U.S. patent application number 12/424581 was filed with the patent office on 2009-08-27 for expression cassettes for vascular tissue-preferential expression in plants.
This patent application is currently assigned to SunGene GmbH. Invention is credited to Karin Herbers, Helke Hillebrand, Ulrich Keetman.
Application Number | 20090217419 12/424581 |
Document ID | / |
Family ID | 36577243 |
Filed Date | 2009-08-27 |
United States Patent
Application |
20090217419 |
Kind Code |
A1 |
Keetman; Ulrich ; et
al. |
August 27, 2009 |
EXPRESSION CASSETTES FOR VASCULAR TISSUE-PREFERENTIAL EXPRESSION IN
PLANTS
Abstract
The present invention relates to expression cassettes comprising
transcription regulating sequences with vascular
tissue-preferential or vascular tissue-specific expression profiles
in plants obtainable from Arabidopsis thaliana genes At4g00140,
At5g45350, At2g39830, At1g68430, or At5g67280.
Inventors: |
Keetman; Ulrich;
(Quedlinburg, 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: |
SunGene GmbH
Catersleben
DE
|
Family ID: |
36577243 |
Appl. No.: |
12/424581 |
Filed: |
April 16, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11296444 |
Dec 7, 2005 |
7524948 |
|
|
12424581 |
|
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Current U.S.
Class: |
800/295 ;
435/320.1; 435/410; 435/6.12; 435/6.13 |
Current CPC
Class: |
C12N 15/8223
20130101 |
Class at
Publication: |
800/295 ;
435/320.1; 435/410; 435/6 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C12N 15/74 20060101 C12N015/74; C12N 5/10 20060101
C12N005/10; C12Q 1/68 20060101 C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 8, 2004 |
EP |
04029025.6 |
Feb 3, 2005 |
EP |
05002263.1 |
Feb 11, 2005 |
EP |
05002854.7 |
Claims
1. An expression cassette for regulating vascular
tissue-preferential or vascular tissue-specific expression in
plants comprising i) at least one transcription regulating
nucleotide sequence of a plant gene, said plant gene selected from
the group of genes described by the Arabidopsis thaliana genome
loci At5g45350, At2g39830, At1g68430, and At5g67280, 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 nucleotide sequence described by SEQ
ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24,
25, 28, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41, 42, 43, 44, or
45, ii) a fragment of at least 50 consecutive bases of a sequence
under 1) which has substantially the same promoter activity as the
corresponding transcription regulating nucleotide sequence
described by SEQ ID NO: 8, 0, 10, 11, 12, 13, 14, 15, 18, 19, 20,
21, 22, 23, 24, 25, 28, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41,
42, 43, 44, or 45: iii) a nucleotide sequence having substantial
similarity with a sequence identity of at least 40% to a
transcription regulating nucleotide sequence described by SEQ ID
NO: 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25,
28, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41, 42, 43, 44, or 45;
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. to a transcription
regulating nucleotide sequence described by SEQ ID NO: 8, 9, 10,
11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25, 28, 29, 30, 31,
32, 33, 34, 35, 38, 30, 40, 41, 42, 43, 44, or 45, 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 NaPO.sub.4, 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: 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25,
28, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41, 42, 43, 44, or 45,
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 a polypeptide comprising SEQ ID NO: 17, 27, 37, or 47.
4. The expression cassette of claim 1, wherein expression of the
nucleic acid sequence results in expression of a protein, or
expression of a antisense RNA, sense or 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 the
expression cassette.
8. A transgenic plant comprising the expression cassette of claim
1, a vector comprising the expression cassette, a cell comprising
the expression cassette, or a cell comprising the vector.
9. A method for identifying and/or isolating a sequence with
vascular tissue-preferential or vascular tissue-specific
transcription regulating activity comprising utilizing a nucleic
acid sequence encoding a amino acid sequence as described by SEQ ID
NO: 17, 27, 37, or 47, or a part of at least 15 bases thereof for
identifying and/or isolating a sequence with vascular
tissue-preferential or vascular tissue-specific transcription
regulating activity.
10. The method of claim 9, wherein the nucleic acid sequences is
described by SEQ ID NO: 16, 26, 36, or 46, or a part of at least 15
bases thereof.
11. The method of claim 9, wherein said identification and/or
isolation is realized by a method selected from polymerase chain
reaction, hybridization, or database screening.
12. A method for providing a transgenic expression cassette for
vascular tissue-preferential or vascular tissue-specific expression
comprising the steps of: I. isolating of a vascular
tissue-preferential or vascular tissue-specific transcription
regulating nucleotide sequence utilizing at least one nucleic acid
sequence or a part thereof wherein said sequence is encoding a
polypeptide comprising SEQ ID NO: 17, 27, 37, or 47, or a part of
at least 15 bases thereof, and II. functionally linking said
vascular tissue-preferential or vascular tissue-specific
transcription regulating nucleotide sequence to another nucleotide
sequence of interest, which is heterologous in relation to said
vascular tissue-preferential or vascular tissue-specific
transcription regulating nucleotide sequence.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/296,444, filed Dec. 7, 2005 which claims priority to
European Application No. 04029025.6, filed Dec. 8, 2004, European
Application No. 05002263.1, filed Feb. 3, 2005 and European
Application No. 05002854.7, filed Feb. 11, 2005 The entire contents
of each of these applications are hereby incorporated by reference
herein.
SUBMISSION OF SEQUENCE LISTING
[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_List.sub.--13173.sub.--00032_US. The size of the text tile
is 143 KB, and the text file was created on Apr. 15, 2009.
FIELD OF THE INVENTION
[0003] The present invention relates to expression cassettes
comprising transcription regulating nucleotide sequences with
vascular tissue-preferential or vascular tissue-specific expression
profiles in plants obtainable from Arabidopsis thaliana genes
At4g00140, At5g45350, At2g39830, At1g68430, or At5g67280.
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 vascular tissue-preferential or vascular tissue-specific
promoters are useful for improving the transport capacities within
a plant. The number of promoters which are capable to regulate
expression in vascular tissue is limited (see e.g., WO2004048595;
Gittins 2003; Liu Z Z 2003; Ramos 2004).
[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 vascular tissue-preferential
or vascular tissue-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 vascular tissue-specific or vascular
tissue-preferential transcription 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 At4g00140, At5g45350,
At2g39830, At1g68430, or At5g67280, 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, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20,
21, 22, 23, 24, 25, 28, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41,
42, 43, 44, and 45, [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, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25,
28, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41, 42, 43, 44, or 45;
[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, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20,
21, 22, 23, 24, 25, 28, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41,
42, 43, 44, or 45; [0014] iv) a nucleotide sequence capable of
hybridizing (preferably 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, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20,
21, 22, 23, 24, 25, 28, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41,
42, 43, 44, or 45, or the complement thereof; [0015] v) a
nucleotide sequence capable of hybridizing (preferably 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, 8, 9, 10, 11, 12,
13, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25, 28, 29, 30, 31, 32, 33,
34, 35, 38, 39, 40, 41, 42, 43, 44, and 45 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: 7, 17, 27, 37, and 47, 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 vascular
tissue-specific or vascular tissue-preferential transcription
regulating activity characterized that said identification and/or
isolation utilizes; nucleic acid sequence encoding a amino acid
sequence as described by SEQ ID NO: 7, 17, 27, 37, or 47 or a part
of at least 15 bases thereof. Preferably the nucleic acid sequences
is described by SEQ ID NO: 6, 16, 26, 36, or 46 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 vascular
tissue-specific or vascular tissue-preferential expression
comprising the steps of: [0022] I. isolating of a vascular
tissue-preferential or vascular tissue-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: 7, 17, 27, 37, or 47, or a part
of at least 15 bases thereof, and [0023] II. functionally linking
said vascular tissue-preferential or vascular tissue specific
transcription regulating nucleotide sequence to another nucleotide
sequence of interest, which is heterologous in relation to said
vascular tissue-preferential or vascular tissue-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.
A "marker gene" encodes a selectable or screenable trait.
[0029] The term "chimeric gene" refers to any gene that
contains
[0030] 1) DNA sequences, including regulatory and coding sequences,
that are not found together in nature, or
[0031] 2) sequences encoding parts of proteins not naturally
adjoined, or
[0032] 3) parts of promoters that are not naturally adjoined.
[0033] 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.
[0034] A "transgene" refers to a gene that has been introduced into
the genome by transformation 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.
[0035] 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.
[0036] The terms "protein," "peptide" and "polypeptide" are used
interchangeably herein.
[0037] 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 vascular
tissue-specific or vascular tissue-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 re-ligation 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",
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, 8, 9, 10, 11, 12, 13, 14, 16,
18, 19, 20, 21, 22, 23, 24, 25, 28, 20, 30, 31, 32, 33, 34, 35, 38,
39, 40, 41, 42, 43, 44, or 45, a nucleotide sequence comprising an
open reading frame having any one of SEQ ID NOs: 6, 16, 26, 36, or
46, which encodes one of SEQ ID NOs: 7, 17, 27, 37, or 47. 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 transgenic 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 1907) 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 trans-genic 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.
[0097] 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."
[0098] "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 vascular tissue-specific or
vascular tissue preferential promoters of the invention).
[0099] "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).
[0100] 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).
[0101] 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
bi-functional 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.
[0102] "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.
[0103] A "transgenic plant" is a plant having one or more plant
cells that contain an expression vector.
[0104] "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.
[0105] 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". [0106] (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.
[0107] (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.
[0108] 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.
[0109] 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.
[0110] Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information
(www.ncbi.nlm.nih.gov/). 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.
[0111] 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.
[0112] 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). See
www.ncbi.nlm.nih.gov. Alignment may also be performed manually by
inspection.
[0113] 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. [0114] (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.). [0115] (d) As used herein, "percentage or 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. [0116] (e) (i) The term
"substantial identity" or "substantial similarity" of
polynucleotide sequences means (preferably for a protein encoding
sequence) 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
(preferably 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%.
[0117] 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. [0118] (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.
[0119] 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 sequencers) relative to the
reference sequence, based on the designated program parameters.
[0120] 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.
[0121] "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
[0122] 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.
[0123] 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.
[0124] 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% form amide, 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.
[0125] 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.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.
[0126] "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.
[0127] "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.
[0128] 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.
[0129] 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, Carophyllacese,
Chenopodiaceae, Compositae, Cucurbitace ae, Labiatae, Leguminosae,
Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae,
Saxifragaceae, Scrophulariaceae, Solanaceae, Tetragoniaceae.
[0130] 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 Chloroptiyceae, Phaeophpyceae,
Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophyceae
(diatoms) and Euglenophyceae.
[0131] 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,
Gesnedaceae 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.
[0132] 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 Cruciferae,
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
saliva (lettuce) and many others.
[0133] 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,
linseed, potato and tagetes.
[0134] "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.
[0135] "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
[0136] The present invention thus provides for isolated nucleic
acid molecules comprising a plant nucleotide sequence that directs
vascular tissue-preferential or vascular tissue-specific
transcription of an operably linked nucleic acid fragment in a
plant cell.
[0137] Specifically, the present invention provides transgenic
expression cassettes for regulating vascular tissue-preferential or
vascular tissue-specific expression in plants comprising [0138] 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
At4g00140, At5g45350, At2g39830, At1g68430, or At5g67280, or a
functional equivalent thereof, and functionally linked thereto
[0139] ii) at least one nucleic acid sequence which is heterologous
in relation to said transcription regulating nucleotide
sequence.
[0140] The term "vasculature-specific" or "vascular
tissue-preferential" in the context of the inventions means a
expression in one or more vascular tissue of a plant. Vascular
tissue are is the tissue in which long distance transport of water
and various dissolved substances is located. Vascular tissue can
essentially be divided into phloem and xylem and a cambial region
separating both tissue sub-types. Phloem constitutes cells in which
photosynthates (i.e. photosynthetic products as e.g. sugars and
amino acids, signal molecules, as e.g. hormones and micro RNAs) are
transported from their site of production (i.e. source tissue as
e.g. fully grown leaves) to their site of storage and consumption
(i.e. sink tissue as e.g. flowers, tubers, seeds). This fluid is
also referred to as "phloem sap". The transport of solutes in the
sieve elements of the phloem is highly dependent on the neighboring
companion cells that provide energy to the sieve element cells and
also function in loading and unloading solutes into and from the
sieve elements, respectively Xylem vessels consist of fused cells
and are surrounded by parenchyma cells. Xylem vessels are
instrumental in transporting water and minerals from the root to
the shoot, a process that is driven by transpiration of water vapor
in the above-ground organs of a plant. Vascular tissue in the
context of the invention comprises all of the cell types described
afore regardless of different anatomical organization of phloem and
xylem in roots, stems/stalks, flowers or leaves. In leaves,
vascular bundles of different order are formed during the
development. This is also referred to as leave veins of different
order.
[0141] The vascular tissue-preferential or vascular tissue-specific
promoters may be useful for improving the transport capacities
within a plant. Promoters specifically active in the vascular
tissue of plants could serve in driving effect genes, e.g. encoding
transporters, in the phloem involved in loading or unloading
solutes. One might manipulate this process in order to make it more
efficient or more selective for particular solutes and by this
alter storage compound allocation. Promoters active in the xylem
might be useful in enhancing water use efficiency of plants (see
e.g., Chaves 2004). Furthermore it is known that micro RNAs and
other signal molecules (as e.g. hormones) implied in the systemic
or directed spread of signals are also transported in the phloem.
One might engineer constructs interfering with these processes by
using vascular tissue-specific promoters (see e.g., Sobeih 2004).
It is also proposed that pathogens as e.g. viruses make use of the
vascular system when infecting plants (see e.g., Decroocq 2001).
Promoters specifically active in vascular tissue might therefore
also be useful in preventing pathogens from systemic spread.
[0142] One might combine approaches in which vascular
tissue-specific promoters drive certain effect genes with
approaches in which root-specific promoters are employed, e.g. in
order to enhance nutrient uptake from the soil. By this
combinatorial approach uptake into and distribution within the
plant might be improved. Another field of application for vascular
tissue-specific promoters might be the alteration of fiber
composition, structure or content, by driving trait genes coding
for e.g. cell wall modifying enzymes possibly involved in
lignification. Theses approaches would aim at the improvement of
food or feed quality, or at modified fiber characteristics
important for industrial applications.
[0143] "Vascular tissue-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.
All of the transcription regulating nucleotide sequences specified
herein (designated pSUK416, pSUK416 GB, pSUK418, pSUK418 GB,
pSUK418LGB, pSUK420, pSUK420 GB, pSUK422, pSUK422GB, pSUK424L,
pSUK424LGB, pSUK424S, pSUK424SGB, pSUK426L, pSUK426LGB, pSUK426S,
pSUK426SGB, pSUK428L, pSUK428LGB, pSUK428S, pSUK428SGB, pSUK430L,
pSUK430LGB, pSUK430S, pSUK430SGB, pSUK436L, pSUK436LGB, pSUK436S,
pSUK436SGB, pSUK438L, pSUK438LGB, pSUK438S, pSUK438SGB) are
considered to be vascular tissue-specific transcription regulating
nucleotide sequences.
[0144] "Vascular tissue-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.
[0145] 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). The 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 vascular tissue-preferential or vascular
tissue-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.
[0146] 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 At4g00140 encoding Arabidopsis SEQ ID NO: NM_116231
NP_191925 thaliana expressed protein 1, 2, 3, 4, 5 SEQ ID NO: 6 SEQ
ID NO: 7 At5g45350 encoding Arabidopsis SEQ ID NO: NM_123903
NP_568642.1 thaliana proline-rich family 8, 9, 10, 11, 12, 13, SEQ
ID NO: 16 SEQ ID NO: 17 protein 14, 15 At2g39830 encoding LIM
domain- SEQ ID NO: NM_129542 NP_181513 containing protein 18, 19,
20, 21, 22, SEQ ID NO: 26 SEQ ID NO: 27 23, 24, 25, At1g68430
encoding expressed protein SEQ ID NO: NM_105514 NP_564929 28, 29,
30, 31, 32, SEQ ID NO: 36 SEQ ID NO: 37 33, 34, 35, At5g67280
encoding putative leucine- SEQ ID NO: NM_126128 NP_201529 rich
repeat transmembrane 38, 39, 40, 41, 42, SEQ ID NO: 46 SEQ ID NO:
47 protein kinase 43, 44, 45
[0147] Preferably the transcription regulating nucleotide sequence
(or the functional equivalent thereof) is selected from the group
of sequences consisting of [0148] i) the sequences described by SEQ
ID NOs: 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20,
21, 22, 23, 24, 25, 28, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41,
42, 43, 44, and 45 [0149] 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, 8, 9, 10, 11, 12,
13, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25, 28, 29, 30, 31, 32, 33,
34, 35, 38, 39, 40, 41, 42, 43, 44, or 45; [0150] 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, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25,
28, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41, 42, 43, 44, or 45;
[0151] iv) a nucleotide sequence capable of hybridizing (preferably
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, 8, 9, 10, 11, 12, 13, 14,
15, 18, 19, 20, 21, 22, 23, 24, 25, 28, 29, 30, 31, 32, 33, 34, 35,
38, 39, 40, 41, 42, 43, 44, or 45, or the complement thereof;
[0152] v) a nucleotide sequence capable of hybridizing (preferably
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% SODS 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, 8, 9, 10, 11, 12,
13, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25, 28, 29, 30, 31, 32, 33,
34, 35, 38, 39, 40, 41, 42, 43, 44, or 45, or the complement
thereof;
[0153] vi) a nucleotide sequence which is the complement or reverse
complement of any of the previously mentioned nucleotide sequences
under i) to v).
[0154] 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: 7, 17, 27, 37, or 47,
respectively, or a fragment of said transcription regulating
nucleotide sequence which exhibits promoter activity in a vascular
tissue-preferential or vascular tissue-specific fashion.
[0155] The activity of a transcription regulating nucleotide
sequence is considered equivalent if transcription is initiated in
a vascular tissue-preferential or vascular tissue-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
.beta.-galactosidase. Especially preferred is .beta.-glucuronidase
(Jefferson 1987).
[0156] 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%.
[0157] 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:
6, 16, 26, 36, or 46, respectively, or a fragment of said
transcription regulating nucleotide sequence which exhibits
promoter activity in a vascular tissue-preferential or vascular
tissue-specific fashion.
[0158] Such functional equivalent of the transcription regulating
nucleotide sequence may be obtained from other plant species by
using the vascular tissue-preferential or vascular tissue-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 vascular tissue-preferential or vascular tissue-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 vascular
tissue-preferential or vascular tissue-specific promoter sequences
could be employed to identify structurally related sequences in a
database using computer algorithms.
[0159] 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.
[0160] 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.
[0161] 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.
[0162] 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.
[0163] 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.
[0164] Thus, another embodiment of the invention relates to a
method for identifying and/or isolating a sequence with vascular
tissue-preferential or vascular tissue-specific transcription
regulating activity utilizing a nucleic acid sequence encoding a
amino acid sequence as described by SEQ ID NO: 7, 17, 27, 37, or 47
or a part thereof. Preferred are nucleic acid sequences described
by SEQ ID NO: 6, 16, 26, 36, or 46 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").
[0165] Another embodiment of the invention is related to a method
for providing a trans-genic expression cassette for vascular
tissue-preferential or vascular tissue-specific expression
comprising the steps of: [0166] I. isolating of a vascular
tissue-preferential or vascular tissue-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: 7, 17, 27, 37, or 47, or a part
of at least 15 bases thereof, and [0167] II. functionally linking
said vascular tissue-preferential or vascular tissue-specific
transcription regulating nucleotide sequence to another nucleotide
sequence of interest, which is heterologous in relation to said
vascular tissue-preferential or vascular tissue-specific
transcription regulating nucleotide sequence.
[0168] 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: 6, 16, 26, 36, or 46. Preferably, the isolation of the vascular
tissue-preferential or vascular tissue-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.
[0169] 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, 8,
9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25, 28, 29,
30, 31, 32, 33, 34, 35, 38, 39, 40, 41, 42, 43, 44, and 45, or the
promoter orthologs thereof, which include the minimal promoter
region.
[0170] 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, 8, 9, 10, 11, 12, 13, 14,
15, 18, 19, 20, 21, 22, 23, 24, 25, 28, 29, 30, 31, 32, 33, 34, 35,
38, 39, 40, 41, 42, 43, 44, and 45, 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.
[0171] The transcription regulating nucleotide sequences of the
invention or their functional equivalents are capable of driving
vascular tissue-preferential or vascular tissue-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 vascular tissue-preferential or vascular
tissue-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.
[0172] The transcription regulating nucleotide sequences and
promoters of the invention arc 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.
[0173] 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.
[0174] 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, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23,
24, 25, 28, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41, 42, 43, 44,
or 45) 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.
[0175] 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).
[0176] 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, 8, 9,
10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25, 28, 29, 30,
31, 32, 33, 34, 35, 38, 39, 40, 41, 42, 43, 44, or 45) 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 vascular tissue-preferential or vascular
tissue-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.
[0177] 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.
[0178] 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.
[0179] Vascular tissue-preferential or vascular tissue-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. Vascular tissue-preferential or vascular
tissue-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 arc useful to
generate synthetic transcription regulating nucleotide sequences
(e.g., promoters).
[0180] 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.
[0181] 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.
[0182] 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.
[0183] 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, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22,
23, 24, 25, 28, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41, 42, 43,
44, or 45 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).
[0184] 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).
[0185] 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.
[0186] 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.
[0187] 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."
[0188] 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.
[0189] 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.
[0190] 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.
[0191] 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
[0192] 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.
[0193] 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.
[0194] 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.
[0195] 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 prepare 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.
[0196] 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 phage 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.
[0197] 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.
[0198] 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.
[0199] 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.
[0200] 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 which 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.
[0201] 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 BIOBASE
database "Transfac" (Biologische Datenbanken GmbH, Braunschweig;
Wingender 2001) or the database PlantCARE (Lescot 2002).
[0202] 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, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25,
28, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41, 42, 43, 44, or 45.
More preferably this fragment is starting from the 3'-end of the
indicated sequences.
[0203] 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: 5 (3977
bp) SEQ ID NO: 1 (1043 bp) SEQ ID NO: 2 (1057 bp) SEQ ID NO: 3
(2464 bp) SEQ ID NO: 4 (2478 bp) SEQ ID NO: 12 (1931 bp) SEQ ID NO:
SEQ ID NO: 8 (1147 bp) 13 (1945 bp) SEQ ID NO: 9 (1161 bp) SEQ ID
NO: 10 (304 bp) SEQ ID NO: 11 (316 bp) SEQ ID NO: 14 (1088 bp) SEQ
ID NO: 15 (1100 bp) SEQ ID NO: 22 (2399 bp) SEQ ID NO: SEQ ID NO:
18 (1235 bp) 23 (2413 bp) SEQ ID NO: 19 (1249 bp) SEQ ID NO: 20
(1135 bp) SEQ ID NO: 21 (1147 bp) SEQ ID NO: 24 (2299 bp) SEQ ID
NO: 25 (2311 bp) SEQ ID NO: 32 (2096 bp) SEQ ID NO: SEQ ID NO: 28
(1051 bp) 33 (2110 bp) SEQ ID NO: 29 (1065 bp) SEQ ID NO: 30 (1038
bp) SEQ ID NO: 31 (1050 bp) SEQ ID NO: 34 (2083 bp) SEQ ID NO: 35
(2095 bp) SEQ ID NO: 42 (2022 bp) SEQ ID NO: SEQ ID NO: 38 (1030
bp) 43 (2036 bp) SEQ ID NO: 39 (1044 bp) SEQ ID NO: 40 (968 bp) SEQ
ID NO: 41 (980 bp) SEQ ID NO: 44 (1960 bp) SEQ ID NO: 45 (1972
bp)
[0204] 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.
[0205] 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).
[0206] Additional regulatory elements may comprise additional
promoter, minimal promoters, or promoter elements which may modify
the expression regulating properties. For ex ample 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 realized expression in other organisms (such as E. coli or
Agrobacterium). Such regulatory elements can be find 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).
[0207] 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 spatialtemporally regulated.
[0208] 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.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] 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, Della-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 loci At4g00140, At5g453sn,
At2g39830, At1g68430, or At5g67280, or of functional equivalent
thereof.
[0213] 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).
[0214] 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 (EIlis 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.
[0215] 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).
[0216] 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 vascular tissue-preferential or
vascular tissue-specific manner.
[0217] 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.
[0218] 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.
[0219] 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
tinal polypeptide. Targeting of certain proteins may be desirable
in order to enhance the stability of the protein (U.S. Pat. No.
5,545,818).
[0220] 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 an 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.
[0221] 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.
[0222] 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.
[0223] 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.
[0224] 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.
[0225] 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 cotransformation.
[0226] 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.
[0227] 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 Insect Resistance
[0228] An important aspect of the present invention concerns the
introduction of insert resistance-conferring genes into plants.
Especially for preventing damages caused by sucking insects
expression of insecticidal protein in vascular tissues is
contemplated to be useful. 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 CrylA(b) and CrylA(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 II 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. This group may be exemplified by
cystatin and amylase inhibitors, such as those from wheat and
barley.
[0229] 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.
[0230] 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).
[0231] 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.
[0232] 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.
[0233] 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.
[0234] Tripsacum dactyloides is a species of grass that is
resistant to certain insects, including corn root worm. 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 & Cuss, 1972).
[0235] 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.
1.2 Environment or Stress Resistance
[0236] 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. Especially expression of genes
improving transport capabilities in vascular tissue is contemplated
to be useful in the context of the present invention. 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 chloroplasts (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.
[0237] 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).
[0238] 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 & Bohncrt 1002), and raffinose (Dermal-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.
[0239] 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, 1003). 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.
[0240] 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 or
tissue-specific expression 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.
[0241] 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).
[0242] 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 cross a full range of
stresses relating to water availability, yield stability or
consistency of yield performance may be realized.
[0243] 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; Poi Z M et al. (1998) Science
282:287-200), 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 B 1A"; 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).
1.3 Disease Resistance
[0244] It is proposed that increased resistance to diseases may be
realized through introduction of genes into plants period.
Especially expression of genes that confer resistance against
systemically acting pathogens (such as bacteria or virus), which
utilize the vascular tissue to migrate throughout the plant, is
thought to be useful. 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.
[0245] 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.
[0246] 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.
[0247] 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.
[0248] 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
P450 (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 nivalis) lectin agglutinin (Rao
et al. (1998) Plant J 15(4):469-77). The expression of synthetic
cryIA(b) and crylA(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).
1.4 Plant Agronomic Characteristics
[0249] 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, plant 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
trans-formation 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. Promoters, which can mediate
vascular-tissue specific or -preferential expression are especially
useful in this context to enhance resistance against draught.
[0250] 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.
[0251] Delay of late season vegetative senescence would increase
the flow of assimilate 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.
1.5 Nutrient Utilization
[0252] 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.
1.6. Non-Protein-Expressing Sequences
1.6.1 RNA-Expressing
[0253] 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.
[0254] 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.
[0255] 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 arte 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).
[0256] 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 possees
reduced levels of polypeptides including but not limited to the
polypeptides cited above that may be affected by antisense RNA.
[0257] 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.
1.6.2 Non-RNA-Expressing
[0258] 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.
[0259] 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.
[0260] 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).
[0261] 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., vascular-, root, green tissue (leaf and
stem), panicle-, or pollen, or is expressed constitutively.
2. Marker Genes
[0262] 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.
[0263] 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).
[0264] 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.
[0265] 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.
[0266] 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.
[0267] 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
[0268] Various selectable markers are known in the art suitable for
plant transformation. Such markers can be expressed under a
transcription regulating nucleotide sequence of the invention but
can also be employed in operable linkage with another promoter
(e.g., a constitutive promoter) as a separate expression cassette
in an expression construct or vector of the invention. Such markers
may include but are not limited to:
2.1.1 Negative Selection Markers
[0269] Negative selection markers confer a resistance to a hiocidal
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: [0270] 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. [0271] 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). [0272] Glyphosate.RTM. degrading enzymes
(Glyphosate.RTM. oxidoreductase; gox), [0273] Dalapon.RTM.
inactivating dehalogenases (deh) [0274] 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) [0275] Bromoxynil.RTM.
degrading nitrilases (bxn; Stalker 1988) [0276] Kanamycin- or,
geneticin (G3418) resistance genes (NPTII; NPT or neo; Potrykus
1985) coding e.g., for neomycin phosphotransferases (Fraley 1983;
Nehra 1994) [0277] 2-Desoxyglucose-6-phosphate phosphatase
(DOG.sup.R1-Gene product; WO 98/45456; EP 0 807 836) conferring
resistance against 2-desoxyglucose (Randez-Gil 1995). [0278]
hygromycin phosphotransferase (HPT), which mediates resistance to
hygromycin (Vanden Elzen 1985). [0279] altered dihydrofolate
reductase (Eichholtz 1987) conferring resistance against
methotrexat (Thillet 1988); [0280] mutated anthranilate synthase
genes that confers resistance to 5-methyl tryptophan.
[0281] Additional negative selectable marker genes of bacterial
origin that confer resistance to antibiotics include the aadA gene,
which conters 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).
[0282] 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 daol 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; GenBank Acc.-No.: J01603).
[0283] 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).
[0284] 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.
[0285] 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.
[0286] 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/l may be
included in the medium. Typical concentrations for selection are 20
to 40 mg/l.
2.1.2 Positive Selection Marker
[0287] 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)
.beta.-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
[0288] 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
.alpha.-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., prevent 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
[0289] 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 xylE gene (Zukowsky 1983)
which encodes a catechol dioxygenase that can convert chromogenic
catechols; an .alpha.-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 torm the easily
detectable compound melanin; .beta.-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).
[0290] 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, P1. Alternatively any
genotype of maize can be utilized if the C1 and R alleles are
introduced together.
[0291] 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 present 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.
[0292] 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
[0293] 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.
[0294] 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.
[0295] 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.
[0296] 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).
[0297] 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.
[0298] 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.
[0299] 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 vascular tissue-preferential or vascular tissue-specific
expression of the antisense sequence produces an RNA transcript
that interferes with translation of the mRNA of the native DNA
sequence.
[0300] 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 (betaGAL), and
luciferase.
[0301] 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.
[0302] 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 shidy. 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.
[0303] 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.
[0304] 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 EXM5 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 biusynthetic enzyme promoter, an
Sadenosyl-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
Sadenosyl-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 clialcone 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
[0305] 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.
[0306] 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).
[0307] 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.
[0308] 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, mituchondria
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, 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.
[0309] 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
organism. Both microorganism and higher organisms are comprised.
Preferred microorganism are bacteria, yeast, algae, and fungi.
Preferred bacteria are those of the genus Escherichia, Erwinia,
Agrobacterium, Flavobacterium, Alcali-genes, 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).
[0310] 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.
[0311] 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.
[0312] 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).
[0313] 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).
[0314] 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.
[0315] 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).
[0316] 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 (Cross-way 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); Datta
1990 (rice); Klein 1988 (maize); Klein 1988 (maize), Klein 1988
(maize); Fromm 1990 (miaize), 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).
[0317] 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
95/16783, 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 present invention are obtained, and are preferentially capable
of high expression of the nucleotide sequence.
[0318] 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.
[0319] 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 CS8C1[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
Tiplasmids or helper plasmids, it is preferred that the virF gene
be deleted or inactivated (Jarschow 1991).
[0320] 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).
[0321] 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).
[0322] 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 g/l peptone, 5 g/l NaCl, 15 g/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.
[0323] 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 to 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.
[0324] 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.
[0325] 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 1990)
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
thiolsulfate, 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 thiolsulfate. 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.
[0326] 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.
[0327] 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
[0328] 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
[0329] 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 and plantlets
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.
[0330] 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; "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.
[0331] 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.
[0332] The presence of nucleic acid elements introduced through the
methods of this invention may be determined by polymerase chain
reaction (PCR). Using this 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.
[0333] 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.
[0334] 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.
[0335] 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 transformiants (R.sub.0) was
suggested by germlne 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.
[0336] 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.
[0337] 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.
[0338] 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.
[0339] 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.
[0340] 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
[0341] 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.
[0342] 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.
[0343] 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 anti-bodies (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.
[0344] 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.
[0345] 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.
[0346] 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 to dispense 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
[0347] 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, trans-formation 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).
[0348] 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.
Example 1
Growth Conditions for Plants for Tissue-Specific Expression
Analysis
[0349] 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 .mu.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.
[0350] 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 VM, 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 flowers are harvested in stage 15
to 16. Green and yellow shoots used for the analysis have a length
of 10 to 13 mm.
Example 2
Demonstration of Expression Profile
[0351] 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 .beta.-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).
[0352] 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 3
Cloning of the Promoter Fragments
[0353] To isolate the promoter fragments described by SEQ ID NO: 1,
2, 3, 4, 5, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23,
24, 25, 28, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41, 42, 43, 44,
and 45 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 pSUK416 SUK416for SUK416rev SpeI/NcoI SEQ ID
NO: 48 SEQ ID NO: 49 SEQ ID NO: 2 pSUK416GB SUK416for SUK416rev
SpeI/NcoI SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 3 pSUK418
SUK418for SUK418rev SpeI/NcoI SEQ ID NO: 50 SEQ ID NO: 51 SEQ ID
NO: 4 pSUK418GB SUK418for SUK418rev SpeI/NcoI SEQ ID NO: 50 SEQ ID
NO: 51 SEQ ID NO: 5 pSUK418LGB SUK418Lfor SUK418Lrev BamHI/NcoI SEQ
ID NO: 52 SEQ ID NO: 53 SEQ ID NO: 8 pSUK420L SUK420for SUK420Lrev
EcoRI/NcoI SEQ ID NO: 54 SEQ ID NO: 55 SEQ ID NO: 9 pSUK420LGB
SUK420for SUK420Lrev EcoRI/NcoI SEQ ID NO: 54 SEQ ID NO: 55 SEQ ID
NO: 10 pSUK420S SUK420for SUK420Srev EcoRI/NcoI SEQ ID NO: 54 SEQ
ID NO: 56 SEQ ID NO: 11 pSUK420SGB SUK420for SUK420Srev EcoRI/NcoI
SEQ ID NO: 54 SEQ ID NO: 56 SEQ ID NO: 12 pSUK422L SUK422for
SUK422Lrev BamHI/NcoI SEQ ID NO: 57 SEQ ID NO: 58 SEQ ID NO: 13
pSUK422LGB SUK422for SUK422Lrev BamHI/NcoI SEQ ID NO: 57 SEQ ID NO:
58 SEQ ID NO: 14 pSUK422S SUK422for SUK422Srev BamHI/NcoI SEQ ID
NO: 57 SEQ ID NO: 59 SEQ ID NO: 15 pSUK422SGB SUK422for SUK422Srev
BamHI/NcoI SEQ ID NO: 57 SEQ ID NO: 59 SEQ ID NO: 18 pSUK424L
SUK424for SUK424Lrev EcoRI/NcoI SEQ ID NO: 60 SEQ ID NO: 61 SEQ ID
NO: 19 pSUK424LGB SUK424for SUK424Lrev EcoRI/NcoI SEQ ID NO: 60 SEQ
ID NO: 61 SEQ ID NO: 20 pSUK424S SUK424for SUK424Srev EcoRI/NcoI
SEQ ID NO: 60 SEQ ID NO: 62 SEQ ID NO: 21 pSUK424SGB SUK424for
SUK424Srev EcoRI/NcoI SEQ ID NO: 60 SEQ ID NO: 62 SEQ ID NO: 22
pSUK426L SUK426for SUK426Lrev BamHI/NcoI SEQ ID NO: 63 SEQ ID NO:
64 SEQ ID NO: 23 pSUK426LGB SUK426for SUK426Lrev BamHI/NcoI SEQ ID
NO: 63 SEQ ID NO: 64 SEQ ID NO: 24 pSUK426S SUK426for SUK426Srev
BamHI/NcoI SEQ ID NO: 63 SEQ ID NO: 65 SEQ ID NO: 25 pSUK426SGB
SUK426for SUK426Srev BamHI/NcoI SEQ ID NO: 63 SEQ ID NO: 65 SEQ ID
NO: 28 pSUK428L SUK428for SUK428Lrev SpeI/NcoI SEQ ID NO: 66 SEQ ID
NO: 67 SEQ ID NO: 29 pSUK428LGB SUK428for SUK428Lrev SpeI/NcoI SEQ
ID NO: 66 SEQ ID NO: 67 SEQ ID NO: 30 pSUK428S SUK428for SUK428Srev
SpeI/NcoI SEQ ID NO: 66 SEQ ID NO: 68 SEQ ID NO: 31 pSUK428SGB
SUK428for SUK428Srev SpeI/NcoI SEQ ID NO: 66 SEQ ID NO: 68 SEQ ID
NO: 32 pSUK430L SUK430for SUK430Lrev SpeI/NcoI SEQ ID NO: 69 SEQ ID
NO: 70 SEQ ID NO: 33 pSUK430LGB SUK430for SUK430Lrev SpeI/NcoI SEQ
ID NO: 69 SEQ ID NO: 70 SEQ ID NO: 34 pSUK430S SUK430for SUK430Srev
SpeI/NcoI SEQ ID NO: 69 SEQ ID NO: 71 SEQ ID NO: 35 pSUK430SGB
SUK430for SUK430Srev SpeI/NcoI SEQ ID NO: 69 SEQ ID NO: 71 SEQ ID
NO: 38 pSUK436L SUK436for SUK436Lrev BamHI/NcoI SEQ ID NO: 72 SEQ
ID NO: 73 SEQ ID NO: 39 pSUK436LGB SUK436for SUK436Lrev BamHI/NcoI
SEQ ID NO: 72 SEQ ID NO: 73 SEQ ID NO: 40 pSUK436S SUK436for
SUK436Srev BamHI/NcoI SEQ ID NO: 72 SEQ ID NO: 74 SEQ ID NO: 41
pSUK436SGB SUK436for SUK436Srev BamHI/NcoI SEQ ID NO: 72 SEQ ID NO:
74 SEQ ID NO: 42 pSUK438L SUK438for SUK438Lrev BamHI/NcoI SEQ ID
NO: 75 SEQ ID NO: 76 SEQ ID NO: 43 pSUK438LGB SUK438for SUK438Lrev
BamHI/NcoI SEQ ID NO: 75 SEQ ID NO: 76 SEQ ID NO: 44 pSUK438S
SUK438for SUK438Srev BamHI/NcoI SEQ ID NO: 75 SEQ ID NO: 77 SEQ ID
NO: 45 pSUK438SGB SUK438for SUK438Srev BamHI/NcoI SEQ ID NO: 75 SEQ
ID NO: 77
[0354] Amplification is carried out as follows:
[0355] 100 ng genomic DNA
[0356] 1.times.PCR buffer
[0357] 2.5 mM MgCl.sub.2,
[0358] 200 .mu.M each of dATP, dCTP, dGTP und dTTP
[0359] 10 pmol of each oligonucleotide primers
[0360] 2.5 Units Pfu DNA Polymerase (Stratagene)
[0361] in a final volume of 50 .mu.l
[0362] The following temperature program is employed for the
various amplifications (BIORAD Thermocycler):
[0363] 1. 95.degree. C. for 5 min
[0364] 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.
[0365] 3. 54.degree. C. for 1 min, followed by 72.degree. C. for 10
min.
[0366] 4. Storage at 4.degree. C.
[0367] 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: 78) (predigested 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 4
Expression Profile of the Various Promoter::GUS Constructs in
Stably Transformed A. thaliana Plants
[0368] 4.1 pSUK416, pSUK416 GB, pSUK418, pSUK418 GB, pSUK418LGB
[0369] This vascular tissue-specific promoter is mainly active in
the parenchymatic cells of the xylem. Activity is stronger in above
ground organs but also detectable in roots. GUS expression driven
by this promoter was detected in all organs of seedlings and adult
plants tested and includes leaf veins of lower order.
4.2 pSUK420, pSUK420 GB, pSUK422, pSUK422 GB
[0370] Strong vascular tissue-specific expression is conferred by
this promoter in all organs of seedlings and adult plants analyzed.
Expression is not confined to parenchymatic cells of xylem but was
also detectable in the phloem. The promoter is active in leave
veins including vessels of the lowest order.
4.3 pSUK424L, pSUK424LGB, pSUK424S, pSUK424SGB, pSUK426L,
pSUK426LGB, pSUK426S, pSUK426SGB
[0371] This vascular tissue-specific promoter is mainly active in
the parenchymatic cells of the xylem. The tissue-specific activity
is strong in all organs of seedlings and adult plants analyzed. The
promoter is stronger in leave veins of higher order than in their
lower order counterparts.
4.4 pSUK428L, pSUK428LGB, pSUK428S, pSUK428SGB, pSUK430L, pSUK
30LGB, pSUK430S, pSUK430SGB
[0372] This vascular tissue-specific promoter is mainly active in
the parenchymatic cells of the xylem but weaker activity was also
detected in the phloem. The promoter is active in all organs of
seedlings and adult plants analyzed. Lower order leave veins
revealed somewhat weaker promoter strength than veins of higher
order.
4.5 pSUK436L, pSUK436LGB, pSUK436S, pSUK436SGB, pSUK438L,
pSUK438LGB, pSUK438S, pSUK438SGB
[0373] The activity of this vascular tissue-specific promoter is
confined to above ground organs. It is mainly active in the
parenchymatic tissue of the xylem. Leave veins of higher as well as
of lower order revealed reporter gene expression driven by the
promoter.
Example 5
Vector Construction for Overexpression and Gene "Knockout"
Experiments
5.1 Overexpression
[0374] 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.
[0375] For biolistic transformation (biolistic vectors), the
requirements are as follows:
[0376] 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
[0377] 2. a plant-specific portion consisting of:
[0378] 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);
[0379] 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).
[0380] Vectors designed for transformation by Agrobacterium
tumefaciens (A. tumefaciens; binary vectors) consist of:
[0381] 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;
[0382] 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.
5.2 Gene Silencing Vectors
[0383] 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
[0384] 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
[0385] For dsRNAi vectors, a partial gene fragment (typically, 300
to 500 base pairs 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 base pairing of the
two complementary gene fragments in planta.
[0386] Biolistic or binary vectors designed for overexpression or
knockout can vary in a number of different ways, including e.g.,
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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[0652] All publications, patents and patent 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
7811043DNAArabidopsis thalianapromoter(1)..(1043)transcription
regulating sequence from Arabidopsis thaliana gene At4g00140
1gtccttgaaa atcagttaca tgtttctttt gttcttatct ttagtctttt gtgttttctc
60ttttggcctt ttttcttcct ttatctattt acaagtcaag tcagcttatt aacaacgcgg
120tatttccatc cccacaaaat cttcttatgg ctacttttat atatatatac
atatcaaact 180atactgacga caaaataaat aaattaagaa aaaaaaagaa
agagaaagtg ggtgtaggcc 240atgcattatc ttaccaataa cacgtttcac
accattcgca ttgcgctctc tccttccttc 300atatgatcat atagaacgaa
aggtggaaac tttcttattt tgtatggatc gtatacgcaa 360tatggttgga
caaaactaat ctgtttcaaa caaaatacat gaaatatccc acaagaaaca
420aagcaaacta taaaaaaaat aatgcggtaa acacggaaaa tatcacatat
acgaatcctt 480tttcggatgt gttttcaatc ctttgaatta aatatgtcca
cactcgatat gtaaaaacaa 540cttgacccat cacaattatg atcataaaat
accaacgtac tgttagccta atgataaatc 600tcccaagcag aggtgtttag
ttcgagttat gttgtaaggg attttttctc ctaaggaaat 660aaatttaatt
tgatgtgaat tctaggaaat atagggcctc tcgacttaaa ctttcaaata
720ttaaaaaaaa taaaaataat gatcatcatg agctattggg ttttcaatga
tttatcagca 780gagtgttcaa tgagagtcca agtatttggg ctgtaatatc
aatatgggcc caaggtaaag 840cccaacataa tcaatcggct attgggtatt
ctaaattctc tcattataga agtgttcaat 900gagggcccaa gtaattggtc
ttaatatggg cccaagtaat tggtcttaat atgagcccaa 960gttaaaccca
acttaaacga ttgttggagg cggcaaaata aacaaatccc aaactctgga
1020gaatcaaatt cctaattagc taa 104321057DNAArabidopsis
thalianapromoter(1)..(1057)transcription regulating sequence from
Arabidopsis thaliana gene At4g00140 2gaccatactc atgtccttga
aaatcagtta catgtttctt ttgttcttat ctttagtctt 60ttgtgttttc tcttttggcc
ttttttcttc ctttatctat ttacaagtca agtcagctta 120ttaacaacgc
ggtatttcca tccccacaaa atcttcttat ggctactttt atatatatat
180acatatcaaa ctatactgac gacaaaataa ataaattaag aaaaaaaaag
aaagagaaag 240tgggtgtagg ccatgcatta tcttaccaat aacacgtttc
acaccattcg cattgcgctc 300tctccttcct tcatatgatc atatagaacg
aaaggtggaa actttcttat tttgtatgga 360tcgtatacgc aatatggttg
gacaaaacta atctgtttca aacaaaatac atgaaatatc 420ccacaagaaa
caaagcaaac tataaaaaaa ataatgcggt aaacacggaa aatatcacat
480atacgaatcc tttttcggat gtgttttcaa tcctttgaat taaatatgtc
cacactcgat 540atgtaaaaac aacttgaccc atcacaatta tgatcataaa
ataccaacgt actgttagcc 600taatgataaa tctcccaagc agaggtgttt
agttcgagtt atgttgtaag ggattttttc 660tcctaaggaa ataaatttaa
tttgatgtga attctaggaa atatagggcc tctcgactta 720aactttcaaa
tattaaaaaa aataaaaata atgatcatca tgagctattg ggttttcaat
780gatttatcag cagagtgttc aatgagagtc caagtatttg ggctgtaata
tcaatatggg 840cccaaggtaa agcccaacat aatcaatcgg ctattgggta
ttctaaattc tctcattata 900gaagtgttca atgagggccc aagtaattgg
tcttaatatg ggcccaagta attggtctta 960atatgagccc aagttaaacc
caacttaaac gattgttgga ggcggcaaaa taaacaaatc 1020ccaaactctg
gagaatcaaa ttcctaatta gctaaca 105732464DNAArabidopsis
thalianapromoter(1)..(2464)transcription regulating sequence from
Arabidopsis thaliana gene At4g00140 3cacacatcga agcatactct
tgataatata tggacaggga aatacatatt agaaagctat 60caaactttct cttccttttt
tttttgtttg ctgggtttca tctttcacta tattgtatgt 120gtgacatgtc
attaagaaat gaataacaac aggctattca ttgctttttt ctattcattg
180cttacaaaaa gtaaaatgat gataaaatac gcagcttttg gtgttatttg
tccttcaact 240aattgagtta tcatttttct tttactcctt ttagacaccg
catatgtact tttgtctcaa 300agaagactct cctaggagtt aggaagattt
tgattttgtt ataatcagac tttaactcac 360atctgtcttc catcatccca
ctgtttttca agtattttat tattagttgt taaaaacttc 420tacgtccaaa
atctcaatca ctacgagtat tgcaatgttg atattgtgcg ttaaatgtat
480atgtatataa ttccaaattt ccaacaattg gtttataaac gatggcacta
acataacctc 540atatattgtg atacaacgaa aggcaccgga tggtggaacg
catcacgcat catgcatcct 600cgatcccttt tcaaagtggt atgtaagaga
tcattatttc aacataacat acatttgcta 660tgatgacgca ttttagtcaa
attagttgtt tgttcaatca tattaataag ggtcaacaga 720tatttatacg
aactgtaatt aaaatgataa aatccaaagc gacaagtttg ctactaccaa
780atgtgtgttt aatatatata atttagaaac attgaatttg agaaaatgag
tatacggttg 840cacaaaagaa gaagaagaag aggagtatac caacaaaagg
ttgtacgtaa tataattccg 900accccctaaa aagatatata ataaggtgga
gagcgaggga tctgaaattg atgggtgtgt 960ttttgtgtgc ataacaaaag
ttaaaaatgt atttatatta tttttaattt taaaattctt 1020ttcttacttg
tgtttattta ttagttaaat aaataattta atgagagaag cttttgatac
1080aatttttact ctctttcaat ctccatctca acttttgtag atgccaagct
tgcccaccat 1140atctgctcct caacctctca ctttacaagt acttcattta
ttttcttagt tagtctattt 1200atttcaataa acaaactcta ctcaagttca
tcaagattcg taacagtgga atcctttatt 1260aatgttagat catcaaactc
tacacatgtt tatttccatc tctatgcatg cctttatatt 1320gcttaagttg
tatacacgta catatacact tcactactct tattatctag ttccatatta
1380cgctatatat ttatgaattt atatacatag accatactca tgtccttgaa
aatcagttac 1440atgtttcttt tgttcttatc tttagtcttt tgtgttttct
cttttggcct tttttcttcc 1500tttatctatt tacaagtcaa gtcagcttat
taacaacgcg gtatttccat ccccacaaaa 1560tcttcttatg gctactttta
tatatatata catatcaaac tatactgacg acaaaataaa 1620taaattaaga
aaaaaaaaga aagagaaagt gggtgtaggc catgcattat cttaccaata
1680acacgtttca caccattcgc attgcgctct ctccttcctt catatgatca
tatagaacga 1740aaggtggaaa ctttcttatt ttgtatggat cgtatacgca
atatggttgg acaaaactaa 1800tctgtttcaa acaaaataca tgaaatatcc
cacaagaaac aaagcaaact ataaaaaaaa 1860taatgcggta aacacggaaa
atatcacata tacgaatcct ttttcggatg tgttttcaat 1920cctttgaatt
aaatatgtcc acactcgata tgtaaaaaca acttgaccca tcacaattat
1980gatcataaaa taccaacgta ctgttagcct aatgataaat ctcccaagca
gaggtgttta 2040gttcgagtta tgttgtaagg gattttttct cctaaggaaa
taaatttaat ttgatgtgaa 2100ttctaggaaa tatagggcct ctcgacttaa
actttcaaat attaaaaaaa ataaaaataa 2160tgatcatcat gagctattgg
gttttcaatg atttatcagc agagtgttca atgagagtcc 2220aagtatttgg
gctgtaatat caatatgggc ccaaggtaaa gcccaacata atcaatcggc
2280tattgggtat tctaaattct ctcattatag aagtgttcaa tgagggccca
agtaattggt 2340cttaatatgg gcccaagtaa ttggtcttaa tatgagccca
agttaaaccc aacttaaacg 2400attgttggag gcggcaaaat aaacaaatcc
caaactctgg agaatcaaat tcctaattag 2460ctaa 246442478DNAArabidopsis
thalianapromoter(1)..(2478)transcription regulating sequence from
Arabidopsis thaliana gene At4g00140 4gacgggactg atcacacatc
gaagcatact cttgataata tatggacagg gaaatacata 60ttagaaagct atcaaacttt
ctcttccttt tttttttgtt tgctgggttt catctttcac 120tatattgtat
gtgtgacatg tcattaagaa atgaataaca acaggctatt cattgctttt
180ttctattcat tgcttacaaa aagtaaaatg atgataaaat acgcagcttt
tggtgttatt 240tgtccttcaa ctaattgagt tatcattttt cttttactcc
ttttagacac cgcatatgta 300cttttgtctc aaagaagact ctcctaggag
ttaggaagat tttgattttg ttataatcag 360actttaactc acatctgtct
tccatcatcc cactgttttt caagtatttt attattagtt 420gttaaaaact
tctacgtcca aaatctcaat cactacgagt attgcaatgt tgatattgtg
480cgttaaatgt atatgtatat aattccaaat ttccaacaat tggtttataa
acgatggcac 540taacataacc tcatatattg tgatacaacg aaaggcaccg
gatggtggaa cgcatcacgc 600atcatgcatc ctcgatccct tttcaaagtg
gtatgtaaga gatcattatt tcaacataac 660atacatttgc tatgatgacg
cattttagtc aaattagttg tttgttcaat catattaata 720agggtcaaca
gatatttata cgaactgtaa ttaaaatgat aaaatccaaa gcgacaagtt
780tgctactacc aaatgtgtgt ttaatatata taatttagaa acattgaatt
tgagaaaatg 840agtatacggt tgcacaaaag aagaagaaga agaggagtat
accaacaaaa ggttgtacgt 900aatataattc cgacccccta aaaagatata
taataaggtg gagagcgagg gatctgaaat 960tgatgggtgt gtttttgtgt
gcataacaaa agttaaaaat gtatttatat tatttttaat 1020tttaaaattc
ttttcttact tgtgtttatt tattagttaa ataaataatt taatgagaga
1080agcttttgat acaattttta ctctctttca atctccatct caacttttgt
agatgccaag 1140cttgcccacc atatctgctc ctcaacctct cactttacaa
gtacttcatt tattttctta 1200gttagtctat ttatttcaat aaacaaactc
tactcaagtt catcaagatt cgtaacagtg 1260gaatccttta ttaatgttag
atcatcaaac tctacacatg tttatttcca tctctatgca 1320tgcctttata
ttgcttaagt tgtatacacg tacatataca cttcactact cttattatct
1380agttccatat tacgctatat atttatgaat ttatatacat agaccatact
catgtccttg 1440aaaatcagtt acatgtttct tttgttctta tctttagtct
tttgtgtttt ctcttttggc 1500cttttttctt cctttatcta tttacaagtc
aagtcagctt attaacaacg cggtatttcc 1560atccccacaa aatcttctta
tggctacttt tatatatata tacatatcaa actatactga 1620cgacaaaata
aataaattaa gaaaaaaaaa gaaagagaaa gtgggtgtag gccatgcatt
1680atcttaccaa taacacgttt cacaccattc gcattgcgct ctctccttcc
ttcatatgat 1740catatagaac gaaaggtgga aactttctta ttttgtatgg
atcgtatacg caatatggtt 1800ggacaaaact aatctgtttc aaacaaaata
catgaaatat cccacaagaa acaaagcaaa 1860ctataaaaaa aataatgcgg
taaacacgga aaatatcaca tatacgaatc ctttttcgga 1920tgtgttttca
atcctttgaa ttaaatatgt ccacactcga tatgtaaaaa caacttgacc
1980catcacaatt atgatcataa aataccaacg tactgttagc ctaatgataa
atctcccaag 2040cagaggtgtt tagttcgagt tatgttgtaa gggatttttt
ctcctaagga aataaattta 2100atttgatgtg aattctagga aatatagggc
ctctcgactt aaactttcaa atattaaaaa 2160aaataaaaat aatgatcatc
atgagctatt gggttttcaa tgatttatca gcagagtgtt 2220caatgagagt
ccaagtattt gggctgtaat atcaatatgg gcccaaggta aagcccaaca
2280taatcaatcg gctattgggt attctaaatt ctctcattat agaagtgttc
aatgagggcc 2340caagtaattg gtcttaatat gggcccaagt aattggtctt
aatatgagcc caagttaaac 2400ccaacttaaa cgattgttgg aggcggcaaa
ataaacaaat cccaaactct ggagaatcaa 2460attcctaatt agctaaca
247853977DNAArabidopsis thalianapromoter(1)..(3977)transcription
regulating sequence from Arabidopsis thaliana gene At4g00140
5tccaccggag tttcaattat taaaaaaata ttttccttaa ttcaatttat cttaaatgac
60aaatttttag tttctgattt tattttgctc agtgcgatgg atttttaaat ttaagtttca
120cacaaatata taaatttttg tgagaagtta attaattgtc tgattatcaa
acacttattg 180tataacacat tcaatatata ttaattgtgg ggattatttt
tgatcgacta aataacgtga 240tagaataatg cttggattag ctcaatacta
tatttttcta attaaaaatg aatggggtgt 300tgattttgat gcgaggcaga
aagctactgc tcattattgt gattatatga ttatataatg 360gttagagttt
gttgtggttt gtgtctttgc gaggggtcta tttttaattt ataaacatat
420gtctccatga tgtcacatgg gtcttgtatt attttattta tttggaccac
aatacatttt 480tgtgtgcgcc gtttctaacc tttttttttc tccgacgaaa
caaaggtttg ctttttctcc 540tttaaatggt ccatacgcat tacatatata
atgcgcccat ttgctttacg cattaactgc 600atttctctat agaaatattt
cagaaacaaa ccaagtgtat acaatacatc aacatgtttt 660ttttggggtt
ttaatgtgaa gatttcattt caataaaaag attacaagct aatctaagta
720atacaaacca acatattttg gtttactctt tatattaaaa catcatttag
gataattatt 780taaaatatat attcaaacat ctaaggtcct aattaagcat
tacaaaagat ttccgtttta 840gaaacatatc aagagttgat taaatggaga
aagacgacta aaatttgtga gtgaaaaatg 900acaatatgcc gcaagttatt
gtctcttgtg caataaacgt tggttggata cagacacatt 960tgattgaatg
tgaatagttt agttttgaca gatcataagc cgtgtcctca agcagaagac
1020cggtcgtgat cggtcaatct acacgtgtac ggcagaaaca catgtgtcgt
ttccctgtga 1080gagatgatca accaaatcaa cggtcagttt ttgtcaacta
atgtgtgtat tgatttgtaa 1140catgcctacg tgaacataag ctagtcacgc
aacaagcaag gcctgggtca cgcaggctcg 1200gctccactaa gacgcgccga
cgtagtcact catcatatat cacatgtcta gattcaaatg 1260gtaaccgttt
gatgagcgca gtaagtagaa acattgaccg gtccaggcga ggtctagcaa
1320atactattag aattaaaatt gatgtaatca atcactgata aatactatta
gaactactct 1380actttacact cacgtattcc atcatttaat atacatacgt
gagtgtaaag tatttcttca 1440caatttattt ttgaaaatag cttttaaccc
atagcaaatg cctatgttgc catggggtag 1500acgggactga tcacacatcg
aagcatactc ttgataatat atggacaggg aaatacatat 1560tagaaagcta
tcaaactttc tcttcctttt ttttttgttt gctgggtttc atctttcact
1620atattgtatg tgtgacatgt cattaagaaa tgaataacaa caggctattc
attgcttttt 1680tctattcatt gcttacaaaa agtaaaatga tgataaaata
cgcagctttt ggtgttattt 1740gtccttcaac taattgagtt atcatttttc
ttttactcct tttagacacc gcatatgtac 1800ttttgtctca aagaagactc
tcctaggagt taggaagatt ttgattttgt tataatcaga 1860ctttaactca
catctgtctt ccatcatccc actgtttttc aagtatttta ttattagttg
1920ttaaaaactt ctacgtccaa aatctcaatc actacgagta ttgcaatgtt
gatattgtgc 1980gttaaatgta tatgtatata attccaaatt tccaacaatt
ggtttataaa cgatggcact 2040aacataacct catatattgt gatacaacga
aaggcaccgg atggtggaac gcatcacgca 2100tcatgcatcc tcgatccctt
ttcaaagtgg tatgtaagag atcattattt caacataaca 2160tacatttgct
atgatgacgc attttagtca aattagttgt ttgttcaatc atattaataa
2220gggtcaacag atatttatac gaactgtaat taaaatgata aaatccaaag
cgacaagttt 2280gctactacca aatgtgtgtt taatatatat aatttagaaa
cattgaattt gagaaaatga 2340gtatacggtt gcacaaaaga agaagaagaa
gaggagtata ccaacaaaag gttgtacgta 2400atataattcc gaccccctaa
aaagatatat aataaggtgg agagcgaggg atctgaaatt 2460gatgggtgtg
tttttgtgtg cataacaaaa gttaaaaatg tatttatatt atttttaatt
2520ttaaaattct tttcttactt gtgtttattt attagttaaa taaataattt
aatgagagaa 2580gcttttgata caatttttac tctctttcaa tctccatctc
aacttttgta gatgccaagc 2640ttgcccacca tatctgctcc tcaacctctc
actttacaag tacttcattt attttcttag 2700ttagtctatt tatttcaata
aacaaactct actcaagttc atcaagattc gtaacagtgg 2760aatcctttat
taatgttaga tcatcaaact ctacacatgt ttatttccat ctctatgcat
2820gcctttatat tgcttaagtt gtatacacgt acatatacac ttcactactc
ttattatcta 2880gttccatatt acgctatata tttatgaatt tatatacata
gaccatactc atgtccttga 2940aaatcagtta catgtttctt ttgttcttat
ctttagtctt ttgtgttttc tcttttggcc 3000ttttttcttc ctttatctat
ttacaagtca agtcagctta ttaacaacgc ggtatttcca 3060tccccacaaa
atcttcttat ggctactttt atatatatat acatatcaaa ctatactgac
3120gacaaaataa ataaattaag aaaaaaaaag aaagagaaag tgggtgtagg
ccatgcatta 3180tcttaccaat aacacgtttc acaccattcg cattgcgctc
tctccttcct tcatatgatc 3240atatagaacg aaaggtggaa actttcttat
tttgtatgga tcgtatacgc aatatggttg 3300gacaaaacta atctgtttca
aacaaaatac atgaaatatc ccacaagaaa caaagcaaac 3360tataaaaaaa
ataatgcggt aaacacggaa aatatcacat atacgaatcc tttttcggat
3420gtgttttcaa tcctttgaat taaatatgtc cacactcgat atgtaaaaac
aacttgaccc 3480atcacaatta tgatcataaa ataccaacgt actgttagcc
taatgataaa tctcccaagc 3540agaggtgttt agttcgagtt atgttgtaag
ggattttttc tcctaaggaa ataaatttaa 3600tttgatgtga attctaggaa
atatagggcc tctcgactta aactttcaaa tattaaaaaa 3660aataaaaata
atgatcatca tgagctattg ggttttcaat gatttatcag cagagtgttc
3720aatgagagtc caagtatttg ggctgtaata tcaatatggg cccaaggtaa
agcccaacat 3780aatcaatcgg ctattgggta ttctaaattc tctcattata
gaagtgttca atgagggccc 3840aagtaattgg tcttaatatg ggcccaagta
attggtctta atatgagccc aagttaaacc 3900caacttaaac gattgttgga
ggcggcaaaa taaacaaatc ccaaactctg gagaatcaaa 3960ttcctaatta gctaaca
39776774DNAArabidopsis thalianaCDS(1)..(774) 6atg gcg agg gga gaa
tcg gag gga gag agc tca gga agt gaa cga gag 48Met Ala Arg Gly Glu
Ser Glu Gly Glu Ser Ser Gly Ser Glu Arg Glu1 5 10 15agt tcg agc tcg
agt tcc ggc aac gaa tcg gag ccg ata aag ggg aaa 96Ser Ser Ser Ser
Ser Ser Gly Asn Glu Ser Glu Pro Ile Lys Gly Lys 20 25 30atc tcg gaa
tat gag aag cag agg ttg tca agg atc gct gag aac aaa 144Ile Ser Glu
Tyr Glu Lys Gln Arg Leu Ser Arg Ile Ala Glu Asn Lys 35 40 45gcg aga
ttg gat gca ctc gga att ccg gca ata gcg ctt tct cta cag 192Ala Arg
Leu Asp Ala Leu Gly Ile Pro Ala Ile Ala Leu Ser Leu Gln 50 55 60ggc
tct gtt gca gga ggc tct cgt acg aaa aat acg aga agc gat aaa 240Gly
Ser Val Ala Gly Gly Ser Arg Thr Lys Asn Thr Arg Ser Asp Lys65 70 75
80gag gct gca act atg aag aag aaa aga cag gaa ggt gga aaa ggg ttc
288Glu Ala Ala Thr Met Lys Lys Lys Arg Gln Glu Gly Gly Lys Gly Phe
85 90 95att act cgg aga gat gtg gcg aaa atg gca acg gtg cat gac ttc
aca 336Ile Thr Arg Arg Asp Val Ala Lys Met Ala Thr Val His Asp Phe
Thr 100 105 110tgg aca gaa gag gaa tta caa gac atg att cgt tcc ttt
gac atg gac 384Trp Thr Glu Glu Glu Leu Gln Asp Met Ile Arg Ser Phe
Asp Met Asp 115 120 125aag gac gga aag gtg ggt act aag ttg aag gta
ttc acg ttt att aga 432Lys Asp Gly Lys Val Gly Thr Lys Leu Lys Val
Phe Thr Phe Ile Arg 130 135 140gaa aca tta ttg cta aac atc ttt aat
ttt ggg cag cta agc tgc cga 480Glu Thr Leu Leu Leu Asn Ile Phe Asn
Phe Gly Gln Leu Ser Cys Arg145 150 155 160tcg tct gac gga acg ggt
cgg aca gac cga tcg tcg gat ggg atg gat 528Ser Ser Asp Gly Thr Gly
Arg Thr Asp Arg Ser Ser Asp Gly Met Asp 165 170 175cga gaa aag tgt
tcg agg aag cac aag gat aag tgc atc cga aca cac 576Arg Glu Lys Cys
Ser Arg Lys His Lys Asp Lys Cys Ile Arg Thr His 180 185 190cca agg
gtc gcc cat gga tgc aat ctt acc ggt aaa ggg aga aag cac 624Pro Arg
Val Ala His Gly Cys Asn Leu Thr Gly Lys Gly Arg Lys His 195 200
205caa gaa tgg gta agg aac gtg att cac cat ata gga tca aca tcg cgg
672Gln Glu Trp Val Arg Asn Val Ile His His Ile Gly Ser Thr Ser Arg
210 215 220ccg cct tca tgt atc tat ctc ctc agc cat ggt gaa acc gat
caa aga 720Pro Pro Ser Cys Ile Tyr Leu Leu Ser His Gly Glu Thr Asp
Gln Arg225 230 235 240tcg gta aga cca aag ggt gac tcg atg gat aac
caa cgg gaa agt cca 768Ser Val Arg Pro Lys Gly Asp Ser Met Asp Asn
Gln Arg Glu Ser Pro 245 250 255cgg tga 774Arg7257PRTArabidopsis
thaliana 7Met Ala Arg Gly Glu Ser Glu Gly Glu Ser Ser Gly Ser Glu
Arg Glu1 5 10 15Ser Ser Ser Ser Ser Ser Gly Asn Glu Ser Glu Pro Ile
Lys Gly Lys 20 25 30Ile Ser Glu Tyr Glu Lys Gln Arg Leu Ser Arg Ile
Ala Glu Asn Lys 35 40 45Ala Arg Leu Asp Ala Leu Gly Ile Pro Ala Ile
Ala Leu Ser Leu Gln 50 55 60Gly Ser Val Ala Gly Gly Ser Arg Thr Lys
Asn Thr Arg Ser Asp Lys65 70 75 80Glu Ala Ala Thr Met Lys Lys Lys
Arg Gln Glu Gly Gly Lys Gly Phe 85 90 95Ile Thr Arg Arg Asp Val Ala
Lys Met Ala
Thr Val His Asp Phe Thr 100 105 110Trp Thr Glu Glu Glu Leu Gln Asp
Met Ile Arg Ser Phe Asp Met Asp 115 120 125Lys Asp Gly Lys Val Gly
Thr Lys Leu Lys Val Phe Thr Phe Ile Arg 130 135 140Glu Thr Leu Leu
Leu Asn Ile Phe Asn Phe Gly Gln Leu Ser Cys Arg145 150 155 160Ser
Ser Asp Gly Thr Gly Arg Thr Asp Arg Ser Ser Asp Gly Met Asp 165 170
175Arg Glu Lys Cys Ser Arg Lys His Lys Asp Lys Cys Ile Arg Thr His
180 185 190Pro Arg Val Ala His Gly Cys Asn Leu Thr Gly Lys Gly Arg
Lys His 195 200 205Gln Glu Trp Val Arg Asn Val Ile His His Ile Gly
Ser Thr Ser Arg 210 215 220Pro Pro Ser Cys Ile Tyr Leu Leu Ser His
Gly Glu Thr Asp Gln Arg225 230 235 240Ser Val Arg Pro Lys Gly Asp
Ser Met Asp Asn Gln Arg Glu Ser Pro 245 250
255Arg81147DNAArabidopsis thalianapromoter(1)..(1147)transcription
regulating sequence from Arabidopsis thaliana gene At5g45350
8tctcctttgc ccccacagtt taatatattt cataaacacc cccacgagaa attaaaaacc
60gttccaaatt tgaaaaaata caacaaacaa ttcatgaaaa tattcaatct ataagcaaaa
120cattggtagt gttacatgtg tcgtccttcg tgctgaaaat ttggatagca
ttgttaatta 180tgacaacgta agtgtagcct gtgattcata gtaaaatata
aggactaaaa ataaaaatat 240atcttttttg gaaatcgtac cgcgtaagaa
acatgtataa atacctataa ggtcttattt 300ttttctcttc caatttcgtt
tcgacatatt tcgcgattcg tctaaggtaa aaaaaaactc 360atcttttttt
cttttagatc gttaattttt gatcagcgat tcgctcttct gatctgtgtt
420ctttaagctt gtcttctctc ttgattcgat ctgctgaaaa cctagaaatt
tttgattttt 480ttgtttgttt tgctccatgt gtatgggtat atttacgatt
ttaacaaaac aaaaatatga 540attgaggttt tttatttagc gaattgggtt
ttaattgttc acattcgttt ggctctctcg 600aggtgagtga taaagtatag
aactttctta tgcttaggat cttaaattcg agttctttga 660tttacctgtc
atgtgttatt gattgatctc atttatattg tgtctgcttg atgtttaaag
720cttggtgtat gcaatttgat tgggtttact ggagattgat ctgtgaccta
atcaatgagt 780gataacattg ctggttcatc tgatttctca tctggtgtgt
ctgctcgatt ccctgaagaa 840agtttgaaac tcaggcttgg tttgtgcagt
ttgattgatt tatctcatta cttgactctg 900gtgtacttga ttctttgatg
aaagcttgaa gttaatgttt ggtttgtgca atatgattgg 960gattaactgc
atttgatctc tgaccaaaat cagtgattaa ttacattgct tggttatctc
1020attacttgag tttggtgttc ctgcatgatc ccctgagctt gaagttcatg
cttggtttgt 1080ccaattgtga cctaaattga tgttttcttg tggtatcttt
tcgtaggttt tgttactgat 1140tgagaaa 114791161DNAArabidopsis
thalianapromoter(1)..(1161)transcription regulating sequence from
Arabidopsis thaliana gene At5g45350 9ttctcataat tctctccttt
gcccccacag tttaatatat ttcataaaca cccccacgag 60aaattaaaaa ccgttccaaa
tttgaaaaaa tacaacaaac aattcatgaa aatattcaat 120ctataagcaa
aacattggta gtgttacatg tgtcgtcctt cgtgctgaaa atttggatag
180cattgttaat tatgacaacg taagtgtagc ctgtgattca tagtaaaata
taaggactaa 240aaataaaaat atatcttttt tggaaatcgt accgcgtaag
aaacatgtat aaatacctat 300aaggtcttat ttttttctct tccaatttcg
tttcgacata tttcgcgatt cgtctaaggt 360aaaaaaaaac tcatcttttt
ttcttttaga tcgttaattt ttgatcagcg attcgctctt 420ctgatctgtg
ttctttaagc ttgtcttctc tcttgattcg atctgctgaa aacctagaaa
480tttttgattt ttttgtttgt tttgctccat gtgtatgggt atatttacga
ttttaacaaa 540acaaaaatat gaattgaggt tttttattta gcgaattggg
ttttaattgt tcacattcgt 600ttggctctct cgaggtgagt gataaagtat
agaactttct tatgcttagg atcttaaatt 660cgagttcttt gatttacctg
tcatgtgtta ttgattgatc tcatttatat tgtgtctgct 720tgatgtttaa
agcttggtgt atgcaatttg attgggttta ctggagattg atctgtgacc
780taatcaatga gtgataacat tgctggttca tctgatttct catctggtgt
gtctgctcga 840ttccctgaag aaagtttgaa actcaggctt ggtttgtgca
gtttgattga tttatctcat 900tacttgactc tggtgtactt gattctttga
tgaaagcttg aagttaatgt ttggtttgtg 960caatatgatt gggattaact
gcatttgatc tctgaccaaa atcagtgatt aattacattg 1020cttggttatc
tcattacttg agtttggtgt tcctgcatga tcccctgagc ttgaagttca
1080tgcttggttt gtccaattgt gacctaaatt gatgttttct tgtggtatct
tttcgtaggt 1140tttgttactg attgagaaaa c 116110304DNAArabidopsis
thalianapromoter(1)..(304)transcription regulating sequence from
Arabidopsis thaliana gene At5g45350 10tctcctttgc ccccacagtt
taatatattt cataaacacc cccacgagaa attaaaaacc 60gttccaaatt tgaaaaaata
caacaaacaa ttcatgaaaa tattcaatct ataagcaaaa 120cattggtagt
gttacatgtg tcgtccttcg tgctgaaaat ttggatagca ttgttaatta
180tgacaacgta agtgtagcct gtgattcata gtaaaatata aggactaaaa
ataaaaatat 240atcttttttg gaaatcgtac cgcgtaagaa acatgtataa
atacctataa ggtcttattt 300tttt 30411316DNAArabidopsis
thalianapromoter(1)..(316)transcription regulating sequence from
Arabidopsis thaliana gene At5g45350 11ttctcataat tctctccttt
gcccccacag tttaatatat ttcataaaca cccccacgag 60aaattaaaaa ccgttccaaa
tttgaaaaaa tacaacaaac aattcatgaa aatattcaat 120ctataagcaa
aacattggta gtgttacatg tgtcgtcctt cgtgctgaaa atttggatag
180cattgttaat tatgacaacg taagtgtagc ctgtgattca tagtaaaata
taaggactaa 240aaataaaaat atatcttttt tggaaatcgt accgcgtaag
aaacatgtat aaatacctat 300aaggtcttat tttttt 316121931DNAArabidopsis
thalianapromoter(1)..(1931)transcription regulating sequence from
Arabidopsis thaliana gene At5g45350 12gctgagcaaa agtctcgacc
ttttgtccaa tttcataagg cctactttct gtaaggtctt 60ctctctctct ctctctctga
cctgattctg cctgggttct gtggagttga aatgaatcaa 120atgtagctct
tgttttaatc agtcagcttt gtaacgtata catatatatc attgtatgtg
180attaacattg tccttaaaaa gtgttacacc tagtaattac gttccaactt
tatcagtaaa 240cctattggta actagtaatt tgctcccgat attggttttc
tcattaattt ttatagccat 300tctttgttca tttaaaccat attacaaatt
tgtcctggtt agaactgatt atgatgacaa 360atcgatgatt agttcaaaat
gaagtttata gcaatatatt ctaataacta tacatcaatt 420atcaccactg
atgtgtttga atgaaaaatg aattgataat cttaataaat gtttgatttt
480atagataagt ataaagtatg aatggattta gaaaatttta gtatttagtg
agataagcat 540aagcaaaact agaaaaatga aattattatt aaataaattc
attaatacac attggcaatg 600tgacattgcc taaaatcaaa tatgtactaa
ttcaattaat gttaataaga attttttgga 660ttcgattctc ttgcgaaagt
gcaaattaaa agacattctt atctcctacg taaacagaat 720cacgcgtcca
ttgcgcgttc tcgcaacgct aaaatattta cgttggaaaa tattctcata
780attctctcct ttgcccccac agtttaatat atttcataaa cacccccacg
agaaattaaa 840aaccgttcca aatttgaaaa aatacaacaa acaattcatg
aaaatattca atctataagc 900aaaacattgg tagtgttaca tgtgtcgtcc
ttcgtgctga aaatttggat agcattgtta 960attatgacaa cgtaagtgta
gcctgtgatt catagtaaaa tataaggact aaaaataaaa 1020atatatcttt
tttggaaatc gtaccgcgta agaaacatgt ataaatacct ataaggtctt
1080atttttttct cttccaattt cgtttcgaca tatttcgcga ttcgtctaag
gtaaaaaaaa 1140actcatcttt ttttctttta gatcgttaat ttttgatcag
cgattcgctc ttctgatctg 1200tgttctttaa gcttgtcttc tctcttgatt
cgatctgctg aaaacctaga aatttttgat 1260ttttttgttt gttttgctcc
atgtgtatgg gtatatttac gattttaaca aaacaaaaat 1320atgaattgag
gttttttatt tagcgaattg ggttttaatt gttcacattc gtttggctct
1380ctcgaggtga gtgataaagt atagaacttt cttatgctta ggatcttaaa
ttcgagttct 1440ttgatttacc tgtcatgtgt tattgattga tctcatttat
attgtgtctg cttgatgttt 1500aaagcttggt gtatgcaatt tgattgggtt
tactggagat tgatctgtga cctaatcaat 1560gagtgataac attgctggtt
catctgattt ctcatctggt gtgtctgctc gattccctga 1620agaaagtttg
aaactcaggc ttggtttgtg cagtttgatt gatttatctc attacttgac
1680tctggtgtac ttgattcttt gatgaaagct tgaagttaat gtttggtttg
tgcaatatga 1740ttgggattaa ctgcatttga tctctgacca aaatcagtga
ttaattacat tgcttggtta 1800tctcattact tgagtttggt gttcctgcat
gatcccctga gcttgaagtt catgcttggt 1860ttgtccaatt gtgacctaaa
ttgatgtttt cttgtggtat cttttcgtag gttttgttac 1920tgattgagaa a
1931131945DNAArabidopsis thalianapromoter(1)..(1945)transcription
regulating sequence from Arabidopsis thaliana gene At5g45350
13ccctgaggtt ctgctgagca aaagtctcga ccttttgtcc aatttcataa ggcctacttt
60ctgtaaggtc ttctctctct ctctctctct gacctgattc tgcctgggtt ctgtggagtt
120gaaatgaatc aaatgtagct cttgttttaa tcagtcagct ttgtaacgta
tacatatata 180tcattgtatg tgattaacat tgtccttaaa aagtgttaca
cctagtaatt acgttccaac 240tttatcagta aacctattgg taactagtaa
tttgctcccg atattggttt tctcattaat 300ttttatagcc attctttgtt
catttaaacc atattacaaa tttgtcctgg ttagaactga 360ttatgatgac
aaatcgatga ttagttcaaa atgaagttta tagcaatata ttctaataac
420tatacatcaa ttatcaccac tgatgtgttt gaatgaaaaa tgaattgata
atcttaataa 480atgtttgatt ttatagataa gtataaagta tgaatggatt
tagaaaattt tagtatttag 540tgagataagc ataagcaaaa ctagaaaaat
gaaattatta ttaaataaat tcattaatac 600acattggcaa tgtgacattg
cctaaaatca aatatgtact aattcaatta atgttaataa 660gaattttttg
gattcgattc tcttgcgaaa gtgcaaatta aaagacattc ttatctccta
720cgtaaacaga atcacgcgtc cattgcgcgt tctcgcaacg ctaaaatatt
tacgttggaa 780aatattctca taattctctc ctttgccccc acagtttaat
atatttcata aacaccccca 840cgagaaatta aaaaccgttc caaatttgaa
aaaatacaac aaacaattca tgaaaatatt 900caatctataa gcaaaacatt
ggtagtgtta catgtgtcgt ccttcgtgct gaaaatttgg 960atagcattgt
taattatgac aacgtaagtg tagcctgtga ttcatagtaa aatataagga
1020ctaaaaataa aaatatatct tttttggaaa tcgtaccgcg taagaaacat
gtataaatac 1080ctataaggtc ttattttttt ctcttccaat ttcgtttcga
catatttcgc gattcgtcta 1140aggtaaaaaa aaactcatct ttttttcttt
tagatcgtta atttttgatc agcgattcgc 1200tcttctgatc tgtgttcttt
aagcttgtct tctctcttga ttcgatctgc tgaaaaccta 1260gaaatttttg
atttttttgt ttgttttgct ccatgtgtat gggtatattt acgattttaa
1320caaaacaaaa atatgaattg aggtttttta tttagcgaat tgggttttaa
ttgttcacat 1380tcgtttggct ctctcgaggt gagtgataaa gtatagaact
ttcttatgct taggatctta 1440aattcgagtt ctttgattta cctgtcatgt
gttattgatt gatctcattt atattgtgtc 1500tgcttgatgt ttaaagcttg
gtgtatgcaa tttgattggg tttactggag attgatctgt 1560gacctaatca
atgagtgata acattgctgg ttcatctgat ttctcatctg gtgtgtctgc
1620tcgattccct gaagaaagtt tgaaactcag gcttggtttg tgcagtttga
ttgatttatc 1680tcattacttg actctggtgt acttgattct ttgatgaaag
cttgaagtta atgtttggtt 1740tgtgcaatat gattgggatt aactgcattt
gatctctgac caaaatcagt gattaattac 1800attgcttggt tatctcatta
cttgagtttg gtgttcctgc atgatcccct gagcttgaag 1860ttcatgcttg
gtttgtccaa ttgtgaccta aattgatgtt ttcttgtggt atcttttcgt
1920aggttttgtt actgattgag aaaac 1945141088DNAArabidopsis
thalianapromoter(1)..(1088)transcription regulating sequence from
Arabidopsis thaliana gene At5g45350 14gctgagcaaa agtctcgacc
ttttgtccaa tttcataagg cctactttct gtaaggtctt 60ctctctctct ctctctctga
cctgattctg cctgggttct gtggagttga aatgaatcaa 120atgtagctct
tgttttaatc agtcagcttt gtaacgtata catatatatc attgtatgtg
180attaacattg tccttaaaaa gtgttacacc tagtaattac gttccaactt
tatcagtaaa 240cctattggta actagtaatt tgctcccgat attggttttc
tcattaattt ttatagccat 300tctttgttca tttaaaccat attacaaatt
tgtcctggtt agaactgatt atgatgacaa 360atcgatgatt agttcaaaat
gaagtttata gcaatatatt ctaataacta tacatcaatt 420atcaccactg
atgtgtttga atgaaaaatg aattgataat cttaataaat gtttgatttt
480atagataagt ataaagtatg aatggattta gaaaatttta gtatttagtg
agataagcat 540aagcaaaact agaaaaatga aattattatt aaataaattc
attaatacac attggcaatg 600tgacattgcc taaaatcaaa tatgtactaa
ttcaattaat gttaataaga attttttgga 660ttcgattctc ttgcgaaagt
gcaaattaaa agacattctt atctcctacg taaacagaat 720cacgcgtcca
ttgcgcgttc tcgcaacgct aaaatattta cgttggaaaa tattctcata
780attctctcct ttgcccccac agtttaatat atttcataaa cacccccacg
agaaattaaa 840aaccgttcca aatttgaaaa aatacaacaa acaattcatg
aaaatattca atctataagc 900aaaacattgg tagtgttaca tgtgtcgtcc
ttcgtgctga aaatttggat agcattgtta 960attatgacaa cgtaagtgta
gcctgtgatt catagtaaaa tataaggact aaaaataaaa 1020atatatcttt
tttggaaatc gtaccgcgta agaaacatgt ataaatacct ataaggtctt 1080attttttt
1088151100DNAArabidopsis thalianapromoter(1)..(1100)transcription
regulating sequence from Arabidopsis thaliana gene At5g45350
15ccctgaggtt ctgctgagca aaagtctcga ccttttgtcc aatttcataa ggcctacttt
60ctgtaaggtc ttctctctct ctctctctct gacctgattc tgcctgggtt ctgtggagtt
120gaaatgaatc aaatgtagct cttgttttaa tcagtcagct ttgtaacgta
tacatatata 180tcattgtatg tgattaacat tgtccttaaa aagtgttaca
cctagtaatt acgttccaac 240tttatcagta aacctattgg taactagtaa
tttgctcccg atattggttt tctcattaat 300ttttatagcc attctttgtt
catttaaacc atattacaaa tttgtcctgg ttagaactga 360ttatgatgac
aaatcgatga ttagttcaaa atgaagttta tagcaatata ttctaataac
420tatacatcaa ttatcaccac tgatgtgttt gaatgaaaaa tgaattgata
atcttaataa 480atgtttgatt ttatagataa gtataaagta tgaatggatt
tagaaaattt tagtatttag 540tgagataagc ataagcaaaa ctagaaaaat
gaaattatta ttaaataaat tcattaatac 600acattggcaa tgtgacattg
cctaaaatca aatatgtact aattcaatta atgttaataa 660gaattttttg
gattcgattc tcttgcgaaa gtgcaaatta aaagacattc ttatctccta
720cgtaaacaga atcacgcgtc cattgcgcgt tctcgcaacg ctaaaatatt
tacgttggaa 780aatattctca taattctctc ctttgccccc acagtttaat
atatttcata aacaccccca 840cgagaaatta aaaaccgttc caaatttgaa
aaaatacaac aaacaattca tgaaaatatt 900caatctataa gcaaaacatt
ggtagtgtta catgtgtcgt ccttcgtgct gaaaatttgg 960atagcattgt
taattatgac aacgtaagtg tagcctgtga ttcatagtaa aatataagga
1020ctaaaaataa aaatatatct tttttggaaa tcgtaccgcg taagaaacat
gtataaatac 1080ctataaggtc ttattttttt 110016862DNAArabidopsis
thalianaCDS(66)..(599)encoding Arabidopsis thaliana proline-rich
family protein 16ctcttccaat ttcgtttcga catatttcgc gattcgtcta
aggttttgtt actgattgag 60aaaac atg gga ggt gac aat gat aat gac aaa
gac aaa ggg ttt cat ggg 110 Met Gly Gly Asp Asn Asp Asn Asp Lys Asp
Lys Gly Phe His Gly 1 5 10 15tat cct ccc gct gga tac cca ccc cct
ggg gct tat cca ccc gct gga 158Tyr Pro Pro Ala Gly Tyr Pro Pro Pro
Gly Ala Tyr Pro Pro Ala Gly 20 25 30tac cca caa caa ggt tac cct cca
cca ccc ggt gct tac ccg cct gca 206Tyr Pro Gln Gln Gly Tyr Pro Pro
Pro Pro Gly Ala Tyr Pro Pro Ala 35 40 45ggt tat cct ccg ggt gcc tac
cca cct gct cct ggt ggt tat cct ccc 254Gly Tyr Pro Pro Gly Ala Tyr
Pro Pro Ala Pro Gly Gly Tyr Pro Pro 50 55 60gcc cct ggt tat ggt ggt
tat cct cca gct cct ggt tat gga ggt tat 302Ala Pro Gly Tyr Gly Gly
Tyr Pro Pro Ala Pro Gly Tyr Gly Gly Tyr 65 70 75cct cct gca cct ggt
cat ggt ggt tac cct cct gct ggc tat cct gct 350Pro Pro Ala Pro Gly
His Gly Gly Tyr Pro Pro Ala Gly Tyr Pro Ala80 85 90 95cat cac tca
gga cac gca gga gga att ggg ggt atg att gca ggt gct 398His His Ser
Gly His Ala Gly Gly Ile Gly Gly Met Ile Ala Gly Ala 100 105 110gca
gct gcc tat gga gct cac cac gta gct cat agc tct cac ggt cct 446Ala
Ala Ala Tyr Gly Ala His His Val Ala His Ser Ser His Gly Pro 115 120
125tac gga cat gct gca tat ggt cac ggt ttt ggc cat ggt cat ggc tat
494Tyr Gly His Ala Ala Tyr Gly His Gly Phe Gly His Gly His Gly Tyr
130 135 140ggc tat ggt cat ggt cat ggt aag ttc aag cat ggg aag cac
ggg aag 542Gly Tyr Gly His Gly His Gly Lys Phe Lys His Gly Lys His
Gly Lys 145 150 155ttc aag cat ggg aag cat gga atg ttt gga gga ggc
aag ttc aag aag 590Phe Lys His Gly Lys His Gly Met Phe Gly Gly Gly
Lys Phe Lys Lys160 165 170 175tgg aag tga tctagttaat accttttgtg
aatctgtctg gactgaccaa 639Trp Lystgtttcaaat aagccctaaa cattatataa
gttgactttc gtcggttaga ttgctggttc 699gagttggaat aattgaaact
taattagtat caaatcttat tgtgtacttt aaagctatcg 759ttggctttat
aatgacagat tctggtttcg gtgtgttgtt ttaagatttt tgtatatact
819gttttttaca ttgcttaagc ttatagaagt catgattatg att
86217177PRTArabidopsis thaliana 17Met Gly Gly Asp Asn Asp Asn Asp
Lys Asp Lys Gly Phe His Gly Tyr1 5 10 15Pro Pro Ala Gly Tyr Pro Pro
Pro Gly Ala Tyr Pro Pro Ala Gly Tyr 20 25 30Pro Gln Gln Gly Tyr Pro
Pro Pro Pro Gly Ala Tyr Pro Pro Ala Gly 35 40 45Tyr Pro Pro Gly Ala
Tyr Pro Pro Ala Pro Gly Gly Tyr Pro Pro Ala 50 55 60Pro Gly Tyr Gly
Gly Tyr Pro Pro Ala Pro Gly Tyr Gly Gly Tyr Pro65 70 75 80Pro Ala
Pro Gly His Gly Gly Tyr Pro Pro Ala Gly Tyr Pro Ala His 85 90 95His
Ser Gly His Ala Gly Gly Ile Gly Gly Met Ile Ala Gly Ala Ala 100 105
110Ala Ala Tyr Gly Ala His His Val Ala His Ser Ser His Gly Pro Tyr
115 120 125Gly His Ala Ala Tyr Gly His Gly Phe Gly His Gly His Gly
Tyr Gly 130 135 140Tyr Gly His Gly His Gly Lys Phe Lys His Gly Lys
His Gly Lys Phe145 150 155 160Lys His Gly Lys His Gly Met Phe Gly
Gly Gly Lys Phe Lys Lys Trp 165 170 175Lys181235DNAArabidopsis
thalianapromoter(1)..(1235)transcription regulating sequence from
Arabidopsis thaliana gene At2g39830 18ccaaaaacaa gcagccttaa
tagaaaataa gttatggatc gtccatacag agatcgactt 60tagcaagttg ataaagaaat
ggtcagatct caaggtggaa actgttgctg aaaaaagcta 120gatttgatct
gatgctgtct ttaacccaaa atattaattt gatcccaaaa aaaaggtaat
180ttaatcgaaa gaaagaactt aaacatgaca ataggttagg cttttgttct
gtaaaaattg 240ggtgtctgat tccactttga agcctctctt tcacctttgc
tgcgtcatca ccggtcacga 300ccttcctata gattgatgcc tcaaattata
aataacaata ttattattgg tccgtcgttt
360gtaatgtcat aaaagcttaa ttcccattat aaagttgtct gtggtaacgt
tgcaaaagcg 420catctttatc gtgtattaga gtatgacctt tgttaatacg
cccatattat gtatagtgca 480aacattgtta tactacttct catggattca
tgagtcggat atttgaaacc caaaaacaat 540tacaagaatc atacaaattt
tgaaactaaa gttttagtta aaaaataaaa tgacatatct 600tcaatgtagc
tatagattca ttaaaaactc ggtgagggta tgagaccata aaaacaaaaa
660caaggaaaat ttaacaaaac caaagtttaa aggcaaatag ttagagccga
tggaacgagc 720gtctccatca aaacccaaaa agaagaattt ttttttgctt
tcgtttacaa atctaacttt 780ttggtttttc tccccaacaa aaaaaaaaaa
aataaagtgt aaaaagagag aagcttaaaa 840ggtttcaact atcttcctcc
tccccacacc gttgcttgaa ggattcttcc gcctcagcaa 900aaaccaaaaa
gacaaaaata ttctcttaaa aaaacatctc tttctctctg ttcctttcct
960ttcagaagct aagcatctct tccttttctt tttctctttt aatttttttt
tgcccgatct 1020cttctgcaaa gattctctct ttctttcttc tcttttcatt
tatgttttca ttctctaata 1080acaaattgta atgacttata acttcttctt
cttctcccct cttccttctt cttcctcctc 1140ctcttctctt ctttgcttct
ctctccggcc gtcgttttcg ctttactcac acgttttcaa 1200gtatatttaa
tcacgtgggg gccatttttc catcc 1235191249DNAArabidopsis
thalianapromoter(1)..(1249)transcription regulating sequence from
Arabidopsis thaliana gene At2g39830 19aaatgagaat tcccaaaaac
aagcagcctt aatagaaaat aagttatgga tcgtccatac 60agagatcgac tttagcaagt
tgataaagaa atggtcagat ctcaaggtgg aaactgttgc 120tgaaaaaagc
tagatttgat ctgatgctgt ctttaaccca aaatattaat ttgatcccaa
180aaaaaaggta atttaatcga aagaaagaac ttaaacatga caataggtta
ggcttttgtt 240ctgtaaaaat tgggtgtctg attccacttt gaagcctctc
tttcaccttt gctgcgtcat 300caccggtcac gaccttccta tagattgatg
cctcaaatta taaataacaa tattattatt 360ggtccgtcgt ttgtaatgtc
ataaaagctt aattcccatt ataaagttgt ctgtggtaac 420gttgcaaaag
cgcatcttta tcgtgtatta gagtatgacc tttgttaata cgcccatatt
480atgtatagtg caaacattgt tatactactt ctcatggatt catgagtcgg
atatttgaaa 540cccaaaaaca attacaagaa tcatacaaat tttgaaacta
aagttttagt taaaaaataa 600aatgacatat cttcaatgta gctatagatt
cattaaaaac tcggtgaggg tatgagacca 660taaaaacaaa aacaaggaaa
atttaacaaa accaaagttt aaaggcaaat agttagagcc 720gatggaacga
gcgtctccat caaaacccaa aaagaagaat ttttttttgc tttcgtttac
780aaatctaact ttttggtttt tctccccaac aaaaaaaaaa aaaataaagt
gtaaaaagag 840agaagcttaa aaggtttcaa ctatcttcct cctccccaca
ccgttgcttg aaggattctt 900ccgcctcagc aaaaaccaaa aagacaaaaa
tattctctta aaaaaacatc tctttctctc 960tgttcctttc ctttcagaag
ctaagcatct cttccttttc tttttctctt ttaatttttt 1020tttgcccgat
ctcttctgca aagattctct ctttctttct tctcttttca tttatgtttt
1080cattctctaa taacaaattg taatgactta taacttcttc ttcttctccc
ctcttccttc 1140ttcttcctcc tcctcttctc ttctttgctt ctctctccgg
ccgtcgtttt cgctttactc 1200acacgttttc aagtatattt aatcacgtgg
gggccatttt tccatcctc 1249201135DNAArabidopsis
thalianapromoter(1)..(1135)transcription regulating sequence from
Arabidopsis thaliana gene At2g39830 20ccaaaaacaa gcagccttaa
tagaaaataa gttatggatc gtccatacag agatcgactt 60tagcaagttg ataaagaaat
ggtcagatct caaggtggaa actgttgctg aaaaaagcta 120gatttgatct
gatgctgtct ttaacccaaa atattaattt gatcccaaaa aaaaggtaat
180ttaatcgaaa gaaagaactt aaacatgaca ataggttagg cttttgttct
gtaaaaattg 240ggtgtctgat tccactttga agcctctctt tcacctttgc
tgcgtcatca ccggtcacga 300ccttcctata gattgatgcc tcaaattata
aataacaata ttattattgg tccgtcgttt 360gtaatgtcat aaaagcttaa
ttcccattat aaagttgtct gtggtaacgt tgcaaaagcg 420catctttatc
gtgtattaga gtatgacctt tgttaatacg cccatattat gtatagtgca
480aacattgtta tactacttct catggattca tgagtcggat atttgaaacc
caaaaacaat 540tacaagaatc atacaaattt tgaaactaaa gttttagtta
aaaaataaaa tgacatatct 600tcaatgtagc tatagattca ttaaaaactc
ggtgagggta tgagaccata aaaacaaaaa 660caaggaaaat ttaacaaaac
caaagtttaa aggcaaatag ttagagccga tggaacgagc 720gtctccatca
aaacccaaaa agaagaattt ttttttgctt tcgtttacaa atctaacttt
780ttggtttttc tccccaacaa aaaaaaaaaa aataaagtgt aaaaagagag
aagcttaaaa 840ggtttcaact atcttcctcc tccccacacc gttgcttgaa
ggattcttcc gcctcagcaa 900aaaccaaaaa gacaaaaata ttctcttaaa
aaaacatctc tttctctctg ttcctttcct 960ttcagaagct aagcatctct
tccttttctt tttctctttt aatttttttt tgcccgatct 1020cttctgcaaa
gattctctct ttctttcttc tcttttcatt tatgttttca ttctctaata
1080acaaattgta atgacttata acttcttctt cttctcccct cttccttctt cttcc
1135211147DNAArabidopsis thalianapromoter(1)..(1147)transcription
regulating sequence from Arabidopsis thaliana gene At2g39830
21aaatgagaat tcccaaaaac aagcagcctt aatagaaaat aagttatgga tcgtccatac
60agagatcgac tttagcaagt tgataaagaa atggtcagat ctcaaggtgg aaactgttgc
120tgaaaaaagc tagatttgat ctgatgctgt ctttaaccca aaatattaat
ttgatcccaa 180aaaaaaggta atttaatcga aagaaagaac ttaaacatga
caataggtta ggcttttgtt 240ctgtaaaaat tgggtgtctg attccacttt
gaagcctctc tttcaccttt gctgcgtcat 300caccggtcac gaccttccta
tagattgatg cctcaaatta taaataacaa tattattatt 360ggtccgtcgt
ttgtaatgtc ataaaagctt aattcccatt ataaagttgt ctgtggtaac
420gttgcaaaag cgcatcttta tcgtgtatta gagtatgacc tttgttaata
cgcccatatt 480atgtatagtg caaacattgt tatactactt ctcatggatt
catgagtcgg atatttgaaa 540cccaaaaaca attacaagaa tcatacaaat
tttgaaacta aagttttagt taaaaaataa 600aatgacatat cttcaatgta
gctatagatt cattaaaaac tcggtgaggg tatgagacca 660taaaaacaaa
aacaaggaaa atttaacaaa accaaagttt aaaggcaaat agttagagcc
720gatggaacga gcgtctccat caaaacccaa aaagaagaat ttttttttgc
tttcgtttac 780aaatctaact ttttggtttt tctccccaac aaaaaaaaaa
aaaataaagt gtaaaaagag 840agaagcttaa aaggtttcaa ctatcttcct
cctccccaca ccgttgcttg aaggattctt 900ccgcctcagc aaaaaccaaa
aagacaaaaa tattctctta aaaaaacatc tctttctctc 960tgttcctttc
ctttcagaag ctaagcatct cttccttttc tttttctctt ttaatttttt
1020tttgcccgat ctcttctgca aagattctct ctttctttct tctcttttca
tttatgtttt 1080cattctctaa taacaaattg taatgactta taacttcttc
ttcttctccc ctcttccttc 1140ttcttcc 1147222399DNAArabidopsis
thalianapromoter(1)..(2399)transcription regulating sequence from
Arabidopsis thaliana gene At2g39830 22cttttttggc gggaaaatgt
tgattttttt tttggtggta aactgttaaa tcgcggttta 60ggagaaaaaa atgtgaaatc
ttttttgaca aaaaaatgtt gattcacttg aaaaatgatt 120ttaaaaaaaa
tcgtgggtac ccataaccca atgaggtaaa cccatatgtg ttttatttat
180ttggataccc agcccatttt aaacctgtgt tttatttcca gaaaaaatag
atctatgagc 240ttaaaatttt atgtgtgttt tgggtaccag tggattttaa
cccatcatta acatctctag 300gtacggtact ctagtggtgg cctacattgg
ttgataaagt cataaagtca atatcgttta 360aacatgcaaa tgcaaatttt
atggaccgga ttaagtcgga atgatccgta tgttgaggct 420tgaaagagcc
catcaatgca atggaaaata gtcgtctctt ttcttttcct ttcttttctt
480tacgatcata aagttctctt tttttttggg tgaatattgt aaaggttttg
agtcattctt 540tccaactcaa ttccacgttt ctcatctctc aaggcttttt
actacaaaaa tctttacaga 600tacaaagtta gagatttata acaacttgga
taagatgtct catgcatgag ataagagaaa 660gtaaattctt gtattttagg
tgattaaagg aataaagaca tatgcagcat ttttgcatcg 720gctactcaaa
ctcagctaaa ttcttcgcta tatatatata ctttattttg ttacaataat
780tgtatcagac taatacaact tgatccatgt gtaggaaagt ttcaataaga
ttgatcgttt 840ttatgttacc aaaaataaaa aaagattgat cgtttaaaaa
gtcattctta acaagaaata 900aatacttgtt gaactagaag tctagaacaa
atacatttgt tcagacattt taaattgtaa 960gattaattac attcacaaaa
aaaattgtaa gattaactga ggaatataat gatgtaatag 1020gaaactaacg
caaatgctta aaatgtttta acattttcat gatgaaaaat aagtaaacat
1080ctttgggggt atagatactt aagataaaat atataacttg acaagaaaaa
agaaaagata 1140agcattttct ttaaatgaga attcccaaaa acaagcagcc
ttaatagaaa ataagttatg 1200gatcgtccat acagagatcg actttagcaa
gttgataaag aaatggtcag atctcaaggt 1260ggaaactgtt gctgaaaaaa
gctagatttg atctgatgct gtctttaacc caaaatatta 1320atttgatccc
aaaaaaaagg taatttaatc gaaagaaaga acttaaacat gacaataggt
1380taggcttttg ttctgtaaaa attgggtgtc tgattccact ttgaagcctc
tctttcacct 1440ttgctgcgtc atcaccggtc acgaccttcc tatagattga
tgcctcaaat tataaataac 1500aatattatta ttggtccgtc gtttgtaatg
tcataaaagc ttaattccca ttataaagtt 1560gtctgtggta acgttgcaaa
agcgcatctt tatcgtgtat tagagtatga cctttgttaa 1620tacgcccata
ttatgtatag tgcaaacatt gttatactac ttctcatgga ttcatgagtc
1680ggatatttga aacccaaaaa caattacaag aatcatacaa attttgaaac
taaagtttta 1740gttaaaaaat aaaatgacat atcttcaatg tagctataga
ttcattaaaa actcggtgag 1800ggtatgagac cataaaaaca aaaacaagga
aaatttaaca aaaccaaagt ttaaaggcaa 1860atagttagag ccgatggaac
gagcgtctcc atcaaaaccc aaaaagaaga attttttttt 1920gctttcgttt
acaaatctaa ctttttggtt tttctcccca acaaaaaaaa aaaaaataaa
1980gtgtaaaaag agagaagctt aaaaggtttc aactatcttc ctcctcccca
caccgttgct 2040tgaaggattc ttccgcctca gcaaaaacca aaaagacaaa
aatattctct taaaaaaaca 2100tctctttctc tctgttcctt tcctttcaga
agctaagcat ctcttccttt tctttttctc 2160ttttaatttt tttttgcccg
atctcttctg caaagattct ctctttcttt cttctctttt 2220catttatgtt
ttcattctct aataacaaat tgtaatgact tataacttct tcttcttctc
2280ccctcttcct tcttcttcct cctcctcttc tcttctttgc ttctctctcc
ggccgtcgtt 2340ttcgctttac tcacacgttt tcaagtatat ttaatcacgt
gggggccatt tttccatcc 2399232413DNAArabidopsis
thalianapromoter(1)..(2413)transcription regulating sequence from
Arabidopsis thaliana gene At2g39830 23aattttgaat gtcttttttg
gcgggaaaat gttgattttt tttttggtgg taaactgtta 60aatcgcggtt taggagaaaa
aaatgtgaaa tcttttttga caaaaaaatg ttgattcact 120tgaaaaatga
ttttaaaaaa aatcgtgggt acccataacc caatgaggta aacccatatg
180tgttttattt atttggatac ccagcccatt ttaaacctgt gttttatttc
cagaaaaaat 240agatctatga gcttaaaatt ttatgtgtgt tttgggtacc
agtggatttt aacccatcat 300taacatctct aggtacggta ctctagtggt
ggcctacatt ggttgataaa gtcataaagt 360caatatcgtt taaacatgca
aatgcaaatt ttatggaccg gattaagtcg gaatgatccg 420tatgttgagg
cttgaaagag cccatcaatg caatggaaaa tagtcgtctc ttttcttttc
480ctttcttttc tttacgatca taaagttctc tttttttttg ggtgaatatt
gtaaaggttt 540tgagtcattc tttccaactc aattccacgt ttctcatctc
tcaaggcttt ttactacaaa 600aatctttaca gatacaaagt tagagattta
taacaacttg gataagatgt ctcatgcatg 660agataagaga aagtaaattc
ttgtatttta ggtgattaaa ggaataaaga catatgcagc 720atttttgcat
cggctactca aactcagcta aattcttcgc tatatatata tactttattt
780tgttacaata attgtatcag actaatacaa cttgatccat gtgtaggaaa
gtttcaataa 840gattgatcgt ttttatgtta ccaaaaataa aaaaagattg
atcgtttaaa aagtcattct 900taacaagaaa taaatacttg ttgaactaga
agtctagaac aaatacattt gttcagacat 960tttaaattgt aagattaatt
acattcacaa aaaaaattgt aagattaact gaggaatata 1020atgatgtaat
aggaaactaa cgcaaatgct taaaatgttt taacattttc atgatgaaaa
1080ataagtaaac atctttgggg gtatagatac ttaagataaa atatataact
tgacaagaaa 1140aaagaaaaga taagcatttt ctttaaatga gaattcccaa
aaacaagcag ccttaataga 1200aaataagtta tggatcgtcc atacagagat
cgactttagc aagttgataa agaaatggtc 1260agatctcaag gtggaaactg
ttgctgaaaa aagctagatt tgatctgatg ctgtctttaa 1320cccaaaatat
taatttgatc ccaaaaaaaa ggtaatttaa tcgaaagaaa gaacttaaac
1380atgacaatag gttaggcttt tgttctgtaa aaattgggtg tctgattcca
ctttgaagcc 1440tctctttcac ctttgctgcg tcatcaccgg tcacgacctt
cctatagatt gatgcctcaa 1500attataaata acaatattat tattggtccg
tcgtttgtaa tgtcataaaa gcttaattcc 1560cattataaag ttgtctgtgg
taacgttgca aaagcgcatc tttatcgtgt attagagtat 1620gacctttgtt
aatacgccca tattatgtat agtgcaaaca ttgttatact acttctcatg
1680gattcatgag tcggatattt gaaacccaaa aacaattaca agaatcatac
aaattttgaa 1740actaaagttt tagttaaaaa ataaaatgac atatcttcaa
tgtagctata gattcattaa 1800aaactcggtg agggtatgag accataaaaa
caaaaacaag gaaaatttaa caaaaccaaa 1860gtttaaaggc aaatagttag
agccgatgga acgagcgtct ccatcaaaac ccaaaaagaa 1920gaattttttt
ttgctttcgt ttacaaatct aactttttgg tttttctccc caacaaaaaa
1980aaaaaaaata aagtgtaaaa agagagaagc ttaaaaggtt tcaactatct
tcctcctccc 2040cacaccgttg cttgaaggat tcttccgcct cagcaaaaac
caaaaagaca aaaatattct 2100cttaaaaaaa catctctttc tctctgttcc
tttcctttca gaagctaagc atctcttcct 2160tttctttttc tcttttaatt
tttttttgcc cgatctcttc tgcaaagatt ctctctttct 2220ttcttctctt
ttcatttatg ttttcattct ctaataacaa attgtaatga cttataactt
2280cttcttcttc tcccctcttc cttcttcttc ctcctcctct tctcttcttt
gcttctctct 2340ccggccgtcg ttttcgcttt actcacacgt tttcaagtat
atttaatcac gtgggggcca 2400tttttccatc ctc 2413242299DNAArabidopsis
thalianapromoter(1)..(2299)transcription regulating sequence from
Arabidopsis thaliana gene At2g39830 24cttttttggc gggaaaatgt
tgattttttt tttggtggta aactgttaaa tcgcggttta 60ggagaaaaaa atgtgaaatc
ttttttgaca aaaaaatgtt gattcacttg aaaaatgatt 120ttaaaaaaaa
tcgtgggtac ccataaccca atgaggtaaa cccatatgtg ttttatttat
180ttggataccc agcccatttt aaacctgtgt tttatttcca gaaaaaatag
atctatgagc 240ttaaaatttt atgtgtgttt tgggtaccag tggattttaa
cccatcatta acatctctag 300gtacggtact ctagtggtgg cctacattgg
ttgataaagt cataaagtca atatcgttta 360aacatgcaaa tgcaaatttt
atggaccgga ttaagtcgga atgatccgta tgttgaggct 420tgaaagagcc
catcaatgca atggaaaata gtcgtctctt ttcttttcct ttcttttctt
480tacgatcata aagttctctt tttttttggg tgaatattgt aaaggttttg
agtcattctt 540tccaactcaa ttccacgttt ctcatctctc aaggcttttt
actacaaaaa tctttacaga 600tacaaagtta gagatttata acaacttgga
taagatgtct catgcatgag ataagagaaa 660gtaaattctt gtattttagg
tgattaaagg aataaagaca tatgcagcat ttttgcatcg 720gctactcaaa
ctcagctaaa ttcttcgcta tatatatata ctttattttg ttacaataat
780tgtatcagac taatacaact tgatccatgt gtaggaaagt ttcaataaga
ttgatcgttt 840ttatgttacc aaaaataaaa aaagattgat cgtttaaaaa
gtcattctta acaagaaata 900aatacttgtt gaactagaag tctagaacaa
atacatttgt tcagacattt taaattgtaa 960gattaattac attcacaaaa
aaaattgtaa gattaactga ggaatataat gatgtaatag 1020gaaactaacg
caaatgctta aaatgtttta acattttcat gatgaaaaat aagtaaacat
1080ctttgggggt atagatactt aagataaaat atataacttg acaagaaaaa
agaaaagata 1140agcattttct ttaaatgaga attcccaaaa acaagcagcc
ttaatagaaa ataagttatg 1200gatcgtccat acagagatcg actttagcaa
gttgataaag aaatggtcag atctcaaggt 1260ggaaactgtt gctgaaaaaa
gctagatttg atctgatgct gtctttaacc caaaatatta 1320atttgatccc
aaaaaaaagg taatttaatc gaaagaaaga acttaaacat gacaataggt
1380taggcttttg ttctgtaaaa attgggtgtc tgattccact ttgaagcctc
tctttcacct 1440ttgctgcgtc atcaccggtc acgaccttcc tatagattga
tgcctcaaat tataaataac 1500aatattatta ttggtccgtc gtttgtaatg
tcataaaagc ttaattccca ttataaagtt 1560gtctgtggta acgttgcaaa
agcgcatctt tatcgtgtat tagagtatga cctttgttaa 1620tacgcccata
ttatgtatag tgcaaacatt gttatactac ttctcatgga ttcatgagtc
1680ggatatttga aacccaaaaa caattacaag aatcatacaa attttgaaac
taaagtttta 1740gttaaaaaat aaaatgacat atcttcaatg tagctataga
ttcattaaaa actcggtgag 1800ggtatgagac cataaaaaca aaaacaagga
aaatttaaca aaaccaaagt ttaaaggcaa 1860atagttagag ccgatggaac
gagcgtctcc atcaaaaccc aaaaagaaga attttttttt 1920gctttcgttt
acaaatctaa ctttttggtt tttctcccca acaaaaaaaa aaaaaataaa
1980gtgtaaaaag agagaagctt aaaaggtttc aactatcttc ctcctcccca
caccgttgct 2040tgaaggattc ttccgcctca gcaaaaacca aaaagacaaa
aatattctct taaaaaaaca 2100tctctttctc tctgttcctt tcctttcaga
agctaagcat ctcttccttt tctttttctc 2160ttttaatttt tttttgcccg
atctcttctg caaagattct ctctttcttt cttctctttt 2220catttatgtt
ttcattctct aataacaaat tgtaatgact tataacttct tcttcttctc
2280ccctcttcct tcttcttcc 2299252311DNAArabidopsis
thalianapromoter(1)..(2311)transcription regulating sequence from
Arabidopsis thaliana gene At2g39830 25aattttgaat gtcttttttg
gcgggaaaat gttgattttt tttttggtgg taaactgtta 60aatcgcggtt taggagaaaa
aaatgtgaaa tcttttttga caaaaaaatg ttgattcact 120tgaaaaatga
ttttaaaaaa aatcgtgggt acccataacc caatgaggta aacccatatg
180tgttttattt atttggatac ccagcccatt ttaaacctgt gttttatttc
cagaaaaaat 240agatctatga gcttaaaatt ttatgtgtgt tttgggtacc
agtggatttt aacccatcat 300taacatctct aggtacggta ctctagtggt
ggcctacatt ggttgataaa gtcataaagt 360caatatcgtt taaacatgca
aatgcaaatt ttatggaccg gattaagtcg gaatgatccg 420tatgttgagg
cttgaaagag cccatcaatg caatggaaaa tagtcgtctc ttttcttttc
480ctttcttttc tttacgatca taaagttctc tttttttttg ggtgaatatt
gtaaaggttt 540tgagtcattc tttccaactc aattccacgt ttctcatctc
tcaaggcttt ttactacaaa 600aatctttaca gatacaaagt tagagattta
taacaacttg gataagatgt ctcatgcatg 660agataagaga aagtaaattc
ttgtatttta ggtgattaaa ggaataaaga catatgcagc 720atttttgcat
cggctactca aactcagcta aattcttcgc tatatatata tactttattt
780tgttacaata attgtatcag actaatacaa cttgatccat gtgtaggaaa
gtttcaataa 840gattgatcgt ttttatgtta ccaaaaataa aaaaagattg
atcgtttaaa aagtcattct 900taacaagaaa taaatacttg ttgaactaga
agtctagaac aaatacattt gttcagacat 960tttaaattgt aagattaatt
acattcacaa aaaaaattgt aagattaact gaggaatata 1020atgatgtaat
aggaaactaa cgcaaatgct taaaatgttt taacattttc atgatgaaaa
1080ataagtaaac atctttgggg gtatagatac ttaagataaa atatataact
tgacaagaaa 1140aaagaaaaga taagcatttt ctttaaatga gaattcccaa
aaacaagcag ccttaataga 1200aaataagtta tggatcgtcc atacagagat
cgactttagc aagttgataa agaaatggtc 1260agatctcaag gtggaaactg
ttgctgaaaa aagctagatt tgatctgatg ctgtctttaa 1320cccaaaatat
taatttgatc ccaaaaaaaa ggtaatttaa tcgaaagaaa gaacttaaac
1380atgacaatag gttaggcttt tgttctgtaa aaattgggtg tctgattcca
ctttgaagcc 1440tctctttcac ctttgctgcg tcatcaccgg tcacgacctt
cctatagatt gatgcctcaa 1500attataaata acaatattat tattggtccg
tcgtttgtaa tgtcataaaa gcttaattcc 1560cattataaag ttgtctgtgg
taacgttgca aaagcgcatc tttatcgtgt attagagtat 1620gacctttgtt
aatacgccca tattatgtat agtgcaaaca ttgttatact acttctcatg
1680gattcatgag tcggatattt gaaacccaaa aacaattaca agaatcatac
aaattttgaa 1740actaaagttt tagttaaaaa ataaaatgac atatcttcaa
tgtagctata gattcattaa 1800aaactcggtg agggtatgag accataaaaa
caaaaacaag gaaaatttaa caaaaccaaa 1860gtttaaaggc aaatagttag
agccgatgga acgagcgtct ccatcaaaac ccaaaaagaa 1920gaattttttt
ttgctttcgt ttacaaatct aactttttgg tttttctccc caacaaaaaa
1980aaaaaaaata aagtgtaaaa agagagaagc ttaaaaggtt tcaactatct
tcctcctccc 2040cacaccgttg cttgaaggat tcttccgcct cagcaaaaac
caaaaagaca aaaatattct 2100cttaaaaaaa catctctttc tctctgttcc
tttcctttca gaagctaagc atctcttcct 2160tttctttttc tcttttaatt
tttttttgcc cgatctcttc tgcaaagatt ctctctttct 2220ttcttctctt
ttcatttatg ttttcattct ctaataacaa attgtaatga cttataactt
2280cttcttcttc tcccctcttc cttcttcttc c 2311261787DNAArabidopsis
thalianaCDS(103)..(1614)encoding LIM domain-containing protein
26tcctcctctt ctcttctttg cttctctctc cggccgtcgt tttcgcttta ctcacacgtt
60ttcaagtata tttaatcacg tgggggccat ttttccatcc
tc atg gat tct tct 114 Met Asp Ser Ser 1tcc tct tcc tct tct tct tct
cct tct tct tcc tac ggt gtt gct cgt 162Ser Ser Ser Ser Ser Ser Ser
Pro Ser Ser Ser Tyr Gly Val Ala Arg5 10 15 20gtc agc cat atc tcc
aat cct tgc atc ttc ggg gaa gtt ggg tcg tca 210Val Ser His Ile Ser
Asn Pro Cys Ile Phe Gly Glu Val Gly Ser Ser 25 30 35tct tcg tca acg
tat aga gat aag aaa tgg aag ttg atg aaa tgg gtg 258Ser Ser Ser Thr
Tyr Arg Asp Lys Lys Trp Lys Leu Met Lys Trp Val 40 45 50agt aaa ctt
ttc aag agt ggc tcg aat ggt ggt ggt agt ggt gct cac 306Ser Lys Leu
Phe Lys Ser Gly Ser Asn Gly Gly Gly Ser Gly Ala His 55 60 65act aat
cat cat cct cct cag ttt caa gaa gac gag aat atg gtc ttt 354Thr Asn
His His Pro Pro Gln Phe Gln Glu Asp Glu Asn Met Val Phe 70 75 80cct
cta cct cct tct tct ttg gat gat cgg tca aga ggt gca cgg gac 402Pro
Leu Pro Pro Ser Ser Leu Asp Asp Arg Ser Arg Gly Ala Arg Asp85 90 95
100aaa gaa gaa ctc gac cgt tca att tca ctt tct cta gct gac aac acg
450Lys Glu Glu Leu Asp Arg Ser Ile Ser Leu Ser Leu Ala Asp Asn Thr
105 110 115aag cgc cca cat ggg tat ggt tgg tct atg gat aac aac cga
gat ttt 498Lys Arg Pro His Gly Tyr Gly Trp Ser Met Asp Asn Asn Arg
Asp Phe 120 125 130cca agg cct ttt cac ggt ggc ttg aat cca tca tct
ttc att cca cct 546Pro Arg Pro Phe His Gly Gly Leu Asn Pro Ser Ser
Phe Ile Pro Pro 135 140 145tat gag cct tcc tat caa tat aga cga aga
caa aga ata tgt ggc ggt 594Tyr Glu Pro Ser Tyr Gln Tyr Arg Arg Arg
Gln Arg Ile Cys Gly Gly 150 155 160tgc aat agc gat att gga tcg ggg
aac tat cta gga tgc atg ggc aca 642Cys Asn Ser Asp Ile Gly Ser Gly
Asn Tyr Leu Gly Cys Met Gly Thr165 170 175 180ttc ttt cat cct gaa
tgc ttc cgt tgc cat tct tgt ggt tat gct atc 690Phe Phe His Pro Glu
Cys Phe Arg Cys His Ser Cys Gly Tyr Ala Ile 185 190 195act gag cat
gag ata cca act aat gat gct ggc ttg atc gag tat cga 738Thr Glu His
Glu Ile Pro Thr Asn Asp Ala Gly Leu Ile Glu Tyr Arg 200 205 210tgc
cat ccg ttt tgg aac caa aag tat tgc ccg tct cac gaa tat gat 786Cys
His Pro Phe Trp Asn Gln Lys Tyr Cys Pro Ser His Glu Tyr Asp 215 220
225aaa act gct cgt tgt tgt agc tgc gaa cgt ttg gag tca tgg gat gtg
834Lys Thr Ala Arg Cys Cys Ser Cys Glu Arg Leu Glu Ser Trp Asp Val
230 235 240aga tat tac acg tta gag gat ggg aga agt ttg tgt tta gaa
tgt atg 882Arg Tyr Tyr Thr Leu Glu Asp Gly Arg Ser Leu Cys Leu Glu
Cys Met245 250 255 260gaa acc gcg ata acc gat act gga gaa tgt caa
ccg ctt tac cac gct 930Glu Thr Ala Ile Thr Asp Thr Gly Glu Cys Gln
Pro Leu Tyr His Ala 265 270 275ata aga gac tat tac gaa gga atg tac
atg aaa ctt gat caa cag att 978Ile Arg Asp Tyr Tyr Glu Gly Met Tyr
Met Lys Leu Asp Gln Gln Ile 280 285 290cct atg ctt ctt gtt caa aga
gaa gct ctc aat gat gct atc gta gga 1026Pro Met Leu Leu Val Gln Arg
Glu Ala Leu Asn Asp Ala Ile Val Gly 295 300 305gag aaa aac gga tac
cat cac atg cct gag aca aga ggt tta tgc ttg 1074Glu Lys Asn Gly Tyr
His His Met Pro Glu Thr Arg Gly Leu Cys Leu 310 315 320tct gaa gaa
caa aca gtt aca agt gtt ctt aga aga ccg aga ctt ggt 1122Ser Glu Glu
Gln Thr Val Thr Ser Val Leu Arg Arg Pro Arg Leu Gly325 330 335
340gct cac cgt ctt gtt ggt atg aga act cag cct caa agg ctt aca cgc
1170Ala His Arg Leu Val Gly Met Arg Thr Gln Pro Gln Arg Leu Thr Arg
345 350 355aaa tgt gaa gtc aca gcg att cta gtt ctt tac ggg ctc ccg
cga tta 1218Lys Cys Glu Val Thr Ala Ile Leu Val Leu Tyr Gly Leu Pro
Arg Leu 360 365 370ctg acc gga gca att ctc gcc cat gag ctc atg cat
gga tgg cta agg 1266Leu Thr Gly Ala Ile Leu Ala His Glu Leu Met His
Gly Trp Leu Arg 375 380 385ctt aat ggt aca tac tgg ttt agg aac ctt
aac cct gag gta gag gaa 1314Leu Asn Gly Thr Tyr Trp Phe Arg Asn Leu
Asn Pro Glu Val Glu Glu 390 395 400gga atc tgc caa gtc ctc tct tac
atg tgg ctt gaa tct gaa gtt ctc 1362Gly Ile Cys Gln Val Leu Ser Tyr
Met Trp Leu Glu Ser Glu Val Leu405 410 415 420tca gat cct tca aca
aga aac ttg cct tca aca tca tcg gtg gcc aca 1410Ser Asp Pro Ser Thr
Arg Asn Leu Pro Ser Thr Ser Ser Val Ala Thr 425 430 435tca tca tca
tca tcc ttc tcg aac aag aaa gga gga aaa tca aac gtg 1458Ser Ser Ser
Ser Ser Phe Ser Asn Lys Lys Gly Gly Lys Ser Asn Val 440 445 450gag
aag aaa ctt gga gag ttc ttt aaa cat cag ata gct cat gat gcg 1506Glu
Lys Lys Leu Gly Glu Phe Phe Lys His Gln Ile Ala His Asp Ala 455 460
465tct cca gct tat gga gga ggt ttc agg gca gca aat gca gcg gct tgt
1554Ser Pro Ala Tyr Gly Gly Gly Phe Arg Ala Ala Asn Ala Ala Ala Cys
470 475 480aag tac ggt ctt cgt cga aca ctc gat cat atc cgc tta act
gga act 1602Lys Tyr Gly Leu Arg Arg Thr Leu Asp His Ile Arg Leu Thr
Gly Thr485 490 495 500ttt cct ttg tga tcagatttaa tgttatgtgt
catcttgcta tatgttcttg 1654Phe Pro Leuatttggattt gatggatcac
acagcgtttg tgttaacttt aaatagaatc tcaagacaga 1714ctttatctcc
ccttggattt tgaatgggga tatttgttgc ttggagattt tataagaaca
1774atgactgaat act 178727503PRTArabidopsis thaliana 27Met Asp Ser
Ser Ser Ser Ser Ser Ser Ser Ser Pro Ser Ser Ser Tyr1 5 10 15Gly Val
Ala Arg Val Ser His Ile Ser Asn Pro Cys Ile Phe Gly Glu 20 25 30Val
Gly Ser Ser Ser Ser Ser Thr Tyr Arg Asp Lys Lys Trp Lys Leu 35 40
45Met Lys Trp Val Ser Lys Leu Phe Lys Ser Gly Ser Asn Gly Gly Gly
50 55 60Ser Gly Ala His Thr Asn His His Pro Pro Gln Phe Gln Glu Asp
Glu65 70 75 80Asn Met Val Phe Pro Leu Pro Pro Ser Ser Leu Asp Asp
Arg Ser Arg 85 90 95Gly Ala Arg Asp Lys Glu Glu Leu Asp Arg Ser Ile
Ser Leu Ser Leu 100 105 110Ala Asp Asn Thr Lys Arg Pro His Gly Tyr
Gly Trp Ser Met Asp Asn 115 120 125Asn Arg Asp Phe Pro Arg Pro Phe
His Gly Gly Leu Asn Pro Ser Ser 130 135 140Phe Ile Pro Pro Tyr Glu
Pro Ser Tyr Gln Tyr Arg Arg Arg Gln Arg145 150 155 160Ile Cys Gly
Gly Cys Asn Ser Asp Ile Gly Ser Gly Asn Tyr Leu Gly 165 170 175Cys
Met Gly Thr Phe Phe His Pro Glu Cys Phe Arg Cys His Ser Cys 180 185
190Gly Tyr Ala Ile Thr Glu His Glu Ile Pro Thr Asn Asp Ala Gly Leu
195 200 205Ile Glu Tyr Arg Cys His Pro Phe Trp Asn Gln Lys Tyr Cys
Pro Ser 210 215 220His Glu Tyr Asp Lys Thr Ala Arg Cys Cys Ser Cys
Glu Arg Leu Glu225 230 235 240Ser Trp Asp Val Arg Tyr Tyr Thr Leu
Glu Asp Gly Arg Ser Leu Cys 245 250 255Leu Glu Cys Met Glu Thr Ala
Ile Thr Asp Thr Gly Glu Cys Gln Pro 260 265 270Leu Tyr His Ala Ile
Arg Asp Tyr Tyr Glu Gly Met Tyr Met Lys Leu 275 280 285Asp Gln Gln
Ile Pro Met Leu Leu Val Gln Arg Glu Ala Leu Asn Asp 290 295 300Ala
Ile Val Gly Glu Lys Asn Gly Tyr His His Met Pro Glu Thr Arg305 310
315 320Gly Leu Cys Leu Ser Glu Glu Gln Thr Val Thr Ser Val Leu Arg
Arg 325 330 335Pro Arg Leu Gly Ala His Arg Leu Val Gly Met Arg Thr
Gln Pro Gln 340 345 350Arg Leu Thr Arg Lys Cys Glu Val Thr Ala Ile
Leu Val Leu Tyr Gly 355 360 365Leu Pro Arg Leu Leu Thr Gly Ala Ile
Leu Ala His Glu Leu Met His 370 375 380Gly Trp Leu Arg Leu Asn Gly
Thr Tyr Trp Phe Arg Asn Leu Asn Pro385 390 395 400Glu Val Glu Glu
Gly Ile Cys Gln Val Leu Ser Tyr Met Trp Leu Glu 405 410 415Ser Glu
Val Leu Ser Asp Pro Ser Thr Arg Asn Leu Pro Ser Thr Ser 420 425
430Ser Val Ala Thr Ser Ser Ser Ser Ser Phe Ser Asn Lys Lys Gly Gly
435 440 445Lys Ser Asn Val Glu Lys Lys Leu Gly Glu Phe Phe Lys His
Gln Ile 450 455 460Ala His Asp Ala Ser Pro Ala Tyr Gly Gly Gly Phe
Arg Ala Ala Asn465 470 475 480Ala Ala Ala Cys Lys Tyr Gly Leu Arg
Arg Thr Leu Asp His Ile Arg 485 490 495Leu Thr Gly Thr Phe Pro Leu
500281051DNAArabidopsis thalianapromoter(1)..(1051)transcription
regulating sequence from Arabidopsis thaliana gene At1g68430
28atatgttttt gatataaggg gaggccacca atttgtcaga cggaaaatgt ttcagagttg
60ggccaaccga ggtatcttca aattcagtga tccaatttgt tttactattg agcccaaaaa
120ttgaaggtgt tgtagttttg tcggatgtca ggatgtgtac tgagttaagt
cagaagttcc 180aaaaatatat gaaccaactt aaagtgacgt ggacataacg
agaaaatgtt gtaacaatat 240gcagtatata cttataagat tgtgataatt
actaatggtg atttaaaggt atttctaatc 300tgtgaaatat acttataagg
ttgtgggatt gaatgggaga gtttaataag tttctttttt 360tgttgcaaaa
taatcagctt ttaaaatact tattacattc gaggcacatg aactattttt
420ctatccattg gccgattcta aatataaata gatgttggat aatagagttt
aaagaatttt 480gagttttcta atatacagat tgagaggcca agtgctagat
tcaatatgta acaatatcag 540gctgatattt tcgattgaat atcaccagta
ttcaataata aaatcatcaa taagactaac 600aagtatggtc tcattctaaa
cacagtaatc acgacaagaa ttctaatgag aatctatgac 660caataaagac
taacagtagg tgtcttgcat ctacgctcac attatttcat ttctttcaaa
720aaaggagtaa aattgtaaaa accagagcta atgattcttc cttctatgca
cattaaggaa 780aaagtaaaag agaacaacat gagaaaaagc aaaaggaatc
gaataaacaa acactttatc 840tttttcaaaa tctcatcaat aatgacacaa
tttggaattt cattggaaac tgtgtcctta 900tggtccacaa aaattccaat
tcaaagcaca cactttcgag acaaagactt gcttcaaaag 960aatcaaaagg
acaaagtttt ttgtgttgcc aagttttgga tccttttata atgaacctca
1020cttgaaagca aagtttccac atttccaaat t 1051291065DNAArabidopsis
thalianapromoter(1)..(1065)transcription regulating sequence from
Arabidopsis thaliana gene At1g68430 29ataatcacta gaatatgttt
ttgatataag gggaggccac caatttgtca gacggaaaat 60gtttcagagt tgggccaacc
gaggtatctt caaattcagt gatccaattt gttttactat 120tgagcccaaa
aattgaaggt gttgtagttt tgtcggatgt caggatgtgt actgagttaa
180gtcagaagtt ccaaaaatat atgaaccaac ttaaagtgac gtggacataa
cgagaaaatg 240ttgtaacaat atgcagtata tacttataag attgtgataa
ttactaatgg tgatttaaag 300gtatttctaa tctgtgaaat atacttataa
ggttgtggga ttgaatggga gagtttaata 360agtttctttt tttgttgcaa
aataatcagc ttttaaaata cttattacat tcgaggcaca 420tgaactattt
ttctatccat tggccgattc taaatataaa tagatgttgg ataatagagt
480ttaaagaatt ttgagttttc taatatacag attgagaggc caagtgctag
attcaatatg 540taacaatatc aggctgatat tttcgattga atatcaccag
tattcaataa taaaatcatc 600aataagacta acaagtatgg tctcattcta
aacacagtaa tcacgacaag aattctaatg 660agaatctatg accaataaag
actaacagta ggtgtcttgc atctacgctc acattatttc 720atttctttca
aaaaaggagt aaaattgtaa aaaccagagc taatgattct tccttctatg
780cacattaagg aaaaagtaaa agagaacaac atgagaaaaa gcaaaaggaa
tcgaataaac 840aaacacttta tctttttcaa aatctcatca ataatgacac
aatttggaat ttcattggaa 900actgtgtcct tatggtccac aaaaattcca
attcaaagca cacactttcg agacaaagac 960ttgcttcaaa agaatcaaaa
ggacaaagtt ttttgtgttg ccaagttttg gatcctttta 1020taatgaacct
cacttgaaag caaagtttcc acatttccaa attct 1065301038DNAArabidopsis
thalianapromoter(1)..(1038)transcription regulating sequence from
Arabidopsis thaliana gene At1g68430 30atatgttttt gatataaggg
gaggccacca atttgtcaga cggaaaatgt ttcagagttg 60ggccaaccga ggtatcttca
aattcagtga tccaatttgt tttactattg agcccaaaaa 120ttgaaggtgt
tgtagttttg tcggatgtca ggatgtgtac tgagttaagt cagaagttcc
180aaaaatatat gaaccaactt aaagtgacgt ggacataacg agaaaatgtt
gtaacaatat 240gcagtatata cttataagat tgtgataatt actaatggtg
atttaaaggt atttctaatc 300tgtgaaatat acttataagg ttgtgggatt
gaatgggaga gtttaataag tttctttttt 360tgttgcaaaa taatcagctt
ttaaaatact tattacattc gaggcacatg aactattttt 420ctatccattg
gccgattcta aatataaata gatgttggat aatagagttt aaagaatttt
480gagttttcta atatacagat tgagaggcca agtgctagat tcaatatgta
acaatatcag 540gctgatattt tcgattgaat atcaccagta ttcaataata
aaatcatcaa taagactaac 600aagtatggtc tcattctaaa cacagtaatc
acgacaagaa ttctaatgag aatctatgac 660caataaagac taacagtagg
tgtcttgcat ctacgctcac attatttcat ttctttcaaa 720aaaggagtaa
aattgtaaaa accagagcta atgattcttc cttctatgca cattaaggaa
780aaagtaaaag agaacaacat gagaaaaagc aaaaggaatc gaataaacaa
acactttatc 840tttttcaaaa tctcatcaat aatgacacaa tttggaattt
cattggaaac tgtgtcctta 900tggtccacaa aaattccaat tcaaagcaca
cactttcgag acaaagactt gcttcaaaag 960aatcaaaagg acaaagtttt
ttgtgttgcc aagttttgga tccttttata atgaacctca 1020cttgaaagca aagtttcc
1038311050DNAArabidopsis thalianapromoter(1)..(1050)transcription
regulating sequence from Arabidopsis thaliana gene At1g68430
31ataatcacta gaatatgttt ttgatataag gggaggccac caatttgtca gacggaaaat
60gtttcagagt tgggccaacc gaggtatctt caaattcagt gatccaattt gttttactat
120tgagcccaaa aattgaaggt gttgtagttt tgtcggatgt caggatgtgt
actgagttaa 180gtcagaagtt ccaaaaatat atgaaccaac ttaaagtgac
gtggacataa cgagaaaatg 240ttgtaacaat atgcagtata tacttataag
attgtgataa ttactaatgg tgatttaaag 300gtatttctaa tctgtgaaat
atacttataa ggttgtggga ttgaatggga gagtttaata 360agtttctttt
tttgttgcaa aataatcagc ttttaaaata cttattacat tcgaggcaca
420tgaactattt ttctatccat tggccgattc taaatataaa tagatgttgg
ataatagagt 480ttaaagaatt ttgagttttc taatatacag attgagaggc
caagtgctag attcaatatg 540taacaatatc aggctgatat tttcgattga
atatcaccag tattcaataa taaaatcatc 600aataagacta acaagtatgg
tctcattcta aacacagtaa tcacgacaag aattctaatg 660agaatctatg
accaataaag actaacagta ggtgtcttgc atctacgctc acattatttc
720atttctttca aaaaaggagt aaaattgtaa aaaccagagc taatgattct
tccttctatg 780cacattaagg aaaaagtaaa agagaacaac atgagaaaaa
gcaaaaggaa tcgaataaac 840aaacacttta tctttttcaa aatctcatca
ataatgacac aatttggaat ttcattggaa 900actgtgtcct tatggtccac
aaaaattcca attcaaagca cacactttcg agacaaagac 960ttgcttcaaa
agaatcaaaa ggacaaagtt ttttgtgttg ccaagttttg gatcctttta
1020taatgaacct cacttgaaag caaagtttcc 1050322096DNAArabidopsis
thalianapromoter(1)..(2096)transcription regulating sequence from
Arabidopsis thaliana gene At1g68430 32tttgtaggtt aacatttatt
ttcttatcaa tttgtataaa aagaaaattt gactatatat 60atatatctac aaataaatga
atttgactat gatttcaaca gaaaaagaaa aagaattttc 120gaaactgtat
taaggatttt ctgtttaaat tttggcaaaa actaatatat tatttggaaa
180atatattaga ttcgattaaa tttacaatat ggatgtgagt tttcggacat
atatgaatat 240ttttggaaaa atagtttatt ctattcatga attattattc
ataaataaac acagacaaag 300ggaaaggtac tagaaacact cattatggac
caatttgtga caatttgcaa tgtagaaaca 360acgacatgcg aaccacacaa
gtaatgccac gtgtaatctc ctgaatgaat agaagaaaga 420aaaggcataa
atagccttaa gattttctca ccagaatgac ctggcgttcg atttggtcca
480aatcagtgaa atcaaaataa acgtttcttt ttctggttag atgcacgcca
cgatttcgtc 540taagagaagg cacaatttaa tcttactaga agaaggggct
tatcaattta gttacgtttt 600tggtttttac tcaaaaacaa cttgtatgtt
ctacaagaaa cttcgaagca aatctaactt 660gtaataatgg attgagttag
atctaaaagc aatatgtatt tttaaggcta agaattttca 720acagctatat
accacaataa ttatcatttt gataatttca caaaattaaa gaaacgtgta
780gagagaaact tattatactt ctagaaaaat gatttaactt ttaataaaaa
aatttatact 840taatatatgt atttgatatg cggtttatac aaaacaatca
catgtgttta aataagaata 900gttaatatta aaaatctaaa acaataatta
ttctggaaca taaataatgc ttacataaaa 960tttgatgtga aatggaatta
gtatttattg acgttaaaaa aataaaaaat aattattgac 1020gctaatgggt
tatataatca ctagaatatg tttttgatat aaggggaggc caccaatttg
1080tcagacggaa aatgtttcag agttgggcca accgaggtat cttcaaattc
agtgatccaa 1140tttgttttac tattgagccc aaaaattgaa ggtgttgtag
ttttgtcgga tgtcaggatg 1200tgtactgagt taagtcagaa gttccaaaaa
tatatgaacc aacttaaagt gacgtggaca 1260taacgagaaa atgttgtaac
aatatgcagt atatacttat aagattgtga taattactaa 1320tggtgattta
aaggtatttc taatctgtga aatatactta taaggttgtg ggattgaatg
1380ggagagttta ataagtttct ttttttgttg caaaataatc agcttttaaa
atacttatta 1440cattcgaggc acatgaacta tttttctatc cattggccga
ttctaaatat aaatagatgt 1500tggataatag agtttaaaga attttgagtt
ttctaatata cagattgaga ggccaagtgc 1560tagattcaat atgtaacaat
atcaggctga tattttcgat tgaatatcac cagtattcaa 1620taataaaatc
atcaataaga ctaacaagta tggtctcatt ctaaacacag taatcacgac
1680aagaattcta atgagaatct atgaccaata aagactaaca gtaggtgtct
tgcatctacg 1740ctcacattat ttcatttctt tcaaaaaagg agtaaaattg
taaaaaccag agctaatgat 1800tcttccttct atgcacatta aggaaaaagt
aaaagagaac aacatgagaa aaagcaaaag 1860gaatcgaata aacaaacact
ttatcttttt caaaatctca tcaataatga cacaatttgg 1920aatttcattg
gaaactgtgt ccttatggtc cacaaaaatt ccaattcaaa gcacacactt
1980tcgagacaaa gacttgcttc aaaagaatca aaaggacaaa gttttttgtg
ttgccaagtt 2040ttggatcctt ttataatgaa cctcacttga aagcaaagtt
tccacatttc caaatt 2096332110DNAArabidopsis
thalianapromoter(1)..(2110)transcription regulating sequence from
Arabidopsis thaliana gene At1g68430 33taatacatta attttgtagg
ttaacattta ttttcttatc aatttgtata aaaagaaaat 60ttgactatat atatatatct
acaaataaat gaatttgact atgatttcaa cagaaaaaga 120aaaagaattt
tcgaaactgt attaaggatt ttctgtttaa attttggcaa aaactaatat
180attatttgga aaatatatta gattcgatta aatttacaat atggatgtga
gttttcggac 240atatatgaat atttttggaa aaatagttta ttctattcat
gaattattat tcataaataa 300acacagacaa agggaaaggt actagaaaca
ctcattatgg accaatttgt gacaatttgc 360aatgtagaaa caacgacatg
cgaaccacac aagtaatgcc acgtgtaatc tcctgaatga 420atagaagaaa
gaaaaggcat aaatagcctt aagattttct caccagaatg acctggcgtt
480cgatttggtc caaatcagtg aaatcaaaat aaacgtttct ttttctggtt
agatgcacgc 540cacgatttcg tctaagagaa ggcacaattt aatcttacta
gaagaagggg cttatcaatt 600tagttacgtt tttggttttt actcaaaaac
aacttgtatg ttctacaaga aacttcgaag 660caaatctaac ttgtaataat
ggattgagtt agatctaaaa gcaatatgta tttttaaggc 720taagaatttt
caacagctat ataccacaat aattatcatt ttgataattt cacaaaatta
780aagaaacgtg tagagagaaa cttattatac ttctagaaaa atgatttaac
ttttaataaa 840aaaatttata cttaatatat gtatttgata tgcggtttat
acaaaacaat cacatgtgtt 900taaataagaa tagttaatat taaaaatcta
aaacaataat tattctggaa cataaataat 960gcttacataa aatttgatgt
gaaatggaat tagtatttat tgacgttaaa aaaataaaaa 1020ataattattg
acgctaatgg gttatataat cactagaata tgtttttgat ataaggggag
1080gccaccaatt tgtcagacgg aaaatgtttc agagttgggc caaccgaggt
atcttcaaat 1140tcagtgatcc aatttgtttt actattgagc ccaaaaattg
aaggtgttgt agttttgtcg 1200gatgtcagga tgtgtactga gttaagtcag
aagttccaaa aatatatgaa ccaacttaaa 1260gtgacgtgga cataacgaga
aaatgttgta acaatatgca gtatatactt ataagattgt 1320gataattact
aatggtgatt taaaggtatt tctaatctgt gaaatatact tataaggttg
1380tgggattgaa tgggagagtt taataagttt ctttttttgt tgcaaaataa
tcagctttta 1440aaatacttat tacattcgag gcacatgaac tatttttcta
tccattggcc gattctaaat 1500ataaatagat gttggataat agagtttaaa
gaattttgag ttttctaata tacagattga 1560gaggccaagt gctagattca
atatgtaaca atatcaggct gatattttcg attgaatatc 1620accagtattc
aataataaaa tcatcaataa gactaacaag tatggtctca ttctaaacac
1680agtaatcacg acaagaattc taatgagaat ctatgaccaa taaagactaa
cagtaggtgt 1740cttgcatcta cgctcacatt atttcatttc tttcaaaaaa
ggagtaaaat tgtaaaaacc 1800agagctaatg attcttcctt ctatgcacat
taaggaaaaa gtaaaagaga acaacatgag 1860aaaaagcaaa aggaatcgaa
taaacaaaca ctttatcttt ttcaaaatct catcaataat 1920gacacaattt
ggaatttcat tggaaactgt gtccttatgg tccacaaaaa ttccaattca
1980aagcacacac tttcgagaca aagacttgct tcaaaagaat caaaaggaca
aagttttttg 2040tgttgccaag ttttggatcc ttttataatg aacctcactt
gaaagcaaag tttccacatt 2100tccaaattct 2110342083DNAArabidopsis
thalianapromoter(1)..(2083)transcription regulating sequence from
Arabidopsis thaliana gene At1g68430 34tttgtaggtt aacatttatt
ttcttatcaa tttgtataaa aagaaaattt gactatatat 60atatatctac aaataaatga
atttgactat gatttcaaca gaaaaagaaa aagaattttc 120gaaactgtat
taaggatttt ctgtttaaat tttggcaaaa actaatatat tatttggaaa
180atatattaga ttcgattaaa tttacaatat ggatgtgagt tttcggacat
atatgaatat 240ttttggaaaa atagtttatt ctattcatga attattattc
ataaataaac acagacaaag 300ggaaaggtac tagaaacact cattatggac
caatttgtga caatttgcaa tgtagaaaca 360acgacatgcg aaccacacaa
gtaatgccac gtgtaatctc ctgaatgaat agaagaaaga 420aaaggcataa
atagccttaa gattttctca ccagaatgac ctggcgttcg atttggtcca
480aatcagtgaa atcaaaataa acgtttcttt ttctggttag atgcacgcca
cgatttcgtc 540taagagaagg cacaatttaa tcttactaga agaaggggct
tatcaattta gttacgtttt 600tggtttttac tcaaaaacaa cttgtatgtt
ctacaagaaa cttcgaagca aatctaactt 660gtaataatgg attgagttag
atctaaaagc aatatgtatt tttaaggcta agaattttca 720acagctatat
accacaataa ttatcatttt gataatttca caaaattaaa gaaacgtgta
780gagagaaact tattatactt ctagaaaaat gatttaactt ttaataaaaa
aatttatact 840taatatatgt atttgatatg cggtttatac aaaacaatca
catgtgttta aataagaata 900gttaatatta aaaatctaaa acaataatta
ttctggaaca taaataatgc ttacataaaa 960tttgatgtga aatggaatta
gtatttattg acgttaaaaa aataaaaaat aattattgac 1020gctaatgggt
tatataatca ctagaatatg tttttgatat aaggggaggc caccaatttg
1080tcagacggaa aatgtttcag agttgggcca accgaggtat cttcaaattc
agtgatccaa 1140tttgttttac tattgagccc aaaaattgaa ggtgttgtag
ttttgtcgga tgtcaggatg 1200tgtactgagt taagtcagaa gttccaaaaa
tatatgaacc aacttaaagt gacgtggaca 1260taacgagaaa atgttgtaac
aatatgcagt atatacttat aagattgtga taattactaa 1320tggtgattta
aaggtatttc taatctgtga aatatactta taaggttgtg ggattgaatg
1380ggagagttta ataagtttct ttttttgttg caaaataatc agcttttaaa
atacttatta 1440cattcgaggc acatgaacta tttttctatc cattggccga
ttctaaatat aaatagatgt 1500tggataatag agtttaaaga attttgagtt
ttctaatata cagattgaga ggccaagtgc 1560tagattcaat atgtaacaat
atcaggctga tattttcgat tgaatatcac cagtattcaa 1620taataaaatc
atcaataaga ctaacaagta tggtctcatt ctaaacacag taatcacgac
1680aagaattcta atgagaatct atgaccaata aagactaaca gtaggtgtct
tgcatctacg 1740ctcacattat ttcatttctt tcaaaaaagg agtaaaattg
taaaaaccag agctaatgat 1800tcttccttct atgcacatta aggaaaaagt
aaaagagaac aacatgagaa aaagcaaaag 1860gaatcgaata aacaaacact
ttatcttttt caaaatctca tcaataatga cacaatttgg 1920aatttcattg
gaaactgtgt ccttatggtc cacaaaaatt ccaattcaaa gcacacactt
1980tcgagacaaa gacttgcttc aaaagaatca aaaggacaaa gttttttgtg
ttgccaagtt 2040ttggatcctt ttataatgaa cctcacttga aagcaaagtt tcc
2083352095DNAArabidopsis thalianapromoter(1)..(2095)transcription
regulating sequence from Arabidopsis thaliana gene At1g68430
35taatacatta attttgtagg ttaacattta ttttcttatc aatttgtata aaaagaaaat
60ttgactatat atatatatct acaaataaat gaatttgact atgatttcaa cagaaaaaga
120aaaagaattt tcgaaactgt attaaggatt ttctgtttaa attttggcaa
aaactaatat 180attatttgga aaatatatta gattcgatta aatttacaat
atggatgtga gttttcggac 240atatatgaat atttttggaa aaatagttta
ttctattcat gaattattat tcataaataa 300acacagacaa agggaaaggt
actagaaaca ctcattatgg accaatttgt gacaatttgc 360aatgtagaaa
caacgacatg cgaaccacac aagtaatgcc acgtgtaatc tcctgaatga
420atagaagaaa gaaaaggcat aaatagcctt aagattttct caccagaatg
acctggcgtt 480cgatttggtc caaatcagtg aaatcaaaat aaacgtttct
ttttctggtt agatgcacgc 540cacgatttcg tctaagagaa ggcacaattt
aatcttacta gaagaagggg cttatcaatt 600tagttacgtt tttggttttt
actcaaaaac aacttgtatg ttctacaaga aacttcgaag 660caaatctaac
ttgtaataat ggattgagtt agatctaaaa gcaatatgta tttttaaggc
720taagaatttt caacagctat ataccacaat aattatcatt ttgataattt
cacaaaatta 780aagaaacgtg tagagagaaa cttattatac ttctagaaaa
atgatttaac ttttaataaa 840aaaatttata cttaatatat gtatttgata
tgcggtttat acaaaacaat cacatgtgtt 900taaataagaa tagttaatat
taaaaatcta aaacaataat tattctggaa cataaataat 960gcttacataa
aatttgatgt gaaatggaat tagtatttat tgacgttaaa aaaataaaaa
1020ataattattg acgctaatgg gttatataat cactagaata tgtttttgat
ataaggggag 1080gccaccaatt tgtcagacgg aaaatgtttc agagttgggc
caaccgaggt atcttcaaat 1140tcagtgatcc aatttgtttt actattgagc
ccaaaaattg aaggtgttgt agttttgtcg 1200gatgtcagga tgtgtactga
gttaagtcag aagttccaaa aatatatgaa ccaacttaaa 1260gtgacgtgga
cataacgaga aaatgttgta acaatatgca gtatatactt ataagattgt
1320gataattact aatggtgatt taaaggtatt tctaatctgt gaaatatact
tataaggttg 1380tgggattgaa tgggagagtt taataagttt ctttttttgt
tgcaaaataa tcagctttta 1440aaatacttat tacattcgag gcacatgaac
tatttttcta tccattggcc gattctaaat 1500ataaatagat gttggataat
agagtttaaa gaattttgag ttttctaata tacagattga 1560gaggccaagt
gctagattca atatgtaaca atatcaggct gatattttcg attgaatatc
1620accagtattc aataataaaa tcatcaataa gactaacaag tatggtctca
ttctaaacac 1680agtaatcacg acaagaattc taatgagaat ctatgaccaa
taaagactaa cagtaggtgt 1740cttgcatcta cgctcacatt atttcatttc
tttcaaaaaa ggagtaaaat tgtaaaaacc 1800agagctaatg attcttcctt
ctatgcacat taaggaaaaa gtaaaagaga acaacatgag 1860aaaaagcaaa
aggaatcgaa taaacaaaca ctttatcttt ttcaaaatct catcaataat
1920gacacaattt ggaatttcat tggaaactgt gtccttatgg tccacaaaaa
ttccaattca 1980aagcacacac tttcgagaca aagacttgct tcaaaagaat
caaaaggaca aagttttttg 2040tgttgccaag ttttggatcc ttttataatg
aacctcactt gaaagcaaag tttcc 209536540DNAArabidopsis
thalianaCDS(16)..(456)encoding expressed protein 36acatttccaa attct
atg gct aca ctg cag aga ttc aag ttc ttg ggg acg 51 Met Ala Thr Leu
Gln Arg Phe Lys Phe Leu Gly Thr 1 5 10cag tgc gga gta gca gca caa
agc ccg aca cga agt ccg agt ccg agg 99Gln Cys Gly Val Ala Ala Gln
Ser Pro Thr Arg Ser Pro Ser Pro Arg 15 20 25aca agt cca ttg gta cag
ctt cga cga aag aag aca act tta aag atg 147Thr Ser Pro Leu Val Gln
Leu Arg Arg Lys Lys Thr Thr Leu Lys Met 30 35 40ctt ttg agt ctt gca
tct ccg agt cgc cga gag cag caa ccg ttg att 195Leu Leu Ser Leu Ala
Ser Pro Ser Arg Arg Glu Gln Gln Pro Leu Ile45 50 55 60cat cat cat
cac aag gac gta gcc gga cgg aaa ctt aaa gac tta ttc 243His His His
His Lys Asp Val Ala Gly Arg Lys Leu Lys Asp Leu Phe 65 70 75gtc tct
tcg tct tcc gca gag gaa gaa caa gaa gag gac gag aga cca 291Val Ser
Ser Ser Ser Ala Glu Glu Glu Gln Glu Glu Asp Glu Arg Pro 80 85 90aag
ggg aaa aca aaa gaa gaa gtt ctt gca gcc atg gcg gct aaa ctg 339Lys
Gly Lys Thr Lys Glu Glu Val Leu Ala Ala Met Ala Ala Lys Leu 95 100
105aat gca gct tca aga tta caa tgt gag tct gct gat gca gca cca gtt
387Asn Ala Ala Ser Arg Leu Gln Cys Glu Ser Ala Asp Ala Ala Pro Val
110 115 120tgg ttc gga ttc agc aaa cgg ctt ctt cag cga gct tgg cgt
cct aaa 435Trp Phe Gly Phe Ser Lys Arg Leu Leu Gln Arg Ala Trp Arg
Pro Lys125 130 135 140ctt ggt acc att cac gag taa tgtaacccaa
ttttcttctc tttttttggt 486Leu Gly Thr Ile His Glu 145gtgactttgg
aaaccattag tttcccatat gaatgaatat atatgtttct tctc
54037146PRTArabidopsis thaliana 37Met Ala Thr Leu Gln Arg Phe Lys
Phe Leu Gly Thr Gln Cys Gly Val1 5 10 15Ala Ala Gln Ser Pro Thr Arg
Ser Pro Ser Pro Arg Thr Ser Pro Leu 20 25 30Val Gln Leu Arg Arg Lys
Lys Thr Thr Leu Lys Met Leu Leu Ser Leu 35 40 45Ala Ser Pro Ser Arg
Arg Glu Gln Gln Pro Leu Ile His His His His 50 55 60Lys Asp Val Ala
Gly Arg Lys Leu Lys Asp Leu Phe Val Ser Ser Ser65 70 75 80Ser Ala
Glu Glu Glu Gln Glu Glu Asp Glu Arg Pro Lys Gly Lys Thr 85 90 95Lys
Glu Glu Val Leu Ala Ala Met Ala Ala Lys Leu Asn Ala Ala Ser 100 105
110Arg Leu Gln Cys Glu Ser Ala Asp Ala Ala Pro Val Trp Phe Gly Phe
115 120 125Ser Lys Arg Leu Leu Gln Arg Ala Trp Arg Pro Lys Leu Gly
Thr Ile 130 135 140His Glu145381030DNAArabidopsis
thalianapromoter(1)..(1030)transcription regulating sequence from
Arabidopsis thaliana gene At5g67280 38ttactacgta gtacatgtta
aactacatat ataaggattc cataaatatg aatcaaatcg 60aattcttctt atataactaa
gacattaaat gtctacttgc acttcattaa aaagaatctt 120ctgattttat
ttttggtata tgcaaaatta taactgcatt taaaacagag attagatcat
180gaaacgaata ttatttctga aaaagtaggt atatctagta atgttaattt
attttttgct 240aaggatatct agtaatattt tatatgaaac acttgttttt
atttatgtgt tttgcgcata 300aagtcaatat tataacgaat cactagatta
gttttatttt tatcttatag attagtttaa 360taagcctata tctataagat
gtactatgat cagagaaaca ttaggtgtaa tgagaaaaca 420ataataaaac
ggtcatatat aagtaaatcc aagtcagtga gagacctgca aaaatttcga
480actttttgtg aatgtcttat agcaaagata ctattctccc atctgatcat
gtttccatag 540atattagtta attggctaat aatacaattc ttcacacaca
tcacatgatc gatatgcgta 600atctcacgtc acaatttccg tcataatcaa
aaggagtaac agaattagta tattatatag 660tttctgtgat ctagcaaaga
tttaaatgta aacaaaacta tccagattta tatggttttg 720ttacgtttct
ttgaagatca atgtaaacaa tgatgaacca atattagggt ccacgttgaa
780tataaacgtg aaaaacgaca aagcgacgtc agcataattt acgaatagga
aaacagtgtt 840aacttttgtt atcacattcc gtgcaattta cataggatat
agaatttttt atttacaaaa 900gttatattta taaagaatta ctcaaatcaa
gaattccata aaaagataaa cactttcata 960tatcgttcac aatcacatgg
ccttttcaaa aaatcaatct tttagttctc tatcgatgcg 1020taggcttgaa
1030391044DNAArabidopsis thalianapromoter(1)..(1044)transcription
regulating sequence from Arabidopsis thaliana gene At5g67280
39ccattgggat tattactacg tagtacatgt taaactacat atataaggat tccataaata
60tgaatcaaat cgaattcttc ttatataact aagacattaa atgtctactt gcacttcatt
120aaaaagaatc ttctgatttt atttttggta tatgcaaaat tataactgca
tttaaaacag 180agattagatc atgaaacgaa tattatttct gaaaaagtag
gtatatctag taatgttaat 240ttattttttg ctaaggatat ctagtaatat
tttatatgaa acacttgttt ttatttatgt 300gttttgcgca taaagtcaat
attataacga atcactagat tagttttatt tttatcttat 360agattagttt
aataagccta tatctataag atgtactatg atcagagaaa cattaggtgt
420aatgagaaaa caataataaa acggtcatat ataagtaaat ccaagtcagt
gagagacctg 480caaaaatttc gaactttttg tgaatgtctt atagcaaaga
tactattctc ccatctgatc 540atgtttccat agatattagt taattggcta
ataatacaat tcttcacaca catcacatga 600tcgatatgcg taatctcacg
tcacaatttc cgtcataatc aaaaggagta acagaattag 660tatattatat
agtttctgtg atctagcaaa gatttaaatg taaacaaaac tatccagatt
720tatatggttt tgttacgttt ctttgaagat caatgtaaac aatgatgaac
caatattagg 780gtccacgttg aatataaacg tgaaaaacga caaagcgacg
tcagcataat ttacgaatag 840gaaaacagtg ttaacttttg ttatcacatt
ccgtgcaatt tacataggat atagaatttt 900ttatttacaa aagttatatt
tataaagaat tactcaaatc aagaattcca taaaaagata 960aacactttca
tatatcgttc acaatcacat ggccttttca aaaaatcaat cttttagttc
1020tctatcgatg cgtaggcttg aagc 104440968DNAArabidopsis
thalianapromoter(1)..(968)transcription regulating sequence from
Arabidopsis thaliana gene At5g67280 40ttactacgta gtacatgtta
aactacatat ataaggattc cataaatatg aatcaaatcg 60aattcttctt atataactaa
gacattaaat gtctacttgc acttcattaa aaagaatctt 120ctgattttat
ttttggtata tgcaaaatta taactgcatt taaaacagag attagatcat
180gaaacgaata ttatttctga aaaagtaggt atatctagta atgttaattt
attttttgct 240aaggatatct agtaatattt tatatgaaac acttgttttt
atttatgtgt tttgcgcata 300aagtcaatat tataacgaat cactagatta
gttttatttt tatcttatag attagtttaa 360taagcctata tctataagat
gtactatgat cagagaaaca ttaggtgtaa tgagaaaaca 420ataataaaac
ggtcatatat aagtaaatcc aagtcagtga gagacctgca aaaatttcga
480actttttgtg aatgtcttat agcaaagata ctattctccc atctgatcat
gtttccatag 540atattagtta attggctaat aatacaattc ttcacacaca
tcacatgatc gatatgcgta 600atctcacgtc acaatttccg tcataatcaa
aaggagtaac agaattagta tattatatag 660tttctgtgat ctagcaaaga
tttaaatgta aacaaaacta tccagattta tatggttttg 720ttacgtttct
ttgaagatca atgtaaacaa tgatgaacca atattagggt ccacgttgaa
780tataaacgtg aaaaacgaca aagcgacgtc agcataattt acgaatagga
aaacagtgtt 840aacttttgtt atcacattcc gtgcaattta cataggatat
agaatttttt atttacaaaa 900gttatattta taaagaatta ctcaaatcaa
gaattccata aaaagataaa cactttcata 960tatcgttc 96841980DNAArabidopsis
thalianapromoter(1)..(980)transcription regulating sequence from
Arabidopsis thaliana gene At5g67280 41ccattgggat tattactacg
tagtacatgt taaactacat atataaggat tccataaata 60tgaatcaaat cgaattcttc
ttatataact aagacattaa atgtctactt gcacttcatt 120aaaaagaatc
ttctgatttt atttttggta tatgcaaaat tataactgca tttaaaacag
180agattagatc atgaaacgaa tattatttct gaaaaagtag gtatatctag
taatgttaat 240ttattttttg ctaaggatat ctagtaatat tttatatgaa
acacttgttt ttatttatgt 300gttttgcgca taaagtcaat attataacga
atcactagat tagttttatt tttatcttat 360agattagttt aataagccta
tatctataag atgtactatg atcagagaaa cattaggtgt 420aatgagaaaa
caataataaa acggtcatat ataagtaaat ccaagtcagt gagagacctg
480caaaaatttc gaactttttg tgaatgtctt atagcaaaga tactattctc
ccatctgatc 540atgtttccat agatattagt taattggcta ataatacaat
tcttcacaca catcacatga 600tcgatatgcg taatctcacg tcacaatttc
cgtcataatc aaaaggagta acagaattag 660tatattatat agtttctgtg
atctagcaaa gatttaaatg taaacaaaac tatccagatt 720tatatggttt
tgttacgttt ctttgaagat caatgtaaac aatgatgaac caatattagg
780gtccacgttg aatataaacg tgaaaaacga caaagcgacg tcagcataat
ttacgaatag 840gaaaacagtg ttaacttttg ttatcacatt ccgtgcaatt
tacataggat atagaatttt 900ttatttacaa aagttatatt tataaagaat
tactcaaatc aagaattcca taaaaagata 960aacactttca tatatcgttc
980422022DNAArabidopsis thalianapromoter(1)..(2022)transcription
regulating sequence from Arabidopsis thaliana gene At5g67280
42aaaaacaccc gaccgatcaa gtgacaatgc gcagtgttgt attattatat tatcactgtt
60tgaaaaattg tcgaactcag gattggttta taactttgca agacgataat aactttggag
120ttttgcataa tggtaagtag aaaacgccat ttttcatgca tctcccgtct
ttgtccaccg 180ccaaactagt agccatcctc taattaataa tgtattacta
cagctttgta tatattcgta 240ttggagttta cagactaatc acatagtggt
tacgtttagg aagataaaac atacagaaat 300ggtaaatagc ttgtgaacct
gaatctcgaa acttaccttg tccaatttgt aacatgaatg 360tcgactataa
gtaaatttgc tctgacaatt tacagtcaaa ctcaactatg agtctataat
420atcactgtag tgggtattgt tcacacacaa cgattaatac tgtaattaat
gctgaaactt 480ggactacacg acactcatat gttttattgt ttcacacaca
gcagaagaaa taattggatt 540ttttttcggt ccagtgattg cactggtttg
cagaaaaatt cttaaacgat aaataaacca 600tgttcatctc atgattactt
aatcgtttga gaaaccgtga catatggtgg taacaaacaa 660aaacaaacaa
ataccaaccg gtaaaacata tatacagtat tttatacata aacaattttg
720tgattttggc aatcaaataa caaggaccac
aatgacgctc gcgaaattta attaaaacgg 780ggaccaattt taatcaattt
gacccggtgt acattaatct tgacatgcat ttatttacgt 840gtgatgttga
cctcttggta gatacagtac tacatttata tatttttgat gcaacacata
900actgtttagt gtttttgata tttccttttt ttatcagtct aactctcgtg
agtcgtgagt 960cgtgagttgt gagtcgtgat ccattgggat tattactacg
tagtacatgt taaactacat 1020atataaggat tccataaata tgaatcaaat
cgaattcttc ttatataact aagacattaa 1080atgtctactt gcacttcatt
aaaaagaatc ttctgatttt atttttggta tatgcaaaat 1140tataactgca
tttaaaacag agattagatc atgaaacgaa tattatttct gaaaaagtag
1200gtatatctag taatgttaat ttattttttg ctaaggatat ctagtaatat
tttatatgaa 1260acacttgttt ttatttatgt gttttgcgca taaagtcaat
attataacga atcactagat 1320tagttttatt tttatcttat agattagttt
aataagccta tatctataag atgtactatg 1380atcagagaaa cattaggtgt
aatgagaaaa caataataaa acggtcatat ataagtaaat 1440ccaagtcagt
gagagacctg caaaaatttc gaactttttg tgaatgtctt atagcaaaga
1500tactattctc ccatctgatc atgtttccat agatattagt taattggcta
ataatacaat 1560tcttcacaca catcacatga tcgatatgcg taatctcacg
tcacaatttc cgtcataatc 1620aaaaggagta acagaattag tatattatat
agtttctgtg atctagcaaa gatttaaatg 1680taaacaaaac tatccagatt
tatatggttt tgttacgttt ctttgaagat caatgtaaac 1740aatgatgaac
caatattagg gtccacgttg aatataaacg tgaaaaacga caaagcgacg
1800tcagcataat ttacgaatag gaaaacagtg ttaacttttg ttatcacatt
ccgtgcaatt 1860tacataggat atagaatttt ttatttacaa aagttatatt
tataaagaat tactcaaatc 1920aagaattcca taaaaagata aacactttca
tatatcgttc acaatcacat ggccttttca 1980aaaaatcaat cttttagttc
tctatcgatg cgtaggcttg aa 2022432036DNAArabidopsis
thalianapromoter(1)..(2036)transcription regulating sequence from
Arabidopsis thaliana gene At5g67280 43tataatatat ccaaaaacac
ccgaccgatc aagtgacaat gcgcagtgtt gtattattat 60attatcactg tttgaaaaat
tgtcgaactc aggattggtt tataactttg caagacgata 120ataactttgg
agttttgcat aatggtaagt agaaaacgcc atttttcatg catctcccgt
180ctttgtccac cgccaaacta gtagccatcc tctaattaat aatgtattac
tacagctttg 240tatatattcg tattggagtt tacagactaa tcacatagtg
gttacgttta ggaagataaa 300acatacagaa atggtaaata gcttgtgaac
ctgaatctcg aaacttacct tgtccaattt 360gtaacatgaa tgtcgactat
aagtaaattt gctctgacaa tttacagtca aactcaacta 420tgagtctata
atatcactgt agtgggtatt gttcacacac aacgattaat actgtaatta
480atgctgaaac ttggactaca cgacactcat atgttttatt gtttcacaca
cagcagaaga 540aataattgga ttttttttcg gtccagtgat tgcactggtt
tgcagaaaaa ttcttaaacg 600ataaataaac catgttcatc tcatgattac
ttaatcgttt gagaaaccgt gacatatggt 660ggtaacaaac aaaaacaaac
aaataccaac cggtaaaaca tatatacagt attttataca 720taaacaattt
tgtgattttg gcaatcaaat aacaaggacc acaatgacgc tcgcgaaatt
780taattaaaac ggggaccaat tttaatcaat ttgacccggt gtacattaat
cttgacatgc 840atttatttac gtgtgatgtt gacctcttgg tagatacagt
actacattta tatatttttg 900atgcaacaca taactgttta gtgtttttga
tatttccttt ttttatcagt ctaactctcg 960tgagtcgtga gtcgtgagtt
gtgagtcgtg atccattggg attattacta cgtagtacat 1020gttaaactac
atatataagg attccataaa tatgaatcaa atcgaattct tcttatataa
1080ctaagacatt aaatgtctac ttgcacttca ttaaaaagaa tcttctgatt
ttatttttgg 1140tatatgcaaa attataactg catttaaaac agagattaga
tcatgaaacg aatattattt 1200ctgaaaaagt aggtatatct agtaatgtta
atttattttt tgctaaggat atctagtaat 1260attttatatg aaacacttgt
ttttatttat gtgttttgcg cataaagtca atattataac 1320gaatcactag
attagtttta tttttatctt atagattagt ttaataagcc tatatctata
1380agatgtacta tgatcagaga aacattaggt gtaatgagaa aacaataata
aaacggtcat 1440atataagtaa atccaagtca gtgagagacc tgcaaaaatt
tcgaactttt tgtgaatgtc 1500ttatagcaaa gatactattc tcccatctga
tcatgtttcc atagatatta gttaattggc 1560taataataca attcttcaca
cacatcacat gatcgatatg cgtaatctca cgtcacaatt 1620tccgtcataa
tcaaaaggag taacagaatt agtatattat atagtttctg tgatctagca
1680aagatttaaa tgtaaacaaa actatccaga tttatatggt tttgttacgt
ttctttgaag 1740atcaatgtaa acaatgatga accaatatta gggtccacgt
tgaatataaa cgtgaaaaac 1800gacaaagcga cgtcagcata atttacgaat
aggaaaacag tgttaacttt tgttatcaca 1860ttccgtgcaa tttacatagg
atatagaatt ttttatttac aaaagttata tttataaaga 1920attactcaaa
tcaagaattc cataaaaaga taaacacttt catatatcgt tcacaatcac
1980atggcctttt caaaaaatca atcttttagt tctctatcga tgcgtaggct tgaagc
2036441960DNAArabidopsis thalianapromoter(1)..(1960)transcription
regulating sequence from Arabidopsis thaliana gene At5g67280
44aaaaacaccc gaccgatcaa gtgacaatgc gcagtgttgt attattatat tatcactgtt
60tgaaaaattg tcgaactcag gattggttta taactttgca agacgataat aactttggag
120ttttgcataa tggtaagtag aaaacgccat ttttcatgca tctcccgtct
ttgtccaccg 180ccaaactagt agccatcctc taattaataa tgtattacta
cagctttgta tatattcgta 240ttggagttta cagactaatc acatagtggt
tacgtttagg aagataaaac atacagaaat 300ggtaaatagc ttgtgaacct
gaatctcgaa acttaccttg tccaatttgt aacatgaatg 360tcgactataa
gtaaatttgc tctgacaatt tacagtcaaa ctcaactatg agtctataat
420atcactgtag tgggtattgt tcacacacaa cgattaatac tgtaattaat
gctgaaactt 480ggactacacg acactcatat gttttattgt ttcacacaca
gcagaagaaa taattggatt 540ttttttcggt ccagtgattg cactggtttg
cagaaaaatt cttaaacgat aaataaacca 600tgttcatctc atgattactt
aatcgtttga gaaaccgtga catatggtgg taacaaacaa 660aaacaaacaa
ataccaaccg gtaaaacata tatacagtat tttatacata aacaattttg
720tgattttggc aatcaaataa caaggaccac aatgacgctc gcgaaattta
attaaaacgg 780ggaccaattt taatcaattt gacccggtgt acattaatct
tgacatgcat ttatttacgt 840gtgatgttga cctcttggta gatacagtac
tacatttata tatttttgat gcaacacata 900actgtttagt gtttttgata
tttccttttt ttatcagtct aactctcgtg agtcgtgagt 960cgtgagttgt
gagtcgtgat ccattgggat tattactacg tagtacatgt taaactacat
1020atataaggat tccataaata tgaatcaaat cgaattcttc ttatataact
aagacattaa 1080atgtctactt gcacttcatt aaaaagaatc ttctgatttt
atttttggta tatgcaaaat 1140tataactgca tttaaaacag agattagatc
atgaaacgaa tattatttct gaaaaagtag 1200gtatatctag taatgttaat
ttattttttg ctaaggatat ctagtaatat tttatatgaa 1260acacttgttt
ttatttatgt gttttgcgca taaagtcaat attataacga atcactagat
1320tagttttatt tttatcttat agattagttt aataagccta tatctataag
atgtactatg 1380atcagagaaa cattaggtgt aatgagaaaa caataataaa
acggtcatat ataagtaaat 1440ccaagtcagt gagagacctg caaaaatttc
gaactttttg tgaatgtctt atagcaaaga 1500tactattctc ccatctgatc
atgtttccat agatattagt taattggcta ataatacaat 1560tcttcacaca
catcacatga tcgatatgcg taatctcacg tcacaatttc cgtcataatc
1620aaaaggagta acagaattag tatattatat agtttctgtg atctagcaaa
gatttaaatg 1680taaacaaaac tatccagatt tatatggttt tgttacgttt
ctttgaagat caatgtaaac 1740aatgatgaac caatattagg gtccacgttg
aatataaacg tgaaaaacga caaagcgacg 1800tcagcataat ttacgaatag
gaaaacagtg ttaacttttg ttatcacatt ccgtgcaatt 1860tacataggat
atagaatttt ttatttacaa aagttatatt tataaagaat tactcaaatc
1920aagaattcca taaaaagata aacactttca tatatcgttc
1960451972DNAArabidopsis thalianapromoter(1)..(1972)transcription
regulating sequence from Arabidopsis thaliana gene At5g67280
45tataatatat ccaaaaacac ccgaccgatc aagtgacaat gcgcagtgtt gtattattat
60attatcactg tttgaaaaat tgtcgaactc aggattggtt tataactttg caagacgata
120ataactttgg agttttgcat aatggtaagt agaaaacgcc atttttcatg
catctcccgt 180ctttgtccac cgccaaacta gtagccatcc tctaattaat
aatgtattac tacagctttg 240tatatattcg tattggagtt tacagactaa
tcacatagtg gttacgttta ggaagataaa 300acatacagaa atggtaaata
gcttgtgaac ctgaatctcg aaacttacct tgtccaattt 360gtaacatgaa
tgtcgactat aagtaaattt gctctgacaa tttacagtca aactcaacta
420tgagtctata atatcactgt agtgggtatt gttcacacac aacgattaat
actgtaatta 480atgctgaaac ttggactaca cgacactcat atgttttatt
gtttcacaca cagcagaaga 540aataattgga ttttttttcg gtccagtgat
tgcactggtt tgcagaaaaa ttcttaaacg 600ataaataaac catgttcatc
tcatgattac ttaatcgttt gagaaaccgt gacatatggt 660ggtaacaaac
aaaaacaaac aaataccaac cggtaaaaca tatatacagt attttataca
720taaacaattt tgtgattttg gcaatcaaat aacaaggacc acaatgacgc
tcgcgaaatt 780taattaaaac ggggaccaat tttaatcaat ttgacccggt
gtacattaat cttgacatgc 840atttatttac gtgtgatgtt gacctcttgg
tagatacagt actacattta tatatttttg 900atgcaacaca taactgttta
gtgtttttga tatttccttt ttttatcagt ctaactctcg 960tgagtcgtga
gtcgtgagtt gtgagtcgtg atccattggg attattacta cgtagtacat
1020gttaaactac atatataagg attccataaa tatgaatcaa atcgaattct
tcttatataa 1080ctaagacatt aaatgtctac ttgcacttca ttaaaaagaa
tcttctgatt ttatttttgg 1140tatatgcaaa attataactg catttaaaac
agagattaga tcatgaaacg aatattattt 1200ctgaaaaagt aggtatatct
agtaatgtta atttattttt tgctaaggat atctagtaat 1260attttatatg
aaacacttgt ttttatttat gtgttttgcg cataaagtca atattataac
1320gaatcactag attagtttta tttttatctt atagattagt ttaataagcc
tatatctata 1380agatgtacta tgatcagaga aacattaggt gtaatgagaa
aacaataata aaacggtcat 1440atataagtaa atccaagtca gtgagagacc
tgcaaaaatt tcgaactttt tgtgaatgtc 1500ttatagcaaa gatactattc
tcccatctga tcatgtttcc atagatatta gttaattggc 1560taataataca
attcttcaca cacatcacat gatcgatatg cgtaatctca cgtcacaatt
1620tccgtcataa tcaaaaggag taacagaatt agtatattat atagtttctg
tgatctagca 1680aagatttaaa tgtaaacaaa actatccaga tttatatggt
tttgttacgt ttctttgaag 1740atcaatgtaa acaatgatga accaatatta
gggtccacgt tgaatataaa cgtgaaaaac 1800gacaaagcga cgtcagcata
atttacgaat aggaaaacag tgttaacttt tgttatcaca 1860ttccgtgcaa
tttacatagg atatagaatt ttttatttac aaaagttata tttataaaga
1920attactcaaa tcaagaattc cataaaaaga taaacacttt catatatcgt tc
1972462480DNAArabidopsis thalianaCDS(65)..(2320)encoding putative
leucine-rich repeat transmembrane protein kinase 46acaatcacat
ggccttttca aaaaatcaat cttttagttc tctatcgatg cgtaggcttg 60aagc atg
atg acg aca gtc gcc gcc gat ctc cac cgt tat ctt ttc ctg 109 Met Met
Thr Thr Val Ala Ala Asp Leu His Arg Tyr Leu Phe Leu 1 5 10 15att
acc gtt ttt ctt ttc ttc ctc tgc gac aaa acc tct ctt gct ctg 157Ile
Thr Val Phe Leu Phe Phe Leu Cys Asp Lys Thr Ser Leu Ala Leu 20 25
30acc aca gac ggt gtt ctt ctt ctc tct ttc cgt tac tca atc gtt gac
205Thr Thr Asp Gly Val Leu Leu Leu Ser Phe Arg Tyr Ser Ile Val Asp
35 40 45gat cct ctt tac gtt ttt cgg agc tgg aga ttc gac gac gag act
cct 253Asp Pro Leu Tyr Val Phe Arg Ser Trp Arg Phe Asp Asp Glu Thr
Pro 50 55 60tgc tct tgg cgt ggt gtc acg tgc gat gca tct tcc cgg cac
gtg act 301Cys Ser Trp Arg Gly Val Thr Cys Asp Ala Ser Ser Arg His
Val Thr 65 70 75gtt ctg tca ctt cca agc tcg aac ctt acc ggc aca cta
cct tca aat 349Val Leu Ser Leu Pro Ser Ser Asn Leu Thr Gly Thr Leu
Pro Ser Asn80 85 90 95ttg ggt tca ctc aat tca ctt caa aga ctt gat
ctt tcc aac aat tcc 397Leu Gly Ser Leu Asn Ser Leu Gln Arg Leu Asp
Leu Ser Asn Asn Ser 100 105 110atc aat ggg tct ttc ccg gtt tcg ctt
ctc aac gcg acg gag ctt cga 445Ile Asn Gly Ser Phe Pro Val Ser Leu
Leu Asn Ala Thr Glu Leu Arg 115 120 125ttt ctt gat ctg tcc gat aat
cac atc tcc ggt gca cta ccg gcg agt 493Phe Leu Asp Leu Ser Asp Asn
His Ile Ser Gly Ala Leu Pro Ala Ser 130 135 140ttt ggc gcg ctt tcg
aac ctc caa gtg ttg aat ctc tcc gat aat tcc 541Phe Gly Ala Leu Ser
Asn Leu Gln Val Leu Asn Leu Ser Asp Asn Ser 145 150 155ttc gtc ggc
gaa tta ccg aac aca tta gga tgg aac cgg aac tta acg 589Phe Val Gly
Glu Leu Pro Asn Thr Leu Gly Trp Asn Arg Asn Leu Thr160 165 170
175gag att tca ctt cag aaa aac tat tta tcc ggc ggg att ccg gga ggt
637Glu Ile Ser Leu Gln Lys Asn Tyr Leu Ser Gly Gly Ile Pro Gly Gly
180 185 190ttt aag tcg acg gag tat ctt gat ctc tcg tca aat ttg atc
aaa ggc 685Phe Lys Ser Thr Glu Tyr Leu Asp Leu Ser Ser Asn Leu Ile
Lys Gly 195 200 205tcg ttg ccg tca cat ttc aga ggg aat cgt cta cgc
tat ttc aac gct 733Ser Leu Pro Ser His Phe Arg Gly Asn Arg Leu Arg
Tyr Phe Asn Ala 210 215 220tcg tac aac aga atc tcc ggc gag att ccg
tca ggt ttc gcc gac gaa 781Ser Tyr Asn Arg Ile Ser Gly Glu Ile Pro
Ser Gly Phe Ala Asp Glu 225 230 235atc ccg gaa gac gcc acc gtt gat
ctc tca ttc aac caa ctt aca ggt 829Ile Pro Glu Asp Ala Thr Val Asp
Leu Ser Phe Asn Gln Leu Thr Gly240 245 250 255caa atc ccg ggt ttt
cgg gtt ctc gat aac caa gaa tcc aac tct ttc 877Gln Ile Pro Gly Phe
Arg Val Leu Asp Asn Gln Glu Ser Asn Ser Phe 260 265 270tcc ggt aac
ccg ggt ctc tgc gga tcc gac cat gca aaa cac cct tgt 925Ser Gly Asn
Pro Gly Leu Cys Gly Ser Asp His Ala Lys His Pro Cys 275 280 285cgt
gac ggt gaa gca acc tct cca cct cca tcg ccg act cca aat tct 973Arg
Asp Gly Glu Ala Thr Ser Pro Pro Pro Ser Pro Thr Pro Asn Ser 290 295
300cct cct gca tta gct gct ata cca aat act att ggc tta acc aat cac
1021Pro Pro Ala Leu Ala Ala Ile Pro Asn Thr Ile Gly Leu Thr Asn His
305 310 315cca att agc tcc aaa acc ggt ccg aaa tca aaa tgg gat cat
aaa ccg 1069Pro Ile Ser Ser Lys Thr Gly Pro Lys Ser Lys Trp Asp His
Lys Pro320 325 330 335gtg ctt atc att ggc att gtt gtc ggt gac tta
gcc ggt tta gca atc 1117Val Leu Ile Ile Gly Ile Val Val Gly Asp Leu
Ala Gly Leu Ala Ile 340 345 350ctc ggg att gtg ttt ttc tac att tac
cag tcg aga aaa cgg aag acc 1165Leu Gly Ile Val Phe Phe Tyr Ile Tyr
Gln Ser Arg Lys Arg Lys Thr 355 360 365gta acg gct acg tca aaa tgg
tcc acg tca tca aca gat tcc aag gtc 1213Val Thr Ala Thr Ser Lys Trp
Ser Thr Ser Ser Thr Asp Ser Lys Val 370 375 380tca aaa tgg tac tgt
tta cgc aaa tcc gtt tac gtt gac ggt gac tgc 1261Ser Lys Trp Tyr Cys
Leu Arg Lys Ser Val Tyr Val Asp Gly Asp Cys 385 390 395gaa gaa gaa
gaa gag gaa tct gag aca tcg gaa tcc gaa tcc gac gaa 1309Glu Glu Glu
Glu Glu Glu Ser Glu Thr Ser Glu Ser Glu Ser Asp Glu400 405 410
415gag aac ccg gtc gga cca aat cga cgg tca gga tta gac gat caa gaa
1357Glu Asn Pro Val Gly Pro Asn Arg Arg Ser Gly Leu Asp Asp Gln Glu
420 425 430aaa aag gga acg tta gtg aat ctc gat tca gag aaa gag ctt
gaa atc 1405Lys Lys Gly Thr Leu Val Asn Leu Asp Ser Glu Lys Glu Leu
Glu Ile 435 440 445gaa acg ctt ctc aaa gca tca gct tat att ttg gga
gcc acc ggt tcg 1453Glu Thr Leu Leu Lys Ala Ser Ala Tyr Ile Leu Gly
Ala Thr Gly Ser 450 455 460agc ata atg tat aaa gcg gtg ctt caa gac
gga aca gct gtg gcg gtt 1501Ser Ile Met Tyr Lys Ala Val Leu Gln Asp
Gly Thr Ala Val Ala Val 465 470 475cga cga ata gct gaa tgc ggt tta
gac cgg ttt aga gat ttc gaa gct 1549Arg Arg Ile Ala Glu Cys Gly Leu
Asp Arg Phe Arg Asp Phe Glu Ala480 485 490 495cag gtt cga gcc gtg
gct aag tta ata cat cca aac ctg gta cga att 1597Gln Val Arg Ala Val
Ala Lys Leu Ile His Pro Asn Leu Val Arg Ile 500 505 510cgc ggt ttc
tat tgg gga tcc gac gag aaa ctt gtc att tac gat ttt 1645Arg Gly Phe
Tyr Trp Gly Ser Asp Glu Lys Leu Val Ile Tyr Asp Phe 515 520 525gtc
cct aac ggc agc ctc gct aac gcc cgt tac cgg aaa gtg ggc tcc 1693Val
Pro Asn Gly Ser Leu Ala Asn Ala Arg Tyr Arg Lys Val Gly Ser 530 535
540tct cct tgt cat tta cct tgg gac gct cgg ctc aag ata gca aaa ggc
1741Ser Pro Cys His Leu Pro Trp Asp Ala Arg Leu Lys Ile Ala Lys Gly
545 550 555ata gct cgc ggg cta aca tac gta cac gac aag aag tac gtg
cat ggt 1789Ile Ala Arg Gly Leu Thr Tyr Val His Asp Lys Lys Tyr Val
His Gly560 565 570 575aac ctc aag cct agc aat atc ctt ttg ggc tta
gat atg gag cct aaa 1837Asn Leu Lys Pro Ser Asn Ile Leu Leu Gly Leu
Asp Met Glu Pro Lys 580 585 590gtt gcg gat ttc ggt ctt gag aag ctt
ttg att ggg gac atg agt tat 1885Val Ala Asp Phe Gly Leu Glu Lys Leu
Leu Ile Gly Asp Met Ser Tyr 595 600 605aga acc ggt gga tcg gct cca
ata ttc gga agc aag aga tcc aca acg 1933Arg Thr Gly Gly Ser Ala Pro
Ile Phe Gly Ser Lys Arg Ser Thr Thr 610 615 620tct ctt gag ttt ggg
ccg agt cca agc cca agt cca agt tca gtc ggg 1981Ser Leu Glu Phe Gly
Pro Ser Pro Ser Pro Ser Pro Ser Ser Val Gly 625 630 635tta ccc tac
aat gct cca gaa tct ctt cgg agt att aag ccg aat tcg 2029Leu Pro Tyr
Asn Ala Pro Glu Ser Leu Arg Ser Ile Lys Pro Asn Ser640 645 650
655aaa tgg gat gtg tac tcg ttc gga gtt att ctg ctt gag cta cta acg
2077Lys Trp Asp Val Tyr Ser Phe Gly Val Ile Leu Leu Glu Leu Leu Thr
660 665 670gga aag atc gtg gtg gtc gac gag ctt gga cag gtt aat ggg
ctt gtg 2125Gly Lys Ile Val Val Val Asp Glu Leu Gly Gln Val Asn Gly
Leu Val 675 680 685att gat gac ggt gag cgg gca att cgg atg gcg gac
tct gct ata cgg 2173Ile Asp Asp Gly Glu Arg Ala Ile Arg Met Ala Asp
Ser Ala Ile Arg 690 695 700gct gag tta gaa ggc aaa gaa gaa gct gtg
ttg gca tgt ttg aaa atg 2221Ala Glu Leu Glu Gly Lys Glu Glu Ala Val
Leu Ala Cys Leu Lys Met 705 710 715ggc cta gct tgt gcg tct cca ata
cca cag aga agg ccc aat atc aaa 2269Gly Leu Ala Cys Ala Ser Pro Ile
Pro Gln Arg Arg Pro Asn Ile Lys720 725 730 735gag gct ttg caa gtt
ctc gag aga ttc cct
gtt cat tct agt caa cag 2317Glu Ala Leu Gln Val Leu Glu Arg Phe Pro
Val His Ser Ser Gln Gln 740 745 750taa tgataataat taagaccaag
aaagagttaa ataacttgac tgtgtgtact 2370tagagttaga ttgataggca
agtttgatct tcttttgtgg cttctgattt tgaatttatt 2430tttggttatg
attttagtga caattatatc tctgggcttg ttacaaattt 248047751PRTArabidopsis
thaliana 47Met Met Thr Thr Val Ala Ala Asp Leu His Arg Tyr Leu Phe
Leu Ile1 5 10 15Thr Val Phe Leu Phe Phe Leu Cys Asp Lys Thr Ser Leu
Ala Leu Thr 20 25 30Thr Asp Gly Val Leu Leu Leu Ser Phe Arg Tyr Ser
Ile Val Asp Asp 35 40 45Pro Leu Tyr Val Phe Arg Ser Trp Arg Phe Asp
Asp Glu Thr Pro Cys 50 55 60Ser Trp Arg Gly Val Thr Cys Asp Ala Ser
Ser Arg His Val Thr Val65 70 75 80Leu Ser Leu Pro Ser Ser Asn Leu
Thr Gly Thr Leu Pro Ser Asn Leu 85 90 95Gly Ser Leu Asn Ser Leu Gln
Arg Leu Asp Leu Ser Asn Asn Ser Ile 100 105 110Asn Gly Ser Phe Pro
Val Ser Leu Leu Asn Ala Thr Glu Leu Arg Phe 115 120 125Leu Asp Leu
Ser Asp Asn His Ile Ser Gly Ala Leu Pro Ala Ser Phe 130 135 140Gly
Ala Leu Ser Asn Leu Gln Val Leu Asn Leu Ser Asp Asn Ser Phe145 150
155 160Val Gly Glu Leu Pro Asn Thr Leu Gly Trp Asn Arg Asn Leu Thr
Glu 165 170 175Ile Ser Leu Gln Lys Asn Tyr Leu Ser Gly Gly Ile Pro
Gly Gly Phe 180 185 190Lys Ser Thr Glu Tyr Leu Asp Leu Ser Ser Asn
Leu Ile Lys Gly Ser 195 200 205Leu Pro Ser His Phe Arg Gly Asn Arg
Leu Arg Tyr Phe Asn Ala Ser 210 215 220Tyr Asn Arg Ile Ser Gly Glu
Ile Pro Ser Gly Phe Ala Asp Glu Ile225 230 235 240Pro Glu Asp Ala
Thr Val Asp Leu Ser Phe Asn Gln Leu Thr Gly Gln 245 250 255Ile Pro
Gly Phe Arg Val Leu Asp Asn Gln Glu Ser Asn Ser Phe Ser 260 265
270Gly Asn Pro Gly Leu Cys Gly Ser Asp His Ala Lys His Pro Cys Arg
275 280 285Asp Gly Glu Ala Thr Ser Pro Pro Pro Ser Pro Thr Pro Asn
Ser Pro 290 295 300Pro Ala Leu Ala Ala Ile Pro Asn Thr Ile Gly Leu
Thr Asn His Pro305 310 315 320Ile Ser Ser Lys Thr Gly Pro Lys Ser
Lys Trp Asp His Lys Pro Val 325 330 335Leu Ile Ile Gly Ile Val Val
Gly Asp Leu Ala Gly Leu Ala Ile Leu 340 345 350Gly Ile Val Phe Phe
Tyr Ile Tyr Gln Ser Arg Lys Arg Lys Thr Val 355 360 365Thr Ala Thr
Ser Lys Trp Ser Thr Ser Ser Thr Asp Ser Lys Val Ser 370 375 380Lys
Trp Tyr Cys Leu Arg Lys Ser Val Tyr Val Asp Gly Asp Cys Glu385 390
395 400Glu Glu Glu Glu Glu Ser Glu Thr Ser Glu Ser Glu Ser Asp Glu
Glu 405 410 415Asn Pro Val Gly Pro Asn Arg Arg Ser Gly Leu Asp Asp
Gln Glu Lys 420 425 430Lys Gly Thr Leu Val Asn Leu Asp Ser Glu Lys
Glu Leu Glu Ile Glu 435 440 445Thr Leu Leu Lys Ala Ser Ala Tyr Ile
Leu Gly Ala Thr Gly Ser Ser 450 455 460Ile Met Tyr Lys Ala Val Leu
Gln Asp Gly Thr Ala Val Ala Val Arg465 470 475 480Arg Ile Ala Glu
Cys Gly Leu Asp Arg Phe Arg Asp Phe Glu Ala Gln 485 490 495Val Arg
Ala Val Ala Lys Leu Ile His Pro Asn Leu Val Arg Ile Arg 500 505
510Gly Phe Tyr Trp Gly Ser Asp Glu Lys Leu Val Ile Tyr Asp Phe Val
515 520 525Pro Asn Gly Ser Leu Ala Asn Ala Arg Tyr Arg Lys Val Gly
Ser Ser 530 535 540Pro Cys His Leu Pro Trp Asp Ala Arg Leu Lys Ile
Ala Lys Gly Ile545 550 555 560Ala Arg Gly Leu Thr Tyr Val His Asp
Lys Lys Tyr Val His Gly Asn 565 570 575Leu Lys Pro Ser Asn Ile Leu
Leu Gly Leu Asp Met Glu Pro Lys Val 580 585 590Ala Asp Phe Gly Leu
Glu Lys Leu Leu Ile Gly Asp Met Ser Tyr Arg 595 600 605Thr Gly Gly
Ser Ala Pro Ile Phe Gly Ser Lys Arg Ser Thr Thr Ser 610 615 620Leu
Glu Phe Gly Pro Ser Pro Ser Pro Ser Pro Ser Ser Val Gly Leu625 630
635 640Pro Tyr Asn Ala Pro Glu Ser Leu Arg Ser Ile Lys Pro Asn Ser
Lys 645 650 655Trp Asp Val Tyr Ser Phe Gly Val Ile Leu Leu Glu Leu
Leu Thr Gly 660 665 670Lys Ile Val Val Val Asp Glu Leu Gly Gln Val
Asn Gly Leu Val Ile 675 680 685Asp Asp Gly Glu Arg Ala Ile Arg Met
Ala Asp Ser Ala Ile Arg Ala 690 695 700Glu Leu Glu Gly Lys Glu Glu
Ala Val Leu Ala Cys Leu Lys Met Gly705 710 715 720Leu Ala Cys Ala
Ser Pro Ile Pro Gln Arg Arg Pro Asn Ile Lys Glu 725 730 735Ala Leu
Gln Val Leu Glu Arg Phe Pro Val His Ser Ser Gln Gln 740 745
7504827DNAArtificial Sequenceoligonucleotide primer 48gaccatacta
gtgtccttga aaatcag 274925DNAArtificial Sequenceoligonuceotide
primer 49ccctcgccat ggttagctaa ttagg 255025DNAArtificial
Sequenceoligonuceotide primer 50gacgggacta gtcacacatc gaagc
255125DNAArtificial Sequenceoligonuceotide primer 51ccctcgccat
ggttagctaa ttagg 255230DNAArtificial Sequenceoligonuceotide primer
52tccaccggat cctcaattat taaaaaaata 305325DNAArtificial
Sequenceoligonuceotide primer 53ccctcgccat ggttagctaa ttagg
255424DNAArtificial Sequenceoligonuceotide primer 54ttctcagaat
tctctccttt gccc 245524DNAArtificial Sequenceoligonuceotide primer
55cacctcccat ggtttctcaa tcag 245630DNAArtificial
Sequenceoligonuceotide primer 56aaaaaaccat ggccttatag gtatttatac
305726DNAArtificial Sequenceoligonuceotide primer 57ccctgaggat
ccgctgagca aaagtc 265824DNAArtificial Sequenceoligonuceotide primer
58cacctcccat ggtttctcaa tcag 245930DNAArtificial
Sequenceoligonuceotide primer 59aaaaaaccat ggccttatag gtatttatac
306024DNAArtificial Sequenceoligonuceotide primer 60aaatgagaat
tcccaaaaac aagc 246125DNAArtificial Sequenceoligonuceotide primer
61aagaatccat ggggatggaa aaatg 256228DNAArtificial
Sequenceoligonuceotide primer 62ggaagaccat ggaagagggg agaagaag
286325DNAArtificial Sequenceoligonuceotide primer 63aattttggat
cccttttttg gcggg 256425DNAArtificial Sequenceoligonuceotide primer
64aagaatccat ggggatggaa aaatg 256528DNAArtificial
Sequenceoligonuceotide primer 65ggaagaccat ggaagagggg agaagaag
286623DNAArtificial Sequenceoligonuceotide primer 66ataatcacta
gtatatgttt ttg 236724DNAArtificial Sequenceoligonuceotide primer
67gtgtagccat ggaatttgga aatg 246829DNAArtificial
Sequenceoligonuceotide primer 68ggaaccatgg ctttcaagtg aggttcatt
296925DNAArtificial Sequenceoligonuceotide primer 69taatacacta
gttttgtagg ttaac 257024DNAArtificial Sequenceoligonuceotide primer
70gtgtagccat ggaatttgga aatg 247129DNAArtificial
Sequenceoligonuceotide primer 71ggaaccatgg ctttcaagtg aggttcatt
297226DNAArtificial Sequenceoligonuceotide primer 72ccattgggat
ccttactacg tagtac 267325DNAArtificial Sequenceoligonuceotide primer
73tcgtcaccat ggttcaagcc tacgc 257432DNAArtificial
Sequenceoligonuceotide primer 74gaacgaccat ggaaagtgtt tatcttttta tg
327527DNAArtificial Sequenceoligonuceotide primer 75tataatggat
ccaaaaacac ccgaccg 277625DNAArtificial Sequenceoligonuceotide
primer 76tcgtcaccat ggttcaagcc tacgc 257732DNAArtificial
Sequenceoligonuceotide primer 77gaacgaccat ggaaagtgtt tatcttttta tg
32788986DNAArtificial Sequencebinary vector pSUN0301 78cgttgtaaaa
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 8986
* * * * *
References