtac gcaagcggtg
aggaaaggag cagaagaggg 1380cgtgatacga ggacacgtgt ctcatgttag
tacaaacaaa gattggacaa gagccggacg 1440gcacggtgac ctctgtcaac
aactgttacc gtttcttcta atctatttgt taatctccta 1500agtaatgtaa
ttaattagtt ttcactgttc ataattaatc aagtaataat aacataagaa
1560atgaaatggg agcgttactt gtataccata tgcactcgca aacgagccat
tggttgcgtc 1620agaacggtta cgttcttcat aaaagtcaca ttcgacacat
ggcgatcata tatacgtgcc 1680aatttttccg gttctctcaa atccaatccc
ggtttctttc ttttatgttt tgatcaccgg 1740ttcttcttct aaaccaactt
agctttactc atttggtgat tattattgga ccgtcgtaat 1800cgtcactctt
ttcttttttg aatcaatgat gtgagatatg tatattatac acgcacacgt
1860acacacgtgt atttgatcat tcttgccgtc accaaatttt aaactcattt
atcgtgtaat 1920aatatattgg agatccgcac tgactagact atacattaca
tatatcttgt aatttcggaa 1980agttcaaata tttgtgaaat atatgcacta
gttggagttt ccttatttgt accttaatcg 2040aatattgagt aaacatgatt
ccctgaccca aatatagatt gcgtacatca acttattaat 2100accgtcaata
aatacgagtt tttgcatata atcattacat ggtcatatgt aataaatcta
2160gttgttttgg aaattgcata ttctctccct ccctctttat caattttaca
tatatgtagc 2220atcagcaata tatgtgaatt tgttacatta gtagtttagt
ttgtatttta atttttttgg 2280ataaattatg aattctatta ccaaaattga
acattgtttt ttacaaatac tttggggcag 2340tgaatttcta aaaccaaaaa
aaaaacagtt agtaatttgt ataggtatta tcgccgtggt 2400atacacttaa
ttcaaatatc ataatatact tcgtgtcata tacgacaaaa gtaaaattaa
2460agtttgcaaa aaaaatttta ttcaaagaat ctaacggaaa aaaaccgctt
aaattttcta 2520gaagatattg ccaaatgttg ataaaatatc tgattaacca
aaaagaaaat aacgcatagc 2580ggtgagccgt tacatttcga ctacaaagtt
atcacattaa aatcaccgcc aaaaattagt 2640aattaattag attatatata
tatatttaag ctcattttat acatttattt gtttgtaaaa 2700ttaatttctc
atagtattag attgaatgtt catatattac aattggtata taccataaga
2760tagttgtgtg gttcgatgta acaaaaataa aattaaacca gaagtagaaa
aagtgaagga 2820aagggaataa caatgaagaa gttgaaagat atggaatgtt
ttgcttcacg caaagagctt 2880aacacgagac aagtacgctc catcgcgttg
ctttgaagtc ttttttttct tcttcttctt 2940cttgtcttaa cctctttttt
tttttttagt cttggtaata acaagactaa ttaaattatt 3000attttacttt
ttttattatc taccaacaac caaaccctag ttttgcttac gtccgatctc
3060ctccttctct cttttcttct ctcccaaacg ttgcctttct tttttttctc
taatcacgaa 3120ccaaaaagca tcataatctc gatgagttaa atccatgtat
ctcaaaaccc taatcaccac 3180ttagctagta aaaggaaagg aaaataaaaa
ggacttgtgt ggtagaaaag ga 32321733269DNAArabidopsis
thalianapromoter(1)..(3269)transcription regulating sequence from
Arabidopsis thaliana gene At4g00220 173cttccgctca tcttttatgt
caaagacgta acgttttgtt tgggggaagt tgtgtcgtcg 60tcgaccacag ttattatata
tccaatccaa agagatgttt aagagagaga gatggatggg 120tttgtttatg
aataaatgtt tttaagttga tgtggagcat agaggatcgt gtcaggcaag
180aaaatcatgt cttgcttttg gtggtgggtc atttataatc attacatttt
tctataactt 240gatcatcttt tcttttgtat gtccatattt ttgttttttg
ttttccttgg tgtgtttcac 300ttttctgtac atagttgaat cacacactca
agaaacaagc tattttcaaa caagtgaaag 360aattatctat ttatggactc
ctaaaatcaa tgaaagcata taacacaagg agggataaca 420tatacttgta
aattgaaaag tagacaaatc aacaatatat atcatctaaa ttagcaatca
480taggtttaag acataaaaag attttgtata tgccactgag aatctagaac
taactatcag 540tctatcacac aagcaaggcc tagaaagtag tgaacaagta
aggatttaga attttagata 600gtgagtaatg aagatatata taggctggtg
ggttcagtta acaaggtact aaaatcggat 660tataacgagg gatcaagtgg
tggggattat gaattattat agtaatgaga atgaatgtac 720tagtactaat
tagaatgagt aaagcttgga agcaacggca gagcagattt gtgatatata
780caaaggacaa acaaacacaa atgctttgcg tgaagcaaaa cacaattagc
tgcttgctga 840cgtatttctc tctcaccact cagccgcagg tttccctgtc
acacacacgt gcgatccctc 900accacacgtg tgtaattctt ttatatatgt
tatatactgt atataatttt ttgtcaaact 960agttctctat ggacagcttt
cgtgatagaa ttatagagag aaaatcgaga tgtatgttgc 1020ttcttgtttc
tacaaatcgt atagtcagaa acagacatga cactgatcac tgtttgcttc
1080ccccgcaagt acaacctcag tcagtcagtt actgtcatgt ctctctttct
ctctgtttga 1140caagatccct gacccttgtt cacttcccta atttaaagcc
ttctccttaa tgggccgggc 1200cttgtatctg tttcggatcc ttctttatag
cgaaatgatt aatagtagtg attacaccta 1260aaatgtacgt atatgacaaa
aagaatagtt tttttcttct gtttctcttg tactattagg 1320cctagtgaat
gtattaaata caaacagtgt acattagcgt acgcaagcgg tgaggaaagg
1380agcagaagag ggcgtgatac gaggacacgt gtctcatgtt agtacaaaca
aagattggac 1440aagagccgga cggcacggtg acctctgtca acaactgtta
ccgtttcttc taatctattt 1500gttaatctcc taagtaatgt aattaattag
ttttcactgt tcataattaa tcaagtaata 1560ataacataag aaatgaaatg
ggagcgttac ttgtatacca tatgcactcg caaacgagcc 1620attggttgcg
tcagaacggt tacgttcttc ataaaagtca cattcgacac atggcgatca
1680tatatacgtg ccaatttttc cggttctctc aaatccaatc ccggtttctt
tcttttatgt 1740tttgatcacc ggttcttctt ctaaaccaac ttagctttac
tcatttggtg attattattg 1800gaccgtcgta atcgtcactc ttttcttttt
tgaatcaatg atgtgagata tgtatattat 1860acacgcacac gtacacacgt
gtatttgatc attcttgccg tcaccaaatt ttaaactcat 1920ttatcgtgta
ataatatatt ggagatccgc actgactaga ctatacatta catagatctt
1980gtaatttcgg aaagttcaaa tatttgtgaa atatatgcac tagttggagt
ttccttattt 2040gtaccttaat cgaatattga gtaaacatga ttccctgacc
caaatataga ttgcgtacat 2100caactcatta ataccgtcaa taaatacgag
tttttgcata taatcattac atggtcatat 2160gtaataaatc tagttgtttt
ggaaattgca tattctctcc ctccctcttt atcaatttta 2220catatatgta
gcatcagcaa tatatgtgaa tttgttacat tagtagttta gtttgtattt
2280taattttttg gataaattat gaattctatt accaaaattg aacattgttt
tttacaaata 2340ctttggggca gtgaatttct aaaaccaaaa aaaaaaagtt
agtaatttgt ataggtatta 2400tcgccgtggt atacacttaa ttcaaatatc
ataatataca tgttcgtgtc atatacgaca 2460aaagtaaaat taaagtttgc
aaaaaaaatt ttatttaaag aatctaacgg aaaaaaaccg 2520cttaaatttt
ctagaaaatg ttgccaaatg ttgataaaat atccgattaa ccaaaaagaa
2580aataacgcat agcggtgaac cgttacattt cgactacaaa gttatcacat
taaaatcacc 2640gccaaaaatt agtaattaat tagattatat atatatatat
atatatatat atatatatat 2700ttaagctcat tttatacatt tatttgtttg
taaaattaat ttctcatagt attagattga 2760atgttcatat attacaattg
gtatatacca taagatagtt gtgtggttcg atgtaacaaa 2820aataaaatta
aaccagaagt agaaaaagtg aaggaaaggg aataacaatg aagaagttga
2880aagatatgga atgttttgct tcacgcaaag agcttaacac gagacaagta
cgctccatcg 2940cgttgctttg aagtcttttt tttcttcttc ttcttcttgt
cttaacctct tttttatttt 3000ttagtcttgg taataacaag actaattaaa
ttattatttt acttttttta ttatctacca 3060acaaccaaac cctagttttg
cttacgtccg atctcctcct tctctctttt cttctctccc 3120aaacgttccc
tttctttttt ttctctaatc acgaaccaaa aagcatcata atctcgatga
3180gttaaatcca tgtatctcaa aaccctaatc accacttagc tagtaaaagg
aaaggaaaat 3240aaaaaggact tgtgtggtag aaaaggaag
32691743123DNAArabidopsis thalianapromoter(1)..(3123)transcription
regulating sequence from Arabidopsis thaliana gene At4g00220
174ttttatgtca aagacgtaac gttttgtttg ggggaagttg tgtcgtcgtc
gaccacagtt 60attatatatc caatccaaag agatgtttaa gagagagaga tggatgggtt
tgtttatgaa 120taaatgtttt taagttgatg tggagcatag aggatcgtgt
caggcaagaa aatcatgtct 180tgcttttggt ggtgggtcat ttataatcat
tacatttttc tataacttga tcatcttttc 240ttttgtatgt ccatattttt
gttttttgtt ttccttggtg tgtttcactt ttctgtacat 300agttgaatca
cacactcaag aaacaagcta ttttcaaaca agtgaaagaa ttatctattt
360atggaatcct aaaatcaatg aaagcatata acacaaggag ggataacata
tacttgtaaa 420ttgaaaagta gacaaatcaa caatatatat catctaaatt
agcaatcata ggtttaagac 480ataaaaagat tttgtatatg ccactgagaa
tctagaacta actatcagtc tatcacacaa 540gcaaggccta gaaagtagtg
aacaagtaag gatttagaat tttagatagt gagtaatgaa 600gatatatata
ggctggtggg ttcagttaac aaggtactaa aatcggatta taacgaggga
660tcaagtggtg gggattatga attattatag taatgagaat gaatgtacta
gtactaatta 720gaatgagtaa agcttggaag caacggcaga gcagatttgt
gatatataca aaggacaaac 780aaacacaaat gctttgcgtg aagcaaaaca
caattagctg cttgctgacg tatttctctc 840tcaccactca gccgcaggtt
tccctgtcac acacacgtgc gatccctcac cacacgtgtg 900taattctttt
atatatgtta tatactgtat ataatttttt gtcaaactag ttctctatgg
960acagctttcg tgatagaatt atagagagaa aatcgagatg tatgttgctt
cttgtttcta 1020caaatcgtat agtcagaaac agacatgaca ctgatcactg
tttgcttccc ccgcaagtac 1080aacctcagtc agtcagttac tgtcatgtct
ctctttctct ctgtttgaca agatccctga 1140cccttgttca cttccctaat
ttaaagcctt ctccttaatg ggccgggcct tgtatctgtt 1200tcggatcctt
ctttatagcg aaatgattaa tagtagtgat tacacctaaa atgtacgtat
1260atgacaaaaa gaatagtttt tttcttctgt ttctcttgta ctattaggcc
tagtgaatgt 1320attaaataca aacagtgtac attagcgtac gcaagcggtg
aggaaaggag cagaagaggg 1380cgtgatacga ggacacgtgt ctcatgttag
tacaaacaaa gattggacaa gagccggacg 1440gcacggtgac ctctgtcaac
aactgttacc gtttcttcta atctatttgt taatctccta 1500agtaatgtaa
ttaattagtt ttcactgttc ataattaatc aagtaataat aacataagaa
1560atgaaatggg agcgttactt gtataccata tgcactcgca aacgagccat
tggttgcgtc 1620agaacggtta cgttcttcat aaaagtcaca ttcgacacat
ggcgatcata tatacgtgcc 1680aatttttccg gttctctcaa atccaatccc
ggtttctttc ttttatgttt tgatcaccgg 1740ttcttcttct aaaccaactt
agctttactc atttggtgat tattattgga ccgtcgtaat 1800cgtcactctt
ttcttttttg aatcaatgat gtgagatatg tatattatac acgcacacgt
1860acacacgtgt atttgatcat tcttgccgtc accaaatttt aaactcattt
atcgtgtaat 1920aatatattgg agatccgcac tgactagact atacattaca
tatatcttgt aatttcggaa 1980agttcaaata tttgtgaaat atatgcacta
gttggagttt ccttatttgt accttaatcg 2040aatattgagt aaacatgatt
ccctgaccca aatatagatt gcgtacatca acttattaat 2100accgtcaata
aatacgagtt tttgcatata atcattacat ggtcatatgt aataaatcta
2160gttgttttgg aaattgcata ttctctccct ccctctttat caattttaca
tatatgtagc 2220atcagcaata tatgtgaatt tgttacatta gtagtttagt
ttgtatttta atttttttgg 2280ataaattatg aattctatta ccaaaattga
acattgtttt ttacaaatac tttggggcag 2340tgaatttcta aaaccaaaaa
aaaaacagtt agtaatttgt ataggtatta tcgccgtggt 2400atacacttaa
ttcaaatatc ataatatact tcgtgtcata tacgacaaaa gtaaaattaa
2460agtttgcaaa aaaaatttta ttcaaagaat ctaacggaaa aaaaccgctt
aaattttcta 2520gaagatattg ccaaatgttg ataaaatatc tgattaacca
aaaagaaaat aacgcatagc 2580ggtgagccgt tacatttcga ctacaaagtt
atcacattaa aatcaccgcc aaaaattagt 2640aattaattag attatatata
tatatttaag ctcattttat acatttattt gtttgtaaaa 2700ttaatttctc
atagtattag attgaatgtt catatattac aattggtata taccataaga
2760tagttgtgtg gttcgatgta acaaaaataa aattaaacca gaagtagaaa
aagtgaagga 2820aagggaataa caatgaagaa gttgaaagat atggaatgtt
ttgcttcacg caaagagctt 2880aacacgagac aagtacgctc catcgcgttg
ctttgaagtc ttttttttct tcttcttctt 2940cttgtcttaa cctctttttt
tttttttagt cttggtaata acaagactaa ttaaattatt 3000attttacttt
ttttattatc taccaacaac caaaccctag ttttgcttac gtccgatctc
3060ctccttctct cttttcttct ctcccaaacg ttgcctttct tttttttctc
taatcacgaa 3120cca 31231753158DNAArabidopsis
thalianapromoter(1)..(3158)transcription regulating sequence from
Arabidopsis thaliana gene At4g00220 175cttccgctca tcttttatgt
caaagacgta acgttttgtt tgggggaagt tgtgtcgtcg 60tcgaccacag ttattatata
tccaatccaa agagatgttt aagagagaga gatggatggg 120tttgtttatg
aataaatgtt tttaagttga tgtggagcat agaggatcgt gtcaggcaag
180aaaatcatgt cttgcttttg gtggtgggtc atttataatc attacatttt
tctataactt 240gatcatcttt tcttttgtat gtccatattt ttgttttttg
ttttccttgg tgtgtttcac 300ttttctgtac atagttgaat cacacactca
agaaacaagc tattttcaaa caagtgaaag 360aattatctat ttatggactc
ctaaaatcaa tgaaagcata taacacaagg agggataaca 420tatacttgta
aattgaaaag tagacaaatc aacaatatat atcatctaaa ttagcaatca
480taggtttaag acataaaaag attttgtata tgccactgag aatctagaac
taactatcag 540tctatcacac aagcaaggcc tagaaagtag tgaacaagta
aggatttaga attttagata 600gtgagtaatg aagatatata taggctggtg
ggttcagtta acaaggtact aaaatcggat 660tataacgagg gatcaagtgg
tggggattat gaattattat agtaatgaga atgaatgtac 720tagtactaat
tagaatgagt aaagcttgga agcaacggca gagcagattt gtgatatata
780caaaggacaa acaaacacaa atgctttgcg tgaagcaaaa cacaattagc
tgcttgctga 840cgtatttctc tctcaccact cagccgcagg tttccctgtc
acacacacgt gcgatccctc 900accacacgtg tgtaattctt ttatatatgt
tatatactgt atataatttt ttgtcaaact 960agttctctat ggacagcttt
cgtgatagaa ttatagagag aaaatcgaga tgtatgttgc 1020ttcttgtttc
tacaaatcgt atagtcagaa acagacatga cactgatcac tgtttgcttc
1080ccccgcaagt acaacctcag tcagtcagtt actgtcatgt ctctctttct
ctctgtttga 1140caagatccct gacccttgtt cacttcccta atttaaagcc
ttctccttaa tgggccgggc 1200cttgtatctg tttcggatcc ttctttatag
cgaaatgatt aatagtagtg attacaccta 1260aaatgtacgt atatgacaaa
aagaatagtt tttttcttct gtttctcttg tactattagg 1320cctagtgaat
gtattaaata caaacagtgt acattagcgt acgcaagcgg tgaggaaagg
1380agcagaagag ggcgtgatac gaggacacgt gtctcatgtt agtacaaaca
aagattggac 1440aagagccgga cggcacggtg acctctgtca acaactgtta
ccgtttcttc taatctattt 1500gttaatctcc taagtaatgt aattaattag
ttttcactgt tcataattaa tcaagtaata 1560ataacataag aaatgaaatg
ggagcgttac ttgtatacca tatgcactcg caaacgagcc 1620attggttgcg
tcagaacggt tacgttcttc ataaaagtca cattcgacac atggcgatca
1680tatatacgtg ccaatttttc cggttctctc aaatccaatc ccggtttctt
tcttttatgt 1740tttgatcacc ggttcttctt ctaaaccaac ttagctttac
tcatttggtg attattattg 1800gaccgtcgta atcgtcactc ttttcttttt
tgaatcaatg atgtgagata tgtatattat 1860acacgcacac gtacacacgt
gtatttgatc
attcttgccg tcaccaaatt ttaaactcat 1920ttatcgtgta ataatatatt
ggagatccgc actgactaga ctatacatta catagatctt 1980gtaatttcgg
aaagttcaaa tatttgtgaa atatatgcac tagttggagt ttccttattt
2040gtaccttaat cgaatattga gtaaacatga ttccctgacc caaatataga
ttgcgtacat 2100caactcatta ataccgtcaa taaatacgag tttttgcata
taatcattac atggtcatat 2160gtaataaatc tagttgtttt ggaaattgca
tattctctcc ctccctcttt atcaatttta 2220catatatgta gcatcagcaa
tatatgtgaa tttgttacat tagtagttta gtttgtattt 2280taattttttg
gataaattat gaattctatt accaaaattg aacattgttt tttacaaata
2340ctttggggca gtgaatttct aaaaccaaaa aaaaaaagtt agtaatttgt
ataggtatta 2400tcgccgtggt atacacttaa ttcaaatatc ataatataca
tgttcgtgtc atatacgaca 2460aaagtaaaat taaagtttgc aaaaaaaatt
ttatttaaag aatctaacgg aaaaaaaccg 2520cttaaatttt ctagaaaatg
ttgccaaatg ttgataaaat atccgattaa ccaaaaagaa 2580aataacgcat
agcggtgaac cgttacattt cgactacaaa gttatcacat taaaatcacc
2640gccaaaaatt agtaattaat tagattatat atatatatat atatatatat
atatatatat 2700ttaagctcat tttatacatt tatttgtttg taaaattaat
ttctcatagt attagattga 2760atgttcatat attacaattg gtatatacca
taagatagtt gtgtggttcg atgtaacaaa 2820aataaaatta aaccagaagt
agaaaaagtg aaggaaaggg aataacaatg aagaagttga 2880aagatatgga
atgttttgct tcacgcaaag agcttaacac gagacaagta cgctccatcg
2940cgttgctttg aagtcttttt tttcttcttc ttcttcttgt cttaacctct
tttttatttt 3000ttagtcttgg taataacaag actaattaaa ttattatttt
acttttttta ttatctacca 3060acaaccaaac cctagttttg cttacgtccg
atctcctcct tctctctttt cttctctccc 3120aaacgttccc tttctttttt
ttctctaatc acgaacca 3158176948DNAArabidopsis
thalianaCDS(112)..(798)encoding Arabidopsis thaliana LOB domain
protein 30 / lateral organ boundaries domain protein 30 (LBD30)
176aaaagcatca taatctcgat gagttaaatc catgtatctc aaaaccctaa
tcaccactta 60gctagtaaaa ggaaaggaaa ataaaaagga cttgtgtggt agaaaaggaa
g atg agc 117 Met Ser 1agt agc gga aac cct agc agc agc agc ggt gga
gga gga gga ccg tgc 165Ser Ser Gly Asn Pro Ser Ser Ser Ser Gly Gly
Gly Gly Gly Pro Cys 5 10 15ggc gcg tgc aag ttc ttg aga agg aag tgt
gtc gct ggc tgc atc ttc 213Gly Ala Cys Lys Phe Leu Arg Arg Lys Cys
Val Ala Gly Cys Ile Phe 20 25 30gct cct tac ttt gac tcc gag caa gga
gcg gcg cac ttc gcg gcg gtt 261Ala Pro Tyr Phe Asp Ser Glu Gln Gly
Ala Ala His Phe Ala Ala Val35 40 45 50cac aag gtg ttc gga gcg agc
aac gtc tcc aaa ctc ctc cac cat gtt 309His Lys Val Phe Gly Ala Ser
Asn Val Ser Lys Leu Leu His His Val 55 60 65ccc gag cat aaa cga cca
gac gcc gtc gtt tca atc tgc ttt gag gct 357Pro Glu His Lys Arg Pro
Asp Ala Val Val Ser Ile Cys Phe Glu Ala 70 75 80caa gct cgt ctc aga
gac cct atc tac ggc tgc gtc tct cac atc gtc 405Gln Ala Arg Leu Arg
Asp Pro Ile Tyr Gly Cys Val Ser His Ile Val 85 90 95tct ctt cag caa
cag gtg gta agt ttg caa act gag ctt tca tat cta 453Ser Leu Gln Gln
Gln Val Val Ser Leu Gln Thr Glu Leu Ser Tyr Leu 100 105 110caa gca
cac tta gca act cta gag ctg cca caa cct caa cca cca cag 501Gln Ala
His Leu Ala Thr Leu Glu Leu Pro Gln Pro Gln Pro Pro Gln115 120 125
130gtt ccg gtg tca tct tca gga tct cta cag gct ctt tcc ata acg gac
549Val Pro Val Ser Ser Ser Gly Ser Leu Gln Ala Leu Ser Ile Thr Asp
135 140 145ctc cca aca ata tca ccg tcg gtg tac gac ctc tcc tcc atc
ttc gag 597Leu Pro Thr Ile Ser Pro Ser Val Tyr Asp Leu Ser Ser Ile
Phe Glu 150 155 160ccg gtc atg tcc tcc act tgg gcc atg cag caa caa
cca cga ccg tct 645Pro Val Met Ser Ser Thr Trp Ala Met Gln Gln Gln
Pro Arg Pro Ser 165 170 175gat cat ctc ttc ggc gtc ccg tca tcg tcc
aat atg ggc gga ggc ggt 693Asp His Leu Phe Gly Val Pro Ser Ser Ser
Asn Met Gly Gly Gly Gly 180 185 190gag ctc caa gca ctg gcg cgt gag
ttt att cac ggt ggg caa atg cca 741Glu Leu Gln Ala Leu Ala Arg Glu
Phe Ile His Gly Gly Gln Met Pro195 200 205 210gct cag cca tcg ccg
gga acc agt ggt tct gcc tcg tca gtg ata aaa 789Ala Gln Pro Ser Pro
Gly Thr Ser Gly Ser Ala Ser Ser Val Ile Lys 215 220 225cga gaa tga
agttattttt atcgtatgtt tgtttatttg gaccgtaaga 838Arg Glugatctatagt
tagccatatg tttctccttg taatatatat acttggtcga attcgtattt
898aaattgttgt ctagaatgtt aatataagaa ctatatgatt tacattgatt
948177228PRTArabidopsis thaliana 177Met Ser Ser Ser Gly Asn Pro Ser
Ser Ser Ser Gly Gly Gly Gly Gly1 5 10 15Pro Cys Gly Ala Cys Lys Phe
Leu Arg Arg Lys Cys Val Ala Gly Cys 20 25 30Ile Phe Ala Pro Tyr Phe
Asp Ser Glu Gln Gly Ala Ala His Phe Ala 35 40 45Ala Val His Lys Val
Phe Gly Ala Ser Asn Val Ser Lys Leu Leu His 50 55 60His Val Pro Glu
His Lys Arg Pro Asp Ala Val Val Ser Ile Cys Phe65 70 75 80Glu Ala
Gln Ala Arg Leu Arg Asp Pro Ile Tyr Gly Cys Val Ser His 85 90 95Ile
Val Ser Leu Gln Gln Gln Val Val Ser Leu Gln Thr Glu Leu Ser 100 105
110Tyr Leu Gln Ala His Leu Ala Thr Leu Glu Leu Pro Gln Pro Gln Pro
115 120 125Pro Gln Val Pro Val Ser Ser Ser Gly Ser Leu Gln Ala Leu
Ser Ile 130 135 140Thr Asp Leu Pro Thr Ile Ser Pro Ser Val Tyr Asp
Leu Ser Ser Ile145 150 155 160Phe Glu Pro Val Met Ser Ser Thr Trp
Ala Met Gln Gln Gln Pro Arg 165 170 175Pro Ser Asp His Leu Phe Gly
Val Pro Ser Ser Ser Asn Met Gly Gly 180 185 190Gly Gly Glu Leu Gln
Ala Leu Ala Arg Glu Phe Ile His Gly Gly Gln 195 200 205Met Pro Ala
Gln Pro Ser Pro Gly Thr Ser Gly Ser Ala Ser Ser Val 210 215 220Ile
Lys Arg Glu2251781504DNAArabidopsis
thalianapromoter(1)..(1504)transcription regulating sequence from
Arabidopsis thaliana gene At4g26320 178tcattgtaaa gtttttcatt
ttttttcctt ttgtaaaatt cctctgtttt ctctctgttc 60ttcatgtagt tcttgcgatt
tgccgaaaat tgaatctcgt ttcttgatta cagtttgtat 120ccatgtgaat
atgtctagat tttttttttt tttttttgaa gtatagtcat tgatgattag
180accatcattt cggttttacc tcgagatgtt taagacaaat gttgctgtct
tagatttgtt 240ttgtgtccgt tcataacttt tggggaaaaa aatgagtaaa
gatcaaaagg tttttgagtt 300aatcatccgg tttaggtggt attgaaagta
gctacctcgg tattggtgtt aaaatgtctg 360tactccttga gctctctgcg
agagacagat tttgtttaat gatcaatcaa acatttatca 420aatctctgag
cttagtatat cttcattttt catttaatag caagtctcaa tcgttatggc
480tttgtatcca gatataatca actctaagat caaggacggt gtttgttttt
aagctaactt 540tcttgggtct ctaggtttgg tgtggtgagt ttttggtttc
ttgtcttcca agaaaaaaag 600gctagtgtaa atgggagagt ttcacggttc
ttcatttctt tatttctttt ggtttgaatg 660aaagtcttgt atggccagta
ctgtgtatgg caataggcaa taacatgagc ttgtacctgc 720taatcagaga
aatataatgg aactaagttt gatagcgtga cgaaactatt tataattaaa
780ttgtagtact tcgtagttct aaaacttgaa gaataacaga taaatttcaa
aatgtagaat 840acaaaaaatg ataaatactt ttgttagaaa atacctaatt
catctttatg gagactcacg 900tttataagtt cttgtttcat cgaatattga
gcaataattg ggggagaaat ggaccgagcc 960aattatatac cagagagcac
atggaaccat caatcaaatt ccaaacagct cattgcttct 1020cctatctatt
ttctattcat ttcacagaaa aaaacaaaca gtttcctatt tctttcactc
1080ttattatcag agagaaaacg ttcctactat gaactgtatt agttaaaaaa
tacaaagaac 1140taaaacatat tatattcatt tctacaataa aaaacaaaag
gcgataacat aactaaaatc 1200agctgcagta ctctttccaa ctgtttattt
ttttcttcct ttttcaacta agatctcagt 1260tggctttaca gagattgatg
tgctaatata gttttttttc ctcaccaaat acaaagaatt 1320aaaacaattc
gaaaatgatt aactgtgttg atgttcatca aactgtaaag tgataattac
1380acagttggcc ttacatgcat ctatgtgtat atataaacca caagtcacaa
cacatcagaa 1440cacaccacaa aacactaaac atataatctc ttcgtgcttt
ctcaagaacc acctagagat 1500aacc 15041791492DNAArabidopsis
thalianapromoter(1)..(1492)transcription regulating sequence from
Arabidopsis thaliana gene At4g26320 179ataatgttat cttcattgta
aagtttttca tttttttttc cttttgtaaa attcctcttt 60tttctctctg ttcttaatgt
agttcttgcg atttgccgaa aattgaatct cgtttcttga 120ttacagtttg
tatccatgtg aatatgtcta gatttttttt tttttttttg aagtatagtc
180attgatgatt agaccatcat ttcggtttta cctcgagatg tttaagacaa
atgttgctgt 240cttagatttg ttttgtgttc gttcataact tctggggaaa
aaaatgagta aagatcaaga 300ggttttgagt taatcatccg gtttaagtgg
tattgaaagt agctacctcg gtattggtgt 360taaaatgtct gtactccttg
agcgctctgc gagagacaga tatcttcatt tttcatttaa 420tagcaagtct
caatcgttat ggatttgtat ccagatataa tcaactctaa gatcaaggac
480ggtgtttgtt tttaagctaa ctttcttggg tctctaggtt tggtgtggtg
agtttttggt 540ttcttgtctt ccaagaaaaa aggctagtgt aaatgggaga
gtttcacggt tcttcatttc 600tttatttctt ttggtttgaa tgaaagtctt
gtatggccag tactgtgtat gggcaatagg 660caataacatg agcttgtacc
tgctaatcag agaaatatag tggaactaag tttgatagcg 720tgacgaaact
atttatgatt aaatgatagc acttcgtagt tctaaaactt aaagaataac
780agataaattt caaaatgtag aagtacaaaa aatgataaat acttttgtta
gaaaatacct 840aattcatctt tatgttctct tataaaatgg agacttacgt
ttataagttc ttgtttcatc 900gaatattgag caataattgg gggagaaatg
gattgagcca attatatacc agagagcaca 960tggaaccatc aatcaaattc
caaacagctc attgcttctc ttatctattt tctattcatt 1020tcacgaccgt
cacagaaaaa aaaaaacagt ttcctatttc tttcactctt attatcagag
1080agaaaacgtt cctactatga actgtattag ttaaaaaata caaagaacta
aaacatatta 1140tattcatttc tacaataaaa aacaaaaggc gatgacataa
ctaaaatcag ctgcagtact 1200ctttccaact gtttattttt tctttctttt
tcaactaaga tctcagttgg ctttacagag 1260attgatgtgc taataaagtt
tttgttcacc aaatacaaag aattaaaaca attcgaaaat 1320gattaattgt
gttgatgtgt taatcaaact gtaaagtgat aattacacag ttggccttac
1380atgcatctat gtgtatatat aaaccacaag tcacaacaca tcaaaacaca
ccacaaaaca 1440ctaaacatat agtctcttcg tgctttctca agaaccacct
agagataacc ca 14921801450DNAArabidopsis
thalianapromoter(1)..(1450)transcription regulating sequence from
Arabidopsis thaliana gene At4g26320 180tcattgtaaa gtttttcatt
ttttttcctt ttgtaaaatt cctctgtttt ctctctgttc 60ttcatgtagt tcttgcgatt
tgccgaaaat tgaatctcgt ttcttgatta cagtttgtat 120ccatgtgaat
atgtctagat tttttttttt tttttttgaa gtatagtcat tgatgattag
180accatcattt cggttttacc tcgagatgtt taagacaaat gttgctgtct
tagatttgtt 240ttgtgtccgt tcataacttt tggggaaaaa aatgagtaaa
gatcaaaagg tttttgagtt 300aatcatccgg tttaggtggt attgaaagta
gctacctcgg tattggtgtt aaaatgtctg 360tactccttga gctctctgcg
agagacagat tttgtttaat gatcaatcaa acatttatca 420aatctctgag
cttagtatat cttcattttt catttaatag caagtctcaa tcgttatggc
480tttgtatcca gatataatca actctaagat caaggacggt gtttgttttt
aagctaactt 540tcttgggtct ctaggtttgg tgtggtgagt ttttggtttc
ttgtcttcca agaaaaaaag 600gctagtgtaa atgggagagt ttcacggttc
ttcatttctt tatttctttt ggtttgaatg 660aaagtcttgt atggccagta
ctgtgtatgg caataggcaa taacatgagc ttgtacctgc 720taatcagaga
aatataatgg aactaagttt gatagcgtga cgaaactatt tataattaaa
780ttgtagtact tcgtagttct aaaacttgaa gaataacaga taaatttcaa
aatgtagaat 840acaaaaaatg ataaatactt ttgttagaaa atacctaatt
catctttatg gagactcacg 900tttataagtt cttgtttcat cgaatattga
gcaataattg ggggagaaat ggaccgagcc 960aattatatac cagagagcac
atggaaccat caatcaaatt ccaaacagct cattgcttct 1020cctatctatt
ttctattcat ttcacagaaa aaaacaaaca gtttcctatt tctttcactc
1080ttattatcag agagaaaacg ttcctactat gaactgtatt agttaaaaaa
tacaaagaac 1140taaaacatat tatattcatt tctacaataa aaaacaaaag
gcgataacat aactaaaatc 1200agctgcagta ctctttccaa ctgtttattt
ttttcttcct ttttcaacta agatctcagt 1260tggctttaca gagattgatg
tgctaatata gttttttttc ctcaccaaat acaaagaatt 1320aaaacaattc
gaaaatgatt aactgtgttg atgttcatca aactgtaaag tgataattac
1380acagttggcc ttacatgcat ctatgtgtat atataaacca caagtcacaa
cacatcagaa 1440cacaccacaa 14501811436DNAArabidopsis
thalianapromoter(1)..(1436)transcription regulating sequence from
Arabidopsis thaliana gene At4g26320 181ataatgttat cttcattgta
aagtttttca tttttttttc cttttgtaaa attcctcttt 60tttctctctg ttcttaatgt
agttcttgcg atttgccgaa aattgaatct cgtttcttga 120ttacagtttg
tatccatgtg aatatgtcta gatttttttt tttttttttg aagtatagtc
180attgatgatt agaccatcat ttcggtttta cctcgagatg tttaagacaa
atgttgctgt 240cttagatttg ttttgtgttc gttcataact tctggggaaa
aaaatgagta aagatcaaga 300ggttttgagt taatcatccg gtttaagtgg
tattgaaagt agctacctcg gtattggtgt 360taaaatgtct gtactccttg
agcgctctgc gagagacaga tatcttcatt tttcatttaa 420tagcaagtct
caatcgttat ggatttgtat ccagatataa tcaactctaa gatcaaggac
480ggtgtttgtt tttaagctaa ctttcttggg tctctaggtt tggtgtggtg
agtttttggt 540ttcttgtctt ccaagaaaaa aggctagtgt aaatgggaga
gtttcacggt tcttcatttc 600tttatttctt ttggtttgaa tgaaagtctt
gtatggccag tactgtgtat gggcaatagg 660caataacatg agcttgtacc
tgctaatcag agaaatatag tggaactaag tttgatagcg 720tgacgaaact
atttatgatt aaatgatagc acttcgtagt tctaaaactt aaagaataac
780agataaattt caaaatgtag aagtacaaaa aatgataaat acttttgtta
gaaaatacct 840aattcatctt tatgttctct tataaaatgg agacttacgt
ttataagttc ttgtttcatc 900gaatattgag caataattgg gggagaaatg
gattgagcca attatatacc agagagcaca 960tggaaccatc aatcaaattc
caaacagctc attgcttctc ttatctattt tctattcatt 1020tcacgaccgt
cacagaaaaa aaaaaacagt ttcctatttc tttcactctt attatcagag
1080agaaaacgtt cctactatga actgtattag ttaaaaaata caaagaacta
aaacatatta 1140tattcatttc tacaataaaa aacaaaaggc gatgacataa
ctaaaatcag ctgcagtact 1200ctttccaact gtttattttt tctttctttt
tcaactaaga tctcagttgg ctttacagag 1260attgatgtgc taataaagtt
tttgttcacc aaatacaaag aattaaaaca attcgaaaat 1320gattaattgt
gttgatgtgt taatcaaact gtaaagtgat aattacacag ttggccttac
1380atgcatctat gtgtatatat aaaccacaag tcacaacaca tcaaaacaca ccacaa
14361823019DNAArabidopsis thalianapromoter(1)..(3019)transcription
regulating sequence from Arabidopsis thaliana gene At4g26320
182ttgactgaca agaaaaaaag gactgtagaa gagatgagtt ttgaagaagg
taaccttgtg 60aagactttag aaagaagatg aaggtggtgg cttgaagcca aagcagcgtt
ttttagccgg 120ttagcaccta aatatacaac gtaaacctgg aagacatgcc
aaatccaaac ataaagaaat 180tctgcaaatg aaaaggtata ttcatgcaca
ggattaatga ttagtgattt acatgagaaa 240ttgatgttgt tgcttgaatt
gatgagtgaa gtagtaatgt aagagcaaga aagatccaac 300caagaagtcg
ttccattgat gttatacggc cggagaaaga tctagtttga cacatattgc
360tgctacaaat cactcacact cttataacta attcaaactc aagcttatat
taataatata 420taatcaatca ttgtgacaaa ctcaacaaga cataggctcg
cgagccacat cattctcact 480tttaggtcta aacttgttaa tagctagatc
gtctaattcc tctaacagta ggcctgtttt 540gaaagatgtc tccatttgtt
atgtatagct taacttttat catcaacgct ataacaaatt 600gttaggatca
ataagtagct taggaatgtt cctttgtaga gttttactta gaacaaagag
660tggtaacttg caatttaggt gaaacctata gagaatgtag ctcaatgggt
taaggtgaat 720tctatagaca cattagcttg cgagtctcat cattctcact
tcaactagaa gttaacataa 780tatttcatca attcactaaa gaacataacc
caaaaataca ttaaaaagga actaatatta 840acagatctcc ggtaggttta
cttctaataa tttgcaagaa ttcttattgt tgaagaatcg 900aaacgaaata
tagtgtagaa tcctttaatt ttgttcaaat actaactcaa aaaaggcttc
960atatttgatt atttcctagt tgtttaatca ataaaatact ttaaaaaatg
gattaggaac 1020tattcgatca atgatgatga ctaaacatgg aagtcaacgt
ggcaagatct agaaaagccc 1080ccgacctctg tacattgtat acacatacac
agacaaattg aacgaaaatc acggaatcat 1140cattcagtta cagttttttt
ttctgtgtgt gtatgaacat ggtgttcaac ggccagacgg 1200ttatgtctgt
agcccatttg tcggctgaga tttggcagcg tttacgattg atcccaccgt
1260ccgatcgtat aagcagccga gagatgcttg agctagtctg cttctttccg
ctccagcaat 1320taggtcgatt ggctttatgt ctcttgactt gtctatgtct
tccttctcca ggtttgctct 1380atcctgaaac tgcggaagac gatcgtgatc
acgtttatgt ttatggttcc tcttcttctt 1440ccatcgctac atatcagcat
cactttcgtc tgcattttga gtgatcaaaa tcatcaaact 1500cgataatgtc
atcttcattg taaagttttt catttttttt ccttttgtaa aattcctctg
1560ttttctctct gttcttcatg tagttcttgc gatttgccga aaattgaatc
tcgtttcttg 1620attacagttt gtatccatgt gaatatgtct agattttttt
tttttttttt ttgaagtata 1680gtcattgatg attagaccat catttcggtt
ttacctcgag atgtttaaga caaatgttgc 1740tgtcttagat ttgttttgtg
tccgttcata acttttgggg aaaaaaatga gtaaagatca 1800aaaggttttt
gagttaatca tccggtttag gtggtattga aagtagctac ctcggtattg
1860gtgttaaaat gtctgtactc cttgagctct ctgcgagaga cagattttgt
ttaatgatca 1920atcaaacatt tatcaaatct ctgagcttag tatatcttca
tttttcattt aatagcaagt 1980ctcaatcgtt atggctttgt atccagatat
aatcaactct aagatcaagg acggtgtttg 2040tttttaagct aactttcttg
ggtctctagg tttggtgtgg tgagtttttg gtttcttgtc 2100ttccaagaaa
aaaaggctag tgtaaatggg agagtttcac ggttcttcat ttctttattt
2160cttttggttt gaatgaaagt cttgtatggc cagtactgtg tatggcaata
ggcaataaca 2220tgagcttgta cctgctaatc agagaaatat aatggaacta
agtttgatag cgtgacgaaa 2280ctatttataa ttaaattgta gtacttcgta
gttctaaaac ttgaagaata acagataaat 2340ttcaaaatgt agaatacaaa
aaatgataaa tacttttgtt agaaaatacc taattcatct 2400ttatggagac
tcacgtttat aagttcttgt ttcatcgaat attgagcaat aattggggga
2460gaaatggacc gagccaatta tataccagag agcacatgga accatcaatc
aaattccaaa 2520cagctcattg cttctcctat ctattttcta ttcatttcac
agaaaaaaac aaacagtttc 2580ctatttcttt cactcttatt atcagagaga
aaacgttcct actatgaact gtattagtta 2640aaaaatacaa agaactaaaa
catattatat tcatttctac aataaaaaac aaaaggcgat 2700aacataacta
aaatcagctg cagtactctt tccaactgtt tatttttttc ttcctttttc
2760aactaagatc tcagttggct ttacagagat tgatgtgcta atatagtttt
ttttcctcac 2820caaatacaaa gaattaaaac aattcgaaaa tgattaactg
tgttgatgtt catcaaactg 2880taaagtgata attacacagt tggccttaca
tgcatctatg tgtatatata aaccacaagt 2940cacaacacat cagaacacac
cacaaaacac taaacatata atctcttcgt gctttctcaa
3000gaaccaccta gagataacc 30191833006DNAArabidopsis
thalianapromoter(1)..(3006)transcription regulating sequence from
Arabidopsis thaliana gene At4g26320 183tggacgagat cattgactga
caagaaaaaa aggactgtag aagagatgag ttttgaagaa 60ggtaaccttg tgaagacttt
agaaagaaga tgaaggtggt ggcttgaagc caaagcagcg 120ttttttagcc
ggttagcacc taaatataca acgtaaacct ggaagacatg ccaaatccaa
180acataaagaa attctgcaaa tgaaaaggta tattcatgca caggattaat
gattagtgat 240ttacatgaga aattgatgtt gttgcttgaa ttgatgagtg
aagtagtaat gtaagagcaa 300gaaagatcca accaagaagt cgttccattg
atgttatacg gccggagaaa gatctagttt 360gacacatatt gctgctacaa
atcactcaca ctcttataac taattcaaac tcaagcttat 420attaataata
tataatcaat cattgtgaca aactcaacaa gacataggct cgcgagccac
480atcattctca cttttaggtc taaacttgtt aatagctaga tcgtctaatt
cctctaacag 540taggcctgtt ttgaaagatg tctccatttg ttatgtatag
cttaactttt atcatcaacg 600ctataacaaa ttgttaggat caataagtag
cttaggaatg ttcctttgta gagttttact 660tagaacaaag agtggtaact
tgcaatttag gtgaaaccta tagagaatgt agctcaatgg 720gttaaggtga
attctataga cacattagct tgcgagtctc atcattctca cttcaactag
780aagttaacat aatatttcat caattcacta aagaacataa cccaaaaata
cattaaaaag 840gaactaatat taacagatct ccggtaggtt tacttctaat
aatttgcaag aattcttatt 900gttgaagaat cgaaacgaaa tatagtgtag
aatcctttaa ttttgttcaa atactaactc 960aaaaaaggct tcatatttga
ttatttccta gttgtttaat caataaaata cttttaaaaa 1020tggattagga
actattcgat caatgatgat gactaaacat ggaagtcaac gtggcaagat
1080ctagaaaagc ccccgacctc tgtacattgt atacacatac acagacaaat
tgaacgaaaa 1140tcacggaatc atcattcagt tacagttttt ttttctgtgt
gtgtatgaac atggtgttca 1200acggccagac ggttatgtct gtagcccatt
tgtcggctga gatttggcag cgtttacgat 1260tgatcccacc gtccgatcgt
ataagcagcc gagagatgct tgagctagtc tgcttctttc 1320cgctccagca
attaggtcga ttggctttat gtctcttgac ttgtctatgt cttccttctc
1380caggtttgct ctatcctgaa actgcggaag acgatcgtga tcacgtttat
gtttatggtt 1440cctcttcttc ttccatcgct acatatcagc atcactttcg
tctgcatttt gagtgatcaa 1500aatcatcaaa ctcgataatg ttatcttcat
tgtaaagttt ttcatttttt tttccttttg 1560taaaattcct cttttttctc
tctgttctta atgtagttct tgcgatttgc cgaaaattga 1620atctcgtttc
ttgattacag tttgtatcca tgtgaatatg tctagatttt tttttttttt
1680tttgaagtat agtcattgat gattagacca tcatttcggt tttacctcga
gatgtttaag 1740acaaatgttg ctgtcttaga tttgttttgt gttcgttcat
aacttctggg gaaaaaaatg 1800agtaaagatc aagaggtttt gagttaatca
tccggtttaa gtggtattga aagtagctac 1860ctcggtattg gtgttaaaat
gtctgtactc cttgagcgct ctgcgagaga cagatatctt 1920catttttcat
ttaatagcaa gtctcaatcg ttatggattt gtatccagat ataatcaact
1980ctaagatcaa ggacggtgtt tgtttttaag ctaactttct tgggtctcta
ggtttggtgt 2040ggtgagtttt tggtttcttg tcttccaaga aaaaaggcta
gtgtaaatgg gagagtttca 2100cggttcttca tttctttatt tcttttggtt
tgaatgaaag tcttgtatgg ccagtactgt 2160gtatgggcaa taggcaataa
catgagcttg tacctgctaa tcagagaaat atagtggaac 2220taagtttgat
agcgtgacga aactatttat gattaaatga tagcacttcg tagttctaaa
2280acttaaagaa taacagataa atttcaaaat gtagaagtac aaaaaatgat
aaatactttt 2340gttagaaaat acctaattca tctttatgtt ctcttataaa
atggagactt acgtttataa 2400gttcttgttt catcgaatat tgagcaataa
ttgggggaga aatggattga gccaattata 2460taccagagag cacatggaac
catcaatcaa attccaaaca gctcattgct tctcttatct 2520attttctatt
catttcacga ccgtcacaga aaaaaaaaaa cagtttccta tttctttcac
2580tcttattatc agagagaaaa cgttcctact atgaactgta ttagttaaaa
aatacaaaga 2640actaaaacat attatattca tttctacaat aaaaaacaaa
aggcgatgac ataactaaaa 2700tcagctgcag tactctttcc aactgtttat
tttttctttc tttttcaact aagatctcag 2760ttggctttac agagattgat
gtgctaataa agtttttgtt caccaaatac aaagaattaa 2820aacaattcga
aaatgattaa ttgtgttgat gtgttaatca aactgtaaag tgataattac
2880acagttggcc ttacatgcat ctatgtgtat atataaacca caagtcacaa
cacatcaaaa 2940cacaccacaa aacactaaac atatagtctc ttcgtgcttt
ctcaagaacc acctagagat 3000aaccca 30061842965DNAArabidopsis
thalianapromoter(1)..(2965)transcription regulating sequence from
Arabidopsis thaliana gene At4g26320 184ttgactgaca agaaaaaaag
gactgtagaa gagatgagtt ttgaagaagg taaccttgtg 60aagactttag aaagaagatg
aaggtggtgg cttgaagcca aagcagcgtt ttttagccgg 120ttagcaccta
aatatacaac gtaaacctgg aagacatgcc aaatccaaac ataaagaaat
180tctgcaaatg aaaaggtata ttcatgcaca ggattaatga ttagtgattt
acatgagaaa 240ttgatgttgt tgcttgaatt gatgagtgaa gtagtaatgt
aagagcaaga aagatccaac 300caagaagtcg ttccattgat gttatacggc
cggagaaaga tctagtttga cacatattgc 360tgctacaaat cactcacact
cttataacta attcaaactc aagcttatat taataatata 420taatcaatca
ttgtgacaaa ctcaacaaga cataggctcg cgagccacat cattctcact
480tttaggtcta aacttgttaa tagctagatc gtctaattcc tctaacagta
ggcctgtttt 540gaaagatgtc tccatttgtt atgtatagct taacttttat
catcaacgct ataacaaatt 600gttaggatca ataagtagct taggaatgtt
cctttgtaga gttttactta gaacaaagag 660tggtaacttg caatttaggt
gaaacctata gagaatgtag ctcaatgggt taaggtgaat 720tctatagaca
cattagcttg cgagtctcat cattctcact tcaactagaa gttaacataa
780tatttcatca attcactaaa gaacataacc caaaaataca ttaaaaagga
actaatatta 840acagatctcc ggtaggttta cttctaataa tttgcaagaa
ttcttattgt tgaagaatcg 900aaacgaaata tagtgtagaa tcctttaatt
ttgttcaaat actaactcaa aaaaggcttc 960atatttgatt atttcctagt
tgtttaatca ataaaatact ttaaaaaatg gattaggaac 1020tattcgatca
atgatgatga ctaaacatgg aagtcaacgt ggcaagatct agaaaagccc
1080ccgacctctg tacattgtat acacatacac agacaaattg aacgaaaatc
acggaatcat 1140cattcagtta cagttttttt ttctgtgtgt gtatgaacat
ggtgttcaac ggccagacgg 1200ttatgtctgt agcccatttg tcggctgaga
tttggcagcg tttacgattg atcccaccgt 1260ccgatcgtat aagcagccga
gagatgcttg agctagtctg cttctttccg ctccagcaat 1320taggtcgatt
ggctttatgt ctcttgactt gtctatgtct tccttctcca ggtttgctct
1380atcctgaaac tgcggaagac gatcgtgatc acgtttatgt ttatggttcc
tcttcttctt 1440ccatcgctac atatcagcat cactttcgtc tgcattttga
gtgatcaaaa tcatcaaact 1500cgataatgtc atcttcattg taaagttttt
catttttttt ccttttgtaa aattcctctg 1560ttttctctct gttcttcatg
tagttcttgc gatttgccga aaattgaatc tcgtttcttg 1620attacagttt
gtatccatgt gaatatgtct agattttttt tttttttttt ttgaagtata
1680gtcattgatg attagaccat catttcggtt ttacctcgag atgtttaaga
caaatgttgc 1740tgtcttagat ttgttttgtg tccgttcata acttttgggg
aaaaaaatga gtaaagatca 1800aaaggttttt gagttaatca tccggtttag
gtggtattga aagtagctac ctcggtattg 1860gtgttaaaat gtctgtactc
cttgagctct ctgcgagaga cagattttgt ttaatgatca 1920atcaaacatt
tatcaaatct ctgagcttag tatatcttca tttttcattt aatagcaagt
1980ctcaatcgtt atggctttgt atccagatat aatcaactct aagatcaagg
acggtgtttg 2040tttttaagct aactttcttg ggtctctagg tttggtgtgg
tgagtttttg gtttcttgtc 2100ttccaagaaa aaaaggctag tgtaaatggg
agagtttcac ggttcttcat ttctttattt 2160cttttggttt gaatgaaagt
cttgtatggc cagtactgtg tatggcaata ggcaataaca 2220tgagcttgta
cctgctaatc agagaaatat aatggaacta agtttgatag cgtgacgaaa
2280ctatttataa ttaaattgta gtacttcgta gttctaaaac ttgaagaata
acagataaat 2340ttcaaaatgt agaatacaaa aaatgataaa tacttttgtt
agaaaatacc taattcatct 2400ttatggagac tcacgtttat aagttcttgt
ttcatcgaat attgagcaat aattggggga 2460gaaatggacc gagccaatta
tataccagag agcacatgga accatcaatc aaattccaaa 2520cagctcattg
cttctcctat ctattttcta ttcatttcac agaaaaaaac aaacagtttc
2580ctatttcttt cactcttatt atcagagaga aaacgttcct actatgaact
gtattagtta 2640aaaaatacaa agaactaaaa catattatat tcatttctac
aataaaaaac aaaaggcgat 2700aacataacta aaatcagctg cagtactctt
tccaactgtt tatttttttc ttcctttttc 2760aactaagatc tcagttggct
ttacagagat tgatgtgcta atatagtttt ttttcctcac 2820caaatacaaa
gaattaaaac aattcgaaaa tgattaactg tgttgatgtt catcaaactg
2880taaagtgata attacacagt tggccttaca tgcatctatg tgtatatata
aaccacaagt 2940cacaacacat cagaacacac cacaa
29651852950DNAArabidopsis thalianapromoter(1)..(2950)transcription
regulating sequence from Arabidopsis thaliana gene At4g26320
185tggacgagat cattgactga caagaaaaaa aggactgtag aagagatgag
ttttgaagaa 60ggtaaccttg tgaagacttt agaaagaaga tgaaggtggt ggcttgaagc
caaagcagcg 120ttttttagcc ggttagcacc taaatataca acgtaaacct
ggaagacatg ccaaatccaa 180acataaagaa attctgcaaa tgaaaaggta
tattcatgca caggattaat gattagtgat 240ttacatgaga aattgatgtt
gttgcttgaa ttgatgagtg aagtagtaat gtaagagcaa 300gaaagatcca
accaagaagt cgttccattg atgttatacg gccggagaaa gatctagttt
360gacacatatt gctgctacaa atcactcaca ctcttataac taattcaaac
tcaagcttat 420attaataata tataatcaat cattgtgaca aactcaacaa
gacataggct cgcgagccac 480atcattctca cttttaggtc taaacttgtt
aatagctaga tcgtctaatt cctctaacag 540taggcctgtt ttgaaagatg
tctccatttg ttatgtatag cttaactttt atcatcaacg 600ctataacaaa
ttgttaggat caataagtag cttaggaatg ttcctttgta gagttttact
660tagaacaaag agtggtaact tgcaatttag gtgaaaccta tagagaatgt
agctcaatgg 720gttaaggtga attctataga cacattagct tgcgagtctc
atcattctca cttcaactag 780aagttaacat aatatttcat caattcacta
aagaacataa cccaaaaata cattaaaaag 840gaactaatat taacagatct
ccggtaggtt tacttctaat aatttgcaag aattcttatt 900gttgaagaat
cgaaacgaaa tatagtgtag aatcctttaa ttttgttcaa atactaactc
960aaaaaaggct tcatatttga ttatttccta gttgtttaat caataaaata
cttttaaaaa 1020tggattagga actattcgat caatgatgat gactaaacat
ggaagtcaac gtggcaagat 1080ctagaaaagc ccccgacctc tgtacattgt
atacacatac acagacaaat tgaacgaaaa 1140tcacggaatc atcattcagt
tacagttttt ttttctgtgt gtgtatgaac atggtgttca 1200acggccagac
ggttatgtct gtagcccatt tgtcggctga gatttggcag cgtttacgat
1260tgatcccacc gtccgatcgt ataagcagcc gagagatgct tgagctagtc
tgcttctttc 1320cgctccagca attaggtcga ttggctttat gtctcttgac
ttgtctatgt cttccttctc 1380caggtttgct ctatcctgaa actgcggaag
acgatcgtga tcacgtttat gtttatggtt 1440cctcttcttc ttccatcgct
acatatcagc atcactttcg tctgcatttt gagtgatcaa 1500aatcatcaaa
ctcgataatg ttatcttcat tgtaaagttt ttcatttttt tttccttttg
1560taaaattcct cttttttctc tctgttctta atgtagttct tgcgatttgc
cgaaaattga 1620atctcgtttc ttgattacag tttgtatcca tgtgaatatg
tctagatttt tttttttttt 1680tttgaagtat agtcattgat gattagacca
tcatttcggt tttacctcga gatgtttaag 1740acaaatgttg ctgtcttaga
tttgttttgt gttcgttcat aacttctggg gaaaaaaatg 1800agtaaagatc
aagaggtttt gagttaatca tccggtttaa gtggtattga aagtagctac
1860ctcggtattg gtgttaaaat gtctgtactc cttgagcgct ctgcgagaga
cagatatctt 1920catttttcat ttaatagcaa gtctcaatcg ttatggattt
gtatccagat ataatcaact 1980ctaagatcaa ggacggtgtt tgtttttaag
ctaactttct tgggtctcta ggtttggtgt 2040ggtgagtttt tggtttcttg
tcttccaaga aaaaaggcta gtgtaaatgg gagagtttca 2100cggttcttca
tttctttatt tcttttggtt tgaatgaaag tcttgtatgg ccagtactgt
2160gtatgggcaa taggcaataa catgagcttg tacctgctaa tcagagaaat
atagtggaac 2220taagtttgat agcgtgacga aactatttat gattaaatga
tagcacttcg tagttctaaa 2280acttaaagaa taacagataa atttcaaaat
gtagaagtac aaaaaatgat aaatactttt 2340gttagaaaat acctaattca
tctttatgtt ctcttataaa atggagactt acgtttataa 2400gttcttgttt
catcgaatat tgagcaataa ttgggggaga aatggattga gccaattata
2460taccagagag cacatggaac catcaatcaa attccaaaca gctcattgct
tctcttatct 2520attttctatt catttcacga ccgtcacaga aaaaaaaaaa
cagtttccta tttctttcac 2580tcttattatc agagagaaaa cgttcctact
atgaactgta ttagttaaaa aatacaaaga 2640actaaaacat attatattca
tttctacaat aaaaaacaaa aggcgatgac ataactaaaa 2700tcagctgcag
tactctttcc aactgtttat tttttctttc tttttcaact aagatctcag
2760ttggctttac agagattgat gtgctaataa agtttttgtt caccaaatac
aaagaattaa 2820aacaattcga aaatgattaa ttgtgttgat gtgttaatca
aactgtaaag tgataattac 2880acagttggcc ttacatgcat ctatgtgtat
atataaacca caagtcacaa cacatcaaaa 2940cacaccacaa
2950186371DNAArabidopsis thalianaCDS(57)..(236)encoding Arabidopsis
thaliana arabinogalactan- protein (AGP13) 186aacactaaac atatagtctc
ttcgtgcttt ctcaagaacc acctagagat aaccca atg 59 Met 1gag gca atg aag
atg aga ctc ttt gtg gcg gtt ttg gtg gca gcg atg 107Glu Ala Met Lys
Met Arg Leu Phe Val Ala Val Leu Val Ala Ala Met 5 10 15gct ttc tct
gcg gtg caa cag gct gcc gcg gtg gag gct cca gct ccg 155Ala Phe Ser
Ala Val Gln Gln Ala Ala Ala Val Glu Ala Pro Ala Pro 20 25 30agt cct
acc tcc gat gct tcc ttg gcc atc ccc gct ttc ttc gcc tcg 203Ser Pro
Thr Ser Asp Ala Ser Leu Ala Ile Pro Ala Phe Phe Ala Ser 35 40 45gta
gcc act ttg gcc ttt ggg ttt ctc ttc tag acaaatttgt ttttgtcttt
256Val Ala Thr Leu Ala Phe Gly Phe Leu Phe50 55ttaatgttat
aaaaatcata ttcttgtttt tttatttcgg ttttcattct tatgtactgt
316tctaagattt tccacatttg aatcaataaa attatctcgg tcactttttt tttat
37118759PRTArabidopsis thaliana 187Met Glu Ala Met Lys Met Arg Leu
Phe Val Ala Val Leu Val Ala Ala1 5 10 15Met Ala Phe Ser Ala Val Gln
Gln Ala Ala Ala Val Glu Ala Pro Ala 20 25 30Pro Ser Pro Thr Ser Asp
Ala Ser Leu Ala Ile Pro Ala Phe Phe Ala 35 40 45Ser Val Ala Thr Leu
Ala Phe Gly Phe Leu Phe 50 5518829DNAArtificial
sequenceoligonucleotide primer 188aggatccagc gagtatttga ttttatgtg
2918927DNAArtificial sequenceoligonucleotide primer 189tccatggtat
ccgttgtaga agatatc 2719033DNAArtificial sequenceoligonucleotide
primer 190tcacagccat ggcgttacca ttctttttaa atg 3319129DNAArtificial
sequenceoligonucleotide primer 191tggatccaag tgctctaaac tgacaaaac
2919227DNAArtificial sequenceoligonucleotide primer 192tccatggtat
ccgttgtaga agatatc 2719333DNAArtificial sequenceoligonucleotide
primer 193tcacagccat ggcgttacca ttctttttaa atg 3319426DNAArtificial
sequenceoligonucleotide primer 194cactggatcc ttcttcttct tctacg
2619526DNAArtificial sequenceoligonucleotide primer 195tccatggttg
ttgttgttgt agcagc 2619630DNAArtificial sequenceoligonucleotide
primer 196agaatgccat ggaaggtctc acacctatat 3019729DNAArtificial
sequenceoligonucleotide primer 197tggatccatc aatagaagag taattaatc
2919826DNAArtificial sequenceoligonucleotide primer 198tccatggttg
ttgttgttgt agcagc 2619930DNAArtificial sequenceoligonucleotide
primer 199agaatgccat ggaaggtctc acacctatat 3020029DNAArtificial
sequenceoligonucleotide primer 200aatatactcg agatccgcac tgactagac
2920128DNAArtificial sequenceoligonucleotide primer 201tactgcccat
ggtccttttc taccacac 2820229DNAArtificial sequenceoligonucleotide
primer 202tggttcccat ggagagaaaa aaaagaaag 2920328DNAArtificial
sequenceoligonucleotide primer 203cttccgctcg agttttatgt caaagacg
2820428DNAArtificial sequenceoligonucleotide primer 204tactgcccat
ggtccttttc taccacac 2820529DNAArtificial sequenceoligonucleotide
primer 205tggttcccat ggagagaaaa aaaagaaag 2920629DNAArtificial
sequenceoligonucleotide primer 206ataatgggat cctcattgta aagtttttc
2920725DNAArtificial sequenceoligonucleotide primer 207ttgcctccat
ggggttatct ctagg 2520830DNAArtificial sequenceoligonucleotide
primer 208ttgtggccat ggttgatgtg ttgtgacttg 3020924DNAArtificial
sequenceoligonucleotide primer 209tggacgggat ccttgactga caag
2421025DNAArtificial sequenceoligonucleotide primer 210ttgcctccat
ggggttatct ctagg 2521130DNAArtificial sequenceoligonucleotide
primer 211ttgtggccat ggttgatgtg ttgtgacttg 30
* * * * *