U.S. patent application number 14/505813 was filed with the patent office on 2015-01-29 for expression cassettes for regulation of expression in monocotyledonous plants.
The applicant listed for this patent is BASF Plant Science GmbH. Invention is credited to Christian Dammann, Alleson Dobson, Marc Morra, Christina E. Roche, Hee-Sook Song, Effie Toren.
Application Number | 20150033409 14/505813 |
Document ID | / |
Family ID | 36659859 |
Filed Date | 2015-01-29 |
United States Patent
Application |
20150033409 |
Kind Code |
A1 |
Song; Hee-Sook ; et
al. |
January 29, 2015 |
EXPRESSION CASSETTES FOR REGULATION OF EXPRESSION IN
MONOCOTYLEDONOUS PLANTS
Abstract
The present invention relates to expression cassettes comprising
at least one transcription regulating nucleotide sequence
obtainable from the group of genes of monocotyledonous plants
consisting of caffeoyl-CoA-O-methyltransferase genes, C8,7-sterol
isomerase genes, hydroxyproline-rich glycoprotein (HRGP) genes,
lactate dehydrogenase genes, and chloroplast protein 12 like genes.
More preferably the transcription regulating sequences are
obtainable from Zea mays or Oryza sativa. The transcription
regulating sequences are especially useful for
root/kernel-preferential, leaf/endosperm-preferential,
root/silk/kernel-preferential, or constitutive expression.
Inventors: |
Song; Hee-Sook; (Raleigh,
NC) ; Morra; Marc; (Bronx, NY) ; Dammann;
Christian; (Durham, NC) ; Roche; Christina E.;
(Research Triangle, NC) ; Toren; Effie; (Apex,
NC) ; Dobson; Alleson; (Cary, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BASF Plant Science GmbH |
Ludwigshafen |
|
DE |
|
|
Family ID: |
36659859 |
Appl. No.: |
14/505813 |
Filed: |
October 3, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11883996 |
Aug 8, 2007 |
8884098 |
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PCT/EP06/50781 |
Feb 8, 2006 |
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14505813 |
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60651268 |
Feb 9, 2005 |
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Current U.S.
Class: |
800/278 ;
435/252.2; 435/252.33; 435/320.1; 435/6.12; 800/298 |
Current CPC
Class: |
C12N 15/8218 20130101;
C12Q 2600/158 20130101; C12N 15/8225 20130101; C12N 15/8237
20130101; C12N 15/8234 20130101; C12N 15/8261 20130101; C12Q
2600/13 20130101; C12N 15/8227 20130101; C12N 15/8205 20130101;
C12Q 1/6895 20130101; C12N 15/8216 20130101 |
Class at
Publication: |
800/278 ;
435/320.1; 800/298; 435/6.12; 435/252.33; 435/252.2 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. An expression cassette for regulating expression in a
monocotyledonous plant, said expression cassette comprising: a) at
least one transcription regulating nucleotide sequence functional
in a monocotyledonous plant comprising a sequence selected from the
group consisting of: i) the nucleotide sequence of SEQ ID NO: 2, 3,
6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28,
29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73; ii) a
fragment of at least 50 consecutive bases of the nucleotide
sequence of SEQ ID NO: 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19,
20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68,
71, 72, or 73; iii) a nucleotide sequence having at least 90%
sequence identity to the nucleotide sequence of SEQ ID NO: 2, 3, 6,
7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29,
56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73; and iv) a
nucleotide sequence capable of hybridizing to the nucleotide
sequence of i) or the complement thereof 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.; and b) 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 expression of the
nucleic acid sequence of b) results in expression of a protein, an
antisense RNA, a sense RNA, or a double-stranded RNA.
3. The expression cassette of claim 1, wherein expression of the
nucleic acid sequence of b) in a plant confers an agronomically
valuable trait to said plant.
4. The expression cassette of claim 1, further comprising at least
one element selected from the group consisting of: a) a
5'-untranslated region of a plant expressed gene; b) an intron
sequence from a plant expressed gene; and c) a transcription
termination sequence from a plant expressed gene.
5. The expression cassette of claim 4, wherein the transcription
termination sequence of c) comprises the nucleotide sequence of SEQ
ID NO: 32, 34, or 35.
6. The expression cassette of claim 4, wherein the intron sequence
of b) has expression enhancing properties.
7. The expression cassette of claim 4, wherein the intron sequence
of b) is an intron from an ubiquitin, actin, or alcohol
dehydrogenase gene.
8. A vector comprising the expression cassette of claim 1.
9. A transgenic host cell or non-human organism comprising: a) the
expression cassette of claim 1; or b) a vector comprising the
expression cassette of a).
10. A cell culture, part, organ, tissue or transgenic propagation
material derived from the non-human organism of claim 9, wherein
said cell culture, part, organ, tissue or transgenic propagation
material comprises the expression cassette.
11. A method for the transgenic expression of a nucleic acid, said
method comprising growing or culturing the non-human organism of
claim 9 or cell cultures, parts, organs, tissues or transgenic
propagation material derived therefrom, wherein said cell cultures,
parts, organs, tissues or transgenic propagation material comprise
the expression cassette.
12. A transgenic plant comprising: a) the expression cassette of
claim 1; or b) a vector comprising the expression cassette of
a).
13. A method for providing a transgenic expression cassette for
heterologous expression in a monocotyledonous plant, said method
comprising: a) isolating a transcription regulating nucleotide
sequence from a monocotyledonous plant, wherein the transcription
regulating nucleotide sequence shares at least 90% sequence
identity to the nucleotide sequence of SEQ ID NO: 2, 3, 6, 7, 8,
11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57,
58, 61, 62, 63, 66, 67, 68, 71, 72, or 73; and b) functionally
linking said transcription regulating nucleotide sequence to
another nucleotide sequence of interest, which is heterologous in
relation to said transcription regulating nucleotide sequence.
14. The method of claim 13, further comprising functionally linking
said another nucleotide sequence of interest to a transcription
termination sequence.
15. The method of claim 14, wherein said transcription termination
sequence comprises the nucleotide sequence of SEQ ID NO: 32, 34, or
35.
16. A method for identifying and/or isolating a sequence with
transcription regulating activity, said method comprising: a)
obtaining polynucleotide sequences sharing at least 90% sequence
identity with the nucleotide sequence of SEQ ID NO: 2, 3, 6, 7, 8,
11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57,
58, 61, 62, 63, 66, 67, 68, 71, 72, or 73; b) preparing expression
cassettes comprising any of the polynucleotide sequences of a)
operably linked to a reporter gene or marker and optionally a
terminator sequence; c) testing the expression cassettes for
expression activity of the polynucleotide sequence comprised
therein; and d) identifying and/or isolating a polynucleotide
sequence having transcription regulating activity.
17. A method for directing expression in a monocotyledonous plant,
said method comprising: a) introducing into a plant cell the
expression cassette of claim 1; b) selecting a transgenic cell
which comprises said expression cassette, and c) regenerating a
plant from the transgenic cell, wherein the transcription
regulating nucleotide sequence directs expression of the nucleic
acid sequence which is heterologous in relation to said
transcription regulating nucleotide sequence in the plant.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 11/883,996 filed Aug. 8, 2007, which is a
national stage application (under 35 U.S.C. 371) of
PCT/EP2006/050781 filed Feb. 8, 2006, which claims benefit of U.S.
application 60/651,268 filed Feb. 9, 2005. The entire contents of
each of these applications are hereby incorporated by reference
herein in their entirety.
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_Listing.sub.--074021.sub.--0064.sub.--01. The size of the
text file is 175 KB, and the text file was created on Oct. 3,
2014.
FIELD OF THE INVENTION
[0003] The present invention relates to expression cassettes
comprising at least one transcription regulating nucleotide
sequence obtainable from the group of genes of monocotyledonous
plants consisting of caffeoyl-CoA-O-methyltransferase genes,
C8,7-sterol isomerase genes, hydroxyproline-rich glycoprotein
(HRGP) genes, lactate dehydrogenase genes, and chloroplast protein
12 like genes. More preferably the transcription regulating
sequences are obtainable from Zea mays or Oryza sativa. The
transcription regulating sequences are especially useful for
root/kernel-preferential, leaf/endosperm-preferential,
root/silk/kernel-preferential, or constitutive expression.
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] Constitutive promoters are favored in situations where
expression in all (or most) tissues during all (or most) times of
the plant development is required. The number of constitutive
promoters functional in monocotyledonous plants is limited and
include the rice actin 1 (Wang 1992; U.S. Pat. No. 5,641,876), CaMV
35S (Odell 1985), CaMV 19S (Lawton 1987), and the maize ubiquitin
promoters (Christensen 1996). While several constitutive and
tissue-specific promoters from dicotyledonous plants are described
by sequence (e.g., the promoter from the
caffeoyl-CoA-O-methyltransferase gene from parsley (Grimmig 1997),
poplar (Chen 1998) and pine (Li 1999)) only a very limited number
has been characterized in heterogenous gene expression. In
comparison with dicotyledonous promoters, promoters from
monocotyledonous plants are still very limited. It is advantageous
to have the choice of a variety of different promoters so that the
most suitable promoter may be selected for a particular gene,
construct, cell, tissue, plant, or environment. Moreover, the
increasing interest in cotransforming plants with multiple plant
transcription units (PTU) and the potential problems associated
with using common regulatory sequences for these purposes merit
having a variety of promoter sequences available.
[0006] Root-preferential or root-specific promoters are useful for
alteration of the function of root tissue, modification of growth
rate, improvement of resistance to root preferred pathogens, pests,
herbicides or adverse weather conditions, for detoxification of
soil as well as for broadening the range of soils or environments
in which said plant may grow. Root abundant or root specific gene
expression would provide a mechanism according to which morphology
and metabolism may be altered to improve the yield and to produce
useful proteins in greater amounts. In particular, root specific
promoters may be useful for expressing defense-related genes,
including those conferring insectical resistance and stress
tolerance, e.g. salt, cold or drought tolerance, and genes for
altering nutrient uptake. The number of root preferential and
root-specific promoters functional in monocotyledonous plants is
very limited. These include the MR7 promoter from Zea mays (U.S.
Pat. No. 5,837,848), the ZRP2 promoter of Zea mays (U.S. Pat. No.
5,633,363), and the MTL promoter from Zea mays (U.S. Pat. Nos.
5,466,785 and 6,018,099). Many of these examples disclose promoters
with expression patterns confined to a limited number of root
tissues. Other fail to provide the root specificity needed for
expression of selected genes. It is advantageous to have the choice
of a variety of different promoters so that the most suitable
promoter may be selected for a particular gene, construct, cell,
tissue, plant, or environment. Moreover, the increasing interest in
cotransforming plants with multiple plant transcription units (PTU)
and the potential problems associated with using common regulatory
sequences for these purposes merit having a variety of promoter
sequences available.
[0007] 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 the economically most important
monocotyledonous plants, especially in rice and maize. It is thus
an objective of the present invention to provide new and
alternative expression cassettes for expression of transgenes in
monocotyledonous plants, more preferably with the opportunity to
modulate the tissue specificity of expression
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 Map of Os.CCoAMT1 promoter::Zm.ubiquitin intron::GUS
(PIV2)::CCoAMT1 terminator chimeric construct (pBPSMM325). [0009]
The plasmid comprises an expression construct containing an
Os.CCoAMT1 promoter operably linked to Zm.ubiquitin intron, a
.beta.-glucuronidase gene (GUS including the potato invertase
[PIV]2 intron), and 3' untranslated region and transcriptional
termination region of the Os.CCoAMT1. SM cassette is representing a
selection marker (ahas) cassette.
[0010] FIG. 2 GUS expression controlled by Oryza sativa (Os)
CCoAMT1 promoter construct (pBPSMM325) in maize. The upper panel
(I) represents the original photos with the GUS staining, while the
lower panel (II) indicates areas distinctly stained blue by
overlaid shaded areas. [0011] (A) Leaf+root at the 5 leaf stage
[0012] (B) Leaf at flowering stage [0013] (C) Kernel
(prepollination) [0014] (D) Kernel 30 DAP [0015] Pictures represent
reproducible expression patterns from 15 T.sub.1 single copy
lines.
[0016] FIG. 3 A: Map of Os.CCoAMT1 promoter::Zm.ubiquitin
intron::GUS (PIV2)::NOS terminator fusion construct (pBPSMM271).
[0017] The plasmid comprises an expression construct containing an
Os.CCoAMT1 promoter operably linked to Zm.ubiquitin intron, a
.beta.-glucuronidase gene (GUS including the potato invertase
[PIV]2 intron), and nopaline synthase (NOS) terminator. The SM
cassette is representing a selection marker (ahas) cassette. [0018]
B: GUS expression controlled by Os.CCoAMT1 promoter construct
(pBPSMM271) in maize. The upper panel (I) represents the original
photos with the GUS staining, while the lower panel (II) indicates
areas distinctly stained blue by overlaid shaded areas. [0019] (A)
Leaves and roots at the 5 leaf stage [0020] (B) Leaf at the
flowering stage [0021] (C) Kernel (30 d after pollination: DAP)
[0022] Pictures represent reproducible expression patterns from 15
T.sub.1 single copy lines.
[0023] FIG. 4 A: Map of Os.SI::Zm.ubiquitin intron::GUS (PIV2)::NOS
terminator fusion construct (pBPSMM331). [0024] The plasmid
comprises an expression construct containing an Os.SI promoter
operably linked to Zm.ubiquitin intron, a .beta.-glucuronidase gene
(GUS including the potato invertase [PIV]2 intron), and NOS
terminator. The SM cassette is representing a selection marker
cassette. [0025] B: GUS expression controlled by Os.SI promoter
construct (pBPSMM331) in maize. The upper panel (I) represents the
original photos with the GUS staining, while the lower panel (II)
indicates areas distinctly stained blue by overlaid shaded areas.
[0026] (A) Leaves and roots at the 5 leaf stage [0027] (B) Leaf at
the 5 leaf stage Leaf at the flowering stage [0028] (C) Kernel (30
d after pollination: DAP) [0029] Pictures represent reproducible
expression patterns from 15 T.sub.1 single copy lines.
[0030] FIG. 5 A: Map of Zm.HRGP::Zm.ubiquitin intron::GUS
(PIV2)::Zm.HRGP terminator fusion construct (pBPSET003). [0031] The
plasmid comprises an expression construct containing a Zm.HRGP
promoter operably linked to Zm.ubiquitin intron, a
.beta.-glucuronidase gene (GUS including the potato invertase
[PIV]2 intron), and HRGP terminator. The SM cassette is
representing a selection marker (ahas) cassette. [0032] B: GUS
expression controlled by maize Zm.HRGP promoter construct
(pBPSET003) in maize. The upper panel (I) represents the original
photos with the GUS staining, while the lower panel (II) indicates
areas distinctly stained blue by overlaid shaded areas. [0033] (A)
Leaves and roots at the 5 leaf stage [0034] (B) Leaf at the 5 leaf
stage Leaf at the flowering stage [0035] (C) Kernel (30 d after
pollination: DAP) [0036] Pictures represent reproducible expression
patterns from 15 T.sub.1 single copy lines.
[0037] FIG. 6 A: Map of Zm.LDH::Zm.ubiquitin intron::GUS
(PIV2)::NOS terminator fusion construct (pBPSMM272). [0038] The
plasmid comprises an expression construct containing a Zm.LDH
promoter operably linked to Zm.ubiquitin intron, a
.beta.-glucuronidase gene (GUS including the potato invertase
[PIV]2 intron), and NOS terminator. The SM cassette is representing
a selection marker (ahas) cassette. [0039] B: GUS expression
controlled by maize Zm.LDH promoter construct (pBPSMM272) in maize.
The upper panel (I) represents the original photos with the GUS
staining, while the lower panel (II) indicates areas distinctly
stained blue by overlaid shaded areas. The transgenic plants
containing either pBPSMM272 or pBPSET007 showed the same expression
patterns. [0040] (A) Leaves and roots at the 5 leaf stage [0041]
(B) Leaf at the 5 leaf stage Leaf at the flowering stage [0042] (C)
Kernel (30 d after pollination: DAP) [0043] Pictures represent
reproducible expression patterns from 8 T.sub.1 single copy
lines.
[0044] FIG. 7 A: Map of Zm.LDH::Zm.ubiquitin intron::GUS
(PIV2)::Zm.LDH terminator fusion construct (pBPSET007). [0045] The
plasmid comprises an expression construct containing a Zm.LDH
promoter operably linked to Zm.ubiquitin intron, a
.beta.-glucuronidase gene (GUS including the potato invertase
[PIV]2 intron), and LDH terminator. The SM cassette is representing
a selection marker (ahas) cassette. [0046] B: GUS expression
controlled by maize Zm.LDH promoter construct (pBPSET007) in maize.
The upper panel (I) represents the original photos with the GUS
staining, while the lower panel (II) indicates areas distinctly
stained blue by overlaid shaded areas. [0047] (A) Leaves and roots
at the 5 leaf stage [0048] (B) Leaf at the flowering stage [0049]
(C) Kernel (30 d after pollination: DAP) [0050] Pictures represent
reproducible expression patterns from 15 T.sub.1 single copy
lines.
[0051] FIG. 8 A: Map of Os.CP12::Zm.ubiquitin intron::GUS
(PIV2)::NOS terminator fusion construct (pBPSMM304). [0052] The
plasmid comprises an expression construct containing a Os.CP12
promoter operably linked to Zm.ubiquitin intron, a
.beta.-glucuronidase gene (GUS including the potato invertase
[PIV]2 intron), and NOS terminator. SM cassette is representing a
selection marker cassette. [0053] B: GUS expression controlled by
maize Os.CP12 promoter construct (pBPSMM304) in maize. The upper
panel (I) represents the original photos with the GUS staining,
while the lower panel (II) indicates areas distinctly stained blue
by overlaid shaded areas. [0054] (A) Leaves and roots at the 5 leaf
stage [0055] (B) Leaf at the flowering stage [0056] (C) Kernel (30
d after pollination: DAP) [0057] Pictures represent reproducible
expression patterns from 15 T.sub.1 single copy lines.
[0058] FIG. 9 Drought-stress-induced expression of Zm.LDH promoter
construct (pBPSMM272) in maize. Transgenic plants at 5-leaf stage
were drought-stressed by withholding water. Samples were taken from
leaves at the indicated time points. RNA was isolated from leaf
samples and analyzed with quantitative RT-PCR. GUS expression was
normalized against an internal control gene in each sample. Results
are shown as fold increase of expression levels compared to the
0-timepoint, which is set as 1.
[0059] FIG. 10A-B Protein alignment of rice lactate dehydrogenase
(LDH) protein with the LDH proteins from maize (1) (SEQ ID NO: 26),
rice (2) (SEQ ID NO: 65), barley (3) (SEQ ID NO: 95), rice (4) (SEQ
ID NO: 60), Arabidopsis (5, 6) (SEQ ID NO: 96 and SEQ ID NO: 97),
tomato (7) (SEQ ID NO: 98), potato (8) (SEQ ID NO: 99). [0060] The
sequences motifs distinguishing monocotyledonous LDH proteins from
other dicotyledonous LDH proteins are boxed (relevant different
amino acids are marked with a "+"). Further such sequences motifs
may be readily identified by the person skilled in the art based on
the present alignment.
[0061] FIG. 11. Protein alignment of rice C8,7 sterol isomerase
(SI) protein with the SI proteins from Arabidopsis (1-3) (SEQ ID
NO: 100, SEQ ID NO: 101, SEQ ID NO: 102) and rice (4) (SEQ ID NO:
10).
[0062] FIG. 12. Protein alignment of rice Caffeoyl
CoA-O-methyltransferase 1 (CCoAMT1) with the CCoAMT1 proteins from
tobacco (1) (SEQ ID NO: 103), eucalyptus (2) (SEQ ID NO: 104),
popular (3) (SEQ ID NO: 105), maize (4, 5, 6) (SEQ ID NO: 106, SEQ
ID NO: 107, SEQ ID NO: 70) and rice (7) (SEQ ID NO: 5). The
sequences motifs distinguishing monocotyledonous CCoAMT proteins
from other dicotyledonous CCoAMT proteins are boxed (relevant
different amino acids are marked with a "+"). Further such
sequences motifs may be readily identified by the person skilled in
the art based on the present alignment.
SUMMARY OF THE INVENTION
[0063] Accordingly, a first embodiment of the invention relates to
expression cassettes for regulating expression in monocotyledonous
plants comprising [0064] i) at least one transcription regulating
nucleotide sequence of a monocotyledonous plant gene, said
monocotyledonous plant gene selected from the group of genes
consisting of caffeoyl-CoA-O-methyltransferase genes, C8,7-sterol
isomerase genes, hydroxyproline-rich glycoprotein (HRGP) genes,
lactate dehydrogenase genes, and chloroplast protein like 12 genes,
and functionally linked thereto [0065] ii) at least one nucleic
acid sequence which is heterologous in relation to said
transcription regulating sequence.
[0066] Preferably, the transcription regulating nucleotide sequence
is obtainable from monocotyledonous plant genomic DNA from a gene
encoding a polypeptide which [0067] a1) comprises at least one
sequence motif of a monocotyledonous plant lactate dehydrogenase
protein selected from the group consisting of the amino acid
sequences
TABLE-US-00001 [0067] i) SLSELGFDA, (SEQ ID NO: 76) ii) VIGAGNVGMA,
(SEQ ID NO: 77) iii) IVTAGARQI, (SEQ ID NO: 78) iv) L(F/Y)RKIVP,
(SEQ ID NO: 79) v) GFPASRV, (SEQ ID NO: 80) vi) RF(L/I)AEHL, (SEQ
ID NO: 81) vii) QAYMVGEH, (SEQ ID NO: 82) viii) ALEGIRRAV, (SEQ ID
NO: 83) and ix) GYSVAS(L/I)A, (SEQ ID NO: 84)
[0068] or [0069] b1) is encoding a lactate dehydrogenase protein
from a monocotyledonous plant having an amino acid sequence
identity of at least 90% to a polypeptide selected from the group
described by SEQ ID NO: 26, 60 and 65, or [0070] a2) comprises at
least one sequence motif of a monocotyledonous plant
caffeoyl-CaA-O-methyltransferase protein selected from the group
consisting of the amino acid sequences
TABLE-US-00002 [0070] x) EQKTRHSE, (SEQ ID NO: 85) xi)
L(I/L)KLIGAK, (SEQ ID NO: 86) xii) KTMEIGVY, (SEQ ID NO: 87) xiii)
HERL(L/M)KLV, (SEQ ID NO: 88) xiv) CQLPVGDG, (SEQ ID NO: 89) and
xv) TLCRRVK, (SEQ ID NO: 90)
[0071] or [0072] b2) is encoding a caffeoyl-CaA-O-methyltransferase
protein from a monocotyledonous plant having an amino acid sequence
identity of at least 90% to a polypeptide selected from the group
described by SEQ ID NO: 5, and 70, or [0073] b3) is encoding a
hydroxyproline-rich glycoprotein from a monocotyledonous plant
having an amino acid sequence identity of at least 90% to a
polypeptide selected from the group described by SEQ ID NO: 18, and
75, or [0074] b4) is encoding a C-8,7-stereol-isomerase protein
from a monocotyledonous plant having an amino acid sequence
identity of at least 90% to a polypeptide selected described by SEQ
ID NO: 10, or [0075] b5) is encoding a Chloroplast protein 12 like
protein from a monocotyledonous plant having an amino acid sequence
identity of at least 90% to a polypeptide described by SEQ ID NO:
31.
[0076] Preferably, the transcription regulating nucleotide sequence
is from a corn (Zea mays) or rice (Oryza sativa) plant. Even more
preferably the transcription regulating nucleotide sequence is from
a plant gene selected from the group of genes consisting of Oryza
sativa caffeoyl-CoA-O-methyltransferase genes, Oryza sativa
C8,7-sterol isomerase genes, Zea may hydroxyproline-rich
glycoprotein (HRGP) genes, Zea mays lactate dehydrogenase genes,
Oryza sativa chloroplast protein 12 like genes and functional
equivalents thereof. The functional equivalent gene is preferably
encoding a polypeptide which has at least 90% amino acid sequence
identity to a polypeptide selected from the group described by SEQ
ID NO: 5, 10, 18, 26, 31, 60, 65, 70, and 75.
[0077] In a more preferred embodiment the transcription regulating
nucleotide sequence is selected from the group of sequences
consisting of [0078] i) the sequences described by SEQ ID NOs: 1,
2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27,
28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, and 73, and
[0079] ii) a fragment of at least 50 consecutive bases of a
sequence under i); and [0080] iii) a nucleotide sequence having
substantial similarity (preferably with a sequence identity of at
least 60%; more preferably measured by the BLASTN program with the
default parameters wordlength (W) of 11, an expectation (E) of 10,
a cutoff of 100, M=5, N=-4, and a comparison of both strands) to a
transcription regulating nucleotide sequence described by SEQ ID
NO: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23,
24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73;
and [0081] iv) a nucleotide sequence capable of hybridizing to a
transcription regulating nucleotide sequence described by SEQ ID
NO: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23,
24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73
or the complement thereof; and [0082] v) a nucleotide sequence
capable of hybridizing 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, 6, 7, 8, 11,
12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58,
61, 62, 63, 66, 67, 68, 71, 72, or 73 or the complement thereof
(preferably 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 preferably 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., still more preferably in 7%
sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at
50.degree. C. with washing in 0.5.times.SSC, 0.1% SDS at 50.degree.
C., even 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 50.degree. C., most 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.); and [0083] vi) a nucleotide sequence which is the complement
or reverse complement of any of the previously mentioned nucleotide
sequences under i) to v).
[0084] Another preferred embodiment relates to an expression
cassette for regulating expression in monocotyledonous plants
comprising [0085] a) at least one transcription regulating
nucleotide sequence functional in a monocotyledonous plant
comprising at least one sequence selected from the group of
sequences consisting of [0086] i) the sequences described by SEQ ID
NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23,
24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, and 73,
and [0087] ii) a fragment of at least 50 consecutive bases of a
sequence under i); and [0088] iii) a nucleotide sequence having
substantial similarity (preferably with a sequence identity of at
least 60%; more preferably measured by the BLASTN program with the
default parameters wordlength (W) of 11, an expectation (E) of 10,
a cutoff of 100, M=5, N=-4, and a comparison of both strands) to a
transcription regulating nucleotide sequence described by SEQ ID
NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23,
24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73;
and [0089] iv) a nucleotide sequence capable of hybridizing to a
transcription regulating nucleotide sequence described by SEQ ID
NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23,
24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73
or the complement thereof (preferably 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 preferably 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., still more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M
NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in
0.5.times.SSC, 0.1% SDS at 50.degree. C., even 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 50.degree.
C., most 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.); and [0090] v) a
nucleotide sequence capable of hybridizing to a nucleic acid
comprising 50 to 200 or more consecutive nucleotides of a
transcription regulating nucleotide sequence described by SEQ ID
NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23,
24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73
or the complement thereof; and [0091] vi) a nucleotide sequence
which is the complement or reverse complement of any of the
previously mentioned nucleotide sequences under i) to v), [0092]
and [0093] b) at least one nucleic acid sequence which is
heterologous in relation to said transcription regulating
sequence.
[0094] Preferably, the sequences specified under ii), iii), iv) v)
and vi) in the paragraphs above are capable to modify transcription
in a monocotyledonous plant cell or organism. More preferably said
sequences specified under ii), iii), iv) v) and vi) have
substantially the same transcription regulating activity as the
transcription regulating nucleotide sequence described by SEQ ID
NO: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23,
24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or
73.
[0095] Also preferably the sequences specified under iii) above
have a sequence identity of at least 60%, preferably 70% or 80%,
more preferably 90% or 95% to a sequence described by SEQ ID NO: 1,
2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27,
28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73, wherein
the identity is preferably measured by the BLASTN program with the
default parameters wordlength (W) of 11, an expectation (E) of 10,
a cutoff of 100, M=5, N=-4, and a comparison of both strands.
[0096] Further preferably, the sequences specified under iv) or v)
above are hybridizing under stringent conditions, preferably under
medium stringent conditions, most preferably under high stringent
conditions (such as 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.) with the specified target
sequence.
[0097] In one preferred embodiment of the expression cassette of
the invention, the expression of the nucleic acid sequence results
in expression of a protein, or expression of an antisense RNA,
sense or double-stranded RNA.
[0098] The expression profile of the expression cassettes of the
invention may be modulated depending on the combination of the
transcription regulating nucleotide sequence with expression
enhancing introns and/or transcriptions termination sequences. This
in a preferred embodiment the expression cassette of the inventions
comprises at least one additional element selected from the group
consisting of
a) 5'-untranslated regions, and b) intron encoding sequences, and
c) transcription termination sequences.
[0099] The intron encoding sequences are preferably encoding an
expression enhancing intron from a monocotyledonous plant. More
preferably the intron sequence is an intron from an ubiquitin,
actin or alcohol dehydrogenase gene. Preferably, this intron is
inserted in the expression construct in the 5'-untranslated region
of the nucleic acid sequence, which should be expressed (i.e.,
between the transcription regulating nucleotide sequence and the
protein coding sequence (open reading frame) or the nucleic acid
sequence to be expressed).
[0100] Preferably, the 5'-untranslated region is from the same gene
as the transcription regulating sequences.
[0101] The transcription terminating sequence preferably also
comprises a sequence inducing polyadenylation. The transcription
terminating sequence may be heterologous with respect to the
transcription regulating nucleotide sequence and/or the nucleic
acid sequence to be expressed, but may also be the natural
transcription regulating nucleotide sequence of the gene of said
transcription regulating nucleotide sequence and/or said nucleic
acid sequence to be expressed. In one preferred embodiment of the
invention the transcription regulating nucleotide sequence is the
natural transcription regulating nucleotide sequence of the gene of
the transcription regulating sequence. Preferably the transcription
termination sequence is selected from the group of sequences
described by SEQ ID NO: 32, 34, and 35.
[0102] The transcription regulating sequences of the invention are
especially useful for constitutive or root/kernel-preferential or
root/kernel-specific expression in monocotyledonous plants.
However, a use in other plants (e.g., dicotyledonous or gymnosperm
plants) and other tissues cannot be ruled out. It has been shown
that the tissue specificity of the transcription regulating
sequences of the invention can be advantageously modulated by the
combination with introns and/or transcription termination
sequences.
[0103] The expression cassette may be employed for numerous
expression purposes such as for example expression of a protein, or
expression of an antisense RNA, sense or double-stranded RNA.
Preferably, expression of the nucleic acid sequence confers to the
plant an agronomically valuable trait.
[0104] Some of the transcription regulating sequences of the
invention are novel even as such (i.e. as isolated nucleotide
sequences). Accordingly another embodiment of the invention relates
to an isolated nucleic acid sequence comprising at least one
transcription regulating nucleotide sequence as described by SEQ ID
NO: 6, 7, 8, 11, 12, 13, 19, 20, or 21.
[0105] 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,
more preferably a monocotyledonous plant, most preferably selected
form the group consisting of Zea mays (corn), Oryza sativa (rice),
Triticum aestivum (wheat), Hordeum vulgare (barley), and Avena
sativa (oats).
[0106] Another embodiment of the invention relates to a method for
identifying and/or isolating transcription regulating nucleotide
sequence from a monocotyledonous plant characterized that said
identification and/or isolation utilizes a nucleic acid sequence
encoding an amino acid sequence as described by SEQ ID NOs: 5, 10,
18, 26, 31, 60, 65, 70, or 75, or a part of at least 15 bases of
said nucleic acid sequence. Preferably the employed nucleic acid
sequences is described by SEQ ID NOs: 4, 9, 17, 25, 30, 59, 64, 69,
or 74 or a part of at least 15 bases of said nucleic acid sequence.
Preferably said identification and/or isolation is realized by a
method selected from polymerase chain reaction, hybridization, and
database screening.
[0107] Still another embodiment of the invention relates to a
method for providing a transgenic expression cassette for
heterologous expression in monocotyledonous plants comprising the
steps of: [0108] I. isolating of a transcription regulating
nucleotide sequence from a monocotyledonous plant utilizing at
least one nucleic acid sequence or a part thereof, wherein said
sequence is encoding a polypeptide described by SEQ ID NOs: 5, 10,
18, 26, 31, 60, 65, 70, or 75, or a part of at least 15 bases of
said nucleic acid sequence, and [0109] II. functionally linking
said transcription regulating nucleotide sequence to another
nucleotide sequence of interest, which is heterologous in relation
to said transcription regulating nucleotide sequence.
[0110] For both of the above mentioned methods preferably the
nucleotide sequence utilized for isolation of said transcription
regulating nucleotide sequence is encoding a polypeptide comprising
[0111] a1) at least one sequence motif of a monocotyledonous plant
lactate dehydrogenase protein selected from the group consisting of
the amino acid sequences
TABLE-US-00003 [0111] i) SLSELGFDA, (SEQ ID NO: 76) ii) VIGAGNVGMA,
(SEQ ID NO: 77) iii) IVTAGARQI, (SEQ ID NO: 78) iv) L(F/Y)RKIVP,
(SEQ ID NO: 79) v) GFPASRV, (SEQ ID NO: 80) vi) RF(L/I)AEHL, (SEQ
ID NO: 81) vii) QAYMVGEH, (SEQ ID NO: 82) viii) ALEGIRRAV, (SEQ ID
NO: 83) and ix) GYSVAS(L/I)A, (SEQ ID NO: 84)
[0112] or [0113] a2) at least one sequence motif of a
monocotyledonous plant caffeoyl-CaA-O-methyltransferase protein
selected from the group consisting of the amino acid sequences
TABLE-US-00004 [0113] x) (SEQ ID NO: 85) EQKTRHSE, xi) (SEQ ID NO:
86) L(I/L)KLIGAK, xii) (SEQ ID NO: 87) KTMEIGVY, xiii) (SEQ ID NO:
88) HERL(L/M)KLV, xiv) (SEQ ID NO: 89) CQLPVGDG, and xv) (SEQ ID
NO: 90) TLCRRVK.
DEFINITIONS
[0114] 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.
[0115] 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 percent, preferably 10 percent up or down (higher or lower).
[0116] As used herein, the word "or" means any one member of a
particular list and also includes any combination of members of
that list.
[0117] 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.
[0118] The term "intron" refers to sections of DNA (intervening
sequences) within a gene that do not encode part of the protein
that the gene produces, and that is spliced out of the mRNA that is
transcribed from the gene before it is exported from the cell
nucleus. Intron sequence refers to the nucleic acid sequence of an
intron. Thus, introns are those regions of DNA sequences that are
transcribed along with the coding sequence (exons) but are removed
during the formation of mature mRNA. Introns can be positioned
within the actual coding region or in either the 5' or 3'
untranslated leaders of the pre-mRNA (unspliced mRNA). Introns in
the primary transcript are excised and the coding sequences are
simultaneously and precisely ligated to form the mature mRNA. The
junctions of introns and exons form the splice site. The sequence
of an intron begins with GU and ends with AG. Furthermore, in
plants, two examples of AU-AC introns have been described: intron
14 of the RecA-like protein gene and intron 7 of the G5 gene from
Arabidopsis thaliana are AT-AC introns, Pre-mRNAs containing
introns have three short sequences that are--beside other
sequences--essential for the intron to be accurately spliced. These
sequences are the 5' splice-site, the 3' splice-site, and the
branchpoint. mRNA splicing is the removal of intervening sequences
(introns) present in primary mRNA transcripts and joining or
ligation of exon sequences. This is also known as cis-splicing
which joins two exons on the same RNA with the removal of the
intervening sequence (intron). The functional elements of an intron
comprising sequences that are recognized and bound by the specific
protein components of the spliceosome (e.g. splicing consensus
sequences at the ends of introns). The interaction of the
functional elements with the spliceosome results in the removal of
the intron sequence from the premature mRNA and the rejoining of
the exon sequences. Introns have three short sequences that are
essential--although not sufficient--for the intron to be accurately
spliced. These sequences are the 5' splice site, the 3' splice site
and the branchpoint The branchpoint sequence is important in
splicing and splice-site selection in plants. The branchpoint
sequence is usually located 10-60 nucleotides upstream of the 3'
splice site. Plant sequences exhibit sequence deviations in the
branchpoint, the consensus sequences being CURAY or YURAY.
[0119] 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.
[0120] A "marker gene" encodes a selectable or screenable
trait.
[0121] The term "chimeric gene" refers to any gene that contains
[0122] 1) DNA sequences, including regulatory and coding sequences,
that are not found together in nature, or [0123] 2) sequences
encoding parts of proteins not naturally adjoined, or [0124] 3)
parts of promoters that are not naturally adjoined.
[0125] 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.
[0126] 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.
[0127] 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
1,000's of nucleotides in length.
[0128] The terms "protein," "peptide" and "polypeptide" are used
interchangeably herein.
[0129] 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. 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
Agrobacterium 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).
[0130] "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.
[0131] 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).
[0132] A "functional RNA" refers to an antisense RNA,
double-stranded-RNA, ribozyme, or other RNA that is not
translated.
[0133] 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.
[0134] "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 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 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.
[0135] "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).
[0136] "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.
[0137] 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.
[0138] "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 an
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.
[0139] "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.
[0140] 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.
[0141] 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.
[0142] "Constitutive expression" refers to expression using a
constitutive or regulated promoter.
[0143] By "tissue-independent," "tissue-general," or "constitutive"
is intended expression in the cells throughout a plant at most
times and in most tissues. As with other promoters classified as
"constitutive" (e.g., ubiquitin), some variation in absolute levels
of expression can exist among different tissues or stages. However,
constitutive promoters generally are expressed at high or moderate
levels in most, and preferably all, tissues and most, and
preferably all, developmental stages. "Conditional" and "regulated
expression" refer to expression controlled by a regulated
promoter.
[0144] "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.
[0145] "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.
[0146] "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.
[0147] The term "root" in the context of the inventions means the
usually underground organ of a plant that lacks buds or leaves or
nodes, absorbs water and mineral salts and usually it anchors the
plant to the ground. The plant root consists of many cell types
such as epidermal, root cap, columella, cortex, pericycle, vascular
and root hair forming trichoblasts, organized into tissues or
regions of the root, for example, the root tip, root epidermis,
meristematic zone, primary root, lateral root, root hair, and
vascular tissue. Transcription regulating sequences isolated as
root-specific or root-preferred may regulate expression in one or a
few of these cell types. This cell-specific activity can be useful
for specific applications such as regulating meristematic activity
in only meristematic cell zone or expression of a nematicidal gene
in only the cell type that are contacted by the nematode pest.
[0148] The term "tissue-specific transcription" in the context of
this invention in relation to a certain tissue or a group of tissue
(e.g., root and kernel) means the transcription of a nucleic acid
sequence by a transcription regulating element in a way that
transcription of said nucleic acid sequence in said tissue or group
of tissues 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.
[0149] "Tissue-preferential transcription" in the context of this
invention in relation to a certain tissue or a group of tissue
(e.g., root and kernel) means the transcription of a nucleic acid
sequence by a transcription regulating element in a way that
transcription of said nucleic acid sequence in said tissue or group
of tissues 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.
[0150] "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.
[0151] "Operably-linked" refers 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.
[0152] "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.
[0153] "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.
[0154] 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.
[0155] 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.
[0156] 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.
[0157] 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.
[0158] 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.
[0159] "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.
[0160] "Antisense inhibition" refers to the production of antisense
RNA transcripts capable of suppressing the expression of protein
from an endogenous gene or a transgene.
[0161] "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).
[0162] 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.
[0163] "Homologous to" in the context of nucleotide sequence
identity refers to the similarity between the nucleotide sequences
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.
[0164] The term "substantially similar" refers to nucleotide and
amino acid sequences that represent functional and/or structural
equivalents of the transcription regulating sequences from maize or
rice specifically disclosed herein.
[0165] 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.
[0166] 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
a polypeptide encoded by a gene with a promoter having any one of
SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21,
22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72,
or 73, a nucleotide sequence comprising an open reading frame
having any one of SEQ ID NOs: 4, 9, 17, 25, 30, 59, 64, 69, or 74,
which encodes one of SEQ ID NOs: 5, 10, 18, 26, 31, 60, 65, 70, or
75. 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, specifically binds to the other.
[0167] Sequence comparisons maybe carried out using a
Smith-Waterman sequence alignment algorithm (see e.g., Waterman
(1995) or http://www hto.usc.edu/software/seqaln/index.html). 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.
[0168] 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.
[0169] 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.
[0170] "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.
[0171] 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.
[0172] "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.
[0173] "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.
[0174] "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.
[0175] The term "transformation" refers to the transfer of a
nucleic acid fragment into the genome of a host cell, resulting in
genetically stable inheritance. Host cells containing the
transformed nucleic acid fragments are referred to as "transgenic"
cells, and organisms comprising transgenic cells are referred to as
"transgenic organisms". Examples of methods of transformation of
plants and plant cells include Agrobacterium-mediated
transformation (De Blaere 1987) and particle bombardment technology
(U.S. Pat. No. 4,945,050). Whole plants may be regenerated from
transgenic cells by methods well known to the skilled artisan (see,
for example, Fromm 1990).
[0176] "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.
[0177] "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.
[0178] "Stably transformed" refers to cells that have been selected
and regenerated on a selection media following transformation.
[0179] "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.
[0180] "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.
[0181] "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).
[0182] "Secondary transformants" and the "T1, T2, T3, etc.
generations" refer to transgenic plants derived from primary
transformants through one or more meiotic and fertilization cycles.
They may be derived by self-fertilization of primary or secondary
transformants or crosses of primary or secondary transformants with
other transformed or untransformed plants.
[0183] "Wild-type" refers to a virus or organism found in nature
without any known mutation.
[0184] "Genome" refers to the complete genetic material of an
organism.
[0185] 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.
[0186] 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.
[0187] 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.
[0188] 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.
[0189] "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.
[0190] 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).
[0191] 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.
[0192] 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.
[0193] 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."
[0194] "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.,
root/kernel specific or preferential).
[0195] "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).
[0196] 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).
[0197] 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.
[0198] "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.
[0199] A "transgenic plant" is a plant having one or more plant
cells that contain an expression vector.
[0200] "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.
[0201] 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". [0202] (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.
[0203] (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. 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. [0204] 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. [0205] Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information (http://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. [0206] 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. [0207]
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
http://www.ncbi.nlm.nih.gov. Alignment may also be performed
manually by inspection. [0208] 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. [0209]
(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.). [0210] (d) As
used herein, "percentage of sequence identity" means the value
determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity. [0211] (e) (i) The term "substantial identity" of
polynucleotide sequences means that a polynucleotide comprises a
sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, or 79%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or
94%, and most preferably at least 95%, 96%, 97%, 98%, or 99%
sequence identity, compared to a reference sequence using one of
the alignment programs described using standard parameters. 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%. [0212] 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. [0213] (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.
[0214] For sequence comparison, typically one sequence acts as a
reference sequence to which test sequences are compared. When using
a sequence comparison algorithm, test and reference sequences are
input into a computer, subsequence coordinates are designated if
necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0215] 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.
[0216] "Stringent hybridization conditions" and "stringent
hybridization wash conditions" in the context of nucleic acid
hybridization experiments such as Southern and Northern
hybridization are sequence dependent, and are different under
different environmental parameters. The T.sub.m is the temperature
(under defined ionic strength and pH) at which 50% of the target
sequence hybridizes to a perfectly matched probe. Specificity is
typically the function of post-hybridization washes, the critical
factors being the ionic strength and temperature of the final wash
solution. For DNA-DNA hybrids, the T.sub.m can be approximated from
the equation of Meinkoth and Wahl, 1984:
T.sub.m=81.5.degree. C.+16.6(log.sub.10 M)+0.41(% GC)-0.61(%
form)-500/L
where M is the molarity of monovalent cations, % GC is the
percentage of guanosine and cytosine nucleotides in the DNA, % form
is the percentage of formamide in the hybridization solution, and L
is the length of the hybrid in base pairs. T.sub.m is reduced by
about 1.degree. C. for each 1% of mismatching; thus, T.sub.m,
hybridization, and/or wash conditions can be adjusted to hybridize
to sequences of the desired identity. For example, if sequences
with >90% identity are sought, the T.sub.m can be decreased
10.degree. C. Generally, stringent conditions are selected to be
about 5.degree. C. lower than the thermal melting point I for the
specific sequence and its complement at a defined ionic strength
and pH. However, severely stringent conditions can utilize a
hybridization and/or wash at 1, 2, 3, or 4.degree. C. lower than
the thermal melting point I; moderately stringent conditions can
utilize a hybridization and/or wash at 6, 7, 8, 9, or 10.degree. C.
lower than the thermal melting point I; low stringency conditions
can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or
20.degree. C. lower than the thermal melting point I. Using the
equation, hybridization and wash compositions, and desired T, those
of ordinary skill will understand that variations in the stringency
of hybridization and/or wash solutions are inherently described. If
the desired degree of mismatching results in a T of less than
45.degree. C. (aqueous solution) or 32.degree. C. (formamide
solution), it is preferred to increase the SSC concentration so
that a higher temperature can be used. An extensive guide to the
hybridization of nucleic acids is found in Tijssen, 1993.
Generally, highly stringent hybridization and wash conditions are
selected to be about 5.degree. C. lower than the thermal melting
point T.sub.m for the specific sequence at a defined ionic strength
and pH.
[0217] 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.
[0218] Very stringent conditions are selected to be equal to the
T.sub.m for a particular probe. An example of stringent conditions
for hybridization of complementary nucleic acids which have more
than 100 complementary residues on a filter in a Southern or
Northern blot is 50% formamide, e.g., hybridization in 50%
formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
0.1.times.SSC at 60 to 65.degree. C. Exemplary low stringency
conditions include hybridization with a buffer solution of 30 to
35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at
37.degree. C., and a wash in 1.times. to 2.times.SSC
(20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to
55.degree. C. Exemplary moderate stringency conditions include
hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at
37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to
60.degree. C.
[0219] 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.
[0220] "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.
[0221] "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.
[0222] 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.
[0223] 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.
[0224] Annual, perennial, monocotyledonous and dicotyledonous
plants are preferred host organisms for the generation of
transgenic plants. The use of the recombination system, or method
according to the invention is furthermore advantageous in all
ornamental plants, forestry, fruit, or ornamental trees, flowers,
cut flowers, shrubs or turf. Said plant may include--but shall not
be limited to--bryophytes such as, for example, Hepaticae
(hepaticas) and Musci (mosses); pteridophytes such as ferns,
horsetail and clubmosses; gymnosperms such as conifers, cycads,
ginkgo and Gnetaeae; algae such as Chlorophyceae, Phaeophpyceae,
Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophyceae
(diatoms) and Euglenophyceae.
[0225] Plants for the purposes of the invention may comprise the
families of the Rosaceae such as rose, Ericaceae such as
rhododendrons and azaleas, Euphorbiaceae such as poinsettias and
croton, Caryophyllaceae such as pinks, Solanaceae such as petunias,
Gesneriaceae such as African violet, Balsaminaceae such as
touch-me-not, Orchidaceae such as orchids, Iridaceae such as
gladioli, iris, freesia and crocus, Compositae such as marigold,
Geraniaceae such as geraniums, Liliaceae such as Drachaena,
Moraceae such as ficus, Araceae such as philodendron and many
others.
[0226] The transgenic plants according to the invention are
furthermore selected 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
sativa (lettuce) and many others. 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.
[0227] Most preferably, the transgenic plants according to the
invention may be selected among monocotyledonous crop plants. The
term "monocotyledonous plant" when referring to a transgenic plant
according to the invention or to the source of the transcription
regulating sequences of the invention is intended to comprise all
genera, families and species of monocotyledonous plants. Preferred
are Gramineae plants such as, for example, cereals such as maize,
rice, wheat, barley, sorghum, millet, rye, triticale, or oats, and
other non-cereal monocotyledonous plants such as sugarcane or
banana. Especially preferred are corn (maize), rice, barley, wheat,
rye, and oats. Most preferred are all varieties of the specie Zea
mays and Oryza sativa.
[0228] "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.
[0229] "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
[0230] The present invention provides for isolated nucleic acid
molecules comprising a plant nucleotide sequence that directs
transcription of an operably linked nucleic acid fragment in a
plant cell, preferably in monocotyledonous plants. Specifically,
the present invention provides expression cassettes for regulating
expression in monocotyledonous plants comprising [0231] i) at least
one transcription regulating nucleotide sequence of a
monocotyledonous plant gene, said monocotyledonous plant gene
selected from the group of genes consisting of
caffeoyl-CoA-O-methyltransferase genes, C8,7-sterol isomerase
genes, hydroxyproline-rich glycoprotein (HRGP) genes, lactate
dehydrogenase genes, and chloroplast protein like 12 genes, and
functionally linked thereto [0232] ii) at least one nucleic acid
sequence which is heterologous in relation to said transcription
regulating sequence.
[0233] 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 of the invention 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 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.
[0234] The transcription regulating sequences of the inventions can
be combined with various 5'-untranslated regions, intron
(preferably expression enhancing introns), and transcription
terminations sequences (as described below in more detail). It has
been shown that the tissue specificity of the transcription
regulating sequences of the invention can be advantageously
modulated by the combination with introns and/or transcription
termination sequences. In most combinations the resulting
expression cassettes exhibit a preferential or specific expression
in root and kernel. However other expression specificities (e.g.,
constitutive expression) can be achieved. The transcription
regulating sequences with expression activity in roots may be
useful for alteration of the function of root tissue, modification
of growth rate, improvement of resistance to root preferred
pathogens, pests, herbicides or adverse weather conditions, for
detoxification of soil as well as for broadening the range of soils
or environments in which said plant may grow. Root abundant or root
specific gene expression would provide a mechanism according to
which morphology and metabolism may be altered to improve the yield
and to produce useful proteins in greater amounts.
[0235] However, in some combinations, the transcriptions regulating
sequence may exhibit a strong constitutive expression profile.
Constitutive promoters are favored in situations where expression
in all (or most) tissues during all (or most) times of the plant
development is required. Other tissue specificities may be possible
depending on the regulatory elements used in combination with the
transcription regulating sequences of the invention.
[0236] 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-00005 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. Preferred Promotor mRNA
locus ID Proteine ID Gene Product Specie SEQ ID cDNA SEQ ID Protein
SEQ ID Caffeoyl-CoA-O- Oryza sativa SEQ ID NO: 1, 2, 3 AB023482.2
BAA78733 methyltransferase SEQ ID NO: 4 SEQ ID NO: 5
Caffeoyl-CoA-O- Zea mays SEQ ID NO: 66, 67, 68 SEQ ID NO: 69 SEQ ID
NO: 70 methyltransferase C-8,7-sterol- Oryza sativa SEQ ID NO: 6,
7, 8 NM_183458 NP_908347 isomerase SEQ ID NO: 9 SEQ ID NO: 10
Hydroxyproline- Zea mays SEQ ID NO: S45164 AAB23539 rich
glycoprotein 11, 12, 13, 14, 15, 16 SEQ ID NO: 17 SEQ ID NO: 18
Hydroxyproline- Zea SEQ ID NO: 71, 72, 73 SEQ ID NO: 74 SEQ ID NO:
75 rich glycoprotein diploperennis Lactate- Zea mays SEQ ID NO:
Z11754 CAA77808 dehydrogenase 19, 20, 21, 22, 23, 24 SEQ ID NO: 25
SEQ ID NO: 26 Lactate- Oryza sativa SEQ ID NO: 56, 57, Os06g01590
Os06g01590 dehydrogenase 58, 61, 62, 63 SEQ ID NO: 59 SEQ ID NO: 60
Os02g01510 Os02g01510 SEQ ID NO: 64 SEQ ID NO: 65 Chloroplast
protein Oryza sativa SEQ ID NO: 27, 28, 29 AP002881 BAB19776 12 lie
protein SEQ ID NO: 30 SEQ ID NO: 31
[0237] Preferably, the transcription regulating nucleotide sequence
is obtainable from monocotyledonous plant genomic DNA from a gene
encoding a polypeptide which [0238] a1) comprises at least one
(preferably at least 2 or 3, more preferably at least 4 or 5, most
preferably all) sequence motif of a monocotyledonous plant lactate
dehydrogenase protein selected from the group consisting of the
amino acid sequences
TABLE-US-00006 [0238] i) (SEQ ID NO: 76) SLSELGFDA, ii) (SEQ ID NO:
77) VIGAGNVGMA, iii) (SEQ ID NO: 78) IVTAGARQI, iv) (SEQ ID NO: 79)
L(F/Y)RKIVP, v) (SEQ ID NO: 80) GFPASRV, vi) (SEQ ID NO: 81)
RF(L/I)AEHL, vii) (SEQ ID NO: 82) QAYMVGEH, viii) (SEQ ID NO: 83)
ALEGIRRAV, and ix) (SEQ ID NO: 84) GYSVAS(L/I)A,
[0239] or [0240] b1) is encoding a lactate dehydrogenase protein
from a monocotyledonous plant having an amino acid sequence
identity of at least 90%, preferably at least 95%, more preferably
at least 98% to a polypeptide selected from the group described by
SEQ ID NO: 26, 60 and 65, or [0241] a2) comprises at least one
(preferably at least 2 or 3, more preferably at least 4 or 5, most
preferably all) sequence motif of a monocotyledonous plant
caffeoyl-CaA-O-methyltransferase protein selected from the group
consisting of the amino acid sequences
TABLE-US-00007 [0241] x) (SEQ ID NO: 85) EQKTRHSE, xi) (SEQ ID NO:
86) L(I/L)KLIGAK, xii) (SEQ ID NO: 87) KTMEIGVY, xiii) (SEQ ID NO:
88) HERL(L/M)KLV, xiv) (SEQ ID NO: 89) CQLPVGDG, and xv) (SEQ ID
NO: 90) TLCRRVK,
[0242] or [0243] b2) is encoding a caffeoyl-CaA-O-methyltransferase
protein from a monocotyledonous plant having an amino acid sequence
identity of at least 90%, preferably at least 95%, more preferably
at least 98% to a polypeptide selected from the group described by
SEQ ID NOs: 5 and 70, or [0244] b3) is encoding a
hydroxyproline-rich glycoprotein from a monocotyledonous plant
having an amino acid sequence identity of at least 90%, preferably
at least 95%, more preferably at least 98% to a polypeptide
selected from the group described by SEQ ID NOs: 18 and 75, or
[0245] b4) is encoding a C-8,7-stereol-isomerase protein from a
monocotyledonous plant having an amino acid sequence identity of at
least 90%, preferably at least 95%, more preferably at least 98% to
a polypeptide selected described by SEQ ID NO: 10, or [0246] b5) is
encoding a Chloroplast protein 12 like protein from a
monocotyledonous plant having an amino acid sequence identity of at
least 90%, preferably at least 95%, more preferably at least 98% to
a polypeptide described by SEQ ID NO: 31.
[0247] 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:
4, 9, 17, 25, 30, 59, 64, 69, or 74, respectively, or a fragment of
said transcription regulating nucleotide sequence which exhibits
the same promoter activity (e.g., root/kernel-preferential or
root/kernel-specific or constitutive expression activity).
[0248] Preferably, the transcription regulating nucleotide sequence
is from a corn (Zea mays) or rice (Oryza sativa) plant. Even more
preferably the transcription regulating nucleotide sequence is from
a plant gene selected from the group of genes consisting of Oryza
sativa caffeoyl-CoA-O-methyltransferase genes, Oryza sativa
C8,7-sterol isomerase genes, Zea may hydroxyproline-rich
glycoprotein (HRGP) genes, Zea mays lactate dehydrogenase genes,
Oryza sativa chloroplast protein 12 like genes and functional
equivalents thereof. The functional equivalent gene is preferably
encoding a polypeptide which has at least 90% amino acid sequence
identity, preferably at least 95% amino acid sequence identity,
more preferably at least 98% amino acid sequence identity to a
polypeptide selected from the group described by SEQ ID NOs: 5, 10,
18, 26, 31, 60, 65, 70, and 75.
[0249] Some of the transcription regulating sequences of the
invention provided herein are novel as such (i.e. as isolated
nucleotide sequences). Accordingly another embodiment of the
invention relates to an isolated nucleic acid sequence comprising
at least one transcription regulating nucleotide sequence as
described by SEQ ID NOs: 6, 7, 8, 11, 12, 13, 19, 20, or 21.
[0250] In a more preferred embodiment the transcription regulating
nucleotide sequence is selected from the group of sequences
consisting of [0251] i) the sequences described by SEQ ID NOs: 1,
2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27,
28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, and 73, and
[0252] ii) a fragment of at least 50 (preferably at least 70 or
100, more preferably at least 150 or 200, even more preferably at
least 300 or 400, most preferably at least 500 or 700) consecutive
bases of a sequence under i); and [0253] iii) a nucleotide sequence
having substantial similarity (preferably with a sequence identity
of at least 60%; more preferably measured by the BLASTN program
with the default parameters wordlength (W) of 11, an expectation
(E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both
strands) to a transcription regulating nucleotide sequence
described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16,
19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67,
68, 71, 72, or 73; and [0254] iv) a nucleotide sequence capable of
hybridizing to a transcription regulating nucleotide sequence
described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16,
19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67,
68, 71, 72, or 73 or the complement thereof (preferably 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 preferably 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., still more preferably in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C., even 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
50.degree. C., most 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.); and [0255] v) a
nucleotide sequence capable of hybridizing to a nucleic acid
comprising 50 to 200 or more ((preferably at least 70 or 100, more
preferably at least 150 or 200, even more preferably at least 300
or 400, most preferably at least 500 or 700) consecutive
nucleotides of a transcription regulating nucleotide sequence
described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16,
19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67,
68, 71, 72, or 73 or the complement thereof; and [0256] vii) a
nucleotide sequence which is the complement or reverse complement
of any of the previously mentioned nucleotide sequences under i) to
v).
[0257] Another preferred embodiment relates to an expression
cassette for regulating expression in monocotyledonous plants
comprising [0258] a) at least one transcription regulating
nucleotide sequence functional in a monocotyledonous plant
comprising at least one sequence selected from the group of
sequences consisting of [0259] i) the sequences described by SEQ ID
NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23,
24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, and 73,
and [0260] ii) a fragment of at least 50 (preferably at least 70 or
100, more preferably at least 150 or 200, even more preferably at
least 300 or 400, most preferably at least 500 or 700) consecutive
bases of a sequence under i); and [0261] iii) a nucleotide sequence
having substantial similarity (preferably with a sequence identity
of at least 60%; more preferably measured by the BLASTN program
with the default parameters wordlength (W) of 11, an expectation
(E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both
strands) to a transcription regulating nucleotide sequence
described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16,
19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67,
68, 71, 72, or 73; and [0262] iv) a nucleotide sequence capable of
hybridizing to a transcription regulating nucleotide sequence
described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16,
19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67,
68, 71, 72, or 73 or the complement thereof (preferably 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 preferably 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., still more preferably in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C., even 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
50.degree. C., most 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.); and [0263] v) a
nucleotide sequence capable of hybridizing to a nucleic acid
comprising 50 to 200 or more (preferably at least 70 or 100, more
preferably at least 150 or 200, even more preferably at least 300
or 400, most preferably at least 500 or 700) consecutive
nucleotides of a transcription regulating nucleotide sequence
described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16,
19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67,
68, 71, 72, or 73 or the complement thereof; and [0264] vi) a
nucleotide sequence which is the complement or reverse complement
of any of the previously mentioned nucleotide sequences under i) to
v), [0265] and [0266] b) at least one nucleic acid sequence which
is heterologous in relation to said transcription regulating
sequence.
[0267] Preferably, the sequences specified under ii), iii), iv) v)
and vi) in the paragraphs above are capable to modify transcription
in a monocotyledonous plant cell or organism. More preferably said
sequences specified under ii), iii), iv) v) and vi) have
substantially the same transcription regulating activity as the
transcription regulating nucleotide sequence described by SEQ ID
NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23,
24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or
73.
[0268] Also preferably the sequences specified under iii) above
have a sequence identity of at least 60%, preferably 70% or 80%,
more preferably 90% or 95% to a sequence described by SEQ ID NOs:
1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24,
27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73,
wherein the identity is preferably measured by the BLASTN program
with the default parameters wordlength (W) of 11, an expectation
(E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both
strands.
[0269] Further preferably, the sequences specified under iv) or v)
above are hybridizing under stringent conditions, preferably under
medium stringent conditions, most preferably under high stringent
conditions (such as 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.) with the specified target
sequence.
[0270] The activity of a specific transcription regulating
nucleotide sequence is considered substantially the same or
equivalent if transcription is initiated preferentially or
specifically in the same tissue than the original promoter (e.g.,
in root and kernel or constitutive in all or most tissues) under
otherwise identical conditions (i.e. in combination with the set of
additional regulatory elements (e.g., introns, transcription
terminator sequences and 5'-untranslated regions) and the same
nucleic acid sequence to be expressed in the same plant expression
system). Such expression profile is preferably demonstrated using
reporter genes operably linked to said transcription regulating
sequence. Preferred reporter genes (Schenborn 1999) in this context
are green fluorescence protein (GFP) (Chuff 1996; Leffel 1997),
chloramphenicol transferase, luciferase (Millar 1992),
R-glucuronidase or .beta.-galactosidase. Especially preferred is
R-glucuronidase (Jefferson 1987). With respect to promoters with
constitutive expression activity, the term "at most times" means a
transcription regulating activity (as demonstrated by an
.beta.-glucuronidase assays as described in the examples below)
preferably during at least 50%, preferably at least 70%, more
preferably at least 90% of the development cycle of a plant
comprising the respective expression cassette stably integrated
into its chromosomal DNA.
[0271] With respect to a constitutive transcription regulating
nucleotide sequence (e.g., a constitutive promoter), the term "in
most tissues" means a transcription regulating activity (as
demonstrated by an .beta.-glucuronidase assays as described in the
examples below) in tissues which together account to preferably at
least 50%, preferably at least 70%, more preferably at least 90% of
the entire biomass of the a plant comprising the respective
expression cassette stably integrated into its chromosomal DNA.
[0272] 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%.
[0273] Such functional equivalent of the transcription regulating
nucleotide sequence may be obtained from other monocotyledonous
plant species by using the transcription regulating 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 transcription
regulating sequences of the present invention, which are conserved
among species, can also be used as PCR primers to amplify a segment
from a species other than rice or maize, 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 transcription regulating sequences
could be employed to identify structurally related sequences in a
database using computer algorithms.
[0274] More specifically, based on the transcription regulating
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 transcription regulating sequences of
the invention, e.g., hybridization, PCR or computer generated
sequence comparisons. For example, all or a portion of a particular
transcription regulating nucleotide sequence of the invention is
used as a probe that selectively hybridizes to other gene sequences
present in a population of cloned genomic DNA fragments (i.e.,
genomic libraries) from a chosen source organism. Further, suitable
genomic 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
transcription regulating sequences of the invention 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.
[0275] 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.
[0276] 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.
[0277] Hence, the isolated nucleic acid molecules of the invention
include the orthologs of the transcription regulating sequences
disclosed herein, i.e., the corresponding nucleotide sequences in
other (i.e. other than the host organism where the specific
sequences disclosed herein are derived from) monocotyledonous plant
organisms, preferably, e.g., cereal plants such as corn, wheat,
rye, barley, oats, turfgrass, sorghum, millet, or other
monocotyledonous plants such as sugarcane or banana. An ortholog or
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., 90% 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 maize
or rice sequences disclosed herein, e.g., orthologs in other
monocotyledonous plants such as wheat, barley, oats and others.
Alternatively, recombinant DNA techniques such as hybridization or
PCR may be employed to identify sequences related to the maize and
rice sequences or to clone the equivalent sequences from different
maize or rice DNAs.
[0278] 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.
[0279] Thus, another embodiment of the invention relates to a
method for identifying and/or isolating a transcription regulating
nucleotide sequence from a monocotyledonous plant characterized
that said identification and/or isolation utilizes a nucleic acid
sequence encoding an amino acid sequence as described by SEQ ID
NOs: 5, 10, 18, 26, 31, 60, 65, 70, or 75, or a parts of said
nucleic acid sequence. Preferred are nucleic acid sequences
described by SEQ ID NOs: 4, 9, 17, 25, 30, 59, 64, 69, or 74 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 FOR" ("TAIL
PCR").
[0280] Still another embodiment of the invention relates to a
method for providing a transgenic expression cassette for
heterologous expression in monocotyledonous plants comprising the
steps of: [0281] I. isolating of a transcription regulating
nucleotide sequence from a monocotyledonous plant utilizing at
least one nucleic acid sequence or a part thereof, wherein said
sequence is encoding a polypeptide described by SEQ ID NOs: 5, 10,
18, 26, 31, 60, 65, 70, or 75, or a part of at least 15 bases of
said nucleic acid sequence, and [0282] III. functionally linking
said transcription regulating nucleotide sequence to another
nucleotide sequence of interest, which is heterologous in relation
to said transcription regulating nucleotide sequence.
[0283] 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
NOs: 4, 9, 17, 25, 30, 59, 64, 69, or 74 Preferably, the isolation
of the 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.
[0284] For both of the above mentioned methods preferably the
nucleotide sequence utilized for isolation of said transcription
regulating nucleotide sequence is encoding a polypeptide comprising
[0285] a1) at least one sequence motif of a monocotyledonous plant
lactate dehydrogenase protein selected from the group consisting of
the amino acid sequences
TABLE-US-00008 [0285] i) (SEQ ID NO: 76) SLSELGFDA, ii) (SEQ ID NO:
77) VIGAGNVGMA, iii) (SEQ ID NO: 78) IVTAGARQI, iv) (SEQ ID NO: 79)
L(F/Y)RKIVP, v) (SEQ ID NO: 80) GFPASRV, vi) (SEQ ID NO: 81)
RF(L/I)AEHL, vii) (SEQ ID NO: 82) QAYMVGEH, viii) (SEQ ID NO: 83)
ALEGIRRAV, and ix) (SEQ ID NO: 84) GYSVAS(L/I)A,
[0286] or [0287] a2) at least one sequence motif of a
monocotyledonous plant caffeoyl-CaA-O-methyltransferase protein
selected from the group consisting of the amino acid sequences
TABLE-US-00009 [0287] x) (SEQ ID NO: 85) EQKTRHSE, xi) (SEQ ID NO:
86) L(I/L)KLIGAK, xii) (SEQ ID NO: 87) KTMEIGVY, xiii) (SEQ ID NO:
88) HERL(L/M)KLV, xiv) (SEQ ID NO: 89) CQLPVGDG, and xv) (SEQ ID
NO: 90) TLCRRVK.
[0288] Preferably, the transcription regulating nucleotide
sequences and promoters of the invention include a consecutive
stretch of about 25 to 2,000, including 50 to 500 or 100 to 250,
and up to 1,000 or 1,500, 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, 6,
7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29,
56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, and 73, or the promoter
orthologs thereof, which include the minimal promoter region.
[0289] In a particular embodiment of the invention said consecutive
stretch of about 25 to 2,000, including 50 to 500 or 100 to 250,
and up to 1,000 or 1,500, 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
2,000, including 50 to 500 or 100 to 250, and up to 1,000 or 1,500,
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, 6, 7, 8, 11, 12, 13, 14, 15, 16,
19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67,
68, 71, 72, and 73, 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.
[0290] The transcription regulating nucleotide sequences of the
invention or their functional equivalents are capable of driving
expression in monocotyledonous plants of a coding sequence in a
target cell, particularly in a plant cell. The promoter sequences
and methods disclosed herein are useful in regulating expression in
monocotyledonous plants, 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.
[0291] The transcription regulating nucleotide sequences and
promoters of the invention are useful to modify the phenotype of a
plant. Various changes in the phenotype of a transgenic 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.
[0292] 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.
[0293] 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 NOs:
1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24,
27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73) 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 inbetween 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.
[0294] 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).
[0295] 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 NOs: 1, 2, 3, 6, 7, 8,
11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57,
58, 61, 62, 63, 66, 67, 68, 71, 72, or 73) 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 the
desired way (e.g., root/kernel-preferentially or
root/kernel-specific or constitutive) due to the transcription
regulating properties of the transcription regulating 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.
[0296] 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
sequence, thereby forming an expression cassette of the
invention.
[0297] 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.
[0298] The transcription regulating sequences of the invention with
a constitutive expression profile may be advantageously used 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. Constitutive promoters may be modified so as
to be regulatable, e.g., inducible. The genes and promoters
described hereinabove can be used to identify orthologous genes and
their promoters which are also likely expressed in a particular
tissue and/or development manner. Moreover, the orthologous
promoters are useful to express linked open reading frames. In
addition, by aligning the promoters of these orthologs, novel cis
elements can be identified that are useful to generate synthetic
promoters.
[0299] 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.
[0300] 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.
[0301] 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.
[0302] The transcription regulating 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 NOs:
1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24,
27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73 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).
[0303] 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).
[0304] 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.
[0305] However, this may not always be the case, and the present
invention also encompasses transgenic plants incorporating
non-expressed transgenes.
[0306] 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.
[0307] The nucleotide sequence of interest linked to one or more of
the transcription regulating sequences of the invention can, for
example, code for a ribosomal RNA, an anti-sense 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."
[0308] 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.
[0309] 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.
[0310] 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.
[0311] 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.
[0312] 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.
[0313] 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.
[0314] 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.
[0315] 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.
[0316] In general, the technique of site-specific mutagenesis is
well known in the art, as exemplified by various publications. As
will be appreciated, the technique typically employs a phage vector
which exists in both a single stranded and double stranded form.
Typical vectors useful in site-directed mutagenesis include vectors
such as the M13 phage. These phages are readily commercially
available and their use is generally well known to those skilled in
the art. Double stranded plasmids also are routinely employed in
site directed mutagenesis which eliminates the step of transferring
the gene of interest from a plasmid to a phage.
[0317] 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.
[0318] 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.
[0319] 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.
[0320] 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.
[0321] Functionally equivalent fragments of a transcription
regulating nucleotide sequence of the invention can also be
obtained by removing or deleting non-essential sequences without
deleting the essential one. Narrowing the transcription regulating
nucleotide sequence to its essential, transcription mediating
elements can be realized in vitro by trial-and-arrow deletion
mutations, or in silico using promoter element search routines.
Regions essential for promoter activity often demonstrate clusters
of certain, known promoter elements. Such analysis can be performed
using available computer algorithms such as PLACE ("Plant
Cis-acting Regulatory DNA Elements"; Higo 1999), the B10BASE
database "Transfac" (Biologische Datenbanken GmbH, Braunschweig;
Wingender 2001) or the database PlantCARE (Lescot 2002).
[0322] Preferably, functional equivalent fragments of one of the
transcription regulating 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 NOs: 1, 2, 3, 6, 7, 8,
11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57,
58, 61, 62, 63, 66, 67, 68, 71, 72, or 73. More preferably this
fragment is starting from the 3'-end of the indicated
sequences.
[0323] Especially preferred are equivalent fragments of
transcription regulating 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 sequences of the invention are equivalent
fragments of other sequences (see Table 2 below).
TABLE-US-00010 TABLE 2 Relationship of transcription regulating
sequences of the invention Transcription regulating sequence
Equivalent sequence Equivalent fragment SEQ ID NO: 1 (1034 bp) SEQ
ID NO: 66 (997 bp) SEQ ID NO: 2 (992 bp) SEQ ID NO: 3 (301 bp) SEQ
ID NO: 67 (900 bp) SEQ ID NO: 68 (301 bp) SEQ ID NO: 6 (797 bp) SEQ
ID NO: 7 (766 bp) SEQ ID NO: 8 (301 bp) SEQ ID NO: 11 (1182 bp) SEQ
ID NO: 14 (1270 bp) SEQ ID NO: 12 (1111 bp) SEQ ID NO: 71 (1028 bp)
SEQ ID NO: 13 (301 bp) SEQ ID NO: 15 (1191 bp) SEQ ID NO: 16 (301
bp) SEQ ID NO: 72 (954 bp) SEQ ID NO: 73 (301 bp) SEQ ID NO: 19
(1060 bp) SEQ ID NO: 22 (1093 bp) SEQ ID NO: 20 (946 bp) SEQ ID NO:
56 (1000 bp) SEQ ID NO: 21 (301 bp) SEQ ID NO: 61 (1000 bp) SEQ ID
NO: 23 (948 bp) SEQ ID NO: 24 (301 bp) SEQ ID NO: 57 (945 bp) SEQ
ID NO: 58 (301 bp) SEQ ID NO: 62 (719 bp) SEQ ID NO: 63 (301 bp)
SEQ ID NO: 27 (998 bp) SEQ ID NO: 28 (948 bp) SEQ ID NO: 29 (301
bp)
[0324] 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.
[0325] An expression cassette of the invention may comprise further
regulatory elements. The term in this context is to be understood
in the 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
sequence).
[0326] The expression profile of the expression cassettes of the
invention may be modulated depending on the combination of the
transcription regulating nucleotide sequence with expression
enhancing introns and/or transcriptions termination sequences. This
in a preferred embodiment the expression cassette of the inventions
comprises at least one additional element selected from the group
consisting of
a) 5'-untranslated regions, and b) intron encoding sequences, and
c) transcription termination sequences.
[0327] The intron encoding sequences are preferably encoding an
expression enhancing intron from a monocotyledonous plant. More
preferably the intron sequence is an intron from an ubiquitin,
actin or alcohol dehydrogenase gene. Preferably, this intron is
inserted in the expression construct in the 5'-untranslated region
of the nucleic acid sequence, which should be expressed (i.e.,
between the transcription regulating nucleotide sequence and the
protein coding sequence (open reading frame) or the nucleic acid
sequence to be expressed).
[0328] Preferably, the 5'-untranslated region is from the same gene
as the transcription regulating sequences.
[0329] The transcription terminating sequence preferably also
comprises a sequence inducing polyadenylation. The transcription
terminating sequence may be heterologous with respect to the
transcription regulating nucleotide sequence and/or the nucleic
acid sequence to be expressed, but may also be the natural
transcription regulating nucleotide sequence of the gene of said
transcription regulating nucleotide sequence and/or said nucleic
acid sequence to be expressed. In one preferred embodiment of the
invention the transcription regulating nucleotide sequence is the
natural transcription regulating nucleotide sequence of the gene of
the transcription regulating sequence. Preferably the transcription
termination sequence is selected from the group of sequences
described by SEQ ID NOs: 32, 34, and 35.
[0330] Additional regulatory elements may comprise additional
promoter, minimal promoters, or promoter elements, which may modify
the expression regulating properties. For example the expression
may be made depending on certain stress factors such water stress,
abscisin (Lam 1991) or heat stress (Schoffl 1989). Furthermore
additional promoters or promoter elements may be employed, which
may 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).
[0331] Furthermore, it is contemplated that promoters combining
elements from more than one promoter may be useful. For example,
U.S. Pat. No. 5,491,288 discloses combining a Cauliflower Mosaic
Virus promoter with a histone promoter. Thus, the elements from the
promoters disclosed herein may be combined with elements from other
promoters. Promoters which are useful for plant transgene
expression include those that are inducible, viral, synthetic,
constitutive (Odell 1985), temporally regulated, spatially
regulated, tissue-specific, and spatial-temporally regulated.
[0332] 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.
[0333] 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.
[0334] 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.
[0335] 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.
[0336] 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.
[0337] Especially preferred are the 5'-untranslated region, introns
and the 3'-untranslated region from genes selected from the group
of genes consisting of caffeoyl-CoA-O-methyltransferase genes,
C8,7-sterol isomerase genes, hydroxyproline-rich glycoprotein
(HRGP) genes, lactate dehydrogenase genes, chloroplast protein 12
like genes. More preferably, the 5'-untranslated region, introns
and the 3'-untranslated region utilized in an expression cassette
of the invention is from a plant gene selected from the group of
genes consisting of Oryza sativa caffeoyl-CoA-O-methyltransferase
genes, Oryza sativa C8,7-sterol isomerase genes, Zea may
hydroxyproline-rich glycoprotein (HRGP) genes, Zea mays lactate
dehydrogenase genes, Oryza sativa chloroplast protein 12 like genes
and functional equivalents thereof.
[0338] Most preferred are the 5'-untranslated regions comprised at
the 3'-end of the sequences described by SEQ ID NOs: 1, 6, 11, 14,
19, 22, and 27. Especially preferred are the sequences described by
nucleotide 993 to 1034 of SEQ ID NO: 1, nucleotide 767 to 797 of
SEQ ID NO: 6, nucleotide 1112 to 1182 of SEQ ID NO 11, nucleotide
1192 to 1270 of SEQ ID NO 14, nucleotide 947 to 1060 of SEQ ID NO:
19, nucleotide 949 to 1093 of SEQ ID NO: 22, and nucleotide 949 to
998 of SEQ ID NO: 27.
[0339] The intron encoding sequences is preferably encoding an
expression enhancing intron from a monocotyledonous plant.
Preferably, this intron is inserted in the expression construct in
the 5'-untranslated region of the nucleic acid sequence, which
should be expressed (i.e., between the transcription regulating
nucleotide sequence and the protein coding sequence (open reading
frame) or the nucleic acid sequence to be expressed). Most
preferred as intron sequences are: [0340] a) the introns of the Zea
mays ubiquitin gene, preferably intron I thereof, most preferably
the intron sequence as described by nucleotide 1,082 to 2,091 of
SEQ ID NO: 36, [0341] b) the introns of the rice actin gene,
preferably intron I thereof, most preferably the intron sequence as
described by nucleotide 121 to 568 of the sequence described by
GenBank Acc.-No.: X63830, [0342] c) the introns of the Zea mays
alcohol dehydrogenase (adh) gene, preferably intron 6 thereof, most
preferably the intron sequence as described by nucleotide 3,135 to
3,476 of the sequence described by GenBank Acc.-No.: X04049,
[0343] The transcription terminating sequence preferably also
comprises a sequence inducing polyadenylation. The transcription
terminating sequence may be heterologous with respect to the
transcription regulating nucleotide sequence and/or the nucleic
acid sequence to be expressed, but may also be the natural
transcription regulating nucleotide sequence of the gene of said
transcription regulating nucleotide sequence and/or said nucleic
acid sequence to be expressed. In one preferred embodiment of the
invention the transcription regulating nucleotide sequence is the
natural transcription regulating nucleotide sequence of the gene of
the transcription regulating sequence. Preferred as transcription
termination sequences are the transcription termination sequences
from a plant gene selected from the group of genes consisting of
Oryza sativa caffeoyl-CoA-O-methyltransferase genes, Oryza sativa
C8,7-sterol isomerase genes, Zea may hydroxyproline-rich
glycoprotein (HRGP) genes, Zea mays lactate dehydrogenase genes,
Oryza sativa chloroplast protein 12 like genes and functional
equivalents thereof. Most preferred are the transcription
termination sequence of the Zea mays lactate dehydrogenase gene as
described by SEQ ID NO: 32, of the Oryza sativa
caffeoyl-CoA-O-methyltransferase gene as described by SEQ ID NO:
34, and of the Zea may hydroxyproline-rich glycoprotein (HRGP) gene
as described by SEQ ID NO: 35. By the combination of the
transcription regulating sequences with specific 5'-untranslated
regions, introns, and/or transcription termination sequences it is
possible to modulate the expression specificity, especially tissue
specificity.
TABLE-US-00011 Promoter 5'-UTR Intron Terminator Tissue Specificity
Oryza sativa own Zea mays Own all (constitutive) Caffeoyl-CoA-O-
Ubiquitin methyltransferase Oryza sativa own Zea mays NOS root
(kernel, Caffeoyl-CoA-O- Ubiquitin pollen) methyltransferase Oryza
sativa own Zea mays NOS root, kernel C-8,7-sterol- Ubiquitin
isomerase Zea maize own Zea mays Own root, silk Hydroxy-proline-
Ubiquitin (kernel: rich glyco- embryo) protein (HRGP) Zea maize own
Zea mays NOS or own root, kernel Lactate- Ubiquitin dehydrogenase
Chloroplast own Zea mays NOS Leaf, protein 12 Ubiquitin endosperm
lie protein own: element of the same gene to which the promoter
naturally belongs; NOS: nopaline synthase.
[0344] 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).
[0345] Examples of enhancers include elements from the CaMV 35S
promoter, octopine synthase genes (Ellis et al., 1987), the rice
actin I gene, the maize alcohol dehydrogenase gene (Callis 1987),
the maize shrunken I gene (Vasil 1989), TMV Omega element (Gallie
1989) and promoters from non-plant eukaryotes (e.g. yeast; Ma
1988). Vectors for use in accordance with the present invention may
be constructed to include the ocs enhancer element. This element
was first identified as a 16 bp palindromic enhancer from the
octopine synthase (ocs) gene of ultilane (Ellis 1987), and is
present in at least 10 other promoters (Bouchez 1989). The use of
an enhancer element, such as the ocs elements and particularly
multiple copies of the element, will act to increase the level of
transcription from adjacent promoters when applied in the context
of plant transformation.
[0346] 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).
[0347] Ultimately, the most desirable DNA segments for introduction
into, for example, a monocotyledonous 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
constitutive manner or a root/kernel-preferential or
root/kernel-specific manner.
[0348] 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.
[0349] 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.
[0350] By facilitating the transport of the protein into
compartments inside and outside the cell, these sequences may
increase the accumulation of gene product protecting them from
proteolytic degradation. These sequences also allow for additional
mRNA sequences from highly expressed genes to be attached to the
coding sequence of the genes. Since mRNA being translated by
ribosomes is more stable than naked mRNA, the presence of
translatable mRNA in front of the gene may increase the overall
stability of the mRNA transcript from the gene and thereby increase
synthesis of the gene product. Since transit and signal sequences
are usually post-translationally removed from the initial
translation product, the use of these sequences allows for the
addition of extra translated sequences that may not appear on the
final polypeptide. Targeting of certain proteins may be desirable
in order to enhance the stability of the protein (U.S. Pat. No.
5,545,818).
[0351] It may be useful to target DNA itself within a cell. For
example, it may be useful to target introduced DNA to the nucleus
as this may increase the frequency of transformation. Within the
nucleus itself it would be useful to target a gene in order to
achieve site-specific integration. For example, it would be useful
to have a gene introduced through transformation replace an
existing gene in the cell. Other elements include those that can be
regulated by endogenous or exogenous agents, e.g., by zinc finger
proteins, including naturally occurring zinc finger proteins or
chimeric zinc finger proteins (see, e.g., U.S. Pat. No. 5,789,538,
WO 99/48909; WO 99/45132; WO 98/53060; WO 98/53057; WO 98/53058; WO
00/23464; WO 95/19431; and WO 98/54311) or myb-like transcription
factors. For example, a chimeric zinc finger protein may include
amino acid sequences which bind to a specific DNA sequence (the
zinc finger) and amino acid sequences that activate (e.g., GAL 4
sequences) or repress the transcription of the sequences linked to
the specific DNA sequence.
[0352] 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.
[0353] 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.
[0354] 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.
[0355] 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.
[0356] 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.
[0357] 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.
[0358] 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
[0359] The transcription regulating sequences of the invention are
especially useful for expression (preferably constitutive or
root/kernel-preferential or root/kernel-specific expression) in
monocotyledonous plants (as defined above in the DEFINITION
section), especially in cereal plants such as corn, rice, wheat,
rye, barley and oats. However, a use in other plants (e.g.,
dicotyledonous or gymnosperm plants) and other tissues cannot be
ruled out.
[0360] The transcription regulating nucleotide sequences and
expression cassettes of the invention may be employed for numerous
expression purposes such as for example expression of a protein, or
expression of an antisense RNA, sense or double-stranded RNA.
Preferably, expression of the nucleic acid sequence confers to the
plant an agronomically valuable trait.
1.1. Herbicide Resistance
[0361] The genes encoding phosphinothricin acetyltransferase (bar
and pat), glyphosate tolerant EPSP synthase genes, the glyphosate
degradative enzyme gene gox encoding glyphosate oxidoreductase, deh
(encoding a dehalogenase enzyme that inactivates dalapon),
herbicide resistant (e.g., sulfonylurea and imidazolinone)
acetolactate synthase, and bxn genes (encoding a nitrilase enzyme
that degrades bromoxynil) are good examples of herbicide resistant
genes for use in transformation. The bar and pat genes code for an
enzyme, phosphinothricin acetyltransferase (PAT), which inactivates
the herbicide phosphinothricin and prevents this compound from
inhibiting glutamine synthetase enzymes. The enzyme
5-enolpyruvylshikimate 3-phosphate synthase (EPSP Synthase), is
normally inhibited by the herbicide N-(phosphonomethyl)glycine
(glyphosate). However, genes are known that encode
glyphosate-resistant EPSP Synthase enzymes. The deh gene encodes
the enzyme dalapon dehalogenase and confers resistance to the
herbicide dalapon. The bxn gene codes for a specific nitrilase
enzyme that converts bromoxynil to a non-herbicidal degradation
product.
1.2 Insect Resistance
[0362] An important aspect of the present invention concerns the
introduction of insect resistance-conferring genes into plants.
Potential insect resistance genes which can be introduced include
Bacillus thuringiensis crystal toxin genes or Bt genes (Watrud
1985). Bt genes may provide resistance to lepidopteran or
coleopteran pests such as European Corn Borer (ECB) and corn
rootworm (CRW). Preferred Bt toxin genes for use in such
embodiments include the CryIA(b) and CryIA(c) genes. Endotoxin
genes from other species of B. thuringiensis which affect insect
growth or development may also be employed in this regard. Protease
inhibitors may also provide insect resistance (Johnson 1989), and
will thus have utility in plant transformation. The use of a
protease inhibitor 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.
[0363] 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.
[0364] 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).
[0365] 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.
[0366] 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.
[0367] 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.
[0368] 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 & Guss, 1972).
[0369] Further genes encoding proteins characterized as having
potential insecticidal activity may also be used as transgenes in
accordance herewith. Such genes include, for example, the cowpea
trypsin inhibitor (CpTI; Hilder 1987) which may be used as a
rootworm deterrent; genes encoding avermectin (Campbell 1989; Ikeda
1987) which may prove particularly useful as a corn rootworm
deterrent; ribosome inactivating protein genes; and even genes that
regulate plant structures. Transgenic maize including anti-insect
antibody genes and genes that code for enzymes that can covert a
non-toxic insecticide (pro-insecticide) applied to the outside of
the plant into an insecticide inside the plant are also
contemplated.
1.3 Environment or Stress Resistance
[0370] Improvement of a plant's ability to tolerate various
environmental stresses such as, but not limited to, drought, excess
moisture, chilling, freezing, high temperature, salt, and oxidative
stress, can also be effected through expression of heterologous, or
overexpression of homologous genes. Benefits may be realized in
terms of increased resistance to freezing temperatures through the
introduction of an "antifreeze" protein such as that of the Winter
Flounder (Cutler 1989) or synthetic gene derivatives thereof.
Improved chilling tolerance may also be conferred through increased
expression of glycerol-3-phosphate acetyltransferase in
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.
[0371] 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).
[0372] Similarly, the efficacy of other metabolites in protecting
either enzyme function (e.g. alanopine or propionic acid) or
membrane integrity (e.g., alanopine) has been documented (Loomis
1989), and therefore expression of gene encoding the biosynthesis
of these compounds can confer drought resistance in a manner
similar to or complimentary to mannitol. Other examples of
naturally occurring metabolites that are osmotically active and/or
provide some direct protective effect during drought and/or
desiccation include sugars and sugar derivatives such as fructose,
erythritol (Coxson 1992), sorbitol, dulcitol (Karsten 1992),
glucosylglycerol (Reed 1984; Erdmann 1992), sucrose, stachyose
(Koster & Leopold 1988; Blackman 1992), ononitol and pinitol
(Vernon & Bohnert 1992), and raffinose (Bernal-Lugo &
Leopold 1992). Other osmotically active solutes which are not
sugars include, but are not limited to, proline and glycine-betaine
(Wyn-Jones and Storey, 1981). Continued canopy growth and increased
reproductive fitness during times of stress can be augmented by
introduction and expression of genes such as those controlling the
osmotically active compounds discussed above and other such
compounds, as represented in one exemplary embodiment by the enzyme
myoinositol-O-methyltransferase.
[0373] It is contemplated that the expression of specific proteins
may also increase drought tolerance. Three classes of Late
Embryogenic Proteins have been assigned based on structural
similarities (see Dure 1989). All three classes of these proteins
have been demonstrated in maturing (i.e., desiccating) seeds.
Within these 3 types of proteins, the Type-II (dehydrin-type) have
generally been implicated in drought and/or desiccation tolerance
in vegetative plant parts (e.g. Mundy and Chua, 1988; Piatkowski
1990; Yamaguchi-Shinozaki 1992). Recently, expression of a Type-III
LEA (HVA-1) in tobacco was found to influence plant height,
maturity and drought tolerance (Fitzpatrick, 1993). Expression of
structural genes from all three groups may therefore confer drought
tolerance. Other types of proteins induced during water stress
include thiol proteases, aldolases and transmembrane transporters
(Guerrero 1990), which may confer various protective and/or
repair-type functions during drought stress. The expression of a
gene that effects lipid biosynthesis and hence membrane composition
can also be useful in conferring drought resistance on the
plant.
[0374] Many genes that improve drought resistance have
complementary modes of action. Thus, combinations of these genes
might have additive and/or synergistic effects in improving drought
resistance in maize. Many of these genes also improve freezing
tolerance (or resistance); the physical stresses incurred during
freezing and drought are similar in nature and may be mitigated in
similar fashion. Benefit may be conferred via constitutive
expression or root/kernel-preferential or root/kernel-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.
[0375] 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).
[0376] Given the overall role of water in determining yield, it is
contemplated that enabling plants to utilize water more
efficiently, through the introduction and expression of novel
genes, will improve overall performance even when soil water
availability is not limiting. By introducing genes that improve the
ability of plants to maximize water usage across a full range of
stresses relating to water availability, yield stability or
consistency of yield performance may be realized.
[0377] Improved protection of the plant to abiotic stress factors
such as drought, heat or chill, can also be achieved--for
example--by overexpressing antifreeze polypeptides from
Myoxocephalus Scorpius (WO 00/00512), Myoxocephalus
octodecemspinosus, the Arabidopsis thaliana transcription activator
CBF1, glutamate dehydrogenases (WO 97/12983, WO 98/11240),
calcium-dependent protein kinase genes (WO 98/26045), calcineurins
(WO 99/05902), casein kinase from yeast (WO 02/052012),
farnesyltransferases (WO 99/06580; Pei Z M et al. (1998) Science
282:287-290), ferritin (Deak M et al. (1999) Nature Biotechnology
17:192-196), oxalate oxidase (WO 99/04013; Dunwell J M (1998)
Biotechn Genet Eng Rev 15:1-32), DREB1A factor ("dehydration
response element 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).
1.4 Disease Resistance
[0378] It is proposed that increased resistance to diseases may be
realized through introduction of genes into plants period. It is
possible to produce resistance to diseases caused, by viruses,
bacteria, fungi, root pathogens, insects and nematodes. It is also
contemplated that control of mycotoxin producing organisms may be
realized through expression of introduced genes.
[0379] 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.
[0380] 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.
[0381] 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.
[0382] Furthermore, a resistance to fungi, insects, nematodes and
diseases, can be achieved by targeted accumulation of certain
metabolites or proteins. Such proteins include but are not limited
to glucosinolates (defense against herbivores), chitinases or
glucanases and other enzymes which destroy the cell wall of
parasites, ribosome-inactivating proteins (RIPs) and other proteins
of the plant resistance and stress reaction as are induced when
plants are wounded or attacked by microbes, or chemically, by, for
example, salicylic acid, jasmonic acid or ethylene, or lysozymes
from nonplant sources such as, for example, T4-lysozyme or lysozyme
from a variety of mammals, insecticidal proteins such as Bacillus
thuringiensis endotoxin, a-amylase inhibitor or protease inhibitors
(cowpea trypsin inhibitor), lectins such as wheatgerm agglutinin,
RNAses or ribozymes. Further examples are nucleic acids which
encode the Trichoderma harzianum chit42 endochitinase (GenBank Acc.
No.: S78423) or the N-hydroxylating, multi-functional cytochrome
P-450 (CYP79) protein from Sorghum bicolor (GenBank Acc. No.:
U32624), or functional equivalents of these. The accumulation of
glucosinolates as protection from pests (Rask L et al. (2000) Plant
Mol Biol 42:93-113; Menard R et al. (1999) Phytochemistry
52:29-35), the expression of Bacillus thuringiensis endotoxins
(Vaeck et al. (1987) Nature 328:33-37) or the protection against
attack by fungi, by expression of chitinases, for example from
beans (Broglie et al. (1991) Science 254:1194-1197), is
advantageous. Resistance to pests such as, for example, the rice
pest Nilaparvata lugens in rice plants can be achieved by
expressing the snowdrop (Galanthus nivalis) lectin agglutinin (Rao
et al. (1998) Plant J 15(4):469-77). The expression of synthetic
cryIA(b) and cryIA(c) genes, which encode lepidoptera-specific
Bacillus thuringiensis D-endotoxins can bring about a resistance to
insect pests in various plants (Goyal R K et al. (2000) Crop
Protection 19(5):307-312). Further target genes which are suitable
for pathogen defense comprise "polygalacturonase-inhibiting
protein" (PGIP), thaumatine, invertase and antimicrobial peptides
such as lactoferrin (Lee T J et al. (2002) J Amer Soc Horticult Sci
127(2):158-164).
1.5 Mycotoxin Reduction/Elimination
[0383] Production of mycotoxins, including aflatoxin and fumonisin,
by fungi associated with plants is a significant factor in
rendering the grain not useful. These fungal organisms do not cause
disease symptoms and/or interfere with the growth of the plant, but
they produce chemicals (mycotoxins) that are toxic to animals.
Inhibition of the growth of these fungi would reduce the synthesis
of these toxic substances and, therefore, reduce grain losses due
to mycotoxin contamination. Novel genes may be introduced into
plants that would inhibit synthesis of the mycotoxin without
interfering with fungal growth. Expression of a novel gene which
encodes an enzyme capable of rendering the mycotoxin nontoxic would
be useful in order to achieve reduced mycotoxin contamination of
grain. The result of any of the above mechanisms would be a reduced
presence of mycotoxins on grain.
[0384] 1.6 Grain Composition or Quality
[0385] Genes may be introduced into plants, particularly
commercially important cereals such as maize, wheat or rice, to
improve the grain for which the cereal is primarily grown. A wide
range of novel transgenic plants produced in this manner may be
envisioned depending on the particular end use of the grain.
[0386] For example, the largest use of maize grain is for feed or
food. Introduction of genes that alter the composition of the grain
may greatly enhance the feed or food value. The primary components
of maize grain are starch, protein, and oil. Each of these primary
components of maize grain may be improved by altering its level or
composition. Several examples may be mentioned for illustrative
purposes but in no way provide an exhaustive list of
possibilities.
[0387] The protein of many cereal grains is suboptimal for feed and
food purposes especially when fed to pigs, poultry, and humans. The
protein is deficient in several amino acids that are essential in
the diet of these species, requiring the addition of supplements to
the grain. Limiting essential amino acids may include lysine,
methionine, tryptophan, threonine, valine, arginine, and histidine.
Some amino acids become limiting only after the grain is
supplemented with other inputs for feed formulations. For example,
when the grain is supplemented with soybean meal to meet lysine
requirements, methionine becomes limiting. The levels of these
essential amino acids in seeds and grain may be elevated by
mechanisms which include, but are not limited to, the introduction
of genes to increase the biosynthesis of the amino acids, decrease
the degradation of the amino acids, increase the storage of the
amino acids in proteins, or increase transport of the amino acids
to the seeds or grain.
[0388] One mechanism for increasing the biosynthesis of the amino
acids is to introduce genes that deregulate the amino acid
biosynthetic pathways such that the plant can no longer adequately
control the levels that are produced. This may be done by
deregulating or bypassing steps in the amino acid biosynthetic
pathway which are normally regulated by levels of the amino acid
end product of the pathway. Examples include the introduction of
genes that encode deregulated versions of the enzymes aspartokinase
or dihydrodipicolinic acid (DHDP)-synthase for increasing lysine
and threonine production, and anthranilate synthase for increasing
tryptophan production. Reduction of the catabolism of the amino
acids may be accomplished by introduction of DNA sequences that
reduce or eliminate the expression of genes encoding enzymes that
catalyse steps in the catabolic pathways such as the enzyme
lysine-ketoglutarate reductase.
[0389] The protein composition of the grain may be altered to
improve the balance of amino acids in a variety of ways including
elevating expression of native proteins, decreasing expression of
those with poor composition, changing the composition of native
proteins, or introducing genes encoding entirely new proteins
possessing superior composition. DNA may be introduced that
decreases the expression of members of the zein family of storage
proteins. This DNA may encode ribozymes or antisense sequences
directed to impairing expression of zein proteins or expression of
regulators of zein expression such as the opaque-2 gene product.
The protein composition of the grain may be modified through the
phenomenon of cosuppression, i.e., inhibition of expression of an
endogenous gene through the expression of an identical structural
gene or gene fragment introduced through transformation (Goring
1991). Additionally, the introduced DNA may encode enzymes which
degrade zeins. The decreases in zein expression that are achieved
may be accompanied by increases in proteins with more desirable
amino acid composition or increases in other major seed
constituents such as starch. Alternatively, a chimeric gene may be
introduced that comprises a coding sequence for a native protein of
adequate amino acid composition such as for one of the globulin
proteins or 10 kD zein of maize and a promoter or other regulatory
sequence designed to elevate expression of said protein. The coding
sequence of said gene may include additional or replacement codons
for essential amino acids. Further, a coding sequence obtained from
another species, or, a partially or completely synthetic sequence
encoding a completely unique peptide sequence designed to enhance
the amino acid composition of the seed may be employed.
[0390] The introduction of genes that alter the oil content of the
grain may be of value. Increases in oil content may result in
increases in metabolizable energy content and density of the seeds
for uses in feed and food. The introduced genes may encode enzymes
that remove or reduce rate-limitations or regulated steps in fatty
acid or lipid biosynthesis. Such genes may include, but are not
limited to, those that encode acetyl-CoA carboxylase,
ACP-acyltransferase, beta-ketoacyl-ACP synthase, plus other well
known fatty acid biosynthetic activities. Other possibilities are
genes that encode proteins that do not possess enzymatic activity
such as acyl carrier protein. Additional examples include
2-acetyltransferase, oleosin pyruvate dehydrogenase complex, acetyl
CoA synthetase, ATP citrate lyase, ADP-glucose pyrophosphorylase
and genes of the carnitine-CoA-acetyl-CoA shuttles. It is
anticipated that expression of genes related to oil biosynthesis
will be targeted to the plastid, using a plastid transit peptide
sequence and preferably expressed in the seed embryo. Genes may be
introduced that alter the balance of fatty acids present in the oil
providing a more healthful or nutritive feedstuff. The introduced
DNA may also encode sequences that block expression of enzymes
involved in fatty acid biosynthesis, altering the proportions of
fatty acids present in the grain such as described below.
[0391] Genes may be introduced that enhance the nutritive value of
the starch component of the grain, for example by increasing the
degree of branching, resulting in improved utilization of the
starch in cows by delaying its metabolism.
[0392] Besides affecting the major constituents of the grain, genes
may be introduced that affect a variety of other nutritive,
processing, or other quality aspects of the grain as used for feed
or food. For example, pigmentation of the grain may be increased or
decreased. Enhancement and stability of yellow pigmentation is
desirable in some animal feeds and may be achieved by introduction
of genes that result in enhanced production of xanthophylls and
carotenes by eliminating rate-limiting steps in their production.
Such genes may encode altered forms of the enzymes phytoene
synthase, phytoene desaturase, or lycopene synthase. Alternatively,
unpigmented white corn is desirable for production of many food
products and may be produced by the introduction of DNA which
blocks or eliminates steps in pigment production pathways.
[0393] Feed or food comprising some cereal grains possesses
insufficient quantities of vitamins and must be supplemented to
provide adequate nutritive value. Introduction of genes that
enhance vitamin biosynthesis in seeds may be envisioned including,
for example, vitamins A, E, B.sub.12, choline, and the like. For
example, maize grain also does not possess sufficient mineral
content for optimal nutritive value. Genes that affect the
accumulation or availability of compounds containing phosphorus,
sulfur, calcium, manganese, zinc, and iron among others would be
valuable. An example may be the introduction of a gene that reduced
phytic acid production or encoded the enzyme phytase which enhances
phytic acid breakdown. These genes would increase levels of
available phosphate in the diet, reducing the need for
supplementation with mineral phosphate.
[0394] Numerous other examples of improvement of cereals for feed
and food purposes might be described. The improvements may not even
necessarily involve the grain, but may, for example, improve the
value of the grain for silage. Introduction of DNA to accomplish
this might include sequences that alter lignin production such as
those that result in the "brown midrib" phenotype associated with
superior feed value for cattle.
[0395] In addition to direct improvements in feed or food value,
genes may also be introduced which improve the processing of grain
and improve the value of the products resulting from the
processing. The primary method of processing certain grains such as
maize is via wetmilling. Maize may be improved though the
expression of novel genes that increase the efficiency and reduce
the cost of processing such as by decreasing steeping time.
[0396] Improving the value of wetmilling products may include
altering the quantity or quality of starch, oil, corn gluten meal,
or the components of corn gluten feed. Elevation of starch may be
achieved through the identification and elimination of rate
limiting steps in starch biosynthesis or by decreasing levels of
the other components of the grain resulting in proportional
increases in starch. An example of the former may be the
introduction of genes encoding ADP-glucose pyrophosphorylase
enzymes with altered regulatory activity or which are expressed at
higher level. Examples of the latter may include selective
inhibitors of, for example, protein or oil biosynthesis expressed
during later stages of kernel development.
[0397] The properties of starch may be beneficially altered by
changing the ratio of amylose to amylopectin, the size of the
starch molecules, or their branching pattern. Through these changes
a broad range of properties may be modified which include, but are
not limited to, changes in gelatinization temperature, heat of
gelatinization, clarity of films and pastes, Theological
properties, and the like. To accomplish these changes in
properties, genes that encode granule-bound or soluble starch
synthase activity or branching enzyme activity may be introduced
alone or combination. DNA such as antisense constructs may also be
used to decrease levels of endogenous activity of these enzymes.
The introduced genes or constructs may possess regulatory sequences
that time their expression to specific intervals in starch
biosynthesis and starch granule development. Furthermore, it may be
advisable to introduce and express genes that result in the in vivo
derivatization, or other modification, of the glucose moieties of
the starch molecule. The covalent attachment of any molecule may be
envisioned, limited only by the existence of enzymes that catalyze
the derivatizations and the accessibility of appropriate substrates
in the starch granule. Examples of important derivations may
include the addition of functional groups such as amines,
carboxyls, or phosphate groups which provide sites for subsequent
in vitro derivatizations or affect starch properties through the
introduction of ionic charges. Examples of other modifications may
include direct changes of the glucose units such as loss of
hydroxyl groups or their oxidation to aldehyde or carboxyl
groups.
[0398] Oil is another product of wetmilling of corn and other
grains, the value of which may be improved by introduction and
expression of genes. The quantity of oil that can be extracted by
wetmilling may be elevated by approaches as described for feed and
food above. Oil properties may also be altered to improve its
performance in the production and use of cooking oil, shortenings,
lubricants or other oil-derived products or improvement of its
health attributes when used in the food-related applications. Novel
fatty acids may also be synthesized which upon extraction can serve
as starting materials for chemical syntheses. The changes in oil
properties may be achieved by altering the type, level, or lipid
arrangement of the fatty acids present in the oil. This in turn may
be accomplished by the addition of genes that encode enzymes that
catalyze the synthesis of novel fatty acids and the lipids
possessing them or by increasing levels of native fatty acids while
possibly reducing levels of precursors. Alternatively DNA sequences
may be introduced which slow or block steps in fatty acid
biosynthesis resulting in the increase in precursor fatty acid
intermediates. Genes that might be added include desaturases,
epoxidases, hydratases, dehydratases, and other enzymes that
catalyze reactions involving fatty acid intermediates.
Representative examples of catalytic steps that might be blocked
include the desaturations from stearic to oleic acid and oleic to
linolenic acid resulting in the respective accumulations of stearic
and oleic acids.
[0399] Improvements in the other major cereal wetmilling products,
gluten meal and gluten feed, may also be achieved by the
introduction of genes to obtain novel plants. Representative
possibilities include but are not limited to those described above
for improvement of food and feed value.
[0400] In addition it may further be considered that the plant be
used for the production or manufacturing of useful biological
compounds that were either not produced at all, or not produced at
the same level, in the plant previously. The novel plants producing
these compounds are made possible by the introduction and
expression of genes by transformation methods. The possibilities
include, but are not limited to, any biological compound which is
presently produced by any organism such as proteins, nucleic acids,
primary and intermediary metabolites, carbohydrate polymers, etc.
The compounds may be produced by the plant, extracted upon harvest
and/or processing, and used for any presently recognized useful
purpose such as pharmaceuticals, fragrances, industrial enzymes to
name a few.
[0401] Further possibilities to exemplify the range of grain traits
or properties potentially encoded by introduced genes in transgenic
plants include grain with less breakage susceptibility for export
purposes or larger grit size when processed by dry milling through
introduction of genes that enhance gamma-zein synthesis, popcorn
with improved popping, quality and expansion volume through genes
that increase pericarp thickness, corn with whiter grain for food
uses though introduction of genes that effectively block expression
of enzymes involved in pigment production pathways, and improved
quality of alcoholic beverages or sweet corn through introduction
of genes which affect flavor such as the shrunken gene (encoding
sucrose synthase) for sweet corn.
1.7 Tuber or Seed Composition or Quality
[0402] Various traits can be advantageously expressed especially in
seeds or tubers to improve composition or quality. Such traits
include but are not limited to: [0403] Expression of metabolic
enzymes for use in the food-and-feed sector, for example of
phytases and cellulases. Especially preferred are nucleic acids
such as the artificial cDNA which encodes a microbial phytase
(GenBank Acc. No.: A19451) or functional equivalents thereof.
[0404] Expression of genes which bring about an accumulation of
fine chemicals such as of tocopherols, tocotrienols or carotenoids.
An example which may be mentioned is phytoene desaturase. Preferred
are nucleic acids which encode the Narcissus pseudonarcissus
photoene desaturase (GenBank Acc. No.: X78815) or functional
equivalents thereof. [0405] Production of nutraceuticals such as,
for example, polyunsaturated fatty acids (for example arachidonic
acid, eicosapentaenoic acid or docosahexaenoic acid) by expression
of fatty acid elongases and/or desaturases, or production of
proteins with improved nutritional value such as, for example, with
a high content of essential amino acids (for example the
high-methionine 2S albumin gene of the brazil nut). Preferred are
nucleic acids which encode the Bertholletia excelsa high-methionine
2S albumin (GenBank Acc. No.: AB044391), the Physcomitrella patens
46-acyl-lipid desaturase (GenBank Acc. No.: AJ222980; Girke et al.
(1998) Plant J 15:39-48), the Mortierella alpina 46-desaturase
(Sakuradani et al. 1999 Gene 238:445-453), the Caenorhabditis
elegans 45-desaturase (Michaelson et al. 1998, FEBS Letters
439:215-218), the Caenorhabditis elegans 45-fatty acid desaturase
(des-5) (GenBank Acc. No.: AF078796), the Mortierella alpina
45-desaturase (Michaelson et al. JBC 273:19055-19059), the
Caenorhabditis elegans 46-elongase (Beaudoin et al. 2000, PNAS
97:6421-6426), the Physcomitrella patens 46-elongase (Zank et al.
2000, Biochemical Society Transactions 28:654-657), or functional
equivalents of these. [0406] Production of high-quality proteins
and enzymes for industrial purposes (for example enzymes, such as
lipases) or as pharmaceuticals (such as, for example, antibodies,
blood clotting factors, interferons, lymphokins, colony stimulation
factor, plasminogen activators, hormones or vaccines, as described
by Hood E E, Jilka J M (1999) Curr Opin Biotechnol 10(4):382-6; Ma
J K, Vine N D (1999) Curr Top Microbiol Immunol 236:275-92). For
example, it has been possible to produce recombinant avidin from
chicken albumen and bacterial .beta.-glucuronidase (GUS) on a large
scale in transgenic maize plants (Hood et al. (1999) Adv Exp Med
Biol 464:127-47. Review). [0407] Obtaining an increased storability
in cells which normally comprise fewer storage proteins or storage
lipids, with the purpose of increasing the yield of these
substances, for example by expression of acetyl-CoA carboxylase.
Preferred nucleic acids are those which encode the Medicago sativa
acetyl-CoA carboxylase (ACCase) (GenBank Acc. No.: L25042), or
functional equivalents thereof. [0408] Reducing levels of
.alpha.-glucan L-type tuber phosphorylase (GLTP) or .alpha.-glucan
H-type tuber phosphorylase (GHTP) enzyme activity preferably within
the potato tuber (see U.S. Pat. No. 5,998,701). The conversion of
starches to sugars in potato tubers, particularly when stored at
temperatures below 7.degree. C., is reduced in tubers exhibiting
reduced GLTP or GHTP enzyme activity. Reducing cold-sweetening in
potatoes allows for potato storage at cooler temperatures,
resulting in prolonged dormancy, reduced incidence of disease, and
increased storage life. Reduction of GLTP or GHTP activity within
the potato tuber may be accomplished by such techniques as
suppression of gene expression using homologous antisense or
double-stranded RNA, the use of co-suppression, regulatory
silencing sequences. A potato plant having improved cold-storage
characteristics, comprising a potato plant transformed with an
expression cassette having a TPT promoter sequence operably linked
to a DNA sequence comprising at least 20 nucleotides of a gene
encoding an .alpha.-glucan phosphorylase selected from the group
consisting of .alpha.-glucan L-type tuber phosphorylase (GLTP) and
.alpha.-glucan H-type phosphorylase (GHTP).
[0409] Further examples of advantageous genes are mentioned for
example in Dunwell J M, Transgenic approaches to crop improvement,
J Exp Bot. 2000; 51 Spec No; pages 487-96.
1.7 Plant Agronomic Characteristics
[0410] Two of the factors determining where plants can be grown are
the average daily temperature during the growing season and the
length of time between frosts. Within the areas where it is
possible to grow a particular plant, there are varying limitations
on the maximal time it is allowed to grow to maturity and be
harvested. The plant to be grown in a particular area is selected
for its ability to mature and dry down to harvestable moisture
content within the required period of time with maximum possible
yield. Therefore, plants of varying maturities are developed for
different growing locations. Apart from the need to dry down
sufficiently to permit harvest is the desirability of having
maximal drying take place in the field to minimize the amount of
energy required for additional drying post-harvest. Also the more
readily the grain can dry down, the more time there is available
for growth and kernel fill. Genes that influence maturity and/or
dry down can be identified and introduced into plant lines using
transformation techniques to create new varieties adapted to
different growing locations or the same growing location but having
improved yield to moisture ratio at harvest. Expression of genes
that are involved in regulation of plant development may be
especially useful, e.g., the liguleless and rough sheath genes that
have been identified in plants.
[0411] 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.
[0412] 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.
1.8 Nutrient Utilization
[0413] 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.
1.9 Male Sterility
[0414] Male sterility is useful in the production of hybrid seed.
It is proposed that male sterility may be produced through
expression of novel genes. For example, it has been shown that
expression of genes that encode proteins that interfere with
development of the male inflorescence and/or gametophyte result in
male sterility. Chimeric ribonuclease genes that express in the
anthers of transgenic tobacco and oilseed rape have been
demonstrated to lead to male sterility (Mariani 1990). For example,
a number of mutations were discovered in maize that confer
cytoplasmic male sterility. One mutation in particular, referred to
as T cytoplasm, also correlates with sensitivity to Southern corn
leaf blight. A DNA sequence, designated TURF-13 (Levings 1990), was
identified that correlates with T cytoplasm. It would be possible
through the introduction of TURF-13 via transformation to separate
male sterility from disease sensitivity. As it is necessary to be
able to restore male fertility for breeding purposes and for grain
production, it is proposed that genes encoding restoration of male
fertility may also be introduced.
1.10. Non-Protein-Expressing Sequences
1.10.1 RNA-Expressing
[0415] 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.
[0416] 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 anti-sense 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.
[0417] Expression of antisense-RNA or double-stranded RNA by one of
the expression cassettes of the invention is especially preferred.
Also expression of sense RNA can be employed for gene silencing
(co-suppression). This RNA is preferably a non-translatable RNA.
Gene regulation by double-stranded RNA ("double-stranded RNA
interference"; dsRNAi) is well known in the art and described for
various organism including plants (e.g., Matzke 2000; Fire A et al
1998; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO
00/44895; WO 00/49035; WO 00/63364).
[0418] Genes may also be constructed or isolated, which when
transcribed produce RNA enzymes, or ribozymes, which can act as
endoribonucleases and catalyze the cleavage of RNA molecules with
selected sequences. The cleavage of selected messenger RNA's can
result in the reduced production of their encoded polypeptide
products. These genes may be used to prepare novel transgenic
plants which possess them. The transgenic plants may possess
reduced levels of polypeptides including but not limited to the
polypeptides cited above that may be affected by antisense RNA.
[0419] 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.
1.10.2 Non-RNA-Expressing
[0420] 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.
[0421] 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.
[0422] 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).
[0423] Further nucleotide sequences of interest that may be
contemplated for use within the scope of the present invention in
operable linkage with the promoter sequences according to the
invention are isolated nucleic acid molecules, e.g., DNA or RNA,
comprising a plant nucleotide sequence according to the invention
comprising an open reading frame that is preferentially expressed
in a specific tissue, i.e., seed-, root, green tissue (leaf and
stem), panicle-, or pollen, or is expressed constitutively.
2. Marker Genes
[0424] 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)).
[0425] Of course, many examples of suitable marker genes are known
to the art and can be employed in the practice of the
invention.
[0426] 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).
[0427] 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.
[0428] 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.
[0429] 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.
[0430] 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
[0431] Various selectable markers are known in the art suitable for
plant transformation. Such markers may include but are not limited
to:
2.1.1 Negative Selection Markers
[0432] Negative selection markers confer a resistance to a biocidal
compound such as a metabolic inhibitor (e.g.,
2-deoxyglucose-6-phosphate, WO 98/45456), antibiotics (e.g.,
kanamycin, G 418, bleomycin or hygromycin) or herbicides (e.g.,
phosphinothricin or glyphosate). Transformed plant material (e.g.,
cells, tissues or plantlets), which express marker genes, are
capable of developing in the presence of concentrations of a
corresponding selection compound (e.g., antibiotic or herbicide)
which suppresses growth of an untransformed wild type tissue.
Especially preferred negative selection markers are those which
confer resistance to herbicides. Examples which may be mentioned
are: [0433] 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. [0434] 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). [0435] Glyphosate.RTM. degrading enzymes
(Glyphosate.RTM. oxidoreductase; gox), [0436] Dalapon.RTM.
inactivating dehalogenases (deh) [0437] sulfonylurea- and/or
imidazolinone-inactivating acetolactate synthases (ahas or ALS; for
example mutated ahas/ALS variants with, for example, the S4, X112,
XA17, and/or Hra mutation (EP-A1 154 204) [0438] Bromoxynil.RTM.
degrading nitrilases (bxn; Stalker 1988) [0439] Kanamycin- or.
geneticin (G418) resistance genes (NPTII; NPT or neo; Potrykus
1985) coding e.g., for neomycin phosphotransferases (Fraley 1983;
Nehra 1994) [0440] 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). [0441]
hygromycin phosphotransferase (HPT), which mediates resistance to
hygromycin (Vanden Elzen 1985). [0442] altered dihydrofolate
reductase (Eichholtz 1987) conferring resistance against
methotrexat (Thillet 1988); [0443] mutated anthranilate synthase
genes that confers resistance to 5-methyl tryptophan.
[0444] Additional negative selectable marker genes of bacterial
origin that confer resistance to antibiotics include the aadA gene,
which confers resistance to the antibiotic spectinomycin,
gentamycin acetyl transferase, streptomycin phosphotransferase
(SPT), aminoglycoside-3-adenyl transferase and the bleomycin
resistance determinant (Hayford 1988; Jones 1987; Svab 1990; Hille
1986).
[0445] 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 dao1 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).
[0446] 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).
[0447] 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.
[0448] 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.
[0449] 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
[0450] Furthermore, positive selection marker can be employed.
Genes like isopentenyltransferase 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
[0451] 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
[0452] 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 form 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).
[0453] 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 carries
dominant .quadrature.ultila 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.
[0454] 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.
[0455] 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
[0456] 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 (e.g., roots and kernel) or is expressed
constitutively, or a promoter thereof.
[0457] 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.
[0458] 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.
[0459] 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).
[0460] 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.
[0461] 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 plant's 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.
[0462] Alternatively, it might be desirable to inhibit expression
of a native DNA sequence within a plant's tissues to achieve a
desired phenotype. In this case, such inhibition might be
accomplished with transformation of the plant to comprise a
constitutive, tissue-independent promoter operably linked to an
antisense nucleotide sequence, such that constitutive expression of
the antisense sequence produces an RNA transcript that interferes
with translation of the mRNA of the native DNA sequence.
[0463] To define a minimal promoter region, a DNA segment
representing the promoter region is removed from the 5' region of
the gene of interest and operably linked to the coding sequence of
a marker (reporter) gene by recombinant DNA techniques well known
to the art. The reporter gene is operably linked downstream of the
promoter, so that transcripts initiating at the promoter proceed
through the reporter gene. Reporter genes generally encode proteins
which are easily measured, including, but not limited to,
chloramphenicol acetyl transferase (CAT), .beta.-glucuronidase
(GUS), green fluorescent protein (GFP), .beta.-galactosidase
(.beta.-GAL), and luciferase.
[0464] 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.
[0465] The level of enzyme activity corresponds to the amount of
enzyme that was made, which in turn reveals the level of expression
from the promoter of interest. This level of expression can be
compared to other promoters to determine the relative strength of
the promoter under study. In order to be sure that the level of
expression is determined by the promoter, rather than by the
stability of the mRNA, the level of the reporter mRNA can be
measured directly, such as by Northern blot analysis.
[0466] Once activity is detected, mutational and/or deletion
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.
[0467] In one embodiment, the promoter may be a gamma zein
promoter, an oleosin ole16 promoter, a globulins promoter, an actin
I promoter, an actin cI 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 tapetum-specific gene promoter, tapetum-specific
gene RAB24 promoter, an a nthranilate synthase alpha subunit
promoter, an alpha zein promoter, an anthranilate synthase beta
subunit promoter, a dihydrodipicolinate synthase promoter, a Thil
promoter, an alcohol dehydrogenase promoter, a cab binding protein
promoter, an H3C4 promoter, a RUBISCO SS starch branching enzyme
promoter, an ACCase promoter, an actin3 promoter, an actin7
promoter, a regulatory protein GF14-12 promoter, a ribosomal
protein L9 promoter, a cellulose biosynthetic enzyme promoter, an
S-adenosyl-L-homocysteine hydrolase promoter, a superoxide
dismutase promoter, a C-kinase receptor promoter, a
phosphoglycerate mutase promoter, a root-specific RCc3 mRNA
promoter, a glucose-6 phosphate isomerase promoter, a
pyrophosphate-fructose 6-phosphatelphosphotransferase promoter, an
ubiquitin promoter, a beta-ketoacyl-ACP synthase promoter, a 33 kDa
photosystem 11 promoter, an oxygen evolving protein promoter, a 69
kDa vacuolar ATPase subunit promoter, a metallothionein-like
protein promoter, a glyceraldehyde-3-phosphate dehydrogenase
promoter, an ABA- and ripening-inducible-like protein promoter, a
phenylalanine ammonia lyase promoter, an adenosine triphosphatase
S-adenosyl-L-homocysteine hydrolase promoter, an a-tubulin
promoter, a cab promoter, a PEPCase promoter, an R gene promoter, a
lectin promoter, a light harvesting complex promoter, a heat shock
protein promoter, a chalcone synthase promoter, a zein promoter, a
globulin-1 promoter, an ABA promoter, an auxin-binding protein
promoter, a UDP glucose flavonoid glycosyl-transferase gene
promoter, an NTI promoter, an actin promoter, an opaque 2 promoter,
a b70 promoter, an oleosin promoter, a CaMV 35S promoter, a CaMV
34S promoter, a CaMV 19S promoter, a histone promoter, a
turgor-inducible promoter, a pea small subunit RuBP carboxylase
promoter, a Ti plasmid mannopine synthase promoter, Ti plasmid
nopaline synthase promoter, a petunia chalcone isomerase promoter,
a bean glycine rich protein I promoter, a CaMV 35S transcript
promoter, a potato patatin promoter, or a S-E9 small subunit RuBP
carboxylase promoter.
4. Transformed (Transgenic) Plants of the Invention and Methods of
Preparation
[0468] 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.
[0469] 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).
[0470] 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 nptII) can be associated with the expression cassette to assist
in breeding.
[0471] Thus, the present invention provides a transformed
(transgenic) plant cell, in planta or ex planta, including a
transformed plastid or other organelle, e.g., nucleus, mitochondria
or chloroplast. The present invention may be used for
transformation of any plant species, including, but not limited to,
cells from the plant species specified above in the DEFINITION
section. Preferably, transgenic plants of the present invention are
crop plants and in particular cereals (for example, corn, alfalfa,
sunflower, rice, Brassica, canola, soybean, barley, soybean,
sugarbeet, cotton, safflower, peanut, sorghum, wheat, millet,
tobacco, 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.
[0472] 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 microorganisms are bacteria, yeast, algae, and fungi.
Preferred bacteria are those of the genus Escherichia, Erwinia,
Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus
or Cyanobacterim such as--for example--Synechocystis and other
bacteria described in Brock Biology of Microorganisms Eighth
Edition (pages A-8, A-9, A10 and A11).
[0473] 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.
[0474] 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.
[0475] 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).
[0476] 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).
[0477] 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.
[0478] 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).
[0479] Those skilled in the art will appreciate that the choice of
method might depend on the type of plant, i.e., monocotyledonous or
dicotyledonous, targeted for transformation. Suitable methods of
transforming plant cells include, but are not limited to,
microinjection (Crossway 1986), electroporation (Riggs 1986),
Agrobacterium-mediated transformation (Hinchee 1988), direct gene
transfer (Paszkowski 1984), and ballistic particle acceleration
using devices available from Agracetus, Inc., Madison, Wis. And
BioRad, Hercules, Calif. (see, for example, U.S. Pat. No.
4,945,050; and McCabe 1988). Also see, Weissinger 1988; Sanford
1987 (onion); Christou 1988 (soybean); McCabe 1988 (soybean); Datta
1990 (rice); Klein 1988 (maize); Klein 1988 (maize); Klein 1988
(maize); Fromm 1990 (maize); and Gordon-Kamm 1990 (maize); Svab
1990 (tobacco chloroplast); Koziel 1993 (maize); Shimamoto 1989
(rice); Christou 1991 (rice); European Patent Application EP 0 332
581 (orchardgrass and other Pooideae); Vasil 1993 (wheat); Weeks
1993 (wheat).
[0480] 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.
[0481] 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.
[0482] Various Agrobacterium strains can be employed, preferably
disarmed Agrobacterium tumefaciens or rhizogenes strains. In a
preferred embodiment, Agrobacterium strains for use in the practice
of the invention include octopine strains, e.g., LBA4404 or
agropine strains, e.g., EHA101 or EHA105. Suitable strains of A.
tumefaciens for DNA transfer are for example EHA101[pEHA101] (Hood
1986), EHA105[pEHA105] (Li 1992), LBA4404[pAL4404] (Hoekema 1983),
C58C1[pMP90] (Koncz & Schell 1986), and C58C1[pGV2260]
(Deblaere 1985). Other suitable strains are Agrobacterium
tumefaciens C58, a nopaline strain. Other suitable strains are A.
tumefaciens C58C1 (Van Larebeke 1974), A136 (Watson 1975) or
LBA4011 (Klapwijk 1980). In another preferred embodiment the
soil-borne bacterium is a disarmed variant of Agrobacterium
rhizogenes strain K599 (NCPPB 2659). Preferably, these strains are
comprising a disarmed plasmid variant of a Ti- or Ri-plasmid
providing the functions required for T-DNA transfer into plant
cells (e.g., the vir genes). In a preferred embodiment, the
Agrobacterium strain used to transform the plant tissue
pre-cultured with the plant phenolic compound contains a
L,L-succinamopine type Ti-plasmid, preferably disarmed, such as
pEHA101. In another preferred embodiment, the Agrobacterium strain
used to transform the plant tissue pre-cultured with the plant
phenolic compound contains an octopine-type Ti-plasmid, preferably
disarmed, such as pAL4404. Generally, when using octopine-type
Ti-plasmids or helper plasmids, it is preferred that the virF gene
be deleted or inactivated (Jarschow 1991).
[0483] 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).
[0484] 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).
[0485] 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. 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 bacteria are resuspended in a plant
compatible co-cultivation medium. Supplementation of the co-culture
medium with anti-oxidants (e.g., silver nitrate), phenol-absorbing
compounds (like polyvinylpyrrolidone, Perl 1996) or thiol compounds
(e.g., dithiothreitol, L-cysteine, Olhoft 2001) which can decrease
tissue necrosis due to plant defense 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.
[0486] 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.
[0487] 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.
[0488] 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
[0489] 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.
[0490] 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.
[0491] 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.
[0492] 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.
[0493] 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.
[0494] 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. Both PCR and Southern hybridization techniques can be used
to demonstrate transmission of a preselected DNA segment to
progeny. In most instances the characteristic Southern
hybridization pattern for a given transformant will segregate in
progeny as one or more Mendelian genes (Spencer 1992); Laursen
1994) indicating stable inheritance of the gene. The non-chimeric
nature of the callus and the parental transformants (R.sub.0) was
suggested by germline transmission and the identical Southern blot
hybridization patterns and intensities of the transforming DNA in
callus, R.sub.0 plants and R.sub.1 progeny that segregated for the
transformed gene.
[0495] 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 quantification 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.
[0496] 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.
[0497] 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.
[0498] 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.
[0499] 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
[0500] 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.
[0501] 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.
[0502] The transgenic plants produced herein are thus expected to
be useful for a variety of commercial and research purposes.
Transgenic plants can be created for use in traditional agriculture
to possess traits beneficial to the grower (e.g., agronomic traits
such as resistance to water deficit, pest resistance, herbicide
resistance or increased yield), beneficial to the consumer of the
grain harvested from the plant (e.g., improved nutritive content in
human food or animal feed; increased vitamin, amino acid, and
antioxidant content; the production of antibodies (passive
immunization) and nutriceuticals), or beneficial to the food
processor (e.g., improved processing traits). In such uses, the
plants are generally grown for the use of their grain in human or
animal foods. Additionally, the use of root-specific promoters in
transgenic plants can provide beneficial traits that are localized
in the consumable (by animals and humans) roots of plants such as
carrots, parsnips, and beets. However, other parts of the plants,
including stalks, husks, vegetative parts, and the like, may also
have utility, including use as part of animal silage or for
ornamental purposes. Often, chemical constituents (e.g., oils or
starches) of maize and other crops are extracted for foods or
industrial use and transgenic plants may be created which have
enhanced or modified levels of such components.
[0503] 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.
[0504] 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.
[0505] 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.
Sequences
[0506] 1. SEQ ID NO: 1 Nucleic acid sequence encoding the
transcription regulating nucleotide sequence of Oryza sativa (rice)
caffeoyl CoA-O-methyltransferase (Os.CCoAMT1) gene including
5'-untranslated region [0507] 2. SEQ ID NO: 2 Nucleic acid sequence
encoding the transcription regulating nucleotide sequence of Oryza
sativa (rice) caffeoyl CoA-O-methyltransferase (Os.CCoAMT1) gene
[0508] 3. SEQ ID NO: 3 Nucleic acid sequence encoding the core
promoter region of the transcription regulating nucleotide sequence
of Oryza sativa (rice) caffeoyl CoA-O-methyltransferase
(Os.CCoAMT1) gene comprising clusters of promoter elements. [0509]
4. SEQ ID NO: 4 Nucleic acid sequence encoding Oryza sativa (rice)
caffeoyl CoA-O-methyltransferase (Os.CCoAMT1) [0510] 5. SEQ ID NO:
5 Amino acid sequence encoding Oryza sativa (rice) caffeoyl
CoA-O-methyltransferase (Os.CCoAMT1) [0511] 6. SEQ ID NO: 6 Nucleic
acid sequence encoding a transcription regulating nucleotide
sequence from the Oryza sativa (rice) C8,7-sterol isomerase gene
(Os.SI) including the 5' untranslated region of the gene. [0512] 7.
SEQ ID NO: 7 Nucleic acid sequence encoding a transcription
regulating nucleotide sequence from the Oryza sativa (rice)
C8,7-sterol isomerase gene (Os.SI). [0513] 8. SEQ ID NO: 8 Nucleic
acid sequence encoding the core promoter region of the
transcription regulating nucleotide sequence from the Oryza sativa
(rice) C8,7-sterol isomerase gene (Os.SI) comprising clusters of
promoter elements. [0514] 9. SEQ ID NO: 9 Nucleic acid sequence
encoding Oryza sativa (rice) C8,7-sterol isomerase gene (Os.SI)
[0515] 10. SEQ ID NO: 10 Amino acid sequence encoding Oryza sativa
(rice) C8,7-sterol isomerase gene (Os.SI) [0516] 11. SEQ ID NO: 11
Nucleic acid sequence encoding a transcription regulating
nucleotide sequence from a Zea mays hydroxyproline-rich
glycoprotein (HRGP) (Zm.HRGP) including the 5' untranslated region
of the gene. [0517] 12. SEQ ID NO: 12 Nucleic acid sequence
encoding a transcription regulating nucleotide sequence from a Zea
mays hydroxyproline-rich glycoprotein (HRGP) (Zm.HRGP). [0518] 13.
SEQ ID NO: 13 Nucleic acid sequence encoding the core promoter
region of the transcription regulating nucleotide sequence from a
Zea mays hydroxyproline-rich glycoprotein (HRGP) (Zm.HRGP)
comprising clusters of promoter elements. [0519] 14. SEQ ID NO: 14
Nucleic acid sequence encoding a function equivalent of the
transcription regulating nucleotide sequence from a Zea mays
hydroxyproline-rich glycoprotein (HRGP) (Zm.HRGP) including the 5'
untranslated region of the gene. [0520] 15. SEQ ID NO: 15 Nucleic
acid sequence encoding a function equivalent of the transcription
regulating nucleotide sequence from a Zea mays hydroxyproline-rich
glycoprotein (HRGP) (Zm.HRGP). [0521] 16. SEQ ID NO: 16 Nucleic
acid sequence encoding the core promoter region of a function
equivalent of the transcription regulating nucleotide sequence from
a Zea mays hydroxyproline-rich glycoprotein (HRGP) (Zm.HRGP)
comprising clusters of promoter elements. [0522] 17. SEQ ID NO: 17
Nucleic acid sequence encoding the Zea mays hydroxyproline-rich
glycoprotein (HRGP) [0523] 18. SEQ ID NO: 18 Amino acid sequence
encoding the Zea mays hydroxyproline-rich glycoprotein (HRGP)
[0524] 19. SEQ ID NO: 19 Nucleic acid sequence encoding a
transcription regulating nucleotide sequence from a Zea mays
lactate dehydrogenase (Zm.LDH) including the 5' untranslated region
of the gene. [0525] 20. SEQ ID NO: 20 Nucleic acid sequence
encoding a transcription regulating nucleotide sequence from a Zea
mays lactate dehydrogenase (Zm.LDH). [0526] 21. SEQ ID NO: 21
Nucleic acid sequence encoding the core promoter region of the
transcription regulating nucleotide sequence from a Zea mays
lactate dehydrogenase (Zm.LDH) comprising clusters of promoter
elements. [0527] 22. SEQ ID NO: 22 Nucleic acid sequence encoding a
function equivalent of the transcription regulating nucleotide
sequence from a Zea mays lactate dehydrogenase (Zm.LDH) including
the 5' untranslated region of the gene. [0528] 23. SEQ ID NO: 23
Nucleic acid sequence encoding a function equivalent of the
transcription regulating nucleotide sequence from a Zea mays
lactate dehydrogenase (Zm.LDH). [0529] 24. SEQ ID NO: 24 Nucleic
acid sequence encoding the core promoter region of a function
equivalent of the transcription regulating nucleotide sequence from
a Zea mays lactate dehydrogenase (Zm.LDH) comprising clusters of
promoter elements. [0530] 25. SEQ ID NO: 25 Nucleic acid sequence
encoding the Zea mays lactate dehydrogenase (Zm.LDH) [0531] 26. SEQ
ID NO: 26 Amino acid sequence encoding the Zea mays lactate
dehydrogenase (Zm.LDH) [0532] 27. SEQ ID NO: 27 Nucleic acid
sequence encoding a function equivalent of the transcription
regulating nucleotide sequence from an Oryza sativa (rice)
choroplast 12 (CP12) protein including the 5' untranslated region
of the gene. [0533] 28. SEQ ID NO: 28 Nucleic acid sequence
encoding a function equivalent of the transcription regulating
nucleotide sequence from an Oryza sativa (rice) choroplast 12
(CP12) protein. [0534] 29. SEQ ID NO: 29 Nucleic acid sequence
encoding the core promoter region of a function equivalent of the
transcription regulating nucleotide sequence from an Oryza sativa
(rice) choroplast 12 (CP12) protein comprising clusters of promoter
elements. [0535] 30. SEQ ID NO: 30 Nucleic acid sequence encoding
the Oryza sativa (rice) choroplast 12 (CP12) protein. [0536] 31.
SEQ ID NO: 31 Amino acid sequence encoding the Oryza sativa (rice)
choroplast 12 (CP12) protein. [0537] 32. SEQ ID NO: 32 Nucleic acid
sequence encoding the intergenic sequence comprising
3'-untranslated region of Zea mays lactate dehydrogenase gene with
the transcription termination and polyadenylation sequence. [0538]
33. SEQ ID NO: 33 Nucleic acid sequence encoding construct
pBPSMM304 [Os.CP12 promoter::Zm.ubiquitin intron::GUS (PIV2)::NOS
terminator] [0539] 34. SEQ ID NO: 34 Nucleic acid sequence encoding
the intergenic sequence including the 3' untranslated region of
caffeoyl CoA-O-methyltransferase with the transcription termination
and polyadenylation sequence. [0540] 35. SEQ ID NO: 35 Nucleic acid
sequence encoding the intergenic sequence including the 3'
untranslated region of hydroxyproline-rich glyco-protein gene with
the transcription termination and polyadenylation sequence. [0541]
36. SEQ ID NO: 36 Nucleic acid sequence encoding construct pBPS325
[Os.CCoAMT1 promoter::Zm.ubiquitin intron::GUS (PIV2)::Os.CCoAMT1
terminator] [0542] 37. SEQ ID NO: 37 Nucleic acid sequence encoding
construct pBPS331 [Os.SI promoter::Zm.ubiquitin intron::GUS
(PIV2)::NOS terminator] [0543] 38. SEQ ID NO: 38 Nucleic acid
sequence encoding construct pBPSET003 [Zm.HRGP
promoter::Zm.ubiquitin intron::GUS (PIV2)::Zm.HRGP terminator]
[0544] 39. SEQ ID NO: 39 Nucleic acid sequence encoding construct
pBPSET007 [Zm.LDH promoter::Zm.ubiquitin intron::GUS (PIV2)::Zm.LDH
terminator]
TABLE-US-00012 [0544] 40. Forward Primer Os.CCoAMT1 promoter-5' SEQ
ID NO: 40 5'-CAACTACTGCACGGTAAAAGTGATAGG-3' 41. Reverse primer
Os.CCoAMT1 promoter-3' SEQ ID NO: 41
5'-GCAGCTTGCTTCGATCTCTCGCTCGCC-3' 42. Forward Primer Os.CCoAMT1
3'UTR-5' SEQ ID NO: 42 5'-GCCGATGCCCAAGAACTAGTCATTTTAA-3' 43
Reverse primer Os.CCoAMT1 3'UTR-3' SEQ ID NO: 43
5'-ATTAACACGTCAACCAAACCGCCGTCC-3' 44 Forward Primer Os.SI
promoter-5' SEQ ID NO: 44 5'-TGCCTCGATTCGACCGTGTAATGGAAT-3' 45.
Reverse primer Os.SI promoter-3' SEQ ID NO: 45
5'-ACTCCTGGCTTCCTTCCGATCTGGACT-3' 46. Forward Primer Zm.HRGP
promoter-5' SEQ ID NO: 46 5'-CCGGTGACCTTCTTGCTTCTTCGATCG-3' 47.
Reverse primer Zm.HRGP promoter-3' SEQ ID NO: 47
5'-CCTCTCTCTCACACACACTCTCAGTAA-3' 48. Forward primer ZmLDH
promoter-5' SEQ ID NO: 48 5'-AACAAATGGCGTACTTATATAACCACA-3' 49.
Reverse primer ZmLDH promoter-3' SEQ ID NO: 49
5'-CGGGCGGAATGGGATGGGATTACGTGT-3' 50. Forward primer Zm.HRGP
3'UTR-5' SEQ ID NO: 50 5'-AAAGCGATGCCTACCATACCACACTGC-3' 51.
Reverse primer Zm.HRGP 3'UTR-3' SEQ ID NO: 51
5'-TGCCCACATTTATTATGGTTTTACACCC-3' 52. Forward Primer Zm.LDH
3'UTR-5' SEQ ID NO: 52 5'-TGATCACATCACCGTCTCTCTTCATTAA-3' 53.
Reverse primer Zm.LDH 3'UTR-3' SEQ ID NO: 53
5'-TATCCCAGTCTCGATATTGTCATCCGCT-3' 54. Forward primer Os.CP12-p FP
SEQ ID NO: 54 5'-TTTGTATTTAGGTCCCTAACGCCCTC-3' 55. Reverse primer
Os.CP12-p RP SEQ ID NO: 55 5'-TGTTGATGCGGATTTCTGCGTGTGAT-3'
[0545] 56. SEQ ID NO: 56 Nucleic acid sequence encoding a
transcription regulating nucleotide sequence from a Oryza sativa
lactate dehydrogenase (Os.LDH) including the 5' untranslated region
of the gene. [0546] 57. SEQ ID NO: 57 Nucleic acid sequence
encoding a transcription regulating nucleotide sequence from a
Oryza sativa lactate dehydrogenase (Os.LDH). [0547] 58. SEQ ID NO:
58 Nucleic acid sequence encoding the core promoter region of the
transcription regulating nucleotide sequence from a Oryza sativa
lactate dehydrogenase (Os.LDH) comprising clusters of promoter
elements. [0548] 59. SEQ ID NO: 59 Nucleic acid sequence encoding a
Oryza sativa lactate dehydrogenase (Os.LDH) [0549] 60. SEQ ID NO:
61 Amino acid sequence encoding a Oryza sativa lactate
dehydrogenase (Os.LDH) [0550] 61. SEQ ID NO: 61 Nucleic acid
sequence encoding a transcription regulating nucleotide sequence
from a Oryza sativa lactate dehydrogenase (Os.LDH) including the 5'
untranslated region of the gene. [0551] 62. SEQ ID NO: 62 Nucleic
acid sequence encoding a transcription regulating nucleotide
sequence from a Oryza sativa lactate dehydrogenase (Os.LDH). [0552]
63. SEQ ID NO: 63 Nucleic acid sequence encoding the core promoter
region of the transcription regulating nucleotide sequence from a
Oryza sativa lactate dehydrogenase (Os.LDH) comprising clusters of
promoter elements. [0553] 64. SEQ ID NO: 64 Nucleic acid sequence
encoding a Oryza sativa lactate dehydrogenase (Os.LDH) [0554] 65.
SEQ ID NO: 65 Amino acid sequence encoding a Oryza sativa lactate
dehydrogenase (Os.LDH) [0555] 66. SEQ ID NO: 66 Nucleic acid
sequence encoding the transcription regulating nucleotide sequence
of Zea mays caffeoyl CoA-O-methyltransferase (Zm.CCoAMT1) gene
including 5'-untranslated region [0556] 67. SEQ ID NO: 67 Nucleic
acid sequence encoding the transcription regulating nucleotide
sequence of Zea mays caffeoyl CoA-O-methyltransferase (Zm.CCoAMT1)
gene [0557] 68. SEQ ID NO: 68 Nucleic acid sequence encoding the
core promoter region of the transcription regulating nucleotide
sequence of Zea mays caffeoyl CoA-O-methyltransferase (Zm.CCoAMT1)
gene comprising clusters of promoter elements. [0558] 69. SEQ ID
NO: 69 Nucleic acid sequence encoding Zea mays caffeoyl
CoA-O-methyltransferase (Zm.CCoAMT1) [0559] 70. SEQ ID NO: 70 Amino
acid sequence encoding Zea mays caffeoyl CoA-O-methyltransferase
(Zm.CCoAMT1) [0560] 71. SEQ ID NO: 71 Nucleic acid sequence
encoding a function equivalent of the transcription regulating
nucleotide sequence from a Zea diploperennis hydroxyproline-rich
glycoprotein (HRGP) including the 5' untranslated region of the
gene. [0561] 72. SEQ ID NO: 72 Nucleic acid sequence encoding a
function equivalent of the transcription regulating nucleotide
sequence from a Zea diploperennis hydroxyproline-rich glycoprotein
(HRGP). [0562] 73. SEQ ID NO: 73 Nucleic acid sequence encoding the
core promoter region of a function equivalent of the transcription
regulating nucleotide sequence from a Zea diploperennis
hydroxyproline-rich glycoprotein (HRGP) comprising clusters of
promoter elements. [0563] 74. SEQ ID NO: 74 Nucleic acid sequence
encoding the Zea diploperennis hydroxyproline-rich glycoprotein
(HRGP) [0564] 75. SEQ ID NO: 75 Amino acid sequence encoding the
Zea diploperennis hydroxyproline-rich glycoprotein (HRGP) [0565]
76. SEQ ID NO: 76-84 Amino acid sequence motif of a
monocotyledonous plant lactate dehydrogenase protein [0566] 77. SEQ
ID NO: 85-90 Amino acid sequence motif of a monocotyledonous plant
caffeoyl-CaA-O-methyltransferase protein
TABLE-US-00013 [0566] 78. Oligonucleotide primer GUS-forward: SEQ
ID NO: 91 5'-ttacgtggcaaaggattcgat-3' 79. Oligonucleotide primer
GUS-reverse: SEQ ID NO: 92 5'-gccccaatccagtccattaa-3 80.
Oligonucleotide primer Control gene forward SEQ ID NO: 93
5'-tctgccttgcccttgctt-3' 81. Oligonucleotide primer Control gene
reverse SEQ ID NO: 94 5'-caattgcttggcaggtatattt-3'
EXAMPLES
Materials and General Methods
[0567] Unless indicated otherwise, chemicals and reagents in the
Examples were obtained from Sigma Chemical Company (St. Louis,
Mo.), restriction endonucleases were from New England Biolabs
(Beverly, Mass.) or Roche (Indianapolis, Ind.), oligonucleotides
were synthesized by MWG Biotech Inc. (High Point, N.C.), and other
modifying enzymes or kits regarding biochemicals and molecular
biological assays were from Clontech (Palo Alto, Calif.), Pharmacia
Biotech (Piscataway, N.J.), Promega Corporation (Madison, Wis.), or
Stratagene (La Jolla, Calif.). Materials for cell culture media
were obtained from Gibco/BRL (Gaithersburg, Md.) or DIFCO (Detroit,
Mich.). The cloning steps carried out for the purposes of the
present invention, such as, for example, restriction cleavages,
agarose gel electrophoresis, purification of DNA fragments,
transfer of nucleic acids to nitrocellulose and nylon membranes,
linking DNA fragments, transformation of E. coli cells, growing
bacteria, multiplying phages and sequence analysis of recombinant
DNA, are carried out as described by Sambrook (1989). The
sequencing of recombinant DNA molecules is carried out using ABI
laser fluorescence DNA sequencer following the method of Sanger
(Sanger 1977).
[0568] For generating transgenic 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 a desiccator 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
Isolation of Promoters
[0569] Genomic DNA from maize and rice is extracted using the
Qiagen DNAeasy Plant Mini Kit (Qiagen). The promoter regions were
isolated from genomic DNA using conventional PCR. Approximately 0.1
.mu.g of digested genomic DNA was uses for the regular PCR reaction
(see below). The primers were designed based on the maize or rice
genomic DNA sequences upstream of the EST candidates, maize genomic
sequences, or promoter sequences disclosed in the public database
(e.g. rice caffeoyl CoA-O-methyltransferase [CCoAMT1], GenBank
accession number AB023482; rice unknown protein, GenBank accession
number AP002818; maize hydroxyproline-rich glycoprotein [HRGP],
GenBank accession number AJ131535; maize lactate dehydrogenase
[LDH], GenBank accession number Z11754). One .mu.L of the diluted
digested genomic DNA was used as the DNA template in the primary
PCR reaction. The reaction comprised forward (5') and reverse (3')
primers in a mixture containing Buffer 3 following the protocol
outlined by an Expand Long PCR kit (Cat #1681-842, Roche-Boehringer
Mannheim). The isolated DNA is employed as template DNA in a PCR
amplification reaction using the following primers:
TABLE-US-00014 TABLE 3 Primer sequences for isolation of the
promoter or terminator region Promoter or Size Primer Sequences
Terminator* (bp) Forward Primer (F) & Reverse Primer (R) Oryza
sativa 1,035 F: 5'-CAACTACTGCACGGTAAAAGTGATAGG-3' Caffeoyl-CoA-O-
(SEQ ID NO: 40) methyltransferase R:
5'-GCAGCTTGCTTCGATCTCTCGCTCGCC-3' Promoter (SEQ ID NO: 41)
(Os.CCoAMT1-p) Oryza sativa 1,092 FP:
5'-GCCGATGCCCAAGAACTAGTCATTTTA-3' Caffeoyl-CoA-O- (SEQ ID NO: 42)
methyltransferase RP: 5'-ATTAACACGTCAACCAAACCGCCGTCC-3' Terminator
(SEQ ID NO: 43) (Os.CCoAMT1-t) Oryza sativa 813 FP:
5'-TGCCTCGATTCGACCGTGTAATGGAAT-3' C-8,7-sterol (SEQ ID NO: 44)
isomerase RP: 5'-ACTCCTGGCTTCCTTCCGATCTGGACT-3' Promoter (SEQ ID
NO: 45) (Os.SI-p) Zea maize 1,263 FP:
5'-CCGGTGACCTTCTTGCTTCTTCGATCG-3' Hydroxyproline-rich (SEQ ID NO:
46) glycoprotein RP: 5'-CCTCTCTCTCACACACACTCTCAGTAA-3' Promoter
(SEQ ID NO: 47) (Zm.HRGP-p) Zea maize 541 FP:
5'-AAAGCGATGCCTACCATACCACACTGC-3' Hydroxyproline-rich (SEQ ID NO:
50) glycoprotein RP: 5'-TGCCCACATTTATTATGGTTTTACACCC-3' Terminator
(SEQ ID NO: 51) (Zm.HRGP-t) Zea maize 1,061 FP:
5'-AACAAATGGCGTACTTATATAACCACA-3' Lactate- (SEQ ID NO: 48)
dehydrogenase RP: 5'-CGGGCGGAATGGGATGGGATTACGTGT-3' promoter (SEQ
ID NO: 49) (Zm.LDH-p) Zea maize 475 FP:
5'-TGATCACATCACCGTCTCTCTTCATTAA-3' Lactate- (SEQ ID NO: 52)
dehydrogenase RP: 5'-TATCCCAGTCTCGATATTGTCATCCGCT-3' terminator
(SEQ ID NO: 53) (Zm.LDH-t) Oryza sativa 998 FP:
5'-TTTGTATTTAGGTCCCTAACGCCCTC-3' Chloroplast protein (SEQ ID NO:
54) 12 Promoter RP: 5'-TGTTGATGCGGATTTCTGCGTGTGAT-3' (Os.CP12-p)
(SEQ ID NO: 55) terminator including 3'UTR
[0570] The promoter regions are amplified in the reaction solution
[1.times.PCR reaction buffer (Roche Diagnostics), 5 .mu.L genomic
DNA (corresponds to approximately 80 ng, 2.5 mM of each dATP, dCTP,
dGTP and dTTP (Invitrogen: dNTP mix), 1 .mu.L 5' primer (100 .mu.M)
1 .mu.L 3' primer (100 .mu.M), 1 .mu.L Taq DNA polymerase 5 U/4
(Roche Diagnostics), in a final volume of 100 .mu.L under the
optimized PCR thermocycler program (T3 Thermocycler Biometra; 1
cycle with 180 sec at 95.degree. C., 30 cycles with 40 sec at
95.degree. C., 60 sec at 53.degree. C. and 2 min at 72.degree. C.,
and 1 cycle with 5 min at 72.degree. C. before stop the reaction at
4.degree. C.).
[0571] The PCR product was applied to a 1% (w/v) agarose gel and
separated at 80V followed by excising from the gel and purified
with the aid of the Qiagen Gel Extraction Kit (Qiagen, Hilden,
Germany). If appropriate, the eluate of 50 .mu.L can be evaporated.
The PCR product was cloned directly into vector pCR4-TOPO
(Invitrogen) following the manufacturer's instructions, i.e. the
PCR product obtained is inserted into a vector having T overhangs
with its A overhangs and a topoisomerase.
Example 2
Isolation of Terminator of Interest Including the 3' Untranslated
Region
[0572] Rice genomic DNA fragment (1,092 bp) containing the 3'
untranslated region of caffeoyl CoA-O-methyltransferase
(Os.CCoAMT1) was isolated using sequence specific primers based on
the sequences that disclosed in the public database (GenBank
accession number AB023482). The protocols for plant genomic DNA
isolation and conventional PCR amplification was described in the
Example 1.
TABLE-US-00015 Forward Primer OsCCoAMT1 3'UTR-5': (SEQ ID NO: 42)
5'-GCCGATGCCCAAGAACTAGTCATTTTA-3' Reverse primer OsCCoAMT1
3'UTR-3': (SEQ ID NO: 43) 5'-ATTAACACGTCAACCAAACCGCCGTCC-3'
[0573] SacI for the forward primer and PmeI for the reverse primer
were added to the sequence-specific primers for the further cloning
purpose. (The illustrated primer sequences do not include
restriction enzyme sites.)
[0574] Rice genomic DNA fragment, 519 bp or 473 bp, containing the
3' untranslated region of HRGP or LDH gene was isolated,
respectively using sequence specific primers based on the sequences
that disclosed in the public database (GenBank accession number
AJ131535; Z11754). The protocols for plant genomic DNA isolation
and conventional PCR amplification using sequence specific primers
was described in the Example 1.
[0575] SacI for the forward primer and PmeI for the reverse primer
were added to the sequence-specific primers for the further cloning
purpose. (The illustrated primer sequences do not include
restriction enzyme sites.)
Example 3
Vector Construction
3.1 pUC Based Vector (Promoter of Interest::Intron (IME)::GUS::NOS
or Terminator of Interest)
[0576] The base vector to which the intron candidates were cloned
in was pBPSMM270 at BglI and XmaI. This vector comprises multiple
cloning sites (MCS) followed by Zm.ubiquitin intron, the GUSint ORF
(including the potato invertase [PIV]2 intron to prevent bacterial
expression), and nopaline synthase (NOS) terminator in order (5' to
3'). Maize ubiquitin intron can be replaced with an intron of
interest that functions in intron-mediated enhancement.
[0577] The PCR fragment containing terminator of interest (e.g.
1,092 bp rice genomic DNA including CCoAMT1 terminator; 558 bp
maize genomic DNA including HRGP terminator, 477 bp maize genomic
DNA including LDH terminator) was digested with SacI and PmeI
enzymes. Nopaline synthase terminator region in pBPSMM270 was
replaced with the CCoAMT1 terminator, HRGP terminator or LDH
terminator resulting in pBPSMM270a, pBPSMM270-HRGP3' or
pBPSMM270-LDH3', respectively.
[0578] The genomic DNA fragment containing Os.CCoAMT1 or Os.SI
promoter in the Topo vector (Invitrogen) was digested with PacI and
AscI followed by subcloned upstream of the Zm.ubiquitin intron in
pBPSMM270, respectively.
[0579] The genomic DNA fragment containing CCoAMT1 promoter in the
Topo vector (Invitrogen) was digested with PacI and AscI followed
by subcloned upstream of the Zm.ubiquitin intron in
pBPSMM270-CCoAMT1 3', respectively.
[0580] The genomic DNA fragment containing Zm.HRGP or Zm.LDH
promoter in the Topo vector (Invitrogen) was digested with PacI and
AscI followed by subcloned upstream of the Zm.ubiquitin intron in
pBPSMM270-HRGP3' or pBPSMM270-LDH3', respectively.
3.2 Transformation Binary Vector (Promoter of Interest::Intron
(IME)::GUS::NOS or Terminator of Interest)
[0581] The GUS chimeric cassette (Os.CCoAMT1 promoter::Zm.ubiquitin
intron::GUS (PIV2)::CCoAMT1 terminator, Zm.CCoAMT1
promoter::Zm.ubiquitin intron::GUS (PIV2)::NOS, Os.SI
promoter::Zm.ubiquitin intron::GUS (PIV2)::NOS, Zm.HRGP
promoter::Zm.ubiquitin intron::GUS (PIV2)::Zm.HRGP terminator, or
Zm.LDH promoter::Zm.ubiquitin intron::GUS (PIV2)::Zm.LDH
terminator) in pUC-based vector were digested with AscI or PacI
(5') and PmeI (3') and subcloned into a monocot binary vector
containing a plant selectable marker cassette (pBPSMM344) at AscI
or PacI (5') and PmlI (3') sites to generate pBPSMM325, pBPSMM271,
pBPSMM331, pBPSET003, or pBPSET007, respectively.
Example 4
Agrobacterium-Mediated Transformation in Monocotyledonous
Plants
[0582] The Agrobacterium-mediated plant transformation using
standard transformation and regeneration techniques may also be
carried out for the purposes of transforming crop plants (Gelvin
1995; Glick 1993).
[0583] The transformation of maize or other monocotyledonous plants
can be carried out using, for example, a technique described in
U.S. Pat. No. 5,591,616.
[0584] The transformation of plants using particle bombardment,
polyethylene glycol-mediated DNA uptake or via the silicon
carbonate fiber technique is described, for example, by Freeling
& Walbot (1993) "The maize handbook" ISBN 3-540-97826-7,
Springer Verlag New York).
Example 5
Detection of Reporter Gene Expression
[0585] To identify the characteristics of the promoter and the
essential elements of the latter, which bring about its tissue
specificity, it is necessary to place the promoter itself and
various fragments thereof before what is known as a reporter gene,
which allows the determination of the expression activity. An
example, which may be mentioned, is the bacterial
.beta.-glucuronidase (Jefferson 1987a). The .beta.-glucuronidase
activity can be detected in-planta by means of a chromogenic
substrate such as 5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronic
acid in an activity staining (Jefferson 1987b). To study the tissue
specificity, the plant tissue is cut, embedded, stained and
analyzed as described (for example Baumlein 1991b).
[0586] A second assay permits the quantitative determination of the
GUS activity in the tissue studied. For the quantitative activity
determination, MUG (4-methylumbelliferyl-.beta.-D-glucuronide) is
used as substrate for .beta.-glucuronidase, and the MUG is cleaved
into MU (methylumbelliferone) and glucuronic acid.
[0587] To do this, a protein extract of the desired tissue is first
prepared and the substrate of GUS is then added to the extract. The
substrate can be measured fluorimetrically only after the GUS has
been reacted. Samples which are subsequently measured in a
fluorimeter are taken at various points in time. This assay may be
carried out for example with linseed embryos at various
developmental stages (21, 24 or 30 days after flowering). To this
end, in each case one embryo is ground into a powder in a 2 mL
reaction vessel in liquid nitrogen with the aid of a vibration
grinding mill (Type: Retsch MM 2000). After addition of 100 .mu.L
of EGL buffer (0.1 M KPO.sub.4, pH 7.8; 1 mM EDTA; 5% glycerol; 1 M
DTT), the mixture is centrifuged for 10 minutes at 25.degree. C.
and 14,000.times.g. The supernatant is removed and recentrifuged.
Again, the supernatant is transferred to a new reaction vessel and
kept on ice until further use. 25 .mu.L of this protein extract are
treated with 65 .mu.L of EGL buffer (without DTT) and employed in
the GUS assay. 10 .mu.L of the substrate MUG (10 mM
4-methylumbelliferyl-.beta.-D-glucuronide) are now added, the
mixture is vortexed, and 30 .mu.L are removed immediately as zero
value and treated with 470 .mu.L of Stop buffer (0.2 M
Na.sub.2CO.sub.3). This procedure is repeated for all of the
samples at an interval of 30 seconds. The samples taken were stored
in the refrigerator until measured. Further readings were taken
after 1 h and after 2 h. A calibration series which contained
concentrations from 0.1 mM to 10 mM MU (4-methylumbelliferone) was
established for the fluorimetric measurement. If the sample values
were outside these concentrations, less protein extract was
employed (10 .mu.L, 1 .mu.L, 1 .mu.L from a 1:10 dilution), and
shorter intervals were measured (0 h, 30 min, 1 h). The measurement
was carried out at an excitation of 365 nm and an emission of 445
nm in a Fluoroscan II apparatus (Labsystem). As an alternative, the
substrate cleavage can be monitored fluorimetrically under alkaline
conditions (excitation at 365 nm, measurement of the emission at
455 nm; Spectro Fluorimeter BMG Polarstar+) as described in Bustos
(1989). All the samples were subjected to a protein concentration
determination by the method of Bradford (1976), thus allowing an
identification of the promoter activity and promoter strength in
various tissues and plants.
Example 6
Constitutive Expression in Maize 6.1 Rice
CCoAMT1-Promoter::Zmubiquitin-Intron::GUS::CCoAMT1 terminator
(pBPSMM325)
[0588] CCoAMT1 promoter in combination with CCoAMT1 terminator
shows strong constitutive and ubiquitous expression in all tissues
and organs at different developmental stages. Strong ubiquitous
expression can also be detected in in vitro plants.
TABLE-US-00016 TABLE 4 GUS expression controlled by monocot
constitutive promoter candidates Tissues/Developmental Promoter
(GUS expression levels) stages pBPSMM232* pBPSMM247* pBPSMM272
pBPSMM325 3 days after co-cultivation ++++ +++ ++ +++ Leaves at
5-leaf stage +++++ +++++ ++++ ++++ Roots at 5-leaf stage +++++
+++++ ++++ ++++ Leaves at flowering stage +++++ +++++ ++++ +++ Stem
+++ +++ ++ +++ Pre-pollination +++++ +++++ +++ ++ 5 days after
pollination [DAP] +++++ +++ (7 DAP) ++++ (7 DAP) ND 30 DAP +++++
+++++ ++++ ++ Dry seeds ND +++ ND ++ Imbibition/germination +++++
++++ +++ ND *Positive controls as a constitutive promoter
(pBPSMM232 = Zm.ubiquitin promoter::Zm.ubiquitin intron::GUS
(PIV2)::NOS terminator; pBPSMM247 = sugarcane bacilliform virus
promoter::GUS (PIV2) ::NOS terminator); a range of GUS expression
levels measured by histochemical assay (- to +++++), ND: not
determined yet
Example 7
Root and Kernel Preferable Expression in Maize
7.1 Rice Os.CCoAMT1 Promoter::Zm.Ubiquitin Intron::GUS (PIV2)::NOS
Terminator
[0589] Caffeoyl-CoA-O-methyltransferase (CCoAMT1)
promoter::ubiquitin-intron::NOS terminator (pBPSMM271) showed low
expression in leaves and stem of T1 plants but strong expression in
roots. GUS stain was also detected in kernel and pollen.
7.2 Rice SI Promoter::Zm.Ubiquitin Intron::GUS (PIV2)::NOS
Terminator
[0590] OsC-8,7-sterol-isomerase promoter::Zm.ubiquitinintron::NOS
terminator (pBPSMM331) showed weak expression in most parts of the
plants but good expression in roots and kernels.
7.3 Maize HRGP Promoter::Zm.Ubiquitin Intron::GUS (PIV2)::HRGP
Terminator
[0591] HRGP promoter containing the ubiquitin intron and the HRGP
terminator (pBPSET003) showed no expression in leaves but strong
expression in roots and silk. In kernels expression is
predominantly in the embryo and only weak in the endosperm.
7.4 Maize LDH Promoter::Zm.Ubiquitin Intron::GUS (PIV2)::NOS or LDH
Terminator
[0592] Lactate-dehydrogenase (LDH)
promoter::Zm.ubiquitinintron::NOS or LDH terminator (pBPSMM272 or
pBPSET007, respectively) showed weak expression in leaves but good
expression in roots and kernels.
TABLE-US-00017 TABLE 5 GUS expression controlled by monocot root
and kernel-preferable promoter candidates Promoter (GUS expression
levels) pBPSMM272 Tissues & Developmental or pBP- stages
pBPSMM232* pBPSMM271 pBPSMM331 pBPSET003 SET007 3 days after co-
++++ + ND ND +++ cultivation Leaves at 5-leaf +++++ + + - ++ stage
Roots at 5-leaf +++++ ++++ +++ ++++ ++++ stage Leaves at flowering
+++++ + ++ - ++ stage Stem +++ + ND ND + Pre-pollination +++++ +++
++++ ND +++ 5 days after pollination +++++ +++ ND ND +++ [DAP] 30
DAP ++++ +++ ++ ++ +++ Dry seeds ND ND ND ND ND Imbibition/ +++++
+++ ND ND +++ germination *positive control as a constitutive
promoter (pBPSMM232 = Zm.ubiquitin promoter::Zm.ubiquitin
intron::GUS (PIV2)::NOS terminator); a range of GUS expression
levels measured by histochemical assay (- to +++++), ND: not
determined yet
Example 8
Leaf and Endosperm Preferable Expression in Maize
[0593] Os.CP12 promoter::Zm.ubiquitin intron::GUS (PIV2)::NOS
terminator (pBPSMM304) showed strong expression in leaves and
endosperm, but not in roots or embryo.
TABLE-US-00018 TABLE 6 GUS expression controlled by leaf and
endosperm-preferable monocot promoter Promoter (GUS expression
levels) Tissues/Developmental stages pBPSMM232* pBPSMM304 3 days
after co-cultivation ++++ + In vitro leaves +++++ ++++ In vitro
roots +++++ - Leaves +++++ ++++ Roots +++++ - Kernel
pre-pollination +++++ + Kernel 30 DAP - Endosperm +++++ ++++ Kernel
30 DAP - Embryo +++++ - Dry seeds ++++ ND *positive control as a
constitutive promoter (pBPSMM232 = Znn.ubiquitin
promoter::Zm.ubiquitin intron::GUS (PIV2)::NOS terminator); a range
of GUS expression levels measured by histochemical assay (- to
+++++), ND: not determined yet
Example 9
Analysis of Drought-Inducible Expression Using Real Time RT PCR
Analysis
[0594] Young maize plants were grown from seeds in the greenhouse
under standard conditions. When plants had five true leaves (5-leaf
stage) water was withheld from the drought-stress plants while
watering continued for the water control plants. The 0-timepoint is
taken at the last watering day. After that the first time point is
taken when the soil is dry but plants don't show symptoms of
drought yet (approximately 3 days), the second time point is taken
when at least one leaf of every plant shows "rolling" (mild
symptoms, approximately 5 days) and the last time point is taken
when all leaves show "rolling" (severe symptoms, approximately 7
days).
[0595] Expression levels of the reporter gene was measured at the
mRNA levels using a GeneAmp 5700 Sequence Detection System (Applied
Biosystems). Total nucleic acids were extracted from maize leaf
samples taken at various time points during the drought stress
experiments using the Wizard Magnetic 96 DNA plant System kit
(Promega, FF3661). Subsequently, DNA was removed from the samples
using the DNA-free kit (Ambion, #1906). The resulting DNA-free RNA
solution was used in subsequent PCR reactions. The one-batch
RT/quantitative PCR reactions for expression analysis
contained:
TABLE-US-00019 10 .mu.L RNA solution 15 .mu.L SYBR Green master Mix
(2X; Eurogentec #RTSNRT032X-1) 0.15 .mu.L reverse transcriptase +
inhibitor1 mix (Eurogentec #RTSNRT032X-1) 0.6 .mu.L forward primer
(10 pmol/.mu.L) 0.6 .mu.L reverse primer (10 pmol/.mu.L) 3.65 .mu.L
sterile water
[0596] The PCR program was:
1 cycle 48.degree. C. for 30 min (RT reaction) 40 cycles 90.degree.
C. for 10 min; 95.degree. C. for 15 sec and 60.degree. C. for 1
min
[0597] The amplification was followed by a dissociation
protocol:
95.degree. C. for 15 sec, 60.degree. C. for 20 sec 20 min slow ramp
from 60.degree. C. to 95.degree. C.
TABLE-US-00020 TABLE 7 Primer sequences of the GUS gene and control
gene encoding microtubule-associated protein 1 light chain 3 Gene
Primers SEQ ID GUS Fwd: 5'-ttacgtggcaaaggattcgat SEQ ID NO: 91 Rev:
5'-gccccaatccagtccattaa SEQ ID NO: 92 Control Fwd:
5'-tctgccttgcccttgctt SEQ ID gene NO: 93 Rev:
5'-caattgcttggcaggtcttattt SEQ ID NO: 94
[0598] For each timepoint of the drought-stress experiment RNA
solution of each sample was used in two qPCR reactions that were
run at the same time on the same plate. One reaction contained the
GUS primers the second reaction contained primers for an endogenous
control gene from maize. This endogenous control gene shows stable
expression under stress conditions in a variety of tissues.
[0599] The preset baseline and threshold of the GeneAmp 5700
software were used in all experiments to generate raw data (cycle
numbers). In order to normalize the values obtained with the
GeneAmp 5700 Sequence Detection System the values of the endogenous
primer reactions were subtracted from the values of the GUS primer
reactions. The difference in cycle numbers was then used to
calculate the change of expression levels. As templates are
exponentially amplified during PCR one cycle difference equals a
two-fold difference in template levels. Results are shown as an
x-fold induction compared to the O-timepoint that is set to 1.
9.1 Drought-Inducible Expression Controlled by Maize LDH
Promoter
[0600] In T1 plants containing a single copy shows strong
constitutive expression in roots and in kernels. Expression in
leaves and stem at different developmental stages was weak. Upon
drought-stress expression in leaves was induced two-fold compared
to well-watered control.
Example 10
Utilization of Transgenic Crops
[0601] A reporter gene in pBPSMM325 can be replaced with gene of
interest to express in a constitutive and ubiquitous manner, a
reporter gene in pBPSMM271, pBPSMM331, pBPSET003, and pBPSET007 can
be replaced with gene of interest to express mostly in roots and
kernel, a reporter gene in pBPSMM304 can be replaced with gene of
interest to express mostly in leaves and endosperm (e.g., by
antisense or double-stranded RNA), thereby improving--for
example--biomass and/or yield, or tolerant to biotic and abiotic
environmental stresses. The chimeric constructs are transformed
into monocotyledonous plants. Standard methods for transformation
in the art can be used if required. Transformed plants are
regenerated using known methods. Various phenotypes are measured to
determine improvement of biomass, yield, fatty acid composition,
high oil, disease tolerance, or any other phenotypes that link
yield enhancement or stability. Gene expression levels are
determined at different stages of development and at different
generations (T.sub.0 to T.sub.2 plants or further generations).
Results of the evaluation in plants lead to determine appropriate
genes in combination with this promoter to increase yield, improve
disease tolerance, and/or improve abiotic stress tolerance.
Example 11
Expression of Selectable Marker Gene in Monocotyledonous plants
[0602] A reporter gene in pBPSMM325 can be replaced with a
selectable marker gene and transformed into monocotyledonous plants
such as rice, barley, maize, wheat, or ryegrass but is not
restricted to these plant species. Any methods for improving
expression in monocotyledonous plants are applicable such as
addition of intron or exon with intron in 5'UTR either non-spliced
or spliced. Standard methods for transformation in the art can be
used if required. Transformed plants are selected under the
selection agent of interest and regenerated using known methods.
Selection scheme is examined at early developmental stages of
tissues or tissue culture cells. Gene expression levels can be
determined at different stages of development and at different
generations (T.sub.0 to T.sub.2 plants or further generations).
Results of the evaluation in plants lead to determine appropriate
genes in combination with this promoter.
Example 12
Expression of Transgene for Root Vigor in Monocotyledonous
Plants
[0603] A reporter gene in pBPSMM271, pBPSMM331, pBPSET003,
pBPSMM272, and pBPSET007 can be replaced with gene of interest to
express mostly in roots, which affects root architecture and
transformed into monocotyledonous plants such as rice, barley,
maize, wheat, or ryegrass but is not restricted to these plant
species. Any methods for improving expression in monocotyledonous
plants are applicable such as addition of intron or exon with
intron in 5'UTR either non-spliced or spliced. Standard methods for
transformation in the art can be used if required. Transformed
plants are selected under the selection agent of interest and
regenerated using known methods. Selection scheme is examined at
early developmental stages of tissues or tissue culture cells. Gene
expression levels can be determined at different stages of
development and at different generations (T.sub.0 to T.sub.2 plants
or further generations). Results of the evaluation in plants lead
to determine appropriate genes in combination with this
promoter.
Example 13
Expression of Transgene for Feed and Food in Monocotyledonous
Plants
[0604] A reporter gene in pBPSMM271, pBPSMM331, pBPSET003,
pBPSMM272, pBPSET007, and pBPSMM304 can be replaced with gene of
interest to express mostly in kernel, which improve nutrition in
embryo and endosperm and transformed into monocotyledonous plants
such as rice, barley, maize, wheat, or ryegrass but is not
restricted to these plant species. Any methods for improving
expression in monocotyledonous plants are applicable such as
addition of intron or exon with intron in 5'UTR either non-spliced
or spliced. Standard methods for transformation in the art can be
used if required. Transformed plants are selected under the
selection agent of interest and regenerated using known methods.
Selection scheme is examined at early developmental stages of
tissues or tissue culture cells. Gene expression levels can be
determined at different stages of development and at different
generations (T.sub.0 to T.sub.2 plants or further generations).
Results of the evaluation in plants lead to determine appropriate
genes in combination with this promoter.
Example 14
Deletion Analysis
[0605] The cloning method is described by Rouster (1997) and
Sambrook (1989). Detailed mapping of these promoters (i.e.,
narrowing down of the nucleic acid segments relevant for its
specificity) is performed by generating various reporter gene
expression vectors which firstly contain the entire promoter region
and secondly various fragments thereof. Firstly, the entire
promoter region or fragments thereof are cloned into a binary
vector containing GUS or other reporter gene. To this end,
fragments are employed firstly, which are obtained by using
restriction enzymes for the internal restriction cleavage sites in
the full-length promoter sequence. Secondly, PCR fragments are
employed which are provided with cleavage sites introduced by
primers. The chimeric GUS constructs containing various deleted
promoters are transformed into Zea mays, Arabidopsis and other
plant species using transformation methods in the current art.
Promoter activity is analyzed by using GUS histochemical assays or
other appropriate methods in various tissues and organs at the
different developmental stages. Modification of the promoter
sequences can eliminate leakiness based on our needs.
Example 15
In Vivo Mutagenesis
[0606] The skilled worker is familiar with a variety of methods for
the modification of the promoter activity or identification of
important promoter elements. One of these methods is based on
random mutation followed by testing with reporter genes as
described above. The in vivo mutagenesis of microorganisms can be
achieved by passage of the plasmid (or of another vector) DNA
through E. coli or other microorganisms (for example Bacillus spp.
or yeasts such as Saccharomyces cerevisiae) in which the ability of
maintaining the integrity of the genetic information is disrupted.
Conventional mutator strains have mutations in the genes for the
DNA repair system (for example mutHLS, mutD, mutT and the like; for
reference, see Rupp 1996). The skilled worker is familiar with
these strains. The use of these strains is illustrated for example
by Greener (1994). The transfer of mutated DNA molecules into
plants is preferably effected after selection and testing of the
microorganisms. Transgenic plants are generated and analyzed as
described above.
Example 16
PLACE Analysis for Os.CCoAMT1 Promoter (SEQ ID NO: 1)
[0607] Based on the below given PLACE results are potential TATA
box is localized at base pair 952 to base pair 958 of SEQ ID NO: 1.
In consequence the 5' untranslated region starts at about base pair
993 and extends to base pair 1,035 of SEQ ID NO: 1. The sequence
described by SEQ ID NO: 2 ends 17 base pairs before the ATG start
codon.
[0608] The following clusters of promoter elements were identified
in the Os.CCoAMT1 promoter as described by SEQ ID NO: 1:
TABLE-US-00021 Position IUPAC from-to Str. Sequence LTRECOREATCOR15
33-39 (-) TCCGACC TATABOX3 45-51 (+) TATTAAT CCAATBOX1 86-90 (-)
CCAAT SEF1MOTIF 99-107 (+) ATATTTATA TATAPVTRNALEU 101-113 (+)
ATTTATATATTAA TATABOX2 101-107 (-) TATAAAT TATABOX4 103-109 (-)
TATATAA WBOXATNPR1 132-146 (-) ATTGACGTCGAATTG HEXMOTIFTAH3H4
135-147 (+) TTCGACGTCAATA TGACGTVMAMY 137-149 (-) TCTATTGACGTCG
CGACGOSAMY3 137-141 (+) CGACG ACGTCBOX 138-143 (+) GACGTC ACGTCBOX
138-143 (-) GACGTC BOXIINTPATPB 145-150 (+) ATAGAA SP8BFIBSP8AIB
169-176 (-) ACTGTGTA CIACADIANLELHC 190-199 (-) CAATAATATC
S1FBOXSORPS1L21 221-226 (+) ATGGTA ABRELATERD1 226-238 (+)
ATCAACGTGATCG CIACADIANLELHC 228-237 (+) CAACGTGATC BP5OSWX 228-234
(+) CAACGTG MYBST1 267-273 (+) GGGATAT ABRELATERD1 307-319 (-)
TAAAACGTGTGCT QARBNEXTA 310-316 (-) AACGTGT CCAATBOX1 325-329 (+)
CCAAT SEF3MOTIFGM 333-338 (+) AACCCA TATABOXOSPAL 360-366 (+)
TATTTAA TATABOX2 366-372 (-) TATAAAT QELEMENTZMZM13 375-389 (-)
CAGGTCACGAATTCA WBOXHVISO1 386-400 (+) CCTGACTCACTCACA GCN4OSGLUB1
387-395 (-) GTGAGTCAG WBOXHVISO1 413-427 (-) GGTGACTGAGACAAA
SEBFCONSSTPR10A 414-420 (+) TTGTCTC ARFAT 415-420 (+) TGTCTC
IBOXCORENT 449-455 (+) GATAAGG IBOXCORE 456-462 (-) GATAAAC MYBST1
458-464 (-) AGGATAA CGACGOSAMY3 471-475 (+) CGACG HEXAMERATH4
471-476 (-) CCGTCG IBOXCORE 475-481 (-) GATAACC IBOXCORE 527-533
(+) GATAAAG TAAAGSTKST1 527-533 (+) GATAAAG NTBBF1ARROLB 528-534
(-) ACTTTAT MYB2AT 543-553 (+) GTTTTAACTGC PALBOXLPC 576-586 (+)
CCTCACCAACC MYBPLANT 579-589 (+) CACCAACCTTC MYBPZM 581-586 (+)
CCAACC ARFAT 608-613 (+) TGTCTC AGCBOXNPGLB 623-629 (+) AGCCGCC
RAV1BAT 653-665 (+) ACGCACCTGGCGG ABRELATERD1 689-701 (+)
GAAGACGTGGAGG CCAATBOX1 730-734 (+) CCAAT PALBOXAPC 737-742 (+)
CCGTCC MYB1AT 768-773 (+) AAACCA PALBOXLPC 774-784 (+) CCTCACCAACC
MYBPLANT 777-787 (+) CACCAACCCAA MYBPZM 779-784 (+) CCAACC
SEF3MOTIFGM 781-786 (+) AACCCA CAREOSREP1 785-790 (+) CAACTC
BOXCPSAS1 816-822 (+) CTCCCAC GCBP2ZMGAPC4 831-839 (-) GTGGGCCCG
RAV1BAT 834-846 (+) GCCCACCTGTCGG DRECRTCOREAT 841-847 (-) GCCGACA
CCA1ATLHCB1 880-887 (-) AAAAATCT PYRIMIDINEBOXHVEPB 883-890 (+)
TTTTTTCC MYCATERD1 920-926 (-) CATGTGA MYCATRD22 921-927 (+)
CACATGC BOXCPSAS1 932-938 (+) CTCCCAC TATAPVTRNALEU 948-960 (-)
GTTTATATAGCGC TATABOX4 952-958 (+) TATATAA CGACGOSAMY3 970-974 (-)
CGACG DRECRTCOREAT 981-987 (-) GCCGACG CGACGOSAMY3 981-985 (-)
CGACG WBOXATNPR1 982-996 (-) ATTGACTTCGCCGAC
Example 17
PLACE Analysis for Os.SI Promoter (SEQ ID NO: 6)
[0609] Based on the below given PLACE results, no conventional TATA
box has been found in this promoter region (797 bp). The following
clusters of promoter elements were identified in the Zm.SI promoter
as described by SEQ ID NO: 6:
TABLE-US-00022 Position IUPAC From-to Str. Sequence TBOXATGAPB
66-71 (+) ACTTTG RAV1AAT 96-100 (+) CAACA CATATGGMSAUR 113-118 (+)
CATATG CATATGGMSAUR 113-118 (-) CATATG -300ELEMENT 127-135 (+)
TGCAAAATC POLLEN2LELAT52 159-167 (-) TCCACCATA CGACGOSAMY3 259-263
(+) CGACG HEXAMERATH4 259-264 (-) CCGTCG CGACGOSAMY3 288-292 (+)
CGACG HEXAMERATH4 288-293 (-) CCGTCG GCCCORE 384-390 (-) CGCCGCC
GCCCORE 387-393 (-) CGCCGCC GCCCORE 390-396 (-) TGCCGCC GRAZMRAB28
390-398 (-) CATGCCGCC DRECRTCOREAT 397-403 (-) GCCGACA GCCCORE
401-407 (-) CGCCGCC BS1EGCCR 422-427 (+) AGCGGG CGACGOSAMY3 434-438
(+) CGACG HEXAMERATH4 434-439 (-) CCGTCG GCCCORE 450-456 (-)
CGCCGCC DRECRTCOREAT 456-462 (-) GCCGACC MYBPZM 552-557 (-) CCAACC
CGACGOSAMY3 559-563 (+) CGACG SEF3MOTIFGM 593-598 (-) AACCCA
LRENPCABE 607-619 (-) GCCGACGTGGCAT ABREOSRAB21 608-620 (+)
TGCCACGTCGGCC DRECRTCOREAT 613-619 (-) GCCGACG CGACGOSAMY3 613-617
(-) CGACG TAAAGSTKST1 634-640 (-) GTTAAAG MYBGAHV 637-643 (+)
TAACAAA QELEMENTZMZM13 672-686 (-) TAGGTCAATGCCTCA ELRECOREPCRP1
678-692 (+) ATTGACCTACCTTGG MYBPZM 683-688 (+) CCTACC
LTRECOREATCOR15 733-739 (+) CCCGACG CGACGOSAMY3 735-739 (+) CGACG
CCAATBOX1 756-760 (+) CCAAT
Example 18
PLACE Analysis for Zm.HRGP Promoter (SEQ ID NO: 11)
[0610] Based on the below given PLACE results are potential TATA
box is localized at base pair 1,071 to base pair 1,077 of SEQ ID
NO: 11. In consequence the 5' untranslated region starts at about
base pair 1,112 and extends to base pair 1,182 of SEQ ID NO: 11.
The sequence described by SEQ ID NO: 11 end just before the ATG
start codon.
[0611] The following clusters of promoter elements were identified
in the Zm.HRGP promoter as described by SEQ ID NO: 11:
TABLE-US-00023 Position IUPAC from-to Str. Sequence ACGTCBOX 30-35
(+) GACGTC ACGTCBOX 30-35 (-) GACGTC CGACGOSAMY3 32-36 (-) CGACG
BOXIINTPATPB 95-100 (+) ATAGAA CGACGOSAMY3 138-142 (+) CGACG
HEXAMERATH4 138-143 (-) CCGTCG MYBPZM 146-151 (+) CCAACC GT1CORE
167-177 (+) CGGTTAAATAG TATABOXOSPAL 170-176 (-) TATTTAA
CGACGOSAMY3 179-183 (+) CGACG HEXAMERATH4 179-184 (-) CCGTCG
MYCATERD1 223-229 (-) CATGTGC MYCATRD22 224-230 (+) CACATGC
NTBBF1ARROLB 238-244 (+) ACTTTAT TAAAGSTKST1 239-245 (-) TATAAAG
TATABOX2 242-248 (+) TATAAAT IBOXCORE 276-282 (-) GATAATA
REBETALGLHCB21 291-297 (+) CGGATAG IBOXCORE 296-302 (-) GATAACT
IBOXCORE 325-331 (+) GATAACT NTBBF1ARROLB 329-335 (+) ACTTTAT
TAAAGSTKST1 330-336 (-) TATAAAG TATABOX2 333-339 (+) TATAAAT
GCCCORE 358-364 (-) TGCCGCC ABRELATERD1 366-378 (-) GCAGACGTGTGCG
BOXIINTPATPB 409-414 (+) ATAGAA LTRECOREATCOR15 429-435 (+) TCCGACC
ASF1MOTIFCAMV 447-459 (+) GACATTGACGGAT WBOXATNPR1 450-464 (+)
ATTGACGGATCCAGA ELRECOREPCRP1 466-480 (-) TTTGACCGGATCGCC
CGACGOSAMY3 485-489 (+) CGACG RAV1AAT 537-541 (-) CAACA TATABOX2
560-566 (+) TATAAAT IBOXCORE 595-601 (-) GATAATA IBOXCORE 615-621
(-) GATAACG SV40COREENHAN 643-650 (-) GTGGATCG ABRELATERD1 644-656
(-) AACGACGTGGATC CGACGOSAMY3 650-654 (-) CGACG MYCATERD1 672-678
(-) CATGTGC MYCATRD22 673-679 (+) CACATGG AGCBOXNPGLB 678-684 (-)
AGCCGCC DPBFCOREDCDC3 688-694 (-) ACACAAG ABRELATERD1 744-756 (+)
AATAACGTGAGTA RAV1BAT 791-803 (+) ATCCACCTGCTCC MYBST1 823-829 (-)
TGGATAG AMYBOX2 824-830 (+) TATCCAT TATCCAOSAMY 824-830 (+) TATCCAT
WBOXATNPR1 829-843 (-) GTTGACGAATGGAAT ASF1MOTIFCAMV 834-846 (-)
CATGTTGACGAAT RAV1AAT 840-844 (+) CAACA RYREPEATBNNAPA 841-851 (+)
AACATGCAGGT INTRONLOWER 845-850 (+) TGCAGG CCAATBOX1 895-899 (+)
CCAAT MYCATERD1 944-950 (-) CATGTGG MYCATRD22 945-951 (+) CACATGG
HEXAMERATH4 1006-1011 (+) CCGTCG CGACGOSAMY3 1007-1011 (-) CGACG
ASF1MOTIFCAMV 1014-1026 (-) TCTCGTGACGCCC TATABOX4 1071-1077 (+)
TATATAA CCAATBOX1 1121-1125 (-) CCAAT MYBPZM 1136-1141 (+) CCAACC
MYB2AT 1152-1162 (-) TCAGTAACTGC
Example 19
PLACE Analysis for Zm.LDH Promoter (SEQ ID NO: 19)
[0612] Based on the below given PLACE results are potential TATA
box is localized at base pair 906 to base pair 912 of SEQ ID NO:
19. In consequence the 5' untranslated region starts at bout base
pair 947 and extends to base pair 1,060 of SEQ ID NO: 19. The
sequence described by SEQ ID NO: 19 ends 31 base pairs before the
ATG start codon.
[0613] The following clusters of promoter elements were identified
in the Zm.LDH promoter as described by SEQ ID NO: 19.
TABLE-US-00024 Position IUPAC from-to Str. Sequence TATABOX4 15-21
(-) TATATAA 5256BOXLELAT5256 16-27 (-) TGTGGTTATATA TATABOX4 16-22
(+) TATATAA MYB1AT 20-25 (+) TAACCA ASF1MOTIFCAMV 39-51 (-)
AATAATGACGCAG MYB1AT 93-98 (+) AAACCA AACACOREOSGLUB1 114-120 (-)
AACAAAC LTRE1HVBLT49 119-124 (-) CCGAAA REALPHALGLHCB21 144-154 (-)
AACCAACGATA WBOXHVISO1 172-186 (-) GATGACTCGTACGGC IBOXCORE 201-207
(-) GATAAAA DRE2COREZMRAB17 208-214 (+) ACCGACT RAV1AAT 238-242 (+)
CAACA ASF1MOTIFCAMV 254-266 (-) GTTCGTGACGCTT TAAAGSTKST1 339-345
(+) ATTAAAG NTBBF1ARROLB 340-346 (-) ACTTTAA RAV1AAT 361-365 (+)
CAACA CATATGGMSAUR 378-383 (+) CATATG CATATGGMSAUR 378-383 (-)
CATATG -10PEHVPSBD 397-402 (-) TATTCT TAAAGSTKST1 419-425 (+)
GTTAAAG RAV1AAT 435-439 (-) CAACA CAREOSREP1 448-453 (-) CAACTC
CGACGOSAMY3 456-460 (+) CGACG IBOXCORE 476-482 (+) GATAAAA
TBOXATGAPB 489-494 (-) ACTTTG IBOXCORE 561-567 (+) GATAAAA
TATABOXOSPAL 574-580 (+) TATTTAA TATABOXOSPAL 583-589 (-) TATTTAA
GT1CORE 597-607 (-) AGGTTAAAACT S1FBOXSORPS1L21 667-672 (+) ATGGTA
PROLAMINBOXOSGLUB1 678-686 (+) TGCAAAGAG MYB2AT 732-742 (+)
TGGGTAACTGT WBOXATNPR1 735-749 (-) GTTGACGACAGTTAC ASF1MOTIFCAMV
740-752 (-) CCGGTTGACGACA CCA1ATLHCB1 810-817 (-) AAAAATCT GT1CORE
831-841 (-) TGGTTAAAATT MYB1AT 836-841 (+) TAACCA SV40COREENHAN
861-868 (+) GTGGTTTG MYB1AT 862-867 (-) AAACCA TATABOX3 890-896 (+)
TATTAAT WUSATAg 892-898 (+) TTAATGG TATAPVTRNALEU 902-914 (-)
CTTTATATATTCA TATABOX4 906-912 (+) TATATAA TAAAGSTKST1 908-914 (+)
TATAAAG BOXCPSAS1 937-943 (+) CTCCCAC OCTAMERMOTIFTAH3H4 998-1005
(-) CGCGGATC ELRECOREPCRP1 1010-1024 (+) TTTGACCCAACCAGA MYBPZM
1016-1021 (+) CCAACC CIACADIANLELHC 1017-1026 (+) CAACCAGATC
ABRELATERD1 1031-1043 (-) GATTACGTGTGTG DPBFCOREDCDC3 1032-1038 (+)
ACACACG
Example 20
PLACE Analysis for Os.Cp12 Promoter (SEQ ID NO: 27)
[0614] Based on the below given PLACE results are potential TATA
box is localized at base pair 908 to base pair 914 of SEQ ID NO:27.
In consequence the 5' untranslated region starts at about base pair
960 and extends to base pair 998 of SEQ ID NO:27.
[0615] The following clusters of promoter elements were identified
in the Os.Cp12 promoter as described by SEQ ID NO:27:
TABLE-US-00025 Position IUPAC from-to Str. Sequence
AMMORESIIUDCRNIA1 28-35 (+) GGAAGGGT ABRELATERD1 57-69 (+)
GAGGACGTGAGGC LTRE1HVBLT49 88-93 (-) CCGAAA -300ELEMENT 96-104 (+)
TGAAAAATT SEF1MOTIF 108-116 (-) ATATTTAAA TATABOXOSPAL 109-115 (-)
TATTTAA WBOXATNPR1 173-187 (-) GTTGACTGGGCCTTA MYB2AT 213-223 (+)
GCTGTAACTGG RAV1AAT 244-248 (-) CAACA CIACADIANLELHC 286-295 (+)
CAAGGCCATC MYB1AT 296-301 (-) AAACCA SV40COREENHAN 310-317 (-)
GTGGTAAG CCAATBOX1 352-356 (+) CCAAT -300ELEMENT 362-370 (-)
TGTAAAGTT NTBBF1ARROLB 363-369 (+) ACTTTAC -300CORE 364-370 (-)
TGTAAAG TAAAGSTKST1 364-370 (-) TGTAAAG CCAATBOX1 392-396 (-) CCAAT
-300ELEMENT 403-411 (+) TGAAAAATA RAV1BAT 443-455 (+) CTGCACCTGTACA
MYBPLANT 519-529 (+) CACCAAACTTT EVENINGAT 543-551 (-) AAAATATCT
LTRE1HVBLT49 557-562 (+) CCGAAA RAV1AAT 620-624 (+) CAACA MYBST1
645-651 (-) TGGATAC TATCCAOSAMY 646-652 (+) TATCCAA MYBPZM 649-654
(+) CCAACC WBOXHVISO1 676-690 (-) GATGACTGTGGGTGT ELRECOREPCRP1
698-712 (-) TTTGACCGTGAAAAC ABREAZMRAB28 807-819 (-) TGCCACGTGGGCT
GBOXLERBCS 808-820 (+) GCCCACGTGGCAC UPRMOTIFIIAT 813-831 (-)
CCAATCGTCGTGTGCCACG CGACGOSAMY3 822-826 (+) CGACG CCAATBOX1 827-831
(-) CCAAT IBOXCORENT 842-848 (+) GATAAGA ABRELATERD1 853-865 (-)
GACGACGTGCACT CGACGOSAMY3 859-863 (-) CGACG CGACGOSAMY3 862-866 (-)
CGACG UPRMOTIFIIAT 879-897 (+) CCTTCTCCCCCACCCCACG TATAPVTRNALEU
904-916 (-) GTTTATATATATA TATABOX4 908-914 (+) TATATAA CGACGOSAMY3
948-952 (-) CGACG RAV1AAT 953-957 (+) CAACA RAV1AAT 994-998 (+)
CAACA
Example 21
Vector Construction for Overexpression and Gene "Knockout"
Experiments
21.1 Overexpression
[0616] 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.
[0617] For biolistic transformation (biolistic vectors), the
requirements are as follows: [0618] 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 [0619] 2. a plant-specific portion consisting of:
[0620] a. a gene expression cassette consisting of a promoter (e.g.
ZmUBlint MOD), the gene of interest (typically, a full-length cDNA)
and a transcriptional terminator (e.g., Agrobacterium tumefaciens
nos terminator); [0621] 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).
[0622] Vectors designed for transformation by Agrobacterium
tumefaciens (A. tumefaciens; binary vectors) consist of: [0623] 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; [0624] 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.
21.2 Gene Silencing Vectors
[0625] 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
[0626] 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
[0627] 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 anti-sense orientations,
separated by a spacer region (typically, a plant intron, e.g. the
OsSH1 intron 1, or a selectable marker, e.g. conferring kanamycin
resistance). Vectors of this type are designed to form a
double-stranded mRNA stem, resulting from the basepairing of the
two complementary gene fragments in planta.
[0628] 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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[0891] 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
10711035DNAOryza sativapromoter(1)..(1035)transcription regulating
sequence of caffeoyl CoA-O-methyltransferase gene including
5'-untranslated region 1gcaactactg cacggtaaaa gtgataggaa tcggtcggaa
acagtattaa tgtttttatt 60atttttacaa aaacgaattg aaataattgg aaattttcat
atttatatat taaactattc 120agtatcaact tcaattcgac gtcaatagaa
attagaaaag cataattata cacagtaata 180ggcgttcaag atattattgt
tattatttag ttttgtggaa atggtatcaa cgtgatcgga 240aaattttgta
catgttttca ccctgcggga tatctcaatt ccttctcctc cctctaccgc
300catatcagca cacgttttag agcaccaatc ataacccata aatccgtggg
ctactcactt 360atttaattta tatgtgaatt cgtgacctga ctcactcaca
tactatcaaa aatttgtctc 420agtcacccat ctccttcttt cctggtccga
taagggttta tcctacggtt cgacggttat 480cacgatagtc gtgcggttac
tgaggtatac cgtgatttaa aaatatgata aagttaccgc 540aggttttaac
tgcgcggttt ggtaaacctg ttcctcctca ccaaccttct cctccggtct
600ccttatgtgt ctcaccgagg cgagccgccg cgagaccgca tggacgcggt
ccacgcacct 660ggcggtgcac ctcctcctcc ccggcgaaga agacgtggag
gagagtaaat gagcaatcag 720gcccacggcc caatcgccgt ccaccaccca
ccaccctcag cgacccaaaa ccacctcacc 780aacccaactc tgtaccgtac
tgtacccgcc ctcccctccc actgacactc cgggcccacc 840tgtcggcgcg
actcttccac ggtccccttc tctcctcaga gattttttcc acgcattttt
900taattttttt ttctgcagtt cacatgctct tctcccactc ttccgccgcg
ctatataaac 960cgcgcgaggc gtcgttgcct cgtcggcgaa gtcaatccgg
cgatccccgg cgagcgagag 1020atcgaagcaa gctgc 10352992DNAOryza
sativapromoter(1)..(992)transcription regulating sequence from
caffeoyl CoA-O-methyltransferase gene 2gcaactactg cacggtaaaa
gtgataggaa tcggtcggaa acagtattaa tgtttttatt 60atttttacaa aaacgaattg
aaataattgg aaattttcat atttatatat taaactattc 120agtatcaact
tcaattcgac gtcaatagaa attagaaaag cataattata cacagtaata
180ggcgttcaag atattattgt tattatttag ttttgtggaa atggtatcaa
cgtgatcgga 240aaattttgta catgttttca ccctgcggga tatctcaatt
ccttctcctc cctctaccgc 300catatcagca cacgttttag agcaccaatc
ataacccata aatccgtggg ctactcactt 360atttaattta tatgtgaatt
cgtgacctga ctcactcaca tactatcaaa aatttgtctc 420agtcacccat
ctccttcttt cctggtccga taagggttta tcctacggtt cgacggttat
480cacgatagtc gtgcggttac tgaggtatac cgtgatttaa aaatatgata
aagttaccgc 540aggttttaac tgcgcggttt ggtaaacctg ttcctcctca
ccaaccttct cctccggtct 600ccttatgtgt ctcaccgagg cgagccgccg
cgagaccgca tggacgcggt ccacgcacct 660ggcggtgcac ctcctcctcc
ccggcgaaga agacgtggag gagagtaaat gagcaatcag 720gcccacggcc
caatcgccgt ccaccaccca ccaccctcag cgacccaaaa ccacctcacc
780aacccaactc tgtaccgtac tgtacccgcc ctcccctccc actgacactc
cgggcccacc 840tgtcggcgcg actcttccac ggtccccttc tctcctcaga
gattttttcc acgcattttt 900taattttttt ttctgcagtt cacatgctct
tctcccactc ttccgccgcg ctatataaac 960cgcgcgaggc gtcgttgcct
cgtcggcgaa gt 9923301DNAOryza sativapromoter(1)..(301)Potential
core region of the transcription regulating sequence of caffeoyl
CoA-O-methyltransferase gene comprising clusters of promoter
elements 3acgtggagga gagtaaatga gcaatcaggc ccacggccca atcgccgtcc
accacccacc 60accctcagcg acccaaaacc acctcaccaa cccaactctg taccgtactg
tacccgccct 120cccctcccac tgacactccg ggcccacctg tcggcgcgac
tcttccacgg tccccttctc 180tcctcagaga ttttttccac gcatttttta
attttttttt ctgcagttca catgctcttc 240tcccactctt ccgccgcgct
atataaaccg cgcgaggcgt cgttgcctcg tcggcgaagt 300c 3014783DNAOryza
sativaCDS(1)..(780)coding for caffeoyl CoA-O-methyltransferase 4atg
gcc gag gcg gcg tcg gcg gcg gcg gcg gcg acg acg gag cag gcg 48Met
Ala Glu Ala Ala Ser Ala Ala Ala Ala Ala Thr Thr Glu Gln Ala 1 5 10
15 aat ggg agc agc ggc ggc gag cag aag acg cgg cac tcg gag gtc ggc
96Asn Gly Ser Ser Gly Gly Glu Gln Lys Thr Arg His Ser Glu Val Gly
20 25 30 cac aag agc ctc ctc aag agc gac gat ctc tac cag tac atc
ctg gag 144His Lys Ser Leu Leu Lys Ser Asp Asp Leu Tyr Gln Tyr Ile
Leu Glu 35 40 45 acg agc gtg tac ccg cgc gag cac gag tgc atg aag
gag ctc cgc gag 192Thr Ser Val Tyr Pro Arg Glu His Glu Cys Met Lys
Glu Leu Arg Glu 50 55 60 gtc acc gcc aac cac cca tgg aac ctg atg
acg acg tcg gcg gac gag 240Val Thr Ala Asn His Pro Trp Asn Leu Met
Thr Thr Ser Ala Asp Glu 65 70 75 80 ggg caa ttc ctg aac ctg ctg ctg
aag ctc atc ggc gcc aag aag acc 288Gly Gln Phe Leu Asn Leu Leu Leu
Lys Leu Ile Gly Ala Lys Lys Thr 85 90 95 atg gag atc ggc gtc tac
acc ggc tac tcc ctc ctc gcc acc gcc ctc 336Met Glu Ile Gly Val Tyr
Thr Gly Tyr Ser Leu Leu Ala Thr Ala Leu 100 105 110 gcc atc ccc gac
gac ggc acg atc ttg gcg atg gac atc aac cgg gag 384Ala Ile Pro Asp
Asp Gly Thr Ile Leu Ala Met Asp Ile Asn Arg Glu 115 120 125 aac tac
gag ctg ggg ctc ccg tcg atc gag aag gcg gga gtg gcg cac 432Asn Tyr
Glu Leu Gly Leu Pro Ser Ile Glu Lys Ala Gly Val Ala His 130 135 140
aag atc gac ttc cgg gag gga ccc gcg ctg ccg gtg ctg gac cag ctg
480Lys Ile Asp Phe Arg Glu Gly Pro Ala Leu Pro Val Leu Asp Gln Leu
145 150 155 160 gtg gag gag gag ggc aac cat ggg tcg ttc gac ttc gtg
ttc gtc gac 528Val Glu Glu Glu Gly Asn His Gly Ser Phe Asp Phe Val
Phe Val Asp 165 170 175 gcc gac aag gac aac tac ctc aac tac cac gag
cgg ctg atg aag ctg 576Ala Asp Lys Asp Asn Tyr Leu Asn Tyr His Glu
Arg Leu Met Lys Leu 180 185 190 gtc aag gtc ggc ggc ctc gtc ggc tac
gac aac acg ctc tgg aac ggc 624Val Lys Val Gly Gly Leu Val Gly Tyr
Asp Asn Thr Leu Trp Asn Gly 195 200 205 tcc gtc gtg ctc ccc gcc gac
gcc ccc atg cgc aag tac atc cgc tac 672Ser Val Val Leu Pro Ala Asp
Ala Pro Met Arg Lys Tyr Ile Arg Tyr 210 215 220 tac cgc gac ttc gtg
ctc gag ctc aac aag gcc ctc gcc gcc gac cac 720Tyr Arg Asp Phe Val
Leu Glu Leu Asn Lys Ala Leu Ala Ala Asp His 225 230 235 240 cgc gtc
gag atc tgc cag ctc ccc gtc ggc gac ggc atc acc ctc tgc 768Arg Val
Glu Ile Cys Gln Leu Pro Val Gly Asp Gly Ile Thr Leu Cys 245 250 255
cgc cgc gtc aag tga 783Arg Arg Val Lys 260 5260PRTOryza sativa 5Met
Ala Glu Ala Ala Ser Ala Ala Ala Ala Ala Thr Thr Glu Gln Ala 1 5 10
15 Asn Gly Ser Ser Gly Gly Glu Gln Lys Thr Arg His Ser Glu Val Gly
20 25 30 His Lys Ser Leu Leu Lys Ser Asp Asp Leu Tyr Gln Tyr Ile
Leu Glu 35 40 45 Thr Ser Val Tyr Pro Arg Glu His Glu Cys Met Lys
Glu Leu Arg Glu 50 55 60 Val Thr Ala Asn His Pro Trp Asn Leu Met
Thr Thr Ser Ala Asp Glu 65 70 75 80 Gly Gln Phe Leu Asn Leu Leu Leu
Lys Leu Ile Gly Ala Lys Lys Thr 85 90 95 Met Glu Ile Gly Val Tyr
Thr Gly Tyr Ser Leu Leu Ala Thr Ala Leu 100 105 110 Ala Ile Pro Asp
Asp Gly Thr Ile Leu Ala Met Asp Ile Asn Arg Glu 115 120 125 Asn Tyr
Glu Leu Gly Leu Pro Ser Ile Glu Lys Ala Gly Val Ala His 130 135 140
Lys Ile Asp Phe Arg Glu Gly Pro Ala Leu Pro Val Leu Asp Gln Leu 145
150 155 160 Val Glu Glu Glu Gly Asn His Gly Ser Phe Asp Phe Val Phe
Val Asp 165 170 175 Ala Asp Lys Asp Asn Tyr Leu Asn Tyr His Glu Arg
Leu Met Lys Leu 180 185 190 Val Lys Val Gly Gly Leu Val Gly Tyr Asp
Asn Thr Leu Trp Asn Gly 195 200 205 Ser Val Val Leu Pro Ala Asp Ala
Pro Met Arg Lys Tyr Ile Arg Tyr 210 215 220 Tyr Arg Asp Phe Val Leu
Glu Leu Asn Lys Ala Leu Ala Ala Asp His 225 230 235 240 Arg Val Glu
Ile Cys Gln Leu Pro Val Gly Asp Gly Ile Thr Leu Cys 245 250 255 Arg
Arg Val Lys 260 6797DNAOryza sativapromoter(1)..(797)transcription
regulating sequence of C8,7-sterol isomerase gene comprising
5'untranslated region 6gcctcgattc gaccgtgtaa tggaatgaag gtggtgggcc
cccaccccca caagccactc 60tccacacttt ggtgttcctg gtatgtcacc tagaccaaca
actatgttaa gccatatgtt 120ccacagtgca aaatctacaa gaccacgata
caagtaggta tggtggacta ccacattttc 180acttctcttt cactttcccc
tctctctccc ccctctcttc ctttccccca ccgcagagag 240cctggcgcgc
ggagacggcg acggcgccgg accaagcagt ggtggagcga cggcagggcg
300acagcgccga gcggcgggat gcgctcgccg gcgcaccacc ccctcctctc
ccccccgagc 360ggcggggctg ctcggagcag cagggcggcg gcggcatgtc
ggcggcgggc agacgacttg 420gagcgggaga cggcgacggg cggatgcgag
gcggcggtcg gcgccctcct cccctggagt 480tcggctgctt cgccccctct
cctctctcct ctagcggtgg tgtgggtccc actgagctga 540ggagggcgcg
cggttggacg acgaggcaaa ggaatactag tcttcgcttt tttgggttga
600ggctgaatgc cacgtcggcc cattgtgaat gccctttaac aaaacaaggg
tttatggcta 660tgggatctgg ctgaggcatt gacctacctt ggtccttggc
agagagagag agagactccc 720cctcactcct tccccgacga cctgctcgat
ccgatccaat cagctcctct ccagtccaga 780tcggaaggaa gccagga
7977766DNAOryza sativapromoter(1)..(766)transcription regulating
sequence from C8,7-sterol isomerase gene 7gcctcgattc gaccgtgtaa
tggaatgaag gtggtgggcc cccaccccca caagccactc 60tccacacttt ggtgttcctg
gtatgtcacc tagaccaaca actatgttaa gccatatgtt 120ccacagtgca
aaatctacaa gaccacgata caagtaggta tggtggacta ccacattttc
180acttctcttt cactttcccc tctctctccc ccctctcttc ctttccccca
ccgcagagag 240cctggcgcgc ggagacggcg acggcgccgg accaagcagt
ggtggagcga cggcagggcg 300acagcgccga gcggcgggat gcgctcgccg
gcgcaccacc ccctcctctc ccccccgagc 360ggcggggctg ctcggagcag
cagggcggcg gcggcatgtc ggcggcgggc agacgacttg 420gagcgggaga
cggcgacggg cggatgcgag gcggcggtcg gcgccctcct cccctggagt
480tcggctgctt cgccccctct cctctctcct ctagcggtgg tgtgggtccc
actgagctga 540ggagggcgcg cggttggacg acgaggcaaa ggaatactag
tcttcgcttt tttgggttga 600ggctgaatgc cacgtcggcc cattgtgaat
gccctttaac aaaacaaggg tttatggcta 660tgggatctgg ctgaggcatt
gacctacctt ggtccttggc agagagagag agagactccc 720cctcactcct
tccccgacga cctgctcgat ccgatccaat cagctc 7668301DNAOryza
sativapromoter(1)..(301)potential core region of promoter from
C8,7-sterol isomerase gene comprising clusters of promoer elements
8ctcctcccct ggagttcggc tgcttcgccc cctctcctct ctcctctagc ggtggtgtgg
60gtcccactga gctgaggagg gcgcgcggtt ggacgacgag gcaaaggaat actagtcttc
120gcttttttgg gttgaggctg aatgccacgt cggcccattg tgaatgccct
ttaacaaaac 180aagggtttat ggctatggga tctggctgag gcattgacct
accttggtcc ttggcagaga 240gagagagaga ctccccctca ctccttcccc
gacgacctgc tcgatccgat ccaatcagct 300c 3019660DNAOryza
sativaCDS(1)..(657)coding for C8,7-sterol isomerase 9atg ggg cac
ccc cac ccc cac cct tac gcg ccg gcg gag ctt cac ctc 48Met Gly His
Pro His Pro His Pro Tyr Ala Pro Ala Glu Leu His Leu 1 5 10 15 ccg
ggc ttc gtg cct ctc caa ctg tcc cag gcc caa atc ctc gtg ccc 96Pro
Gly Phe Val Pro Leu Gln Leu Ser Gln Ala Gln Ile Leu Val Pro 20 25
30 tac ctc gcc acc tcc ctc ttc ctc ctc ctc gcc gtc tgg ctc atc tcc
144Tyr Leu Ala Thr Ser Leu Phe Leu Leu Leu Ala Val Trp Leu Ile Ser
35 40 45 ggg aga tgc agt cgt agg ctt tcc gac acc gac cgc tgg ctc
atg tgc 192Gly Arg Cys Ser Arg Arg Leu Ser Asp Thr Asp Arg Trp Leu
Met Cys 50 55 60 tgg tgg gcc ttc acc ggc ctc acc cac att atc atc
gag gga acc ttt 240Trp Trp Ala Phe Thr Gly Leu Thr His Ile Ile Ile
Glu Gly Thr Phe 65 70 75 80 gtc ttt gct cct aat ttc ttc tcc aac caa
aac cct tct tac ttc gat 288Val Phe Ala Pro Asn Phe Phe Ser Asn Gln
Asn Pro Ser Tyr Phe Asp 85 90 95 gaa gtt tgg aaa gag tac agc aaa
ggt gac tcc aga tat gtc gcc aga 336Glu Val Trp Lys Glu Tyr Ser Lys
Gly Asp Ser Arg Tyr Val Ala Arg 100 105 110 gac cct gct act gtt aca
gtt gaa gga att aca gct gtc ttg gaa ggc 384Asp Pro Ala Thr Val Thr
Val Glu Gly Ile Thr Ala Val Leu Glu Gly 115 120 125 cct gct tca ctc
ctt gct gtc tat gcc atc gca tcg ggc aag tcc tac 432Pro Ala Ser Leu
Leu Ala Val Tyr Ala Ile Ala Ser Gly Lys Ser Tyr 130 135 140 agc cat
atc ctc cag ttc act gtc tgt ctt ggt cag ctc tat gga tgc 480Ser His
Ile Leu Gln Phe Thr Val Cys Leu Gly Gln Leu Tyr Gly Cys 145 150 155
160 ctg gtg tac ttt att aca gcc tac ttg gat ggc ttc aac ttc tgg act
528Leu Val Tyr Phe Ile Thr Ala Tyr Leu Asp Gly Phe Asn Phe Trp Thr
165 170 175 agc ccg ttc tac ttc tgg gct tat ttc att ggt gca aac agc
tcg tgg 576Ser Pro Phe Tyr Phe Trp Ala Tyr Phe Ile Gly Ala Asn Ser
Ser Trp 180 185 190 gtt gtt ata cca act atg atc gcc ata agg agc tgg
aag aag att tgt 624Val Val Ile Pro Thr Met Ile Ala Ile Arg Ser Trp
Lys Lys Ile Cys 195 200 205 gca gca ttt caa ggt gaa aag gtg aag act
aaa tag 660Ala Ala Phe Gln Gly Glu Lys Val Lys Thr Lys 210 215
10219PRTOryza sativa 10Met Gly His Pro His Pro His Pro Tyr Ala Pro
Ala Glu Leu His Leu 1 5 10 15 Pro Gly Phe Val Pro Leu Gln Leu Ser
Gln Ala Gln Ile Leu Val Pro 20 25 30 Tyr Leu Ala Thr Ser Leu Phe
Leu Leu Leu Ala Val Trp Leu Ile Ser 35 40 45 Gly Arg Cys Ser Arg
Arg Leu Ser Asp Thr Asp Arg Trp Leu Met Cys 50 55 60 Trp Trp Ala
Phe Thr Gly Leu Thr His Ile Ile Ile Glu Gly Thr Phe 65 70 75 80 Val
Phe Ala Pro Asn Phe Phe Ser Asn Gln Asn Pro Ser Tyr Phe Asp 85 90
95 Glu Val Trp Lys Glu Tyr Ser Lys Gly Asp Ser Arg Tyr Val Ala Arg
100 105 110 Asp Pro Ala Thr Val Thr Val Glu Gly Ile Thr Ala Val Leu
Glu Gly 115 120 125 Pro Ala Ser Leu Leu Ala Val Tyr Ala Ile Ala Ser
Gly Lys Ser Tyr 130 135 140 Ser His Ile Leu Gln Phe Thr Val Cys Leu
Gly Gln Leu Tyr Gly Cys 145 150 155 160 Leu Val Tyr Phe Ile Thr Ala
Tyr Leu Asp Gly Phe Asn Phe Trp Thr 165 170 175 Ser Pro Phe Tyr Phe
Trp Ala Tyr Phe Ile Gly Ala Asn Ser Ser Trp 180 185 190 Val Val Ile
Pro Thr Met Ile Ala Ile Arg Ser Trp Lys Lys Ile Cys 195 200 205 Ala
Ala Phe Gln Gly Glu Lys Val Lys Thr Lys 210 215 111182DNAZea
mayspromoter(1)..(1182)transcription regulating sequence from Zea
mays hydroxyproline-rich glycoprotein (HRGP) gene including
5'-untranslated region 11ggtgaccttc ttgcttcttc gatcgtctgg
acgtcgagga gccccgcggc agcgcacgcg 60tctgcaccgt tatggtggcc gcgctcgcga
tggaatagaa ggggtaatga tggatccggc 120caggaaggcc acgacatcga
cggatccaac cggcaagacg gcgatccggt taaatagacg 180acggatctag
ctgggaaggt agactctaca ttaaatgagg ttgcacatgc cctaataact
240ttataaatct aatttattca gaggcaaggt agtagtatta tctttcccaa
cggatagtta 300tctgatctgc cgttcagctt gatcgataac tttataaatc
taatttattc agaggccggc 360ggcagcgcac acgtctgcac cagtaatgtt
agccgcgcct gtggcgtaat agaaggggta 420acgatggatc cgaccagaaa
ggcctcgaca ttgacggatc cagacggcga tccggtcaaa 480gagacgacga
atctagccga gaaggtagat ctctcgagag agttcatatt aaatgatgtt
540gtacatgcca taataactct ataaatctaa tttattcata ggcgaaggta
gtagtattat 600ctttcccagc ggatcgttat ctgatctgcc gttcagcttg
atcgatccac gtcgtttgat 660ctcggcgagc agcacatggc ggctcttctt
gtgtacaggt ctcactctct gctacttcag 720tgcaaggcgg agtgaatgca
cacaataacg tgagtattgt gggaactact tgtagatgca 780aacgatgtaa
atccacctgc tccaccaagt gcccgcccgg
ctctatccat tccattcgtc 840aacatgcagg ttcagactgg cccgtgctgg
accagtgagc ggtgccggtg aaccccaatg 900caagcgaagc gagtgaccat
cggggaagcc tcccgtgctg cccccacatg gcttgcctga 960atgcctctct
ctcgccgcag tgccctctct ctctcctcct cctctccgtc gaagggcgtc
1020acgagagccc agagggcatc cgaggccccc accccacccc ttcctccgtg
tatataagca 1080gtggcagggt gagcgtctct cctcagacca ccactgcgcc
attggccagc tagagccaac 1140cagaagagct tgcagttact gagagtgtgt
gtgagagaga gg 1182121111DNAZea mayspromoter(1)..(1111)transcription
regulating sequence from Zea mays hydroxyproline-rich glycoprotein
(HRGP) gene 12ggtgaccttc ttgcttcttc gatcgtctgg acgtcgagga
gccccgcggc agcgcacgcg 60tctgcaccgt tatggtggcc gcgctcgcga tggaatagaa
ggggtaatga tggatccggc 120caggaaggcc acgacatcga cggatccaac
cggcaagacg gcgatccggt taaatagacg 180acggatctag ctgggaaggt
agactctaca ttaaatgagg ttgcacatgc cctaataact 240ttataaatct
aatttattca gaggcaaggt agtagtatta tctttcccaa cggatagtta
300tctgatctgc cgttcagctt gatcgataac tttataaatc taatttattc
agaggccggc 360ggcagcgcac acgtctgcac cagtaatgtt agccgcgcct
gtggcgtaat agaaggggta 420acgatggatc cgaccagaaa ggcctcgaca
ttgacggatc cagacggcga tccggtcaaa 480gagacgacga atctagccga
gaaggtagat ctctcgagag agttcatatt aaatgatgtt 540gtacatgcca
taataactct ataaatctaa tttattcata ggcgaaggta gtagtattat
600ctttcccagc ggatcgttat ctgatctgcc gttcagcttg atcgatccac
gtcgtttgat 660ctcggcgagc agcacatggc ggctcttctt gtgtacaggt
ctcactctct gctacttcag 720tgcaaggcgg agtgaatgca cacaataacg
tgagtattgt gggaactact tgtagatgca 780aacgatgtaa atccacctgc
tccaccaagt gcccgcccgg ctctatccat tccattcgtc 840aacatgcagg
ttcagactgg cccgtgctgg accagtgagc ggtgccggtg aaccccaatg
900caagcgaagc gagtgaccat cggggaagcc tcccgtgctg cccccacatg
gcttgcctga 960atgcctctct ctcgccgcag tgccctctct ctctcctcct
cctctccgtc gaagggcgtc 1020acgagagccc agagggcatc cgaggccccc
accccacccc ttcctccgtg tatataagca 1080gtggcagggt gagcgtctct
cctcagacca c 111113301DNAZea mayspromoter(1)..(301)Potential core
region of the promoter from the Zea mays hydroxyproline-rich
glycoprotein (HRGP) gene comprising clusters of promoter elements
13gcccgcccgg ctctatccat tccattcgtc aacatgcagg ttcagactgg cccgtgctgg
60accagtgagc ggtgccggtg aaccccaatg caagcgaagc gagtgaccat cggggaagcc
120tcccgtgctg cccccacatg gcttgcctga atgcctctct ctcgccgcag
tgccctctct 180ctctcctcct cctctccgtc gaagggcgtc acgagagccc
agagggcatc cgaggccccc 240accccacccc ttcctccgtg tatataagca
gtggcagggt gagcgtctct cctcagacca 300c 301141270DNAZea
mayspromoter(1)..(1270)transcription regulating sequence from Zea
mays hydroxyproline-rich glycoprotein (HRGP) gene including
5'-untranslated region 14ggtgaccttc ttgcttcttc gatcgtctgg
acgtcgagga gcccgcggca gcgcacgcgt 60ctgcaccggt aatggtggtc gcgcccgcga
cggaatagaa ggggtaacga tggatcgggc 120caggaaggcc acgacatcga
cggatccaac cggcaagacg gcgatctggt caaatagacg 180acagatctag
ctgggaaggt agatccctcg agagactcta tattaaatga ggttgtacat
240gctctaataa ctctataaat ataatttatt cagaggcgaa ggtagtagcc
cttgatgccg 300agatagtcga agtcgaggtg gtcgtggtcg ggagacatgc
ggcaatagcc tattattcgg 360taggggtcga tgttcaagcg tcaatggtcg
gctgggcgac ataaaaatta gcaccagggt 420gaccttcttg cttcttcgat
cgtctggaca tcgaggagcc cgtggcaacg cacgcgtctg 480cacaggtaat
ggtggtcgcg cacaggtaat ggcggaatag aaggggcaac gatggatccg
540gccaggaagg tcacgacatc gacggatcca accggcaaga cggcgatccg
gttaaataga 600cgacggatct tgctggaaag gtagatccct cgagaaactc
tatattaaat gaggttgtac 660ataccctaat aactttataa atctaattta
ttcagaggca aaggtagtaa gtattatctt 720tcccagcgga tcgttatctg
atctgccgtt cagcttgatc gatccacgtc gtttgatctc 780gtcgagcagc
acatggcggc acttcttgtg tacagggctc actctctgct acttcagtgc
840aaggcggagt gaatgcgcac aataacgtga gtattgtggg aactacttgt
agatgcaaac 900gatgtaaatc cacctatgcc cgcccggctc tatccattcc
attcgtcaac acgcaagttc 960agactggacc agtgagcggt gccggtgaac
ccagcccaag cgagtgacca tcggggaagc 1020ctcccgtgct gcccccacat
ggcttgcctg aatgcctctc gccgcagtgc cctctctcct 1080cctctccgtc
gaagggcgtc acgagagccc agagggcatc cgaggccccc accccacccc
1140ttcctccgtg tatataagca gtggcagggt gagcgtctct cctcagacca
ccactgcgcc 1200attggccagc tagagccaac cagaagagct tgcagttact
gagagtgtgt gtgagagaga 1260ggatgggtgg 1270151191DNAZea
mayspromoter(1)..(1191)transcription regulating sequence from Zea
mays hydroxyproline-rich glycoprotein (HRGP) gene 15ggtgaccttc
ttgcttcttc gatcgtctgg acgtcgagga gcccgcggca gcgcacgcgt 60ctgcaccggt
aatggtggtc gcgcccgcga cggaatagaa ggggtaacga tggatcgggc
120caggaaggcc acgacatcga cggatccaac cggcaagacg gcgatctggt
caaatagacg 180acagatctag ctgggaaggt agatccctcg agagactcta
tattaaatga ggttgtacat 240gctctaataa ctctataaat ataatttatt
cagaggcgaa ggtagtagcc cttgatgccg 300agatagtcga agtcgaggtg
gtcgtggtcg ggagacatgc ggcaatagcc tattattcgg 360taggggtcga
tgttcaagcg tcaatggtcg gctgggcgac ataaaaatta gcaccagggt
420gaccttcttg cttcttcgat cgtctggaca tcgaggagcc cgtggcaacg
cacgcgtctg 480cacaggtaat ggtggtcgcg cacaggtaat ggcggaatag
aaggggcaac gatggatccg 540gccaggaagg tcacgacatc gacggatcca
accggcaaga cggcgatccg gttaaataga 600cgacggatct tgctggaaag
gtagatccct cgagaaactc tatattaaat gaggttgtac 660ataccctaat
aactttataa atctaattta ttcagaggca aaggtagtaa gtattatctt
720tcccagcgga tcgttatctg atctgccgtt cagcttgatc gatccacgtc
gtttgatctc 780gtcgagcagc acatggcggc acttcttgtg tacagggctc
actctctgct acttcagtgc 840aaggcggagt gaatgcgcac aataacgtga
gtattgtggg aactacttgt agatgcaaac 900gatgtaaatc cacctatgcc
cgcccggctc tatccattcc attcgtcaac acgcaagttc 960agactggacc
agtgagcggt gccggtgaac ccagcccaag cgagtgacca tcggggaagc
1020ctcccgtgct gcccccacat ggcttgcctg aatgcctctc gccgcagtgc
cctctctcct 1080cctctccgtc gaagggcgtc acgagagccc agagggcatc
cgaggccccc accccacccc 1140ttcctccgtg tatataagca gtggcagggt
gagcgtctct cctcagacca c 119116301DNAZea
mayspromoter(1)..(301)Potential core region of the promoter from
Zea mays hydroxyproline-rich glycoprotein (HRGP) gene comprising
clusters of promoter elements 16agatgcaaac gatgtaaatc cacctatgcc
cgcccggctc tatccattcc attcgtcaac 60acgcaagttc agactggacc agtgagcggt
gccggtgaac ccagcccaag cgagtgacca 120tcggggaagc ctcccgtgct
gcccccacat ggcttgcctg aatgcctctc gccgcagtgc 180cctctctcct
cctctccgtc gaagggcgtc acgagagccc agagggcatc cgaggccccc
240accccacccc ttcctccgtg tatataagca gtggcagggt gagcgtctct
cctcagacca 300c 30117987DNAZea maysCDS(1)..(984)coding for Zea mays
hydroxyproline-rich glycoprotein (HRGP) 17atg ggt ggc agc ggc agg
gct gct ctg ctg ctg gcc ctg gtg gtg gtg 48Met Gly Gly Ser Gly Arg
Ala Ala Leu Leu Leu Ala Leu Val Val Val 1 5 10 15 gcc gtg agc ctg
gcc gtg gag atc cag gcc gac gcc ggg tac ggt tac 96Ala Val Ser Leu
Ala Val Glu Ile Gln Ala Asp Ala Gly Tyr Gly Tyr 20 25 30 ggc ggc
ggg tac acc ccg acg ccg acg ccg gcc acc ccg acc ccg aag 144Gly Gly
Gly Tyr Thr Pro Thr Pro Thr Pro Ala Thr Pro Thr Pro Lys 35 40 45
ccc gag aag ccc ccc acc aag ggg ccg aag ccg gac aag ccg ccc aag
192Pro Glu Lys Pro Pro Thr Lys Gly Pro Lys Pro Asp Lys Pro Pro Lys
50 55 60 gag cac ggg ccc aag ccg gag aag ccg ccc aag gag cac aag
ccg acg 240Glu His Gly Pro Lys Pro Glu Lys Pro Pro Lys Glu His Lys
Pro Thr 65 70 75 80 ccg ccc acg tac acc ccg agc ccc aaa ccc acg ccg
ccg acg tac act 288Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro Thr Pro
Pro Thr Tyr Thr 85 90 95 ccc acc ccg acg ccc ccc aag ccg acg cca
ccc aca tac act ccc gcc 336Pro Thr Pro Thr Pro Pro Lys Pro Thr Pro
Pro Thr Tyr Thr Pro Ala 100 105 110 cct acg ccc cac aaa ccc aca cca
aaa ccc act ccc act cct ccg acg 384Pro Thr Pro His Lys Pro Thr Pro
Lys Pro Thr Pro Thr Pro Pro Thr 115 120 125 tac acc ccg acc ccc aag
cct ccg aca cct aag ccg acc ccg ccg acg 432Tyr Thr Pro Thr Pro Lys
Pro Pro Thr Pro Lys Pro Thr Pro Pro Thr 130 135 140 tac act cca agc
ccc aaa cct ccg acg ccc aag ccg acc cca ccg acg 480Tyr Thr Pro Ser
Pro Lys Pro Pro Thr Pro Lys Pro Thr Pro Pro Thr 145 150 155 160 tac
acc cct tcc ccc aag cct ccg aca cct aag ccg acc ccg cct acg 528Tyr
Thr Pro Ser Pro Lys Pro Pro Thr Pro Lys Pro Thr Pro Pro Thr 165 170
175 tac act cca agc cct aag cca ccg gct acc aag cct ccc acg ccc aag
576Tyr Thr Pro Ser Pro Lys Pro Pro Ala Thr Lys Pro Pro Thr Pro Lys
180 185 190 ccg acc ccg cca acg tac acc cct tcg cca aag cct ccg aca
ccc aag 624Pro Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro Pro Thr
Pro Lys 195 200 205 ccg acc ccg ccg acg tac acc cct tct ccc aag cct
ccg acg ccc aag 672Pro Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro
Pro Thr Pro Lys 210 215 220 ccg acc ccg ccg acg tac act cca agc ccc
aag cct ccc aca cac ccg 720Pro Thr Pro Pro Thr Tyr Thr Pro Ser Pro
Lys Pro Pro Thr His Pro 225 230 235 240 acg ccc aag ccg acc cca ccg
acg tac acc cct tcc cca aag cct ccg 768Thr Pro Lys Pro Thr Pro Pro
Thr Tyr Thr Pro Ser Pro Lys Pro Pro 245 250 255 acg ccc aag ccg acc
cca ccg acg tac acc cct tcc cca aag cct ccg 816Thr Pro Lys Pro Thr
Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro Pro 260 265 270 aca ccc aag
ccg acc cca ccg acg tac acc cct tcc cca aag cct ccg 864Thr Pro Lys
Pro Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro Pro 275 280 285 aca
ccc aag ccg acc cca ccg acg tac act ccc aca ccg aag ccg ccg 912Thr
Pro Lys Pro Thr Pro Pro Thr Tyr Thr Pro Thr Pro Lys Pro Pro 290 295
300 gcc acc aag ccg ccc acc tac act ccg acg ccg ccg gtg tct cac acc
960Ala Thr Lys Pro Pro Thr Tyr Thr Pro Thr Pro Pro Val Ser His Thr
305 310 315 320 ccc agc ccg ccg cca cca tac tac tag 987Pro Ser Pro
Pro Pro Pro Tyr Tyr 325 18328PRTZea mays 18Met Gly Gly Ser Gly Arg
Ala Ala Leu Leu Leu Ala Leu Val Val Val 1 5 10 15 Ala Val Ser Leu
Ala Val Glu Ile Gln Ala Asp Ala Gly Tyr Gly Tyr 20 25 30 Gly Gly
Gly Tyr Thr Pro Thr Pro Thr Pro Ala Thr Pro Thr Pro Lys 35 40 45
Pro Glu Lys Pro Pro Thr Lys Gly Pro Lys Pro Asp Lys Pro Pro Lys 50
55 60 Glu His Gly Pro Lys Pro Glu Lys Pro Pro Lys Glu His Lys Pro
Thr 65 70 75 80 Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro Thr Pro Pro
Thr Tyr Thr 85 90 95 Pro Thr Pro Thr Pro Pro Lys Pro Thr Pro Pro
Thr Tyr Thr Pro Ala 100 105 110 Pro Thr Pro His Lys Pro Thr Pro Lys
Pro Thr Pro Thr Pro Pro Thr 115 120 125 Tyr Thr Pro Thr Pro Lys Pro
Pro Thr Pro Lys Pro Thr Pro Pro Thr 130 135 140 Tyr Thr Pro Ser Pro
Lys Pro Pro Thr Pro Lys Pro Thr Pro Pro Thr 145 150 155 160 Tyr Thr
Pro Ser Pro Lys Pro Pro Thr Pro Lys Pro Thr Pro Pro Thr 165 170 175
Tyr Thr Pro Ser Pro Lys Pro Pro Ala Thr Lys Pro Pro Thr Pro Lys 180
185 190 Pro Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro Pro Thr Pro
Lys 195 200 205 Pro Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro Pro
Thr Pro Lys 210 215 220 Pro Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys
Pro Pro Thr His Pro 225 230 235 240 Thr Pro Lys Pro Thr Pro Pro Thr
Tyr Thr Pro Ser Pro Lys Pro Pro 245 250 255 Thr Pro Lys Pro Thr Pro
Pro Thr Tyr Thr Pro Ser Pro Lys Pro Pro 260 265 270 Thr Pro Lys Pro
Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro Pro 275 280 285 Thr Pro
Lys Pro Thr Pro Pro Thr Tyr Thr Pro Thr Pro Lys Pro Pro 290 295 300
Ala Thr Lys Pro Pro Thr Tyr Thr Pro Thr Pro Pro Val Ser His Thr 305
310 315 320 Pro Ser Pro Pro Pro Pro Tyr Tyr 325 191060DNAZea
mayspromoter(1)..(1060)transcription regulating sequence from Zea
mays lactate dehydrogenase gene including 5'-untranslated region
19aacaaatggc gtacttatat aaccacaatg tactggtgct gcgtcattat tttatactac
60gcatatatta ttataagtag agaaagctca caaaaccatg cgcgcgcccc cctgtttgtt
120tcggtcgcta attacaccct ttgtatcgtt ggttgatgat ggtctccacc
ggccgtacga 180gtcatcgatc gttgatttat ttttatcacc gacttgcacg
cctttcgaac aaagacgcaa 240caaaggaaag cgaaagcgtc acgaacgagg
ttgttccctg acagttgttc gactaataca 300actgcaagac actgaataag
cagtaaaaat caatatagat taaagttaaa cgaacatgct 360caacatcgaa
tactactcat atgtgttatt attaagagaa taccaccaag gtagaaaagt
420taaaggacct aaactgttgt gccgggagag ttgtgcgacg aacagatgta
aatatgataa 480aataagttca aagttcatat agatagcacg atcacactta
gggctagttt gaagccataa 540aaatggaaga gattaaatga gataaaattc
acttatttaa ttttaaataa gaagagagtt 600ttaacctcta attctctcca
gtattttagc tcctaaacta gctcttacag cagtaaaaga 660cccttgatgg
tagcgtatgc aaagagaagg aactattcaa tgaattgttt ttttaatcac
720tagtagtatg gtgggtaact gtcgtcaacc ggccctatct acttcagttt
agtgaagcac 780taaaccgcac cttggtatgt tcaaatttaa gatttttttt
gaaacgaaac aattttaacc 840agcggctcca aaccggtgaa gtggtttggt
ctttggtgtg gggccagggt attaatggaa 900ttgaatatat aaagagcagg
gtggtggacc tttcccctcc cacgagtcga gtagccattg 960cccattgcca
ttccttcctt cctccacaga gaaatccgat ccgcggagat ttgacccaac
1020cagatcatat cacacacgta atcccatccc attccgcccg 106020946DNAZea
mayspromoter(1)..(946)transcription regulating sequence from Zea
mays lactate dehydrogenase gene 20aacaaatggc gtacttatat aaccacaatg
tactggtgct gcgtcattat tttatactac 60gcatatatta ttataagtag agaaagctca
caaaaccatg cgcgcgcccc cctgtttgtt 120tcggtcgcta attacaccct
ttgtatcgtt ggttgatgat ggtctccacc ggccgtacga 180gtcatcgatc
gttgatttat ttttatcacc gacttgcacg cctttcgaac aaagacgcaa
240caaaggaaag cgaaagcgtc acgaacgagg ttgttccctg acagttgttc
gactaataca 300actgcaagac actgaataag cagtaaaaat caatatagat
taaagttaaa cgaacatgct 360caacatcgaa tactactcat atgtgttatt
attaagagaa taccaccaag gtagaaaagt 420taaaggacct aaactgttgt
gccgggagag ttgtgcgacg aacagatgta aatatgataa 480aataagttca
aagttcatat agatagcacg atcacactta gggctagttt gaagccataa
540aaatggaaga gattaaatga gataaaattc acttatttaa ttttaaataa
gaagagagtt 600ttaacctcta attctctcca gtattttagc tcctaaacta
gctcttacag cagtaaaaga 660cccttgatgg tagcgtatgc aaagagaagg
aactattcaa tgaattgttt ttttaatcac 720tagtagtatg gtgggtaact
gtcgtcaacc ggccctatct acttcagttt agtgaagcac 780taaaccgcac
cttggtatgt tcaaatttaa gatttttttt gaaacgaaac aattttaacc
840agcggctcca aaccggtgaa gtggtttggt ctttggtgtg gggccagggt
attaatggaa 900ttgaatatat aaagagcagg gtggtggacc tttcccctcc cacgag
94621301DNAZea mayspromoter(1)..(301)Potential core region of the
promoter from Zea mays lactate dehydrogenase gene comprising
clusters of promoter elements 21tacagcagta aaagaccctt gatggtagcg
tatgcaaaga gaaggaacta ttcaatgaat 60tgttttttta atcactagta gtatggtggg
taactgtcgt caaccggccc tatctacttc 120agtttagtga agcactaaac
cgcaccttgg tatgttcaaa tttaagattt tttttgaaac 180gaaacaattt
taaccagcgg ctccaaaccg gtgaagtggt ttggtctttg gtgtggggcc
240agggtattaa tggaattgaa tatataaaga gcagggtggt ggacctttcc
cctcccacga 300g 301221093DNAZea
mayspromoter(1)..(1093)transcription regulating sequence from Zea
mays lactate dehydrogenase gene including 5'-untranslated region
22aacaaatggc gtacttatat aaccacaatg tactggtgct gcgtcattat tttatactac
60gcatatatta ttataagtag agaaagctca caaaaccatg cgcgcgcccc cctgtttgct
120ttcggtcgct aattacaccc tttgtatcgt tggttgatga tggtctccac
cggccgtacg 180agtcatcgat cgttgattta tttttatcac cgacttgcac
gcctttcgaa caaagacgca 240acaaaggaaa gcgaaagcac gaacgaggtt
gttccctgac agttgggcga ctaatacaac 300tgcaagacac tgaataagca
gtaaaaatca atatagatta aagttaaacg aacatgctca 360acatcgaata
ctactcatat gtgttattat taagagaata ccaccaaggt agaaaagtta
420aaggacctaa actgttgtgc cgggagagtt gtgcgacgaa cagatgtaaa
tatgataaaa 480taagttcaaa gttcatatag atagcacgat cacacttagg
gctagtttga agccataaaa 540atggaagaga ttaaatgaga taaaattcac
ttatttaatt ttaaataaga agagagtttt 600aacccctcta attctctcca
gtattttagc
tcctaaacta gctcttacag cagtaaaaga 660cccttgatgg tagcgtatgc
aaagagaagg aactattcaa tgaattgttt ttttaatcac 720tagtagtatg
gtgggtaacg tgttcgtcaa ccggccctat ctacttcagt ttagtgaagc
780actaaaccgc accttggtat gttcaaattt aagatttttt ttgaaacgaa
acaattttaa 840ccagcggctc caaaccggtg aagtggtttg gtctttggtg
tggggccagg gtattaatgg 900aattgaatat ataaagagca gggtggtgga
cctttcccca cccacgagtc gagtagccat 960tgcccattgc cattccttcc
ttcctccaca gagaaatccg atccgcggag atttgaccca 1020accagatcat
atcacacacg taatcccatc ccattccgcc cgcaacagca gcaccaccgg
1080tgggaagaag aag 109323948DNAZea
mayspromoter(1)..(948)transcription regulating sequence from Zea
mays lactate dehydrogenase gene 23aacaaatggc gtacttatat aaccacaatg
tactggtgct gcgtcattat tttatactac 60gcatatatta ttataagtag agaaagctca
caaaaccatg cgcgcgcccc cctgtttgct 120ttcggtcgct aattacaccc
tttgtatcgt tggttgatga tggtctccac cggccgtacg 180agtcatcgat
cgttgattta tttttatcac cgacttgcac gcctttcgaa caaagacgca
240acaaaggaaa gcgaaagcac gaacgaggtt gttccctgac agttgggcga
ctaatacaac 300tgcaagacac tgaataagca gtaaaaatca atatagatta
aagttaaacg aacatgctca 360acatcgaata ctactcatat gtgttattat
taagagaata ccaccaaggt agaaaagtta 420aaggacctaa actgttgtgc
cgggagagtt gtgcgacgaa cagatgtaaa tatgataaaa 480taagttcaaa
gttcatatag atagcacgat cacacttagg gctagtttga agccataaaa
540atggaagaga ttaaatgaga taaaattcac ttatttaatt ttaaataaga
agagagtttt 600aacccctcta attctctcca gtattttagc tcctaaacta
gctcttacag cagtaaaaga 660cccttgatgg tagcgtatgc aaagagaagg
aactattcaa tgaattgttt ttttaatcac 720tagtagtatg gtgggtaacg
tgttcgtcaa ccggccctat ctacttcagt ttagtgaagc 780actaaaccgc
accttggtat gttcaaattt aagatttttt ttgaaacgaa acaattttaa
840ccagcggctc caaaccggtg aagtggtttg gtctttggtg tggggccagg
gtattaatgg 900aattgaatat ataaagagca gggtggtgga cctttcccca cccacgag
94824301DNAZea maysmisc_feature(1)..(301)Potential core region of
the promoter from Zea mays lactate dehydrogenase gene comprising
clusters of promoter elements 24cagcagtaaa agacccttga tggtagcgta
tgcaaagaga aggaactatt caatgaattg 60tttttttaat cactagtagt atggtgggta
acgtgttcgt caaccggccc tatctacttc 120agtttagtga agcactaaac
cgcaccttgg tatgttcaaa tttaagattt tttttgaaac 180gaaacaattt
taaccagcgg ctccaaaccg gtgaagtggt ttggtctttg gtgtggggcc
240agggtattaa tggaattgaa tatataaaga gcagggtggt ggacctttcc
ccacccacga 300g 301251065DNAZea maysCDS(1)..(1062)coding for Zea
mays lactate dehydrogenase 25atg aag aag gcc act tcg ctc tcc gag
ctg ggc ttc gac gcc ggc gac 48Met Lys Lys Ala Thr Ser Leu Ser Glu
Leu Gly Phe Asp Ala Gly Asp 1 5 10 15 gcg tcg tcg ggc ttc ttc cgc
cct gtg tcc ggc gac tcg tcg acg ccg 96Ala Ser Ser Gly Phe Phe Arg
Pro Val Ser Gly Asp Ser Ser Thr Pro 20 25 30 acg tcg cag cac cac
cgg cgg agg ctg acc aag gtg tcg gtc atc ggc 144Thr Ser Gln His His
Arg Arg Arg Leu Thr Lys Val Ser Val Ile Gly 35 40 45 gcg ggc aac
gtg ggg atg gcc atc gcg cag acc atc ctc acg cgc gac 192Ala Gly Asn
Val Gly Met Ala Ile Ala Gln Thr Ile Leu Thr Arg Asp 50 55 60 ctg
gcc gac gag atc gcg ctg gtg gac gcg gtc ccg gac aag ctc cgc 240Leu
Ala Asp Glu Ile Ala Leu Val Asp Ala Val Pro Asp Lys Leu Arg 65 70
75 80 ggg gag atg ctg gac ctg cag cac gcg gcg gcg ttc ctg ccg cgc
acg 288Gly Glu Met Leu Asp Leu Gln His Ala Ala Ala Phe Leu Pro Arg
Thr 85 90 95 cgc ctc gtg tcc ggc acc gac atg tcc gtg acc agg ggc
tcg gac ctg 336Arg Leu Val Ser Gly Thr Asp Met Ser Val Thr Arg Gly
Ser Asp Leu 100 105 110 gtc atc gtg acg gcc ggg gcg cgg cag atc cag
ggc gag acg agg ctc 384Val Ile Val Thr Ala Gly Ala Arg Gln Ile Gln
Gly Glu Thr Arg Leu 115 120 125 gac ctg ctc cag cgg aac gtg gcg ctg
ttc cgc aag atc gtg ccg ccg 432Asp Leu Leu Gln Arg Asn Val Ala Leu
Phe Arg Lys Ile Val Pro Pro 130 135 140 ctg gcg gag cag tcc cac gac
gcg ctg ctg ctc gtc gtg tcc aac ccc 480Leu Ala Glu Gln Ser His Asp
Ala Leu Leu Leu Val Val Ser Asn Pro 145 150 155 160 gtg gac gtg ctc
acc tac gtc gcc tgg aag ctt tcg ggc ttc ccg gcc 528Val Asp Val Leu
Thr Tyr Val Ala Trp Lys Leu Ser Gly Phe Pro Ala 165 170 175 agc cgc
gtc atc gga tcc ggc acc aac ctc gac tcg tcc agg ttc agg 576Ser Arg
Val Ile Gly Ser Gly Thr Asn Leu Asp Ser Ser Arg Phe Arg 180 185 190
ttc ctt ctc gcc gag cac ctc gac gtc aac gcc cag gac gta cag gcg
624Phe Leu Leu Ala Glu His Leu Asp Val Asn Ala Gln Asp Val Gln Ala
195 200 205 tac atg gtc ggc gag cac ggc gac agc tcg gtg gcg gtg tgg
tcg agc 672Tyr Met Val Gly Glu His Gly Asp Ser Ser Val Ala Val Trp
Ser Ser 210 215 220 gtg agc gtg gcg ggg atg ccg gtg ctc aag tcg ctg
cag gag agc cac 720Val Ser Val Ala Gly Met Pro Val Leu Lys Ser Leu
Gln Glu Ser His 225 230 235 240 cgc tgc ttc gac gag gag gcg ctg gag
ggc atc cgc cgc gcc gtc gtc 768Arg Cys Phe Asp Glu Glu Ala Leu Glu
Gly Ile Arg Arg Ala Val Val 245 250 255 gac agc gcc tac gag gtc atc
agc ctc aag ggc tac acc tcc tgg gcc 816Asp Ser Ala Tyr Glu Val Ile
Ser Leu Lys Gly Tyr Thr Ser Trp Ala 260 265 270 atc ggc tac tcc gtc
gcc agc ctc gcc gcg tcg ctg ctc cgg gac cag 864Ile Gly Tyr Ser Val
Ala Ser Leu Ala Ala Ser Leu Leu Arg Asp Gln 275 280 285 cgc cgc atc
cac ccg gtc tcc gtc ctc gcc agg ggg ttc cac ggc atc 912Arg Arg Ile
His Pro Val Ser Val Leu Ala Arg Gly Phe His Gly Ile 290 295 300 ccc
gac gga acg acg tct tcc tca gcc tgc ccg cca cgt cgg ccg cgc 960Pro
Asp Gly Thr Thr Ser Ser Ser Ala Cys Pro Pro Arg Arg Pro Arg 305 310
315 320 cgg cgt cca ggg cgt cgc gag atg gag ctc acc gag gag gag gcc
aaa 1008Arg Arg Pro Gly Arg Arg Glu Met Glu Leu Thr Glu Glu Glu Ala
Lys 325 330 335 cga ctg cgc cgc tcc gcc aag acc atc tgg gag aac tgc
cag ctc ctc 1056Arg Leu Arg Arg Ser Ala Lys Thr Ile Trp Glu Asn Cys
Gln Leu Leu 340 345 350 ggc ctc tga 1065Gly Leu 26354PRTZea mays
26Met Lys Lys Ala Thr Ser Leu Ser Glu Leu Gly Phe Asp Ala Gly Asp 1
5 10 15 Ala Ser Ser Gly Phe Phe Arg Pro Val Ser Gly Asp Ser Ser Thr
Pro 20 25 30 Thr Ser Gln His His Arg Arg Arg Leu Thr Lys Val Ser
Val Ile Gly 35 40 45 Ala Gly Asn Val Gly Met Ala Ile Ala Gln Thr
Ile Leu Thr Arg Asp 50 55 60 Leu Ala Asp Glu Ile Ala Leu Val Asp
Ala Val Pro Asp Lys Leu Arg 65 70 75 80 Gly Glu Met Leu Asp Leu Gln
His Ala Ala Ala Phe Leu Pro Arg Thr 85 90 95 Arg Leu Val Ser Gly
Thr Asp Met Ser Val Thr Arg Gly Ser Asp Leu 100 105 110 Val Ile Val
Thr Ala Gly Ala Arg Gln Ile Gln Gly Glu Thr Arg Leu 115 120 125 Asp
Leu Leu Gln Arg Asn Val Ala Leu Phe Arg Lys Ile Val Pro Pro 130 135
140 Leu Ala Glu Gln Ser His Asp Ala Leu Leu Leu Val Val Ser Asn Pro
145 150 155 160 Val Asp Val Leu Thr Tyr Val Ala Trp Lys Leu Ser Gly
Phe Pro Ala 165 170 175 Ser Arg Val Ile Gly Ser Gly Thr Asn Leu Asp
Ser Ser Arg Phe Arg 180 185 190 Phe Leu Leu Ala Glu His Leu Asp Val
Asn Ala Gln Asp Val Gln Ala 195 200 205 Tyr Met Val Gly Glu His Gly
Asp Ser Ser Val Ala Val Trp Ser Ser 210 215 220 Val Ser Val Ala Gly
Met Pro Val Leu Lys Ser Leu Gln Glu Ser His 225 230 235 240 Arg Cys
Phe Asp Glu Glu Ala Leu Glu Gly Ile Arg Arg Ala Val Val 245 250 255
Asp Ser Ala Tyr Glu Val Ile Ser Leu Lys Gly Tyr Thr Ser Trp Ala 260
265 270 Ile Gly Tyr Ser Val Ala Ser Leu Ala Ala Ser Leu Leu Arg Asp
Gln 275 280 285 Arg Arg Ile His Pro Val Ser Val Leu Ala Arg Gly Phe
His Gly Ile 290 295 300 Pro Asp Gly Thr Thr Ser Ser Ser Ala Cys Pro
Pro Arg Arg Pro Arg 305 310 315 320 Arg Arg Pro Gly Arg Arg Glu Met
Glu Leu Thr Glu Glu Glu Ala Lys 325 330 335 Arg Leu Arg Arg Ser Ala
Lys Thr Ile Trp Glu Asn Cys Gln Leu Leu 340 345 350 Gly Leu
27998DNAOryza sativapromoter(1)..(998)transcription regulating
sequence of chloroplast protein 12 gene including 5'-untranslated
region 27tttgtattta ggtccctaac gccctctgga agggtgattt gaaaaaagcc
ctctaggagg 60acgtgaggca cgactagatt tgtaaatttt cggaatgaaa aattattttt
aaatatttta 120atcaaaaaat gtaaaaataa aaaaaattct cggcagggtg
gcagcatggg cctaaggccc 180agtcaactgt gggcctataa gcgactaatc
cggctgtaac tgggccttgc aagaggcttg 240tcttgttggt ccgaactcag
gaagtccagg ttgcggggac aacttcaagg ccatctggtt 300tccacttctc
ttaccacctc aattccgctc ttgatccgag ctagcttagt cccaatctaa
360aaactttaca aagaaagaac catacgcacc tattgggcaa aatgaaaaat
aatttgctac 420tcaccaaata atttgagcac ctctgcacct gtacactaaa
taactctgtt ccaccaaaat 480agttgagata tctaggacgt ttcattttgt
ccgttcttca ccaaactttt ccatagtatc 540tcagatattt tcgagaccga
aagtgatctt tctggcctta gaccgagttc acttccctac 600aagccattct
ttgctggcac aacacgaacc tctacatcaa tttcgtatcc aacctgaact
660tctgcataca tgtacacacc cacagtcatc tgctcatgtt ttcacggtca
aattaaaact 720gcttctctca ccttagattc acccaaggga aaagaaaaag
atctcctttg ccaagtcccc 780atttcgcatg aaatatctca aaatacagcc
cacgtggcac acgacgattg gctgaggagg 840cgataagaaa cgagtgcacg
tcgtcgaatc ctctctcccc ttctccccca ccccacggag 900ctatatatat
ataaacccca tctcttcaat ccgtgcaacg aacgcctcgt cgcaacagct
960acaaacgccc acatcacacg cagaaatccg catcaaca 99828948DNAOryza
sativapromoter(1)..(948)transcription regulating sequence of
chloroplast protein 12 gene 28tttgtattta ggtccctaac gccctctgga
agggtgattt gaaaaaagcc ctctaggagg 60acgtgaggca cgactagatt tgtaaatttt
cggaatgaaa aattattttt aaatatttta 120atcaaaaaat gtaaaaataa
aaaaaattct cggcagggtg gcagcatggg cctaaggccc 180agtcaactgt
gggcctataa gcgactaatc cggctgtaac tgggccttgc aagaggcttg
240tcttgttggt ccgaactcag gaagtccagg ttgcggggac aacttcaagg
ccatctggtt 300tccacttctc ttaccacctc aattccgctc ttgatccgag
ctagcttagt cccaatctaa 360aaactttaca aagaaagaac catacgcacc
tattgggcaa aatgaaaaat aatttgctac 420tcaccaaata atttgagcac
ctctgcacct gtacactaaa taactctgtt ccaccaaaat 480agttgagata
tctaggacgt ttcattttgt ccgttcttca ccaaactttt ccatagtatc
540tcagatattt tcgagaccga aagtgatctt tctggcctta gaccgagttc
acttccctac 600aagccattct ttgctggcac aacacgaacc tctacatcaa
tttcgtatcc aacctgaact 660tctgcataca tgtacacacc cacagtcatc
tgctcatgtt ttcacggtca aattaaaact 720gcttctctca ccttagattc
acccaaggga aaagaaaaag atctcctttg ccaagtcccc 780atttcgcatg
aaatatctca aaatacagcc cacgtggcac acgacgattg gctgaggagg
840cgataagaaa cgagtgcacg tcgtcgaatc ctctctcccc ttctccccca
ccccacggag 900ctatatatat ataaacccca tctcttcaat ccgtgcaacg aacgcctc
94829301DNAOryza sativapromoter(1)..(301)Potential core region of
the promoter of chloroplast protein 12 gene comprising clusters of
promoter elements 29tccaacctga acttctgcat acatgtacac acccacagtc
atctgctcat gttttcacgg 60tcaaattaaa actgcttctc tcaccttaga ttcacccaag
ggaaaagaaa aagatctcct 120ttgccaagtc cccatttcgc atgaaatatc
tcaaaataca gcccacgtgg cacacgacga 180ttggctgagg aggcgataag
aaacgagtgc acgtcgtcga atcctctctc cccttctccc 240ccaccccacg
gagctatata tatataaacc ccatctcttc aatccgtgca acgaacgcct 300c
30130375DNAOryza sativaCDS(1)..(372)coding for putative Choroplast
12 (CP12) protein 30atg gcg tcc acg ctg acc aac gtc ggc ctg tct acc
ccg gcg gcg gcg 48Met Ala Ser Thr Leu Thr Asn Val Gly Leu Ser Thr
Pro Ala Ala Ala 1 5 10 15 gcg tcg tcc ctc gtt agg ccg gtc gcc gga
gct gga cgc gtg gtg ttt 96Ala Ser Ser Leu Val Arg Pro Val Ala Gly
Ala Gly Arg Val Val Phe 20 25 30 ccc cgt gtt ggc cgc ggc ggg ttc
gcg gcg gtg agg gcg agc ggg ccg 144Pro Arg Val Gly Arg Gly Gly Phe
Ala Ala Val Arg Ala Ser Gly Pro 35 40 45 gcg acg ccg ccg gac atc
tcg gac aag atg tcg gag agc atc gac aag 192Ala Thr Pro Pro Asp Ile
Ser Asp Lys Met Ser Glu Ser Ile Asp Lys 50 55 60 gcg aag gag gcg
tgc gcg gag gac acg gcg agc ggc gag tgc gcg gcg 240Ala Lys Glu Ala
Cys Ala Glu Asp Thr Ala Ser Gly Glu Cys Ala Ala 65 70 75 80 gcg tgg
gac gag gtg gag gag ctg agc gcg gcg gcg agc cac gcg cgc 288Ala Trp
Asp Glu Val Glu Glu Leu Ser Ala Ala Ala Ser His Ala Arg 85 90 95
gac aag ctc aag gag acc tcc gac ccg ctc gag gcc tac tgc aag gac
336Asp Lys Leu Lys Glu Thr Ser Asp Pro Leu Glu Ala Tyr Cys Lys Asp
100 105 110 aac ccg gag acc gac gag tgc cgc acc tac gac aac tga
375Asn Pro Glu Thr Asp Glu Cys Arg Thr Tyr Asp Asn 115 120
31124PRTOryza sativa 31Met Ala Ser Thr Leu Thr Asn Val Gly Leu Ser
Thr Pro Ala Ala Ala 1 5 10 15 Ala Ser Ser Leu Val Arg Pro Val Ala
Gly Ala Gly Arg Val Val Phe 20 25 30 Pro Arg Val Gly Arg Gly Gly
Phe Ala Ala Val Arg Ala Ser Gly Pro 35 40 45 Ala Thr Pro Pro Asp
Ile Ser Asp Lys Met Ser Glu Ser Ile Asp Lys 50 55 60 Ala Lys Glu
Ala Cys Ala Glu Asp Thr Ala Ser Gly Glu Cys Ala Ala 65 70 75 80 Ala
Trp Asp Glu Val Glu Glu Leu Ser Ala Ala Ala Ser His Ala Arg 85 90
95 Asp Lys Leu Lys Glu Thr Ser Asp Pro Leu Glu Ala Tyr Cys Lys Asp
100 105 110 Asn Pro Glu Thr Asp Glu Cys Arg Thr Tyr Asp Asn 115 120
32479DNAZea maysterminator(1)..(473)Intergenic sequence comprising
3'-untranslated region of Zea mays lactate dehydrogenase gene
32gatgatcaca tcaccgtctc tcttcattaa ttaattattg tatcaatttc cacaacctag
60cagcagcatc cggtacccgt gttcaataaa aacaaaccgc tacaatgtgt gctttctagc
120tgcattaagc tgcttactac gagtatttgg gctgcggctt tctttttcat
gtatctcacc 180aaatcgttat tgttgtgaga gctatactac acggtggtat
caagagtatc acaatgccca 240acaggcgatg gattgagctt tcctaatttt
ttcatgataa attaagttct actccctccg 300tccacataaa tttgtctttc
tagatttttt cgtaagtcaa aatatttaaa ctttgatcaa 360cgatatatat
aaaagaataa attgttttaa actaaaaaat ttattccctc agttcttttt
420tatttgtcgc agtttagttc aaaaataaac tagcggatga caatatcgag actgggata
479332088DNAArtificial sequenceconstruct pBPSMM304 [Os.CP12
promoter::Zm.ubiquitin intron] 33tttgtattta ggtccctaac gccctctgga
agggtgattt gaaaaaagcc ctctaggagg 60acgtgaggca cgactagatt tgtaaatttt
cggaatgaaa aattattttt aaatatttta 120atcaaaaaat gtaaaaataa
aaaaaattct cggcagggtg gcagcatggg cctaaggccc 180agtcaactgt
gggcctataa gcgactaatc cggctgtaac tgggccttgc aagaggcttg
240tcttgttggt ccgaactcag gaagtccagg ttgcggggac aacttcaagg
ccatctggtt 300tccacttctc ttaccacctc aattccgctc ttgatccgag
ctagcttagt cccaatctaa 360aaactttaca aagaaagaac catacgcacc
tattgggcaa aatgaaaaat aatttgctac 420tcaccaaata atttgagcac
ctctgcacct gtacactaaa taactctgtt ccaccaaaat 480agttgagata
tctaggacgt ttcattttgt ccgttcttca ccaaactttt ccatagtatc
540tcagatattt tcgagaccga aagtgatctt tctggcctta gaccgagttc
acttccctac 600aagccattct ttgctggcac aacacgaacc tctacatcaa
tttcgtatcc aacctgaact 660tctgcataca tgtacacacc cacagtcatc
tgctcatgtt ttcacggtca aattaaaact 720gcttctctca ccttagattc
acccaaggga aaagaaaaag atctcctttg ccaagtcccc
780atttcgcatg aaatatctca aaatacagcc cacgtggcac acgacgattg
gctgaggagg 840cgataagaaa cgagtgcacg tcgtcgaatc ctctctcccc
ttctccccca ccccacggag 900ctatatatat ataaacccca tctcttcaat
ccgtgcaacg aacgcctcgt cgcaacagct 960acaaacgccc acatcacacg
cagaaatccg catcaacagg cgcgccaagc ttgcatgcct 1020gcaggtcgac
tctagaggat ctcccccaaa tccacccgtc ggcacctccg cttcaaggta
1080cgccgctcgt cctccccccc cccccctctc taccttctct agatcggcgt
tccggtccat 1140ggttagggcc cggtagttct acttctgttc atgtttgtgt
tagatccgtg tttgtgttag 1200atccgtgctg ctagcgttcg tacacggatg
cgacctgtac gtcagacacg ttctgattgc 1260taacttgcca gtgtttctct
ttggggaatc ctgggatggc tctagccgtt ccgcagacgg 1320gatcgatttc
atgatttttt ttgtttcgtt gcatagggtt tggtttgccc ttttccttta
1380tttcaatata tgccgtgcac ttgtttgtcg ggtcatcttt tcatgctttt
ttttgtcttg 1440gttgtgatga tgtggtctgg ttgggcggtc gttctagatc
ggagtagaat tctgtttcaa 1500actacctggt ggatttatta attttggatc
tgtatgtgtg tgccatacat attcatagtt 1560acgaattgaa gatgatggat
ggaaatatcg atctaggata ggtatacatg ttgatgcggg 1620ttttactgat
gcatatacag agatgctttt tgttcgcttg gttgtgatga tgtggtgtgg
1680ttgggcggtc gttcattcgt tctagatcgg agtagaatac tgtttcaaac
tacctggtgt 1740atttattaat tttggaactg tatgtgtgtg tcatacatct
tcatagttac gagtttaaga 1800tggatggaaa tatcgatcta ggataggtat
acatgttgat gtgggtttta ctgatgcata 1860tacatgatgg catatgcagc
atctattcat atgctctaac cttgagtacc tatctattat 1920aataaacaag
tatgttttat aattattttg atcttgatat acttggatga tggcatatgc
1980agcagctata tgtggatttt tttagccctg ccttcatacg ctatttattt
gcttggtact 2040gtttcttttg tcgatgctca ccctgttgtt tggtgttact tctgcagc
2088341093DNAOryza sativaterminator(1)..(1093)Intergenic sequence
including the 3' untranslated region of caffeoyl
CoA-O-methyltransferase functional as transcription terminator
34gccgatgccc aagaactagt cattttaaat ttataaatta taactaaatt gtataatttt
60tgcctttttt ttttaatttg caagctactg gaaaatgtta tttaatatat gtataaatgt
120cgagacaata atattattgc attataaatt gacctggttt ttgttagctt
tcgttgcgcg 180tgagaatggt gagtgtgtgc tgctgatgaa actcgaatgt
tcacttttgt tgtcttgtcc 240agctttgcta aactttggca gcattagcaa
agctgttttg ttctgtttct gaattgttct 300tggattgaaa tctctaatat
tgacttgata attaaatttg accggttttt ggcagtaaat 360tccttcagta
ttggcagctc aaactgaatt cttctactaa tagtttagtg cttgtggaga
420gtgtagtcgt gtagagtgga ctggactaat tcagatcttt acttggttag
ctgaagatgg 480catccgttat ctgaattctt cagtagttgg tggataatga
caactcgatg tagagagata 540atttgctgca tgttagtttg gaagtagctt
caaaggattt aattctcagc gtccgaagtc 600ttaagtacag ttggtttgga
gagctgttcc tgtgaagact tttatgattt gtgctagtta 660tgtcatcaca
tgatcacttc aattatcact tatcgatcta gtgagaccaa ccatacatac
720catacaaagg taaaaagtgt tcaaactgga ttgaacaagt tctgtctcca
tatagatcct 780actaaaatgc atacatgttg tagcaatccc atttcatcca
aaccaaacaa aatctctttc 840attcggctct aaccaatcaa acagagccat
ttgtatcccc cgaaccaaac cagccgagca 900tggatgaggg atcgagggca
tccgagcaac caaacaggcc caaagtgaat gctttggtcg 960attttcgatt
tgttccctac gaatccaatt ttagtccttc agccagaaga ccagatacaa
1020ttgatccctc aactatcaaa acaagtgcag atgagctccc tcgacagttt
ggacggcggt 1080ttggttgacg tgg 109335558DNAZea
maysterminator(1)..(558)Intergenic sequence including the 3'
untranslated region of hydroxyproline-rich glyco-protein gene
functional as transcription terminator sequence 35gccaccatac
tactagaaag cgatgcctac cataccacac tgctgtcagt ctctggagca 60tttaggtacg
tactaatact acgtacaacg gtacaagaat ggagcatgca atatgcatgc
120acactacata catttagtat gcttgtgtca aatgtatcgt cagtatcata
ctgatctcct 180ggcatagtct ggcactaacc ataggctctc cttttctttt
gtgttgggac aggtggtctc 240gatcgatgga agaattgtgt cctagccagc
cggcaaaggt gacctgctga tgatgatgat 300gagaggcgag tcctacgccc
tagtcctact actaccctct ttgtgtgctg ccatccatcc 360gtccccgcta
gacgatcgag gagagaataa cgcagagctc tgtgctcccg gcctctgtct
420tctgccgtcc cggccgttta atttatagtc tctactgtgt gttcgtccca
tgtgtttagc 480agcagcagca ggtgtattgt gcgggtatgt aatggtattg
caactatatt gggtgtaaaa 540ccataataaa tgtgggca 558365238DNAArtificial
sequenceconstruct pBPSMM325 [Os.CCoAMT1 promoter::Zm.ubiquitin
intron::GUS::Os.CCoAMT1 terminator] 36gcaactactg cacggtaaaa
gtgataggaa tcggtcggaa acagtattaa tgtttttatt 60atttttacaa aaacgaattg
aaataattgg aaattttcat atttatatat taaactattc 120agtatcaact
tcaattcgac gtcaatagaa attagaaaag cataattata cacagtaata
180ggcgttcaag atattattgt tattatttag ttttgtggaa atggtatcaa
cgtgatcgga 240aaattttgta catgttttca ccctgcggga tatctcaatt
ccttctcctc cctctaccgc 300catatcagca cacgttttag agcaccaatc
ataacccata aatccgtggg ctactcactt 360atttaattta tatgtgaatt
cgtgacctga ctcactcaca tactatcaaa aatttgtctc 420agtcacccat
ctccttcttt cctggtccga taagggttta tcctacggtt cgacggttat
480cacgatagtc gtgcggttac tgaggtatac cgtgatttaa aaatatgata
aagttaccgc 540aggttttaac tgcgcggttt ggtaaacctg ttcctcctca
ccaaccttct cctccggtct 600ccttatgtgt ctcaccgagg cgagccgccg
cgagaccgca tggacgcggt ccacgcacct 660ggcggtgcac ctcctcctcc
ccggcgaaga agacgtggag gagagtaaat gagcaatcag 720gcccacggcc
caatcgccgt ccaccaccca ccaccctcag cgacccaaaa ccacctcacc
780aacccaactc tgtaccgtac tgtacccgcc ctcccctccc actgacactc
cgggcccacc 840tgtcggcgcg actcttccac ggtccccttc tctcctcaga
gattttttcc acgcattttt 900taattttttt ttctgcagtt cacatgctct
tctcccactc ttccgccgcg ctatataaac 960cgcgcgaggc gtcgttgcct
cgtcggcgaa gtcaatccgg cgatccccgg cgagcgagag 1020atcgaagcaa
gctgcgagct cgatctcccc caaatccacc cgtcggcacc tccgcttcaa
1080ggtacgccgc tcgtcctccc cccccccccc tctctacctt ctctagatcg
gcgttccggt 1140ccatggttag ggcccggtag ttctacttct gttcatgttt
gtgttagatc cgtgtttgtg 1200ttagatccgt gctgctagcg ttcgtacacg
gatgcgacct gtacgtcaga cacgttctga 1260ttgctaactt gccagtgttt
ctctttgggg aatcctggga tggctctagc cgttccgcag 1320acgggatcga
tttcatgatt ttttttgttt cgttgcatag ggtttggttt gcccttttcc
1380tttatttcaa tatatgccgt gcacttgttt gtcgggtcat cttttcatgc
ttttttttgt 1440cttggttgtg atgatgtggt ctggttgggc ggtcgttcta
gatcggagta gaattctgtt 1500tcaaactacc tggtggattt attaattttg
gatctgtatg tgtgtgccat acatattcat 1560agttacgaat tgaagatgat
ggatggaaat atcgatctag gataggtata catgttgatg 1620cgggttttac
tgatgcatat acagagatgc tttttgttcg cttggttgtg atgatgtggt
1680gtggttgggc ggtcgttcat tcgttctaga tcggagtaga atactgtttc
aaactacctg 1740gtgtatttat taattttgga actgtatgtg tgtgtcatac
atcttcatag ttacgagttt 1800aagatggatg gaaatatcga tctaggatag
gtatacatgt tgatgtgggt tttactgatg 1860catatacatg atggcatatg
cagcatctat tcatatgctc taaccttgag tacctatcta 1920ttataataaa
caagtatgtt ttataattat tttgatcttg atatacttgg atgatggcat
1980atgcagcagc tatatgtgga tttttttagc cctgccttca tacgctattt
atttgcttgg 2040tactgtttct tttgtcgatg ctcaccctgt tgtttggtgt
tacttctgca gccccgggga 2100tccatgttac gtcctgtaga aaccccaacc
cgtgaaatca aaaaactcga cggcctgtgg 2160gcattcagtc tggatcgcga
aaactgtgga attgatcagc gttggtggga aagcgcgtta 2220caagaaagcc
gggcaattgc tgtgccaggc agttttaacg atcagttcgc cgatgcagat
2280attcgtaatt atgcgggcaa cgtctggtat cagcgcgaag tctttatacc
gaaaggttgg 2340gcaggccagc gtatcgtgct gcgtttcgat gcggtcactc
attacggcaa agtgtgggtc 2400aataatcagg aagtgatgga gcatcagggc
ggctatacgc catttgaagc cgatgtcacg 2460ccgtatgtta ttgccgggaa
aagtgtacgt aagtttctgc ttctaccttt gatatatata 2520taataattat
cattaattag tagtaatata atatttcaaa tatttttttc aaaataaaag
2580aatgtagtat atagcaattg cttttctgta gtttataagt gtgtatattt
taatttataa 2640cttttctaat atatgaccaa aatttgttga tgtgcaggta
tcaccgtttg tgtgaacaac 2700gaactgaact ggcagactat cccgccggga
atggtgatta ccgacgaaaa cggcaagaaa 2760aagcagtctt acttccatga
tttctttaac tatgccggaa tccatcgcag cgtaatgctc 2820tacaccacgc
cgaacacctg ggtggacgat atcaccgtgg tgacgcatgt cgcgcaagac
2880tgtaaccacg cgtctgttga ctggcaggtg gtggccaatg gtgatgtcag
cgttgaactg 2940cgtgatgcgg atcaacaggt ggttgcaact ggacaaggca
ctagcgggac tttgcaagtg 3000gtgaatccgc acctctggca accgggtgaa
ggttatctct atgaactgtg cgtcacagcc 3060aaaagccaga cagagtgtga
tatctacccg cttcgcgtcg gcatccggtc agtggcagtg 3120aagggccaac
agttcctgat taaccacaaa ccgttctact ttactggctt tggtcgtcat
3180gaagatgcgg acttacgtgg caaaggattc gataacgtgc tgatggtgca
cgaccacgca 3240ttaatggact ggattggggc caactcctac cgtacctcgc
attaccctta cgctgaagag 3300atgctcgact gggcagatga acatggcatc
gtggtgattg atgaaactgc tgctgtcggc 3360tttaacctct ctttaggcat
tggtttcgaa gcgggcaaca agccgaaaga actgtacagc 3420gaagaggcag
tcaacgggga aactcagcaa gcgcacttac aggcgattaa agagctgata
3480gcgcgtgaca aaaaccaccc aagcgtggtg atgtggagta ttgccaacga
accggatacc 3540cgtccgcaag tgcacgggaa tatttcgcca ctggcggaag
caacgcgtaa actcgacccg 3600acgcgtccga tcacctgcgt caatgtaatg
ttctgcgacg ctcacaccga taccatcagc 3660gatctctttg atgtgctgtg
cctgaaccgt tattacggat ggtatgtcca aagcggcgat 3720ttggaaacgg
cagagaaggt actggaaaaa gaacttctgg cctggcagga gaaactgcat
3780cagccgatta tcatcaccga atacggcgtg gatacgttag ccgggctgca
ctcaatgtac 3840accgacatgt ggagtgaaga gtatcagtgt gcatggctgg
atatgtatca ccgcgtcttt 3900gatcgcgtca gcgccgtcgt cggtgaacag
gtatggaatt tcgccgattt tgcgacctcg 3960caaggcatat tgcgcgttgg
cggtaacaag aaagggatct tcactcgcga ccgcaaaccg 4020aagtcggcgg
cttttctgct gcaaaaacgc tggactggca tgaacttcgg tgaaaaaccg
4080cagcagggag gcaaacaatg aagatcctct agagtcgacc tgcaggcatg
caagcttggc 4140gcgccgccga tgcccaagaa ctagtcattt taaatttata
aattataact aaattgtata 4200atttttgcct ttttttttta atttgcaagc
tactggaaaa tgttatttaa tatatgtata 4260aatgtcgaga caataatatt
attgcattat aaattgacct ggtttttgtt agctttcgtt 4320gcgcgtgaga
atggtgagtg tgtgctgctg atgaaactcg aatgttcact tttgttgtct
4380tgtccagctt tgctaaactt tggcagcatt agcaaagctg ttttgttctg
tttctgaatt 4440gttcttggat tgaaatctct aatattgact tgataattaa
atttgaccgg tttttggcag 4500taaattcctt cagtattggc agctcaaact
gaattcttct actaatagtt tagtgcttgt 4560ggagagtgta gtcgtgtaga
gtggactgga ctaattcaga tctttacttg gttagctgaa 4620gatggcatcc
gttatctgaa ttcttcagta gttggtggat aatgacaact cgatgtagag
4680agataatttg ctgcatgtta gtttggaagt agcttcaaag gatttaattc
tcagcgtccg 4740aagtcttaag tacagttggt ttggagagct gttcctgtga
agacttttat gatttgtgct 4800agttatgtca tcacatgatc acttcaatta
tcacttatcg atctagtgag accaaccata 4860cataccatac aaaggtaaaa
agtgttcaaa ctggattgaa caagttctgt ctccatatag 4920atcctactaa
aatgcataca tgttgtagca atcccatttc atccaaacca aacaaaatct
4980ctttcattcg gctctaacca atcaaacaga gccatttgta tcccccgaac
caaaccagcc 5040gagcatggat gagggatcga gggcatccga gcaaccaaac
aggcccaaag tgaatgcttt 5100ggtcgatttt cgatttgttc cctacgaatc
caattttagt ccttcagcca gaagaccaga 5160tacaattgat ccctcaacta
tcaaaacaag tgcagatgag ctccctcgac agtttggacg 5220gcggtttggt tgacgtgg
5238371891DNAArtificial sequenceconstruct pBPSMM331 [Os.SI
promoter::Zm.ubiquitin intron] 37gcctcgattc gaccgtgtaa tggaatgaag
gtggtgggcc cccaccccca caagccactc 60tccacacttt ggtgttcctg gtatgtcacc
tagaccaaca actatgttaa gccatatgtt 120ccacagtgca aaatctacaa
gaccacgata caagtaggta tggtggacta ccacattttc 180acttctcttt
cactttcccc tctctctccc ccctctcttc ctttccccca ccgcagagag
240cctggcgcgc ggagacggcg acggcgccgg accaagcagt ggtggagcga
cggcagggcg 300acagcgccga gcggcgggat gcgctcgccg gcgcaccacc
ccctcctctc ccccccgagc 360ggcggggctg ctcggagcag cagggcggcg
gcggcatgtc ggcggcgggc agacgacttg 420gagcgggaga cggcgacggg
cggatgcgag gcggcggtcg gcgccctcct cccctggagt 480tcggctgctt
cgccccctct cctctctcct ctagcggtgg tgtgggtccc actgagctga
540ggagggcgcg cggttggacg acgaggcaaa ggaatactag tcttcgcttt
tttgggttga 600ggctgaatgc cacgtcggcc cattgtgaat gccctttaac
aaaacaaggg tttatggcta 660tgggatctgg ctgaggcatt gacctacctt
ggtccttggc agagagagag agagactccc 720cctcactcct tccccgacga
cctgctcgat ccgatccaat cagctcctct ccagtccaga 780tcggaaggaa
gccaggagtt aggcgcgcca agcttgcatg cctgcaggtc gactctagag
840gatctccccc aaatccaccc gtcggcacct ccgcttcaag gtacgccgct
cgtcctcccc 900ccccccccct ctctaccttc tctagatcgg cgttccggtc
catggttagg gcccggtagt 960tctacttctg ttcatgtttg tgttagatcc
gtgtttgtgt tagatccgtg ctgctagcgt 1020tcgtacacgg atgcgacctg
tacgtcagac acgttctgat tgctaacttg ccagtgtttc 1080tctttgggga
atcctgggat ggctctagcc gttccgcaga cgggatcgat ttcatgattt
1140tttttgtttc gttgcatagg gtttggtttg cccttttcct ttatttcaat
atatgccgtg 1200cacttgtttg tcgggtcatc ttttcatgct tttttttgtc
ttggttgtga tgatgtggtc 1260tggttgggcg gtcgttctag atcggagtag
aattctgttt caaactacct ggtggattta 1320ttaattttgg atctgtatgt
gtgtgccata catattcata gttacgaatt gaagatgatg 1380gatggaaata
tcgatctagg ataggtatac atgttgatgc gggttttact gatgcatata
1440cagagatgct ttttgttcgc ttggttgtga tgatgtggtg tggttgggcg
gtcgttcatt 1500cgttctagat cggagtagaa tactgtttca aactacctgg
tgtatttatt aattttggaa 1560ctgtatgtgt gtgtcataca tcttcatagt
tacgagttta agatggatgg aaatatcgat 1620ctaggatagg tatacatgtt
gatgtgggtt ttactgatgc atatacatga tggcatatgc 1680agcatctatt
catatgctct aaccttgagt acctatctat tataataaac aagtatgttt
1740tataattatt ttgatcttga tatacttgga tgatggcata tgcagcagct
atatgtggat 1800ttttttagcc ctgccttcat acgctattta tttgcttggt
actgtttctt ttgtcgatgc 1860tcaccctgtt gtttggtgtt acttctgcag c
1891384835DNAArtificial sequenceconstruct pBPSET003 [Zm.HRGP
promoter::Zm.ubiquitin intron::GUS::Zm.HRGP terminator]
38ggtgaccttc ttgcttcttc gatcgtctgg acgtcgagga gccccgcggc agcgcacgcg
60tctgcaccgt tatggtggcc gcgctcgcga tggaatagaa ggggtaatga tggatccggc
120caggaaggcc acgacatcga cggatccaac cggcaagacg gcgatccggt
taaatagacg 180acggatctag ctgggaaggt agactctaca ttaaatgagg
ttgcacatgc cctaataact 240ttataaatct aatttattca gaggcaaggt
agtagtatta tctttcccaa cggatagtta 300tctgatctgc cgttcagctt
gatcgataac tttataaatc taatttattc agaggccggc 360ggcagcgcac
acgtctgcac cagtaatgtt agccgcgcct gtggcgtaat agaaggggta
420acgatggatc cgaccagaaa ggcctcgaca ttgacggatc cagacggcga
tccggtcaaa 480gagacgacga atctagccga gaaggtagat ctctcgagag
agttcatatt aaatgatgtt 540gtacatgcca taataactct ataaatctaa
tttattcata ggcgaaggta gtagtattat 600ctttcccagc ggatcgttat
ctgatctgcc gttcagcttg atcgatccac gtcgtttgat 660ctcggcgagc
agcacatggc ggctcttctt gtgtacaggt ctcactctct gctacttcag
720tgcaaggcgg agtgaatgca cacaataacg tgagtattgt gggaactact
tgtagatgca 780aacgatgtaa atccacctgc tccaccaagt gcccgcccgg
ctctatccat tccattcgtc 840aacatgcagg ttcagactgg cccgtgctgg
accagtgagc ggtgccggtg aaccccaatg 900caagcgaagc gagtgaccat
cggggaagcc tcccgtgctg cccccacatg gcttgcctga 960atgcctctct
ctcgccgcag tgccctctct ctctcctcct cctctccgtc gaagggcgtc
1020acgagagccc agagggcatc cgaggccccc accccacccc ttcctccgtg
tatataagca 1080gtggcagggt gagcgtctct cctcagacca ccactgcgcc
attggccagc tagagccaac 1140cagaagagct tgcagttact gagagtgtgt
gtgagagaga gggagctcga tctcccccaa 1200atccacccgt cggcacctcc
gcttcaaggt acgccgctcg tcctcccccc cccccctctc 1260tctaccttct
ctagatcggc gttccggtcc atggttaggg cccggtagtt ctacttctgt
1320tcatgtttgt gttagatccg tgtttgtgtt agatccgtgc tgctagcgtt
cgtacacgga 1380tgcgacctgt acgtcagaca cgttctgatt gctaacttgc
cagtgtttct ctttggggaa 1440tcctgggatg gctctagccg ttccgcagac
gggatcgatt tcatgatttt ttttgtttcg 1500ttgcataggg tttggtttgc
ccttttcctt tatttcaata tatgccgtgc acttgtttgt 1560cgggtcatct
tttcatgctt ttttttgtct tggttgtgat gatgtggtct ggttgggcgg
1620tcgttctaga tcggagtaga attctgtttc aaactacctg gtggatttat
taattttgga 1680tctgtatgtg tgtgccatac atattcatag ttacgaattg
aagatgatgg atggaaatat 1740cgatctagga taggtataca tgttgatgcg
ggttttactg atgcatatac agagatgctt 1800tttgttcgct tggttgtgat
gatgtggtgt ggttgggcgg tcgttcattc gttctagatc 1860ggagtagaat
actgtttcaa actacctggt gtatttatta attttggaac tgtatgtgtg
1920tgtcatacat cttcatagtt acgagtttaa gatggatgga aatatcgatc
taggataggt 1980atacatgttg atgtgggttt tactgatgca tatacatgat
ggcatatgca gcatctattc 2040atatgctcta accttgagta cctatctatt
ataataaaca agtatgtttt ataattattt 2100tgatcttgat atacttggat
gatggcatat gcagcagcta tatgtggatt tttttagccc 2160tgccttcata
cgctatttat ttgcttggta ctgtttcttt tgtcgatgct caccctgttg
2220tttggtgtta cttctgcagc cccggggatc catgttacgt cctgtagaaa
ccccaacccg 2280tgaaatcaaa aaactcgacg gcctgtgggc attcagtctg
gatcgcgaaa actgtggaat 2340tggtcagcgt tggtgggaaa gcgcgttaca
agaaagccgg gcaattgctg tgccaggcag 2400ttttaacgat cagttcgccg
atgcagatat tcgtaattat gcgggcaacg tctggtatca 2460gcgcgaagtc
tttataccga aaggttgggc aggccagcgt atcgtgctgc gtttcgatgc
2520ggtcactcat tacggcaaag tgtgggtcaa taatcaggaa gtgatggagc
atcagggcgg 2580ctatacgcca tttgaagccg atgtcacgcc gtatgttatt
gccgggaaaa gtgtacgtaa 2640gtttctgctt ctacctttga tatatatata
ataattatca ttaattagta gtaatataat 2700atttcaaata tttttttcaa
aataaaagaa tgtagtatat agcaattgct tttctgtagt 2760ttataagtgt
gtatatttta atttataact tttctaatat atgaccaaaa tttgttgatg
2820tgcaggtatc accgtttgtg tgaacaacga actgaactgg cagactatcc
cgccgggaat 2880ggtgattacc gacgaaaacg gcaagaaaaa gcagtcttac
ttccatgatt tctttaacta 2940tgccggaatc catcgcagcg taatgctcta
caccacgccg aacacctggg tggacgatat 3000caccgtggtg acgcatgtcg
cgcaagactg taaccacgcg tctgttgact ggcaggtggt 3060ggccaatggt
gatgtcagcg ttgaactgcg tgatgcggat caacaggtgg ttgcaactgg
3120acaaggcact agcgggactt tgcaagtggt gaatccgcac ctctggcaac
cgggtgaagg 3180ttatctctat gaactgtgcg tcacagccaa aagccagaca
gagtgtgata tctacccgct 3240tcgcgtcggc atccggtcag tggcagtgaa
gggcgaacag ttcctgatta accacaaacc 3300gttctacttt actggctttg
gtcgtcatga agatgcggac ttgcgtggca aaggattcga 3360taacgtgctg
atggtgcacg accacgcatt aatggactgg attggggcca actcctaccg
3420tacctcgcat tacccttacg ctgaagagat gctcgactgg gcagatgaac
atggcatcgt 3480ggtgattgat gaaactgctg ctgtcggctt taacctctct
ttaggcattg gtttcgaagc 3540gggcaacaag ccgaaagaac tgtacagcga
agaggcagtc aacggggaaa ctcagcaagc 3600gcacttacag gcgattaaag
agctgatagc gcgtgacaaa aaccacccaa gcgtggtgat 3660gtggagtatt
gccaacgaac cggatacccg tccgcaaggt gcacgggaat atttcgcgcc
3720actggcggaa gcaacgcgta aactcgaccc gacgcgtccg atcacctgcg
tcaatgtaat 3780gttctgcgac gctcacaccg ataccatcag cgatctcttt
gatgtgctgt gcctgaaccg 3840ttattacgga tggtatgtcc aaagcggcga
tttggaaacg gcagagaagg tactggaaaa 3900agaacttctg gcctggcagg
agaaactgca tcagccgatt atcatcaccg aatacggcgt 3960ggatacgtta
gccgggctgc actcaatgta caccgacatg tggagtgaag agtatcagtg
4020tgcatggctg gatatgtatc accgcgtctt tgatcgcgtc agcgccgtcg
tcggtgaaca 4080ggtatggaat ttcgccgatt ttgcgacctc gcaaggcata
ttgcgcgttg gcggtaacaa 4140gaaagggatc
ttcactcgcg accgcaaacc gaagtcggcg gcttttctgc tgcaaaaacg
4200ctggactggc atgaacttcg gtgaaaaacc gcagcaggga ggcaaacaat
gaagatcctc 4260tagagtcgac ctgcagggcc accatactac tagaaagcga
tgcctaccat accacactgc 4320tgtcagtctc tggagcattt aggtacgtac
taatactacg tacaacggta caagaatgga 4380gcatgcaata tgcatgcaca
ctacatacat ttagtatgct tgtgtcaaat gtatcgtcag 4440tatcatactg
atctcctggc atagtctggc actaaccata ggctctcctt ttcttttgtg
4500ttgggacagg tggtctcgat cgatggaaga attgtgtcct agccagccgg
caaaggtgac 4560ctgctgatga tgatgatgag aggcgagtcc tacgccctag
tcctactact accctctttg 4620tgtgctgcca tccatccgtc cccgctagac
gatcgaggag agaataacgc agagctctgt 4680gctcccggcc tctgtcttct
gccgtcccgg ccgtttaatt tatagtctct actgtgtgtt 4740cgtcccatgt
gtttagcagc agcagcaggt gtattgtgcg ggtatgtaat ggtattgcaa
4800ctatattggg tgtaaaacca taataaatgt gggca 4835394632DNAArtificial
sequenceconstruct pBPSET007 [Zm.LDH promoter::Zm.ubiquitin
intron::GUS::Zm.LDH terminator] 39aacaaatggc gtacttatat aaccacaatg
tactggtgct gcgtcattat tttatactac 60gcatatatta ttataagtag agaaagctca
caaaaccatg cgcgcgcccc cctgtttgtt 120tcggtcgcta attacaccct
ttgtatcgtt ggttgatgat ggtctccacc ggccgtacga 180gtcatcgatc
gttgatttat ttttatcacc gacttgcacg cctttcgaac aaagacgcaa
240caaaggaaag cgaaagcgtc acgaacgagg ttgttccctg acagttgttc
gactaataca 300actgcaagac actgaataag cagtaaaaat caatatagat
taaagttaaa cgaacatgct 360caacatcgaa tactactcat atgtgttatt
attaagagaa taccaccaag gtagaaaagt 420taaaggacct aaactgttgt
gccgggagag ttgtgcgacg aacagatgta aatatgataa 480aataagttca
aagttcatat agatagcacg atcacactta gggctagttt gaagccataa
540aaatggaaga gattaaatga gataaaattc acttatttaa ttttaaataa
gaagagagtt 600ttaacctcta attctctcca gtattttagc tcctaaacta
gctcttacag cagtaaaaga 660cccttgatgg tagcgtatgc aaagagaagg
aactattcaa tgaattgttt ttttaatcac 720tagtagtatg gtgggtaact
gtcgtcaacc ggccctatct acttcagttt agtgaagcac 780taaaccgcac
cttggtatgt tcaaatttaa gatttttttt gaaacgaaac aattttaacc
840agcggctcca aaccggtgaa gtggtttggt ctttggtgtg gggccagggt
attaatggaa 900ttgaatatat aaagagcagg gtggtggacc tttcccctcc
cacgagtcga gtagccattg 960cccattgcca ttccttcctt cctccacaga
gaaatccgat ccgcggagat ttgacccaac 1020cagatcatat cacacacgta
atcccatccc attccgcccg gagctcgatc tcccccaaat 1080ccacccgtcg
gcacctccgc ttcaaggtac gccgctcgtc ctcccccccc cccctctctc
1140taccttctct agatcggcgt tccggtccat ggttagggcc cggtagttct
acttctgttc 1200atgtttgtgt tagatccgtg tttgtgttag atccgtgctg
ctagcgttcg tacacggatg 1260cgacctgtac gtcagacacg ttctgattgc
taacttgcca gtgtttctct ttggggaatc 1320ctgggatggc tctagccgtt
ccgcagacgg gatcgatttc atgatttttt ttgtttcgtt 1380gcatagggtt
tggtttgccc ttttccttta tttcaatata tgccgtgcac ttgtttgtcg
1440ggtcatcttt tcatgctttt ttttgtcttg gttgtgatga tgtggtctgg
ttgggcggtc 1500gttctagatc ggagtagaat tctgtttcaa actacctggt
ggatttatta attttggatc 1560tgtatgtgtg tgccatacat attcatagtt
acgaattgaa gatgatggat ggaaatatcg 1620atctaggata ggtatacatg
ttgatgcggg ttttactgat gcatatacag agatgctttt 1680tgttcgcttg
gttgtgatga tgtggtgtgg ttgggcggtc gttcattcgt tctagatcgg
1740agtagaatac tgtttcaaac tacctggtgt atttattaat tttggaactg
tatgtgtgtg 1800tcatacatct tcatagttac gagtttaaga tggatggaaa
tatcgatcta ggataggtat 1860acatgttgat gtgggtttta ctgatgcata
tacatgatgg catatgcagc atctattcat 1920atgctctaac cttgagtacc
tatctattat aataaacaag tatgttttat aattattttg 1980atcttgatat
acttggatga tggcatatgc agcagctata tgtggatttt tttagccctg
2040ccttcatacg ctatttattt gcttggtact gtttcttttg tcgatgctca
ccctgttgtt 2100tggtgttact tctgcagccc ggggatccat gttacgtcct
gtagaaaccc caacccgtga 2160aatcaaaaaa ctcgacggcc tgtgggcatt
cagtctggat cgcgaaaact gtggaattgg 2220tcagcgttgg tgggaaagcg
cgttacaaga aagccgggca attgctgtgc caggcagttt 2280taacgatcag
ttcgccgatg cagatattcg taattatgcg ggcaacgtct ggtatcagcg
2340cgaagtcttt ataccgaaag gttgggcagg ccagcgtatc gtgctgcgtt
tcgatgcggt 2400cactcattac ggcaaagtgt gggtcaataa tcaggaagtg
atggagcatc agggcggcta 2460tacgccattt gaagccgatg tcacgccgta
tgttattgcc gggaaaagtg tacgtaagtt 2520tctgcttcta cctttgatat
atatataata attatcatta attagtagta atataatatt 2580tcaaatattt
ttttcaaaat aaaagaatgt agtatatagc aattgctttt ctgtagttta
2640taagtgtgta tattttaatt tataactttt ctaatatatg accaaaattt
gttgatgtgc 2700aggtatcacc gtttgtgtga acaacgaact gaactggcag
actatcccgc cgggaatggt 2760gattaccgac gaaaacggca agaaaaagca
gtcttacttc catgatttct ttaactatgc 2820cggaatccat cgcagcgtaa
tgctctacac cacgccgaac acctgggtgg acgatatcac 2880cgtggtgacg
catgtcgcgc aagactgtaa ccacgcgtct gttgactggc aggtggtggc
2940caatggtgat gtcagcgttg aactgcgtga tgcggatcaa caggtggttg
caactggaca 3000aggcactagc gggactttgc aagtggtgaa tccgcacctc
tggcaaccgg gtgaaggtta 3060tctctatgaa ctgtgcgtca cagccaaaag
ccagacagag tgtgatatct acccgcttcg 3120cgtcggcatc cggtcagtgg
cagtgaaggg cgaacagttc ctgattaacc acaaaccgtt 3180ctactttact
ggctttggtc gtcatgaaga tgcggacttg cgtggcaaag gattcgataa
3240cgtgctgatg gtgcacgacc acgcattaat ggactggatt ggggccaact
cctaccgtac 3300ctcgcattac ccttacgctg aagagatgct cgactgggca
gatgaacatg gcatcgtggt 3360gattgatgaa actgctgctg tcggctttaa
cctctcttta ggcattggtt tcgaagcggg 3420caacaagccg aaagaactgt
acagcgaaga ggcagtcaac ggggaaactc agcaagcgca 3480cttacaggcg
attaaagagc tgatagcgcg tgacaaaaac cacccaagcg tggtgatgtg
3540gagtattgcc aacgaaccgg atacccgtcc gcaaggtgca cgggaatatt
tcgcgccact 3600ggcggaagca acgcgtaaac tcgacccgac gcgtccgatc
acctgcgtca atgtaatgtt 3660ctgcgacgct cacaccgata ccatcagcga
tctctttgat gtgctgtgcc tgaaccgtta 3720ttacggatgg tatgtccaaa
gcggcgattt ggaaacggca gagaaggtac tggaaaaaga 3780acttctggcc
tggcaggaga aactgcatca gccgattatc atcaccgaat acggcgtgga
3840tacgttagcc gggctgcact caatgtacac cgacatgtgg agtgaagagt
atcagtgtgc 3900atggctggat atgtatcacc gcgtctttga tcgcgtcagc
gccgtcgtcg gtgaacaggt 3960atggaatttc gccgattttg cgacctcgca
aggcatattg cgcgttggcg gtaacaagaa 4020agggatcttc actcgcgacc
gcaaaccgaa gtcggcggct tttctgctgc aaaaacgctg 4080gactggcatg
aacttcggtg aaaaaccgca gcagggaggc aaacaatgaa gatcctctag
4140agtcgacctg caggatgatc acatcaccgt ctctcttcat taattaatta
ttgtatcaat 4200ttccacaacc tagcagcagc atccggtacc cgtgttcaat
aaaaacaaac cgctacaatg 4260tgtgctttct agctgcatta agctgcttac
tacgagtatt tgggctgcgg ctttcttttt 4320catgtatctc accaaatcgt
tattgttgtg agagctatac tacacggtgg tatcaagagt 4380atcacaatgc
ccaacaggcg atggattgag ctttcctaat tttttcatga taaattaagt
4440tctactccct ccgtccacat aaatttgtct ttctagattt tttcgtaagt
caaaatattt 4500aaactttgat caacgatata tataaaagaa taaattgttt
taaactaaaa aatttattcc 4560ctcagttctt ttttatttgt cgcagtttag
ttcaaaaata aactagcgga tgacaatatc 4620gagactggga ta
46324027DNAArtificial sequenceoligonucleotide primer 40caactactgc
acggtaaaag tgatagg 274127DNAArtificial sequenceoligonucleotide
primer 41gcagcttgct tcgatctctc gctcgcc 274228DNAArtificial
sequenceoligonucleotide primer 42gccgatgccc aagaactagt cattttaa
284327DNAArtificial sequenceoligonucleotide primer 43attaacacgt
caaccaaacc gccgtcc 274427DNAArtificial sequenceoligonucleotide
primer 44tgcctcgatt cgaccgtgta atggaat 274527DNAArtificial
sequenceoligonucleotide primer 45actcctggct tccttccgat ctggact
274627DNAArtificial sequenceoligonucleotide primer 46ccggtgacct
tcttgcttct tcgatcg 274727DNAArtificial sequenceoligonucleotide
primer 47cctctctctc acacacactc tcagtaa 274827DNAArtificial
sequenceoligonucleotide primer 48aacaaatggc gtacttatat aaccaca
274927DNAArtificial sequenceoligonucleotide primer 49cgggcggaat
gggatgggat tacgtgt 275027DNAArtificial sequenceoligonucleotide
primer 50aaagcgatgc ctaccatacc acactgc 275128DNAArtificial
sequenceoligonucleotide primer 51tgcccacatt tattatggtt ttacaccc
285228DNAArtificial sequenceoligonucleotide primer 52tgatcacatc
accgtctctc ttcattaa 285328DNAArtificial sequenceoligonucleotide
primer 53tatcccagtc tcgatattgt catccgct 285426DNAArtificial
sequenceOligonucleotide primer 54tttgtattta ggtccctaac gccctc
265526DNAArtificial sequenceOligonucleotide primer 55tgttgatgcg
gatttctgcg tgtgat 26561000DNAOryza
sativapromoter(1)..(1000)transcription regulating sequence from
Oryza sativa Lactate-dehydrogenase gene comprising 5'-untranslated
region 56aaccgtgcgc gctctcaaat taaaccagcg gcagctcgta gtacctagga
tcatgcttgg 60ccacgaactt ataattactg tgttaagtag tggcccctac agctacttgt
agcttgtttc 120gcaaggccct tcatgcattt acttagtagt actactgatt
atcatatcaa gggtgtctct 180gtcaaggatg atctgagctt gaatcgatct
atgtaatcag cagtcatgca tgatgtttct 240gtgttggtgg acgccacggt
tttctccctc agctcatcgt ttcaaaccga aaaaaaagaa 300tggaatcttg
atcaagattc ttaagaccta attaccacta cagccgcgac ctcgattttg
360ttttcttccg gcctgatcca acaggcatcg caagttaagt acgtggtttt
catccccatc 420aattcaattc tacccaaatc aagtcttatg ttaacggcat
gcgttggtcg tctaattcat 480gctgaccact aattaagtta ctggctagta
gcttagctac tgacaacatt ctttcttaaa 540aagggagatc tatatacatt
catatatatg tacatgtgtg tgtcgcttta ttaaagtgga 600tcgatgaacc
gacgaagaaa ttaggtacca caaattaaag gtagcagtac tgccgccatt
660gtgccaaaaa ccagctaatt aaccatcggt tttcactaat caagcacaat
catgctacta 720attacccttt tcaacctata aatttatcac ggtcagatca
atttttctaa ttactttaaa 780gataatataa tagtagtaat caaccctata
aatacacaag gggtgcaacg accatgcatg 840tatagcaaaa acacatttcc
aacattcggt cttcactact agctagtagt gtttttttcc 900tacatatata
aacaataatt attcattgac aggcatcaag ctagctagct agccaactgt
960ctgcaagaag aagaagaaga agaaggtgca ggataaatcg 100057945DNAOryza
sativapromoter(1)..(945)transcription regulating sequence from
Oryza sativa Lactate-dehydrogenase gene 57aaccgtgcgc gctctcaaat
taaaccagcg gcagctcgta gtacctagga tcatgcttgg 60ccacgaactt ataattactg
tgttaagtag tggcccctac agctacttgt agcttgtttc 120gcaaggccct
tcatgcattt acttagtagt actactgatt atcatatcaa gggtgtctct
180gtcaaggatg atctgagctt gaatcgatct atgtaatcag cagtcatgca
tgatgtttct 240gtgttggtgg acgccacggt tttctccctc agctcatcgt
ttcaaaccga aaaaaaagaa 300tggaatcttg atcaagattc ttaagaccta
attaccacta cagccgcgac ctcgattttg 360ttttcttccg gcctgatcca
acaggcatcg caagttaagt acgtggtttt catccccatc 420aattcaattc
tacccaaatc aagtcttatg ttaacggcat gcgttggtcg tctaattcat
480gctgaccact aattaagtta ctggctagta gcttagctac tgacaacatt
ctttcttaaa 540aagggagatc tatatacatt catatatatg tacatgtgtg
tgtcgcttta ttaaagtgga 600tcgatgaacc gacgaagaaa ttaggtacca
caaattaaag gtagcagtac tgccgccatt 660gtgccaaaaa ccagctaatt
aaccatcggt tttcactaat caagcacaat catgctacta 720attacccttt
tcaacctata aatttatcac ggtcagatca atttttctaa ttactttaaa
780gataatataa tagtagtaat caaccctata aatacacaag gggtgcaacg
accatgcatg 840tatagcaaaa acacatttcc aacattcggt cttcactact
agctagtagt gtttttttcc 900tacatatata aacaataatt attcattgac
aggcatcaag ctagc 94558301DNAOryza sativapromoter(1)..(301)core
transcription regulating sequence from Oryza sativa
Lactate-dehydrogenase gene comprising clusters of promoter elements
58cagtactgcc gccattgtgc caaaaaccag ctaattaacc atcggttttc actaatcaag
60cacaatcatg ctactaatta cccttttcaa cctataaatt tatcacggtc agatcaattt
120ttctaattac tttaaagata atataatagt agtaatcaac cctataaata
cacaaggggt 180gcaacgacca tgcatgtata gcaaaaacac atttccaaca
ttcggtcttc actactagct 240agtagtgttt ttttcctaca tatataaaca
ataattattc attgacaggc atcaagctag 300c 301591083DNAOryza
sativaCDS(1)..(1080)coding for Oryza sativa Lactate-dehydrogenase
59atg aag aag gcg tcg tcg ctg tct gag ctg ggg ttc gac gcc gat ggc
48Met Lys Lys Ala Ser Ser Leu Ser Glu Leu Gly Phe Asp Ala Asp Gly 1
5 10 15 ccg tca ttc ttc cgg cac ctg acg ctg acc gat ggc gac gac ggc
acg 96Pro Ser Phe Phe Arg His Leu Thr Leu Thr Asp Gly Asp Asp Gly
Thr 20 25 30 ctg ccc cgg cgg cgg ctg atc aag atc tcg gtg atc ggc
gcg ggc aac 144Leu Pro Arg Arg Arg Leu Ile Lys Ile Ser Val Ile Gly
Ala Gly Asn 35 40 45 gtg ggc atg gcc atc gcg cag acg atc ctg acg
cag gac ctc gcc gac 192Val Gly Met Ala Ile Ala Gln Thr Ile Leu Thr
Gln Asp Leu Ala Asp 50 55 60 gag atc gtg ctg atc gac gcg gtg gcg
gac aag gtg cgg ggc gag atg 240Glu Ile Val Leu Ile Asp Ala Val Ala
Asp Lys Val Arg Gly Glu Met 65 70 75 80 ctg gac ctg cag cac gcg gcg
gcg ttc ctc ccc cgc gtg aac atc gtg 288Leu Asp Leu Gln His Ala Ala
Ala Phe Leu Pro Arg Val Asn Ile Val 85 90 95 tcc ggc acg gag gtg
tcg ctg acg agg agc tcg gac ctg gtg atc gtg 336Ser Gly Thr Glu Val
Ser Leu Thr Arg Ser Ser Asp Leu Val Ile Val 100 105 110 acg gcg ggg
gct cgg cag atc ccg ggg gag acg cgg ctg aac ctg ctg 384Thr Ala Gly
Ala Arg Gln Ile Pro Gly Glu Thr Arg Leu Asn Leu Leu 115 120 125 cag
cgg aac gtg tcg ctg ttc cgg aag atc gtg ccg gcg gcg gcg gag 432Gln
Arg Asn Val Ser Leu Phe Arg Lys Ile Val Pro Ala Ala Ala Glu 130 135
140 gcg tcg ccg gag tcg gtg ctg gtg atc gtg tcg aac ccg gtg gac gtg
480Ala Ser Pro Glu Ser Val Leu Val Ile Val Ser Asn Pro Val Asp Val
145 150 155 160 ctg acg tac gtg gcg tgg aag ctg tcg ggg ttc ccg gcg
agc agg gtg 528Leu Thr Tyr Val Ala Trp Lys Leu Ser Gly Phe Pro Ala
Ser Arg Val 165 170 175 atc ggg tcg ggg acg aac ctg gac tcg tct cgg
ttc agg ttc ctc ctc 576Ile Gly Ser Gly Thr Asn Leu Asp Ser Ser Arg
Phe Arg Phe Leu Leu 180 185 190 gcc gag cac ctg gag gtg agc gcg cag
gac gtg cag gcg tac atg gtg 624Ala Glu His Leu Glu Val Ser Ala Gln
Asp Val Gln Ala Tyr Met Val 195 200 205 ggg gag cac ggg gac agc tcg
gtg gcg ctg tgg tcg agc atc agc gtg 672Gly Glu His Gly Asp Ser Ser
Val Ala Leu Trp Ser Ser Ile Ser Val 210 215 220 ggg ggg atg ccg gtg
ctg gcg cac ctg cag aag aac cac cgg tcg gcg 720Gly Gly Met Pro Val
Leu Ala His Leu Gln Lys Asn His Arg Ser Ala 225 230 235 240 gcg acg
gcg aag aag ttc gac gag gcg gcg ctg gag ggg atc cgg cgg 768Ala Thr
Ala Lys Lys Phe Asp Glu Ala Ala Leu Glu Gly Ile Arg Arg 245 250 255
gcg gtg gtg ggg agc gcg tac gag gtg atc aag ctc aag ggg tac acg
816Ala Val Val Gly Ser Ala Tyr Glu Val Ile Lys Leu Lys Gly Tyr Thr
260 265 270 tcg tgg gcc atc ggc tac tcc gtc gcc agc atc gcc tgg tcg
ctg ctc 864Ser Trp Ala Ile Gly Tyr Ser Val Ala Ser Ile Ala Trp Ser
Leu Leu 275 280 285 cgt gac cag cgc cgc atc cac ccg gtc tcc gtc ctc
gcc aag ggc ctt 912Arg Asp Gln Arg Arg Ile His Pro Val Ser Val Leu
Ala Lys Gly Leu 290 295 300 gtc cgt ggc gtc ccc gcc gac cgc gag ctc
ttc ctc agc ctg ccc gct 960Val Arg Gly Val Pro Ala Asp Arg Glu Leu
Phe Leu Ser Leu Pro Ala 305 310 315 320 cgc ctc ggc cgc gcc ggc gtg
ctt ggc gtc gcc gcc gag ctg gtg ctc 1008Arg Leu Gly Arg Ala Gly Val
Leu Gly Val Ala Ala Glu Leu Val Leu 325 330 335 acc gac gag gag gag
agg agg ctt cgc atc tcc gcc gaa acc ctc tgg 1056Thr Asp Glu Glu Glu
Arg Arg Leu Arg Ile Ser Ala Glu Thr Leu Trp 340 345 350 gga tac tgc
cac gcc ctc ggc ctc taa 1083Gly Tyr Cys His Ala Leu Gly Leu 355 360
60360PRTOryza sativa 60Met Lys Lys Ala Ser Ser Leu Ser Glu Leu Gly
Phe Asp Ala Asp Gly 1 5 10 15 Pro Ser Phe Phe Arg His Leu Thr Leu
Thr Asp Gly Asp Asp Gly Thr 20 25 30 Leu Pro Arg Arg Arg Leu Ile
Lys Ile Ser Val Ile Gly Ala Gly Asn 35 40 45 Val Gly Met Ala Ile
Ala Gln Thr Ile Leu Thr Gln Asp Leu Ala Asp 50 55 60 Glu Ile Val
Leu Ile Asp Ala Val Ala Asp Lys Val Arg Gly Glu Met 65 70 75 80 Leu
Asp Leu Gln His Ala Ala Ala Phe Leu Pro Arg Val Asn Ile Val 85 90
95 Ser Gly Thr
Glu Val Ser Leu Thr Arg Ser Ser Asp Leu Val Ile Val 100 105 110 Thr
Ala Gly Ala Arg Gln Ile Pro Gly Glu Thr Arg Leu Asn Leu Leu 115 120
125 Gln Arg Asn Val Ser Leu Phe Arg Lys Ile Val Pro Ala Ala Ala Glu
130 135 140 Ala Ser Pro Glu Ser Val Leu Val Ile Val Ser Asn Pro Val
Asp Val 145 150 155 160 Leu Thr Tyr Val Ala Trp Lys Leu Ser Gly Phe
Pro Ala Ser Arg Val 165 170 175 Ile Gly Ser Gly Thr Asn Leu Asp Ser
Ser Arg Phe Arg Phe Leu Leu 180 185 190 Ala Glu His Leu Glu Val Ser
Ala Gln Asp Val Gln Ala Tyr Met Val 195 200 205 Gly Glu His Gly Asp
Ser Ser Val Ala Leu Trp Ser Ser Ile Ser Val 210 215 220 Gly Gly Met
Pro Val Leu Ala His Leu Gln Lys Asn His Arg Ser Ala 225 230 235 240
Ala Thr Ala Lys Lys Phe Asp Glu Ala Ala Leu Glu Gly Ile Arg Arg 245
250 255 Ala Val Val Gly Ser Ala Tyr Glu Val Ile Lys Leu Lys Gly Tyr
Thr 260 265 270 Ser Trp Ala Ile Gly Tyr Ser Val Ala Ser Ile Ala Trp
Ser Leu Leu 275 280 285 Arg Asp Gln Arg Arg Ile His Pro Val Ser Val
Leu Ala Lys Gly Leu 290 295 300 Val Arg Gly Val Pro Ala Asp Arg Glu
Leu Phe Leu Ser Leu Pro Ala 305 310 315 320 Arg Leu Gly Arg Ala Gly
Val Leu Gly Val Ala Ala Glu Leu Val Leu 325 330 335 Thr Asp Glu Glu
Glu Arg Arg Leu Arg Ile Ser Ala Glu Thr Leu Trp 340 345 350 Gly Tyr
Cys His Ala Leu Gly Leu 355 360 61 1000DNAOryza
sativapromoter(1)..(1000)transcription regulating sequence from
Oryza sativa Lactate-dehydrogenase gene comprising 5'-untranslated
region 61atcaacatcc gtaccaaatt aagtgtttct ttgacagcgt gccagatttg
gatcggtcat 60tcgttgcgga aagtagtacc aatgacttac tgctactatt aatctactac
tgtttcgctt 120aagtataggt aaaggaaatg taaccctatt tcgagaaaat
atattagtgg tagataggat 180agctaggaga tccaaagagc agaatggtat
actgcattag atatatccaa tgtgccacga 240tgagtggttt ttctttatct
gtttgtacta atttgcgcac ttttgaccga taagcgggcc 300ggcatctttt
taaatagaga aacacgcgat atgatgatag gcagtgagat ttcttcacaa
360atactgtaca gaacagtaga gcagtagtag tagggcacaa attgatggaa
tcaacttagc 420aacctttgta gaccgtcgtc aattttacaa gggagagata
tagtagaatg cgggtacaac 480agttcatgct gtgttgagtt atatattgtt
tttgggtttg tgattaaaca gtgtaacaac 540acggaaaggt tcaagacgag
caaacacgtc agcatgtaca aacgcagtgt atgagttaat 600taattatatg
gttagcgtgt atcgacattc tcgataccag tttaaaatat gtgatgatat
660tattagacct tgcaccccta taaattaggg ccggtgaaca aaaccatcaa
cgccttagtt 720tttgattttg aagaagaaat tgttgtcagc atcattagat
cttgtgtgag tatatttgag 780agtcaaatca gcatgagcgg tttggaaaac
cgccgtctgt agtgtagcgg ttttgtctcc 840agagcgacct cttctctata
aagaggagga gaacttccat ggaatgagcc acaaaaccac 900catcgatcat
ttccatttcc attttcagga gagatccaca tcgccgttgg cctttcatcc
960agtgagtgag ggagggagat ctcgatctcg gtctcgcgac 100062719DNAOryza
sativapromoter(1)..(719)transcription regulating sequence from
Oryza sativa Lactate-dehydrogenase gene 62atcaacatcc gtaccaaatt
aagtgtttct ttgacagcgt gccagatttg gatcggtcat 60tcgttgcgga aagtagtacc
aatgacttac tgctactatt aatctactac tgtttcgctt 120aagtataggt
aaaggaaatg taaccctatt tcgagaaaat atattagtgg tagataggat
180agctaggaga tccaaagagc agaatggtat actgcattag atatatccaa
tgtgccacga 240tgagtggttt ttctttatct gtttgtacta atttgcgcac
ttttgaccga taagcgggcc 300ggcatctttt taaatagaga aacacgcgat
atgatgatag gcagtgagat ttcttcacaa 360atactgtaca gaacagtaga
gcagtagtag tagggcacaa attgatggaa tcaacttagc 420aacctttgta
gaccgtcgtc aattttacaa gggagagata tagtagaatg cgggtacaac
480agttcatgct gtgttgagtt atatattgtt tttgggtttg tgattaaaca
gtgtaacaac 540acggaaaggt tcaagacgag caaacacgtc agcatgtaca
aacgcagtgt atgagttaat 600taattatatg gttagcgtgt atcgacattc
tcgataccag tttaaaatat gtgatgatat 660tattagacct tgcaccccta
taaattaggg ccggtgaaca aaaccatcaa cgccttagt 71963301DNAOryza
sativapromoter(1)..(301)core transcription regulating sequence from
Oryza sativa Lactate-dehydrogenase gene comprising clusters of
promoter elements 63gcaacctttg tagaccgtcg tcaattttac aagggagaga
tatagtagaa tgcgggtaca 60acagttcatg ctgtgttgag ttatatattg tttttgggtt
tgtgattaaa cagtgtaaca 120acacggaaag gttcaagacg agcaaacacg
tcagcatgta caaacgcagt gtatgagtta 180attaattata tggttagcgt
gtatcgacat tctcgatacc agtttaaaat atgtgatgat 240attattagac
cttgcacccc tataaattag ggccggtgaa caaaaccatc aacgccttag 300t
301641062DNAOryza sativaCDS(1)..(1059)coding for Oryza sativa
Lactate-dehydrogenase 64atg aag aag gct tcg tct ctg tcg gag ctg ggg
ttc gac gcg gag ggc 48Met Lys Lys Ala Ser Ser Leu Ser Glu Leu Gly
Phe Asp Ala Glu Gly 1 5 10 15 gcg tcg tcg ggg ttc ttc cgt ccg gtg
gcg gac ggc ggg tcg acg ccg 96Ala Ser Ser Gly Phe Phe Arg Pro Val
Ala Asp Gly Gly Ser Thr Pro 20 25 30 acg tcg cac cgg cgt cgg ctg
acg aag ata tcg gtg atc ggc gcg ggc 144Thr Ser His Arg Arg Arg Leu
Thr Lys Ile Ser Val Ile Gly Ala Gly 35 40 45 aac gtg ggg atg gcg
atc gcg cag acc atc ctg acc cgg gac atg gcg 192Asn Val Gly Met Ala
Ile Ala Gln Thr Ile Leu Thr Arg Asp Met Ala 50 55 60 gac gag atc
gcg ctg gtg gac gcg gtg ccg gac aag ctg cgc ggg gag 240Asp Glu Ile
Ala Leu Val Asp Ala Val Pro Asp Lys Leu Arg Gly Glu 65 70 75 80 atg
ctg gac ctg cag cac gcg gcg gcg ttc ctc ccc cgc gtc cgc ctc 288Met
Leu Asp Leu Gln His Ala Ala Ala Phe Leu Pro Arg Val Arg Leu 85 90
95 gtc tcc gac acc gac ctg gcc gtc acg cgc ggc tcc gac ctg gcc atc
336Val Ser Asp Thr Asp Leu Ala Val Thr Arg Gly Ser Asp Leu Ala Ile
100 105 110 gtc acg gcc ggc gcg cgc cag atc ccc ggg gag agc cgc ctg
aac ctg 384Val Thr Ala Gly Ala Arg Gln Ile Pro Gly Glu Ser Arg Leu
Asn Leu 115 120 125 ctg cag cgg aac gtg gcg ctg ttc cgg aag atc gtg
ccg gcg ctg gcg 432Leu Gln Arg Asn Val Ala Leu Phe Arg Lys Ile Val
Pro Ala Leu Ala 130 135 140 gag cac tcg ccg gag gcg ctg ctg ctg atc
gtc tcc aac ccc gtc gac 480Glu His Ser Pro Glu Ala Leu Leu Leu Ile
Val Ser Asn Pro Val Asp 145 150 155 160 gtg ctg acg tac gtg gcg tgg
aag ctg tcg ggg ttc ccg gcg agc cgc 528Val Leu Thr Tyr Val Ala Trp
Lys Leu Ser Gly Phe Pro Ala Ser Arg 165 170 175 gtc atc ggc tcc ggc
acc aac ctc gac tcc tcc agg ttc cgc ttc ctc 576Val Ile Gly Ser Gly
Thr Asn Leu Asp Ser Ser Arg Phe Arg Phe Leu 180 185 190 ctc gcc gag
cac ctc cag gtc aac gcc cag gat gtc cag gcg tac atg 624Leu Ala Glu
His Leu Gln Val Asn Ala Gln Asp Val Gln Ala Tyr Met 195 200 205 gtg
gga gag cac ggg gac agc tcg gtg gcg ata tgg tcg agc atg agc 672Val
Gly Glu His Gly Asp Ser Ser Val Ala Ile Trp Ser Ser Met Ser 210 215
220 gtg gcc ggg atg ccg gtg ctc aag tcg ctg cgg gag agc cac cag agc
720Val Ala Gly Met Pro Val Leu Lys Ser Leu Arg Glu Ser His Gln Ser
225 230 235 240 ttc gac gag gag gcc ctg gag gga atc cgg cga gcg gtg
gtg gac agc 768Phe Asp Glu Glu Ala Leu Glu Gly Ile Arg Arg Ala Val
Val Asp Ser 245 250 255 gcg tac gag gtg atc agc ctc aag ggc tac acc
tcc tgg gcc atc ggc 816Ala Tyr Glu Val Ile Ser Leu Lys Gly Tyr Thr
Ser Trp Ala Ile Gly 260 265 270 tac tcc gtc gcc agc ctc gcc gcc tcc
ctc ctc cgc gac cag cac cgc 864Tyr Ser Val Ala Ser Leu Ala Ala Ser
Leu Leu Arg Asp Gln His Arg 275 280 285 atc cac ccc gtc tcc gtc ctc
gcc tcc ggc ttc cac ggc atc ccc caa 912Ile His Pro Val Ser Val Leu
Ala Ser Gly Phe His Gly Ile Pro Gln 290 295 300 gac cac gag gtc ttc
ctc agc ctc ccc gcc cgc ctc ggc cgc gcc ggc 960Asp His Glu Val Phe
Leu Ser Leu Pro Ala Arg Leu Gly Arg Ala Gly 305 310 315 320 gtc ctc
ggc gtc gcc gag atg gag ctc acc gag gag gag gcc cgc cgc 1008Val Leu
Gly Val Ala Glu Met Glu Leu Thr Glu Glu Glu Ala Arg Arg 325 330 335
ctc cgc cgc tcc gcc aag acg ctc tgg gag aac tgc cag ctg ctc gac
1056Leu Arg Arg Ser Ala Lys Thr Leu Trp Glu Asn Cys Gln Leu Leu Asp
340 345 350 ctc taa 1062Leu 65353PRTOryza sativa 65Met Lys Lys Ala
Ser Ser Leu Ser Glu Leu Gly Phe Asp Ala Glu Gly 1 5 10 15 Ala Ser
Ser Gly Phe Phe Arg Pro Val Ala Asp Gly Gly Ser Thr Pro 20 25 30
Thr Ser His Arg Arg Arg Leu Thr Lys Ile Ser Val Ile Gly Ala Gly 35
40 45 Asn Val Gly Met Ala Ile Ala Gln Thr Ile Leu Thr Arg Asp Met
Ala 50 55 60 Asp Glu Ile Ala Leu Val Asp Ala Val Pro Asp Lys Leu
Arg Gly Glu 65 70 75 80 Met Leu Asp Leu Gln His Ala Ala Ala Phe Leu
Pro Arg Val Arg Leu 85 90 95 Val Ser Asp Thr Asp Leu Ala Val Thr
Arg Gly Ser Asp Leu Ala Ile 100 105 110 Val Thr Ala Gly Ala Arg Gln
Ile Pro Gly Glu Ser Arg Leu Asn Leu 115 120 125 Leu Gln Arg Asn Val
Ala Leu Phe Arg Lys Ile Val Pro Ala Leu Ala 130 135 140 Glu His Ser
Pro Glu Ala Leu Leu Leu Ile Val Ser Asn Pro Val Asp 145 150 155 160
Val Leu Thr Tyr Val Ala Trp Lys Leu Ser Gly Phe Pro Ala Ser Arg 165
170 175 Val Ile Gly Ser Gly Thr Asn Leu Asp Ser Ser Arg Phe Arg Phe
Leu 180 185 190 Leu Ala Glu His Leu Gln Val Asn Ala Gln Asp Val Gln
Ala Tyr Met 195 200 205 Val Gly Glu His Gly Asp Ser Ser Val Ala Ile
Trp Ser Ser Met Ser 210 215 220 Val Ala Gly Met Pro Val Leu Lys Ser
Leu Arg Glu Ser His Gln Ser 225 230 235 240 Phe Asp Glu Glu Ala Leu
Glu Gly Ile Arg Arg Ala Val Val Asp Ser 245 250 255 Ala Tyr Glu Val
Ile Ser Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly 260 265 270 Tyr Ser
Val Ala Ser Leu Ala Ala Ser Leu Leu Arg Asp Gln His Arg 275 280 285
Ile His Pro Val Ser Val Leu Ala Ser Gly Phe His Gly Ile Pro Gln 290
295 300 Asp His Glu Val Phe Leu Ser Leu Pro Ala Arg Leu Gly Arg Ala
Gly 305 310 315 320 Val Leu Gly Val Ala Glu Met Glu Leu Thr Glu Glu
Glu Ala Arg Arg 325 330 335 Leu Arg Arg Ser Ala Lys Thr Leu Trp Glu
Asn Cys Gln Leu Leu Asp 340 345 350 Leu 66997DNAZea
mayspromoter(1)..(997)transcription regulating sequence from Zea
mays Caffeoyl-CoA-O-methyltransferase gene comprising
5'-untranslated region 66cagcccgtac caggtgtcgc tcttgggccg
tgttgttgcc gtccaaccaa gcaaggcaag 60cgtgtagata gataagaaga tagcgtgagt
gagtggcatc cagacatcca gtacgttagc 120ctggcttacg ttacggacac
gggcgtggcg cctttgtcgt gctgcgcacc cgccagccag 180cagccatcca
gcagggcctc cgtttgtccg tctcctctct ctacaggctg tttgtgcccc
240caagcaagca ctacgcgcgc gaaaaaaaat aaaaaaaaaa ggcacgtcat
ctggatctct 300tgctctagct cactcaacct caccaaaatg tgcaggctgc
tccgactccg agaacaggac 360ggtcatgccg cgcgcaccgg tttcccttcc
catgccgcta ccacctcccg actcccgagt 420cacgactggg ggaccacccg
atggacgatg attgatgcac tgaaagcaag agtgggttgg 480tggtggaccg
tggcagtcta ccaaatgtcg ctgctctccc cgcccggccg gaacgaggaa
540ggaaggcgac caccactcca cgccggtcgt gccaggtcgg cagtcagagt
cagccgcgcc 600tgcgcgcgtc cctttcccac caacccctct ccctccccgc
actcgacagc cacggccgcg 660acggcgtccg cgtgcaccgg acaccacggc
cagacccgag gccacacagc cctctgacca 720aacgcacgga tccggccagc
cacccacccc gcccccctag cggccgccac ccacccgtgc 780gcgcggcgca
ccaaggcctg gcccggcagt ggcagccagg ctctcacacg cctcgcctag
840tcgccttgcc gccgcgcgtt atataagagc cgccccggca ggcacggtcg
gtcaatccag 900caatacccga cgcgcaagcc agtgccgcac ccagaccaga
tctccgcgac atatcagtcg 960ttcgtccagc taactgcact gcactgcact gcacgca
99767900DNAZea mayspromoter(1)..(900)transcription regulating
sequence from Zea mays Caffeoyl-CoA-O-methyltransferase gene
67cagcccgtac caggtgtcgc tcttgggccg tgttgttgcc gtccaaccaa gcaaggcaag
60cgtgtagata gataagaaga tagcgtgagt gagtggcatc cagacatcca gtacgttagc
120ctggcttacg ttacggacac gggcgtggcg cctttgtcgt gctgcgcacc
cgccagccag 180cagccatcca gcagggcctc cgtttgtccg tctcctctct
ctacaggctg tttgtgcccc 240caagcaagca ctacgcgcgc gaaaaaaaat
aaaaaaaaaa ggcacgtcat ctggatctct 300tgctctagct cactcaacct
caccaaaatg tgcaggctgc tccgactccg agaacaggac 360ggtcatgccg
cgcgcaccgg tttcccttcc catgccgcta ccacctcccg actcccgagt
420cacgactggg ggaccacccg atggacgatg attgatgcac tgaaagcaag
agtgggttgg 480tggtggaccg tggcagtcta ccaaatgtcg ctgctctccc
cgcccggccg gaacgaggaa 540ggaaggcgac caccactcca cgccggtcgt
gccaggtcgg cagtcagagt cagccgcgcc 600tgcgcgcgtc cctttcccac
caacccctct ccctccccgc actcgacagc cacggccgcg 660acggcgtccg
cgtgcaccgg acaccacggc cagacccgag gccacacagc cctctgacca
720aacgcacgga tccggccagc cacccacccc gcccccctag cggccgccac
ccacccgtgc 780gcgcggcgca ccaaggcctg gcccggcagt ggcagccagg
ctctcacacg cctcgcctag 840tcgccttgcc gccgcgcgtt atataagagc
cgccccggca ggcacggtcg gtcaatccag 90068301DNAZea
mayspromoter(1)..(301)core transcription regulating sequence from
Zea mays Caffeoyl-CoA-O-methyltransferase gene comprising clusters
of promoter elements 68ctgcgcgcgt ccctttccca ccaacccctc tccctccccg
cactcgacag ccacggccgc 60gacggcgtcc gcgtgcaccg gacaccacgg ccagacccga
ggccacacag ccctctgacc 120aaacgcacgg atccggccag ccacccaccc
cgccccccta gcggccgcca cccacccgtg 180cgcgcggcgc accaaggcct
ggcccggcag tggcagccag gctctcacac gcctcgccta 240gtcgccttgc
cgccgcgcgt tatataagag ccgccccggc aggcacggtc ggtcaatcca 300g
30169801DNAZea maysCDS(1)..(798)encoding Zea mays
Caffeoyl-CoA-O-methyltransferase 69atg gcc acc acg gcg acc gag gcg
acc aag acg act gca ccg gcg cag 48Met Ala Thr Thr Ala Thr Glu Ala
Thr Lys Thr Thr Ala Pro Ala Gln 1 5 10 15 gag cag cag gcc aac ggc
aac ggc aac ggc aac ggc gag cag aag acg 96Glu Gln Gln Ala Asn Gly
Asn Gly Asn Gly Asn Gly Glu Gln Lys Thr 20 25 30 cgc cac tcc gag
gtc ggg cac aag agc ctg ctc aag agc gac gac ctc 144Arg His Ser Glu
Val Gly His Lys Ser Leu Leu Lys Ser Asp Asp Leu 35 40 45 tac cag
tac atc ctg gac acg agc gtg tac ccg cgg gag ccg gag agc 192Tyr Gln
Tyr Ile Leu Asp Thr Ser Val Tyr Pro Arg Glu Pro Glu Ser 50 55 60
atg aag gag ctg cgc gag atc acc gcc aag cac cca tgg aac ctg atg
240Met Lys Glu Leu Arg Glu Ile Thr Ala Lys His Pro Trp Asn Leu Met
65 70 75 80 acc acc tcc gcc gac gag ggc cag ttc ctc aac atg ctc atc
aag ctc 288Thr Thr Ser Ala Asp Glu Gly Gln Phe Leu Asn Met Leu Ile
Lys Leu 85 90 95 atc ggc gcc aag aag acc atg gag atc ggc gtc tac
acc ggc tac tcg 336Ile Gly Ala Lys Lys Thr Met Glu Ile Gly Val Tyr
Thr Gly Tyr Ser 100 105 110 ctc ctc gcc acc gcg ctc gca ctc ccg gag
gac ggc acg atc ttg gcc 384Leu Leu Ala Thr Ala Leu Ala Leu Pro Glu
Asp Gly Thr Ile Leu Ala 115 120 125 atg gac atc aac cgc gag aac tac
gag cta ggc ctt ccc tgc atc aac 432Met Asp Ile Asn Arg Glu Asn Tyr
Glu Leu Gly Leu Pro Cys Ile Asn 130 135 140
aag gcc ggc gtg gcc cac aag atc gac ttc cgc gag ggc ccc gcg ctc
480Lys Ala Gly Val Ala His Lys Ile Asp Phe Arg Glu Gly Pro Ala Leu
145 150 155 160 ccc gtc ctg gac gac ctc gtg gcg gac aag gag cag cac
ggg tcg ttc 528Pro Val Leu Asp Asp Leu Val Ala Asp Lys Glu Gln His
Gly Ser Phe 165 170 175 gac ttc gcc ttc gtg gac gcc gac aag gac aac
tac ctc agc tac cac 576Asp Phe Ala Phe Val Asp Ala Asp Lys Asp Asn
Tyr Leu Ser Tyr His 180 185 190 gag cgg ctc ctg aag ctg gtg agg ccc
ggc ggc ctc atc ggc tac gac 624Glu Arg Leu Leu Lys Leu Val Arg Pro
Gly Gly Leu Ile Gly Tyr Asp 195 200 205 aac acg ctg tgg aac ggc tcc
gtc gtg ctc ccc gac gac gcg ccc atg 672Asn Thr Leu Trp Asn Gly Ser
Val Val Leu Pro Asp Asp Ala Pro Met 210 215 220 cgc aag tac atc cgc
ttc tac cgc gac ttc gtc ctc gcc ctc aac agc 720Arg Lys Tyr Ile Arg
Phe Tyr Arg Asp Phe Val Leu Ala Leu Asn Ser 225 230 235 240 gcg ctc
gcc gcc gac gac cgc gtc gag atc tgc cag ctc ccc gtc ggc 768Ala Leu
Ala Ala Asp Asp Arg Val Glu Ile Cys Gln Leu Pro Val Gly 245 250 255
gac ggc gtc acg ctc tgc cgc cgc gtc aag tga 801Asp Gly Val Thr Leu
Cys Arg Arg Val Lys 260 265 70266PRTZea mays 70Met Ala Thr Thr Ala
Thr Glu Ala Thr Lys Thr Thr Ala Pro Ala Gln 1 5 10 15 Glu Gln Gln
Ala Asn Gly Asn Gly Asn Gly Asn Gly Glu Gln Lys Thr 20 25 30 Arg
His Ser Glu Val Gly His Lys Ser Leu Leu Lys Ser Asp Asp Leu 35 40
45 Tyr Gln Tyr Ile Leu Asp Thr Ser Val Tyr Pro Arg Glu Pro Glu Ser
50 55 60 Met Lys Glu Leu Arg Glu Ile Thr Ala Lys His Pro Trp Asn
Leu Met 65 70 75 80 Thr Thr Ser Ala Asp Glu Gly Gln Phe Leu Asn Met
Leu Ile Lys Leu 85 90 95 Ile Gly Ala Lys Lys Thr Met Glu Ile Gly
Val Tyr Thr Gly Tyr Ser 100 105 110 Leu Leu Ala Thr Ala Leu Ala Leu
Pro Glu Asp Gly Thr Ile Leu Ala 115 120 125 Met Asp Ile Asn Arg Glu
Asn Tyr Glu Leu Gly Leu Pro Cys Ile Asn 130 135 140 Lys Ala Gly Val
Ala His Lys Ile Asp Phe Arg Glu Gly Pro Ala Leu 145 150 155 160 Pro
Val Leu Asp Asp Leu Val Ala Asp Lys Glu Gln His Gly Ser Phe 165 170
175 Asp Phe Ala Phe Val Asp Ala Asp Lys Asp Asn Tyr Leu Ser Tyr His
180 185 190 Glu Arg Leu Leu Lys Leu Val Arg Pro Gly Gly Leu Ile Gly
Tyr Asp 195 200 205 Asn Thr Leu Trp Asn Gly Ser Val Val Leu Pro Asp
Asp Ala Pro Met 210 215 220 Arg Lys Tyr Ile Arg Phe Tyr Arg Asp Phe
Val Leu Ala Leu Asn Ser 225 230 235 240 Ala Leu Ala Ala Asp Asp Arg
Val Glu Ile Cys Gln Leu Pro Val Gly 245 250 255 Asp Gly Val Thr Leu
Cys Arg Arg Val Lys 260 265 711028DNAZea
diploperennispromoter(1)..(1028)transcription regulating region
from Zea diploperennis hydroxyproline-rich glycoprotein gene
comprising 5'-untranslated region 71atggacttcg aggaaacgat
aacccctgga tgtcgagata gccgaagtcg aggtggtcat 60ggtcgggaga cacgcggcag
tagcatattc ttcggtaggg ctcgatgttc aagcgacaac 120ggtcggcggg
gcgacacaaa aatttagcac cagcagacct tcttgcttct tcgaccgtct
180ggacatcgag gagcccagcc agggaggccg gcagcagcgc acacgtctgc
accagtaatg 240ttagccgcgc ccgcgacgta atagaagggg caacgataga
tccggtcagg aaggccacga 300catcgacgga tctagacagc gatcaggtca
aagagacgac gaatctagcc gggaaggtag 360atccctcgag agagttcata
ttaaatgatg ttgtacatgc cataataact ctataaatct 420aatttattca
taggcgaagg taattgtatt atctttccca gcggagcatt atctgatctg
480ccgttcagct tgatcgatcc acgtcgtttg atctcggcga gcagcacatg
gcggctcttc 540ttgtgtacag ggctcactct ctgctacttc agtgcaaggc
ggagtgaatg cacaataacg 600tgagtattgt gggaactact tgtagatgca
aacgatgtaa atccacctgc tccaccaagt 660gcccgcccgg ctctatccat
tacattcgtc aacacgcagg ttcagactgg cccgtgctgg 720accagtgagc
ggtgaaccca gcccaagcga gtgaccatcg gggaagcctc ccgcccgtgc
780tgcccccaca tggcttgcct gaatgcctct cgccgcagtg ccctctctcc
tcctcctccc 840cctcccctcc gtcgaagggc gtcacgagag cccagagggt
atccgaggcc cccaccccac 900cccttcctcc gtgtatataa gcagtggcag
ggtgaacgtc tctcctcaga ccacccactg 960cgccattggc cagctagagc
caaccagaag agcttgcagt tactgggaga gtgtgtgtga 1020gagagagg
102872954DNAZea diploperennispromoter(1)..(954)transcription
regulating region from Zea diploperennis hydroxyproline-rich
glycoprotein gene 72atggacttcg aggaaacgat aacccctgga tgtcgagata
gccgaagtcg aggtggtcat 60ggtcgggaga cacgcggcag tagcatattc ttcggtaggg
ctcgatgttc aagcgacaac 120ggtcggcggg gcgacacaaa aatttagcac
cagcagacct tcttgcttct tcgaccgtct 180ggacatcgag gagcccagcc
agggaggccg gcagcagcgc acacgtctgc accagtaatg 240ttagccgcgc
ccgcgacgta atagaagggg caacgataga tccggtcagg aaggccacga
300catcgacgga tctagacagc gatcaggtca aagagacgac gaatctagcc
gggaaggtag 360atccctcgag agagttcata ttaaatgatg ttgtacatgc
cataataact ctataaatct 420aatttattca taggcgaagg taattgtatt
atctttccca gcggagcatt atctgatctg 480ccgttcagct tgatcgatcc
acgtcgtttg atctcggcga gcagcacatg gcggctcttc 540ttgtgtacag
ggctcactct ctgctacttc agtgcaaggc ggagtgaatg cacaataacg
600tgagtattgt gggaactact tgtagatgca aacgatgtaa atccacctgc
tccaccaagt 660gcccgcccgg ctctatccat tacattcgtc aacacgcagg
ttcagactgg cccgtgctgg 720accagtgagc ggtgaaccca gcccaagcga
gtgaccatcg gggaagcctc ccgcccgtgc 780tgcccccaca tggcttgcct
gaatgcctct cgccgcagtg ccctctctcc tcctcctccc 840cctcccctcc
gtcgaagggc gtcacgagag cccagagggt atccgaggcc cccaccccac
900cccttcctcc gtgtatataa gcagtggcag ggtgaacgtc tctcctcaga ccac
95473301DNAZea diploperennispromoter(1)..(301)core transcription
regulating region from Zea diploperennis hydroxyproline-rich
glycoprotein gene comprising clusters of promoter elements
73accaagtgcc cgcccggctc tatccattac attcgtcaac acgcaggttc agactggccc
60gtgctggacc agtgagcggt gaacccagcc caagcgagtg accatcgggg aagcctcccg
120cccgtgctgc ccccacatgg cttgcctgaa tgcctctcgc cgcagtgccc
tctctcctcc 180tcctccccct cccctccgtc gaagggcgtc acgagagccc
agagggtatc cgaggccccc 240accccacccc ttcctccgtg tatataagca
gtggcagggt gaacgtctct cctcagacca 300c 301741053DNAZea
diploperennisCDS(1)..(1050)encoding Zea diploperennis
hydroxyproline-rich glycoprotein 74atg ggt ggc agc ggc acg gct gct
ctg ctg ctg gcc ctg gtg gcc gtg 48Met Gly Gly Ser Gly Thr Ala Ala
Leu Leu Leu Ala Leu Val Ala Val 1 5 10 15 agc ctg gcc gtg gag atc
cag gcc gac gcc ggg tac ggg tac acc ccg 96Ser Leu Ala Val Glu Ile
Gln Ala Asp Ala Gly Tyr Gly Tyr Thr Pro 20 25 30 aca ccg acg ccg
gcc acc ccg acc ccg aag ccg gag aag ccc ccc acc 144Thr Pro Thr Pro
Ala Thr Pro Thr Pro Lys Pro Glu Lys Pro Pro Thr 35 40 45 aag ggg
ccc aag ccg gag aag ccg cca aag gag cac aag ccg ccc aag 192Lys Gly
Pro Lys Pro Glu Lys Pro Pro Lys Glu His Lys Pro Pro Lys 50 55 60
gag cac ggg ccc aag ccg gag aag ccg ccc aag gag cac aag ccg acg
240Glu His Gly Pro Lys Pro Glu Lys Pro Pro Lys Glu His Lys Pro Thr
65 70 75 80 ccg ccc acg tac acc ccg agc ccc aaa ccc acg ccg ccg acg
tac act 288Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro Thr Pro Pro Thr
Tyr Thr 85 90 95 ccc acc ccg acg ccg ccg act ccc aag ccg acg cca
ccc aca tac acc 336Pro Thr Pro Thr Pro Pro Thr Pro Lys Pro Thr Pro
Pro Thr Tyr Thr 100 105 110 cca gcc cct acg ccc cac aaa ccc aca ccc
aca cca aaa ccc act ccc 384Pro Ala Pro Thr Pro His Lys Pro Thr Pro
Thr Pro Lys Pro Thr Pro 115 120 125 act cct ccg acg tac acc cct tcc
ccc aag cct ccg aca cct aag ccg 432Thr Pro Pro Thr Tyr Thr Pro Ser
Pro Lys Pro Pro Thr Pro Lys Pro 130 135 140 acc ccg ccg acg tac gct
cca agc ccc aag cca ccg gct acc aag cct 480Thr Pro Pro Thr Tyr Ala
Pro Ser Pro Lys Pro Pro Ala Thr Lys Pro 145 150 155 160 ccc acg ccc
aag ccg acc ccg ccg acg tac acc cct tcc ccc aaa cct 528Pro Thr Pro
Lys Pro Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro 165 170 175 ccg
aca ccc aag ccg acc ccg ccg acg tac acc cca agc ccc aag ccg 576Pro
Thr Pro Lys Pro Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro 180 185
190 acc ccg ccg acg tac acc cct tct ccc aag cct ccg aca cct aag ccg
624Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro Pro Thr Pro Lys Pro
195 200 205 acc ccg cct acg tac act cca agc cct aag cca ccg gct acc
aag cct 672Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro Pro Ala Thr
Lys Pro 210 215 220 ccc acg ccc aag ccg acc ccg cca acg tac acc cct
tcc cca aag cct 720Pro Thr Pro Lys Pro Thr Pro Pro Thr Tyr Thr Pro
Ser Pro Lys Pro 225 230 235 240 ccg aca ccc aag ccg acc ccg ccg acg
tac acc cct tcc ccc aag cca 768Pro Thr Pro Lys Pro Thr Pro Pro Thr
Tyr Thr Pro Ser Pro Lys Pro 245 250 255 ccg gct acc aag ccg acc cca
ccg acg tac acc cct tcc cca aag cct 816Pro Ala Thr Lys Pro Thr Pro
Pro Thr Tyr Thr Pro Ser Pro Lys Pro 260 265 270 ccg aca cct aag ccg
acc ccg ccg acg tac acc cct tcc ccc aag cct 864Pro Thr Pro Lys Pro
Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro 275 280 285 ccg aca ccc
aag ccg acc cca ccg acg tac act cca agc ccc aaa cca 912Pro Thr Pro
Lys Pro Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro 290 295 300 ccg
gct acc aag cct ccc acg ccc aag ccg acc cca ccg acg tac act 960Pro
Ala Thr Lys Pro Pro Thr Pro Lys Pro Thr Pro Pro Thr Tyr Thr 305 310
315 320 ccc aca ccg aag ccg ccg gcc acc aag ccg ccc acc tac act ccg
acg 1008Pro Thr Pro Lys Pro Pro Ala Thr Lys Pro Pro Thr Tyr Thr Pro
Thr 325 330 335 ccg ccg gtg tct cac acc ccc agc ccg ccg cca cct tac
tac tag 1053Pro Pro Val Ser His Thr Pro Ser Pro Pro Pro Pro Tyr Tyr
340 345 350 75350PRTZea diploperennis 75Met Gly Gly Ser Gly Thr Ala
Ala Leu Leu Leu Ala Leu Val Ala Val 1 5 10 15 Ser Leu Ala Val Glu
Ile Gln Ala Asp Ala Gly Tyr Gly Tyr Thr Pro 20 25 30 Thr Pro Thr
Pro Ala Thr Pro Thr Pro Lys Pro Glu Lys Pro Pro Thr 35 40 45 Lys
Gly Pro Lys Pro Glu Lys Pro Pro Lys Glu His Lys Pro Pro Lys 50 55
60 Glu His Gly Pro Lys Pro Glu Lys Pro Pro Lys Glu His Lys Pro Thr
65 70 75 80 Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro Thr Pro Pro Thr
Tyr Thr 85 90 95 Pro Thr Pro Thr Pro Pro Thr Pro Lys Pro Thr Pro
Pro Thr Tyr Thr 100 105 110 Pro Ala Pro Thr Pro His Lys Pro Thr Pro
Thr Pro Lys Pro Thr Pro 115 120 125 Thr Pro Pro Thr Tyr Thr Pro Ser
Pro Lys Pro Pro Thr Pro Lys Pro 130 135 140 Thr Pro Pro Thr Tyr Ala
Pro Ser Pro Lys Pro Pro Ala Thr Lys Pro 145 150 155 160 Pro Thr Pro
Lys Pro Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro 165 170 175 Pro
Thr Pro Lys Pro Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro 180 185
190 Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro Pro Thr Pro Lys Pro
195 200 205 Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro Pro Ala Thr
Lys Pro 210 215 220 Pro Thr Pro Lys Pro Thr Pro Pro Thr Tyr Thr Pro
Ser Pro Lys Pro 225 230 235 240 Pro Thr Pro Lys Pro Thr Pro Pro Thr
Tyr Thr Pro Ser Pro Lys Pro 245 250 255 Pro Ala Thr Lys Pro Thr Pro
Pro Thr Tyr Thr Pro Ser Pro Lys Pro 260 265 270 Pro Thr Pro Lys Pro
Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro 275 280 285 Pro Thr Pro
Lys Pro Thr Pro Pro Thr Tyr Thr Pro Ser Pro Lys Pro 290 295 300 Pro
Ala Thr Lys Pro Pro Thr Pro Lys Pro Thr Pro Pro Thr Tyr Thr 305 310
315 320 Pro Thr Pro Lys Pro Pro Ala Thr Lys Pro Pro Thr Tyr Thr Pro
Thr 325 330 335 Pro Pro Val Ser His Thr Pro Ser Pro Pro Pro Pro Tyr
Tyr 340 345 350 769PRTArtificial sequenceamino acid motif for
monocotyledonous lactate dehydrogenases 76Ser Leu Ser Glu Leu Gly
Phe Asp Ala 1 5 7710PRTArtificial sequenceamino acid motif for
monocotyledonous lactate dehydrogenases 77Val Ile Gly Ala Gly Asn
Val Gly Met Ala 1 5 10 789PRTArtificial sequenceamino acid motif
for monocotyledonous lactate dehydrogenases 78Ile Val Thr Ala Gly
Ala Arg Gln Ile 1 5 797PRTArtificial sequenceamino acid motif for
monocotyledonous lactate dehydrogenases 79Leu Phe Arg Lys Ile Val
Pro 1 5 807PRTArtificial sequenceamino acid motif for
monocotyledonous lactate dehydrogenases 80Gly Phe Pro Ala Ser Arg
Val 1 5 818PRTArtificial sequenceamino acid motif for
monocotyledonous lactate dehydrogenases 81Arg Phe Leu Leu Ala Glu
His Leu 1 5 828PRTArtificial sequenceamino acid motif for
monocotyledonous lactate dehydrogenases 82Gln Ala Tyr Met Val Gly
Glu His 1 5 839PRTArtificial sequenceamino acid motif for
monocotyledonous lactate dehydrogenases 83Ala Leu Glu Gly Ile Arg
Arg Ala Val 1 5 848PRTArtificial sequenceamino acid motif for
monocotyledonous lactate dehydrogenases 84Gly Tyr Ser Val Ala Ser
Leu Ala 1 5 858PRTArtificial sequenceamino acid motif for
monocotyledonous Caffeoyl CoA-O-methyltransferases 85Glu Gln Lys
Thr Arg His Ser Glu 1 5 868PRTArtificial sequenceamino acid motif
for monocotyledonous Caffeoyl CoA-O-methyltransferases 86Leu Ile
Lys Leu Ile Gly Ala Lys 1 5 878PRTArtificial sequenceamino acid
motif for monocotyledonous Caffeoyl CoA-O-methyltransferases 87Lys
Thr Met Glu Ile Gly Val Tyr 1 5 888PRTArtificial sequenceamino acid
motif for monocotyledonous Caffeoyl CoA-O-methyltransferases 88His
Glu Arg Leu Leu Lys Leu Val 1 5 898PRTArtificial sequenceamino acid
motif for monocotyledonous Caffeoyl CoA-O-methyltransferases 89Cys
Gln Leu Pro Val Gly Asp Gly 1 5 907PRTArtificial sequenceamino acid
motif for monocotyledonous Caffeoyl CoA-O-methyltransferases 90Thr
Leu Cys Arg Arg Val Lys 1 5 9121DNAArtificial
sequenceoligonucleotide primer 91ttacgtggca aaggattcga t
219220DNAArtificial sequenceoligonucleotide primer 92gccccaatcc
agtccattaa 209318DNAArtificial sequenceoligonucleotide primer
93tctgccttgc ccttgctt 189423DNAArtificial sequenceoligonucleotide
primer 94caattgcttg gcaggtctta ttt 2395356PRTHordeum vulgare 95Met
His Lys Ala Ser Ser Leu Ser Glu Leu Gly Phe Asp Ala Gly Gly 1 5
10 15 Ala Ser Ser Gly Phe Phe Arg Pro Val Ala Asp Gly Cys Pro Ala
Thr 20 25 30 Pro Thr Ser Ser Ala Val Pro His Arg Arg Leu Thr Lys
Ile Ser Val 35 40 45 Ile Gly Ala Gly Asn Val Gly Met Ala Ile Ala
Gln Thr Ile Leu Thr 50 55 60 Gln Asn Leu Ala Asp Glu Ile Ala Leu
Val Asp Ala Leu Pro Asp Lys 65 70 75 80 Leu Arg Gly Glu Ala Leu Asp
Leu Gln His Ala Ala Ala Phe Leu Pro 85 90 95 Arg Val Arg Ile Ser
Gly Thr Asp Ala Ala Val Thr Lys Asn Ser Asp 100 105 110 Leu Val Ile
Val Thr Ala Gly Ala Arg Gln Ile Pro Gly Glu Thr Arg 115 120 125 Leu
Asn Leu Leu Gln Arg Asn Val Ala Leu Tyr Arg Lys Ile Val Pro 130 135
140 Pro Val Ala Glu His Ser Pro Asp Ala Leu Leu Leu Val Val Ser Asn
145 150 155 160 Pro Val Asp Val Leu Thr Tyr Val Ala Trp Lys Leu Ser
Gly Phe Pro 165 170 175 Ala Ser Arg Val Ile Gly Ser Gly Thr Asn Leu
Asp Ser Ser Arg Phe 180 185 190 Arg Phe Leu Ile Ala Glu His Leu Asp
Val Asn Ala Gln Asp Val Gln 195 200 205 Ala Tyr Met Val Gly Glu His
Gly Asp Ser Ser Val Ala Ile Trp Ser 210 215 220 Ser Ile Ser Val Gly
Gly Met Pro Ala Phe Lys Ser Leu Arg Asp Ser 225 230 235 240 His Arg
Ser Phe Asp Glu Ala Ala Leu Glu Gly Ile Arg Arg Ala Val 245 250 255
Val Gly Gly Ala Tyr Glu Val Ile Gly Leu Lys Gly Tyr Thr Ser Trp 260
265 270 Ala Ile Gly Tyr Ser Val Ala Ser Leu Ala Ala Ser Leu Leu Arg
Asp 275 280 285 Gln Arg Arg Val His Pro Val Ser Val Leu Ala Ser Gly
Phe His Gly 290 295 300 Ile Ser Asp Gly His Glu Val Phe Leu Ser Leu
Pro Ala Arg Leu Gly 305 310 315 320 Arg Gly Gly Ile Leu Gly Val Ala
Glu Met Asp Leu Thr Glu Ala Glu 325 330 335 Ala Ala Gln Leu Arg Arg
Ser Ala Lys Thr Leu Trp Glu Asn Cys Gln 340 345 350 Leu Leu Asp Leu
355 96353PRTArabidopsis thaliana 96Met Glu Lys Asn Ala Ser Thr Ser
Ser Leu Lys Asp Leu Gly Pro Ser 1 5 10 15 Gly Leu Asp Leu Thr Ser
Ala Phe Phe Lys Pro Ile His Asn Ser Asp 20 25 30 Pro Ser Leu Pro
Ser Asn Arg Arg Thr Lys Val Ser Val Val Gly Val 35 40 45 Gly Asn
Val Gly Met Ala Ile Ala Gln Thr Ile Leu Thr Gln Asp Leu 50 55 60
Ala Asp Glu Ile Ala Leu Val Asp Ala Lys Pro Asp Lys Leu Arg Gly 65
70 75 80 Glu Met Leu Asp Leu Gln His Ala Ala Ala Phe Leu Pro Arg
Thr Lys 85 90 95 Ile Thr Ala Ser Val Asp Tyr Glu Val Thr Ala Gly
Ser Asp Leu Cys 100 105 110 Ile Val Thr Ala Gly Ala Arg Gln Asn Pro
Gly Glu Ser Arg Leu Asn 115 120 125 Leu Leu Gln Arg Asn Val Ala Leu
Phe Arg His Ile Ile Pro Pro Leu 130 135 140 Ala Lys Ala Ser Pro Asp
Ser Ile Leu Ile Val Val Ser Asn Pro Val 145 150 155 160 Asp Val Leu
Thr Tyr Val Ala Trp Lys Leu Ser Gly Phe Pro Val Asn 165 170 175 Arg
Val Leu Gly Ser Gly Thr Asn Leu Asp Ser Ser Arg Phe Arg Phe 180 185
190 Leu Ile Ala Asp His Leu Asp Val Asn Ala Gln Asp Val Gln Ala Phe
195 200 205 Ile Val Gly Glu His Gly Asp Ser Ser Val Ala Leu Trp Ser
Ser Ile 210 215 220 Ser Val Gly Gly Ile Pro Val Leu Ser Phe Leu Glu
Lys Asn Gln Ile 225 230 235 240 Ala Tyr Glu Lys Gln Thr Leu Glu Asp
Ile His Gln Ala Val Val Gly 245 250 255 Ser Ala Tyr Glu Val Ile Gly
Leu Lys Gly Tyr Thr Ser Trp Ala Ile 260 265 270 Gly Tyr Ser Val Ala
Asn Leu Ala Arg Thr Ile Leu Arg Asp Gln Arg 275 280 285 Lys Ile His
Pro Val Thr Val Leu Ala Arg Gly Phe Tyr Gly Val Asp 290 295 300 Gly
Gly Asp Val Phe Leu Ser Leu Pro Ala Leu Leu Gly Arg Asn Gly 305 310
315 320 Val Val Ala Val Thr Asn Val His Met Thr Asp Glu Glu Ala Glu
Lys 325 330 335 Leu Gln Lys Ser Ala Lys Thr Ile Leu Glu Met Gln Ser
Gln Leu Gly 340 345 350 Leu 97353PRTArabidopsis thaliana 97Met Glu
Lys Asn Ala Ser Thr Ser Ser Leu Lys Asp Leu Gly Pro Ser 1 5 10 15
Gly Leu Asp Leu Thr Ser Ala Phe Phe Lys Pro Ile His Asn Ser Asp 20
25 30 Pro Ser Leu Pro Ser Asn Arg Arg Thr Lys Val Ser Val Val Gly
Val 35 40 45 Gly Asn Val Gly Met Ala Ile Ala Gln Thr Ile Leu Thr
Gln Asp Leu 50 55 60 Ala Asp Glu Ile Ala Leu Val Asp Ala Lys Pro
Asp Lys Leu Arg Gly 65 70 75 80 Glu Met Leu Asp Leu Gln His Ala Ala
Ala Phe Leu Pro Arg Thr Lys 85 90 95 Ile Thr Ala Ser Val Asp Tyr
Glu Val Thr Ala Gly Ser Asp Leu Cys 100 105 110 Ile Val Thr Ala Gly
Ala Arg Gln Asn Pro Gly Glu Ser Arg Leu Asn 115 120 125 Leu Leu Gln
Arg Asn Val Ala Leu Phe Arg His Ile Ile Pro Pro Leu 130 135 140 Ala
Lys Ala Ser Pro Asp Ser Ile Leu Ile Ile Val Ser Asn Pro Val 145 150
155 160 Asp Val Leu Thr Tyr Val Ala Trp Lys Leu Ser Gly Phe Pro Val
Asn 165 170 175 Arg Val Leu Gly Ser Gly Thr Asn Leu Asp Ser Ser Arg
Phe Arg Phe 180 185 190 Leu Ile Ala Asp His Leu Asp Val Asn Ala Gln
Asp Val Gln Ala Phe 195 200 205 Ile Val Gly Glu His Gly Asp Ser Ser
Val Ala Leu Trp Ser Ser Ile 210 215 220 Ser Val Gly Gly Ile Pro Val
Leu Ser Phe Leu Glu Lys Asn Gln Ile 225 230 235 240 Ala Tyr Glu Lys
Gln Thr Leu Glu Asp Ile His Gln Ala Val Val Gly 245 250 255 Ser Ala
Tyr Glu Val Ile Gly Leu Lys Gly Tyr Thr Ser Trp Ala Ile 260 265 270
Gly Tyr Ser Val Ala Asn Leu Ala Arg Thr Ile Leu Arg Asp Gln Arg 275
280 285 Lys Ile His Pro Val Thr Val Leu Ala Arg Gly Phe Tyr Gly Val
Asp 290 295 300 Gly Gly Asp Val Phe Leu Ser Leu Pro Ala Leu Leu Gly
Arg Asn Gly 305 310 315 320 Val Val Ala Val Thr Asn Val His Met Thr
Asp Glu Glu Ala Glu Lys 325 330 335 Leu Gln Lys Ser Ala Lys Thr Ile
Leu Glu Met Gln Ser Gln Leu Gly 340 345 350 Leu
98350PRTLycopersicon esculentum 98Met Gln Asn Ser Ser Ser Ser Ser
Ser Leu Gly Pro Gly Gly Leu Asp 1 5 10 15 Leu Thr Gln Ala Phe Phe
Lys Ser Ile Ser Asn Ala Ala Pro Pro Ser 20 25 30 Pro Thr Lys Arg
His Thr Lys Ile Ser Val Ile Gly Val Gly Asn Val 35 40 45 Gly Met
Ala Ile Ala Gln Thr Ile Leu Thr Gln Asp Leu Val Asp Glu 50 55 60
Leu Ala Leu Val Asp Ala Lys Ser Asp Lys Leu Arg Gly Glu Met Leu 65
70 75 80 Asp Leu Gln His Ala Ala Ala Phe Leu Pro Arg Thr Lys Ile
His Ala 85 90 95 Ser Ile Asp Tyr Ser Val Thr Ala Gly Ser Asp Leu
Cys Ile Val Thr 100 105 110 Ala Gly Ala Arg Gln Asn Pro Gly Glu Ser
Arg Leu Asn Leu Leu Gln 115 120 125 Arg Asn Met Ala Leu Phe Arg Ser
Ile Ile Pro Pro Leu Val Lys Tyr 130 135 140 Ser Pro Glu Thr Thr Leu
Leu Val Val Ser Asn Pro Val Asp Val Leu 145 150 155 160 Thr Tyr Val
Ala Trp Lys Leu Ser Gly Phe Pro Ala Asn Arg Val Ile 165 170 175 Gly
Ser Gly Thr Asn Leu Asp Ser Ser Arg Phe Arg Phe Leu Ile Ala 180 185
190 Asp His Leu Asp Val Asn Ala Gln Asp Val Gln Ala Tyr Ile Val Gly
195 200 205 Glu His Gly Asp Ser Ser Val Ala Leu Trp Ser Gly Ile Ser
Val Gly 210 215 220 Gly Val Pro Val Leu Ser Phe Leu Glu Arg Gln Gln
Ile Ala Leu Glu 225 230 235 240 Lys Glu Thr Leu Glu Lys Ile His Gln
Glu Val Val His Ser Ala Tyr 245 250 255 Glu Val Ile Ser Leu Lys Gly
Tyr Thr Ser Trp Ala Ile Gly Tyr Ser 260 265 270 Val Ala Asn Leu Ala
Arg Thr Ile Leu Arg Asp Gln Arg Arg Ile His 275 280 285 Pro Val Ser
Val Leu Ala Lys Gly Phe Tyr Gly Ile Asp Gly Gly Asp 290 295 300 Val
Phe Leu Ser Leu Pro Ala Gln Leu Gly Arg Ser Gly Val Leu Gly 305 310
315 320 Val Thr Asn Val His Leu Thr Asp Glu Glu Ile Glu Gln Leu Arg
Asn 325 330 335 Ser Ala Lys Thr Ile Leu Glu Val Gln Ser Gln Leu Gly
Ile 340 345 350 99346PRTSolanum tuberosum 99Met Ser Ser Ser Ser Pro
Leu Ser Leu Asp Gly Leu Asp Leu Asn Gln 1 5 10 15 Val Phe Phe Lys
Ser Ile Ser Asn Ala Asp Pro Pro Ser Gln Thr Asn 20 25 30 His His
Thr Lys Ile Ser Val Ile Gly Val Gly Asn Val Gly Met Ala 35 40 45
Ile Ala Gln Thr Ile Leu Thr Gln Asp Leu Val Asp Glu Leu Ala Leu 50
55 60 Val Asp Val Asn Ser Asp Lys Leu Arg Gly Glu Met Leu Asp Leu
Gln 65 70 75 80 His Ala Ala Ala Phe Leu Pro Arg Thr Lys Ile Val Ala
Ser Val Asp 85 90 95 Tyr Thr Val Thr Ala Gly Ser Asp Leu Cys Ile
Val Thr Ala Gly Ala 100 105 110 Arg Gln Asn Pro Gly Glu Ser Arg Leu
Asn Leu Leu Gln Arg Asn Leu 115 120 125 Ala Met Tyr Lys Ser Ile Val
Pro Glu Leu Val Lys Tyr Ser Pro Glu 130 135 140 Cys Ile Leu Leu Ile
Val Ser Asn Pro Val Asp Val Leu Thr Tyr Val 145 150 155 160 Ala Trp
Lys Ser Gly Phe Pro Val Asn Arg Val Ile Gly Ser Gly Thr 165 170 175
Asn Leu Asp Ser Ser Arg Phe Arg Phe Leu Ile Ala Asp His Leu Asp 180
185 190 Val Asn Ala Gln Asp Val Gln Ala Tyr Ile Val Gly Glu His Gly
Asp 195 200 205 Ser Ser Val Ala Leu Trp Ser Ser Ile Ser Val Gly Gly
Ile Pro Val 210 215 220 Leu Ser Phe Leu Glu Arg Gln Gln Ile Ala Phe
Glu Lys Asp Thr Leu 225 230 235 240 Glu Lys Ile His Lys Gln Val Val
Gln Ser Ala Tyr Glu Val Ile Asn 245 250 255 Leu Lys Gly Tyr Thr Ser
Trp Ala Ile Gly Tyr Ser Val Ala Asn Leu 260 265 270 Ala Phe Ser Ile
Ile Arg Asp Gln Arg Arg Ile His Pro Val Ser Ile 275 280 285 Leu Val
Lys Gly Phe Tyr Gly Ile Asp Gly Gly Asp Val Phe Leu Ser 290 295 300
Leu Pro Ala Gln Leu Gly Arg Ser Gly Val Leu Gly Val Thr Asn Val 305
310 315 320 His Leu Thr Asp Glu Glu Ile Gln Gln Leu Arg Asn Ser Ala
Glu Thr 325 330 335 Ile Leu Glu Val Gln Asn Gln Leu Gly Ile 340 345
100223PRTArabidopsis thaliana 100Met Glu Glu Leu Ala His Pro Tyr
Val Pro Arg Asp Leu Asn Leu Pro 1 5 10 15 Gly Tyr Val Pro Ile Ser
Met Ser Met Ser Ser Ile Val Ser Ile Tyr 20 25 30 Leu Gly Ser Ser
Leu Leu Val Val Ser Leu Val Trp Leu Leu Phe Gly 35 40 45 Arg Lys
Lys Ala Lys Leu Asp Lys Leu Leu Met Cys Trp Trp Thr Phe 50 55 60
Thr Gly Leu Thr His Val Ile Leu Glu Gly Tyr Phe Val Phe Ser Pro 65
70 75 80 Glu Phe Phe Lys Asp Asn Thr Ser Ala Tyr Leu Ala Glu Val
Trp Lys 85 90 95 Glu Tyr Ser Lys Gly Asp Ser Arg Tyr Val Gly Arg
Asp Ser Ala Val 100 105 110 Val Ser Val Glu Gly Ile Thr Ala Val Ile
Val Gly Pro Ala Ser Leu 115 120 125 Leu Ala Ile Tyr Ala Ile Ala Lys
Glu Lys Ser Tyr Ser Tyr Val Leu 130 135 140 Gln Leu Ala Ile Ser Val
Cys Gln Leu Tyr Gly Cys Leu Val Tyr Phe 145 150 155 160 Ile Thr Ala
Ile Leu Glu Gly Asp Asn Phe Ala Thr Asn Ser Phe Tyr 165 170 175 Tyr
Tyr Ser Tyr Tyr Ile Gly Ala Asn Cys Trp Trp Val Leu Ile Pro 180 185
190 Ser Leu Ile Ser Phe Arg Cys Trp Lys Lys Ile Cys Ala Ala Ala Ala
195 200 205 Ile Ala Asn Asn Asn Val Glu Thr Lys Thr Lys Lys Lys Thr
Arg 210 215 220 101223PRTArabidopsis thaliana 101Met Lys Glu Leu
Ala His Pro Tyr Val Pro Arg Asp Leu Asn Leu Pro 1 5 10 15 Gly Tyr
Val Pro Ile Ser Met Ser Met Ser Ser Ile Val Ser Ile Tyr 20 25 30
Leu Gly Ser Ser Leu Leu Val Val Ser Leu Val Trp Leu Leu Phe Gly 35
40 45 Arg Lys Lys Ala Lys Leu Asp Lys Leu Leu Met Cys Trp Trp Thr
Phe 50 55 60 Thr Gly Leu Thr His Val Ile Leu Glu Gly Tyr Phe Val
Phe Ser Pro 65 70 75 80 Glu Phe Phe Lys Asp Asn Thr Ser Ala Tyr Leu
Ala Glu Val Trp Lys 85 90 95 Glu Tyr Ser Lys Gly Asp Ser Arg Tyr
Val Gly Arg Asp Ser Ala Val 100 105 110 Val Ser Val Glu Gly Ile Thr
Ala Val Ile Val Gly Pro Ala Ser Leu 115 120 125 Leu Ala Ile Tyr Ala
Ile Ala Lys Glu Lys Ser Tyr Ser Tyr Val Leu 130 135 140 Gln Leu Ala
Ile Ser Val Cys Gln Leu Tyr Gly Cys Leu Val Tyr Phe 145 150 155 160
Ile Thr Ala Ile Leu Glu Gly Asp Asn Phe Ala Thr Asn Ser Phe Tyr 165
170 175 Tyr Tyr Ser Tyr Tyr Ile Gly Ala Asn Cys Trp Trp Val Leu Ile
Pro 180 185 190 Ser Leu Ile Ser Phe Arg Cys Trp Lys Lys Ile Cys Ala
Ala Ala Ala 195 200 205 Ile Ala Asn Asn Asn Val Glu Thr Lys Thr Lys
Lys Lys Thr Arg 210 215 220 102223PRTArabidopsis thaliana 102Met
Glu Glu Leu Ala His Pro Tyr Val Pro Arg Asp Leu Asn Leu Pro 1 5 10
15 Gly Tyr Val Pro Ile Ser Met Ser Met Ser Ser Ile Val Ser Ile Tyr
20 25 30 Leu Gly Ser Ser Leu Leu Val Val Ser Leu Val Trp Leu Leu
Phe Gly 35 40 45 Arg Lys Lys Ala Lys Leu Asp Lys Leu Leu Met Cys
Trp Trp Thr Phe 50 55 60 Thr Gly Leu Thr His Val Ile Leu Glu Gly
Tyr Phe Val Phe Ser Pro 65 70 75 80 Glu Phe Phe Lys Asp Asn Thr Ser
Ala Tyr Leu Ala Glu Val Trp Lys
85 90 95 Glu Tyr Ser Lys Gly Asp Ser Arg Tyr Val Gly Arg Asp Ser
Ala Val 100 105 110 Val Ser Val Glu Gly Ile Thr Ala Val Ile Val Gly
Pro Ala Ser Leu 115 120 125 Leu Ala Ile Tyr Ala Ile Ala Lys Glu Lys
Ser Tyr Ser Tyr Val Leu 130 135 140 Gln Leu Ala Ile Ser Val Cys Gln
Leu Tyr Gly Cys Val Val Tyr Phe 145 150 155 160 Ile Thr Ala Ile Leu
Glu Gly Asp Asn Phe Ala Thr Asn Ser Phe Tyr 165 170 175 Tyr Tyr Ser
Tyr Tyr Ile Gly Ala Asn Cys Trp Trp Val Leu Ile Pro 180 185 190 Ser
Leu Ile Ser Phe Arg Cys Trp Lys Lys Ile Cys Ala Ala Ala Ala 195 200
205 Ile Ala Asn Asn Asn Val Glu Thr Lys Thr Lys Lys Lys Thr Arg 210
215 220 103240PRTNicotiana tabacum 103Met Ala Glu Asn Gly Ile Lys
His Gln Glu Val Gly His Lys Ser Leu 1 5 10 15 Leu Gln Ser Asp Ala
Leu Tyr Gln Tyr Ile Leu Glu Thr Ser Val Tyr 20 25 30 Pro Arg Glu
Pro Glu Ser Met Lys Glu Leu Arg Glu Val Thr Ala Lys 35 40 45 His
Pro Trp Asn Leu Met Thr Thr Ser Ala Asp Glu Gly Gln Phe Leu 50 55
60 Asn Met Leu Leu Lys Leu Ile Asn Ala Lys Asn Thr Met Glu Ile Gly
65 70 75 80 Val Tyr Thr Gly Tyr Ser Leu Leu Ala Thr Ala Leu Ala Ile
Pro Asp 85 90 95 Asp Gly Lys Ile Leu Ala Met Asp Ile Asn Arg Glu
Asn Tyr Glu Ile 100 105 110 Gly Leu Pro Ile Ile Glu Lys Ala Gly Val
Ala His Lys Ile Glu Phe 115 120 125 Arg Glu Gly Pro Ala Leu Pro Val
Leu Asp Gln Leu Val Glu Asp Lys 130 135 140 Lys Asn His Gly Thr Tyr
Asp Phe Ile Phe Val Asp Ala Asp Lys Asp 145 150 155 160 Asn Tyr Ile
Asn Tyr His Lys Arg Ile Ile Asp Leu Val Lys Val Gly 165 170 175 Gly
Leu Ile Gly Tyr Asp Asn Thr Leu Trp Asn Gly Ser Val Val Ala 180 185
190 Pro Pro Asp Ala Pro Met Arg Lys Tyr Val Arg Tyr Tyr Arg Asp Phe
195 200 205 Val Leu Glu Leu Asn Lys Ala Leu Ala Ala Asp Pro Arg Ile
Glu Ile 210 215 220 Cys Met Leu Pro Val Gly Asp Gly Ile Thr Leu Cys
Arg Arg Ile Thr 225 230 235 240 104247PRTEucalyptus species 104Met
Ala Ala Asn Ala Glu Pro Gln Gln Thr Gln Pro Ala Lys His Ser 1 5 10
15 Glu Val Gly His Lys Ser Leu Leu Gln Ser Asp Ala Leu Tyr Gln Tyr
20 25 30 Ile Leu Glu Thr Ser Val Tyr Pro Arg Glu Pro Glu Ser Met
Lys Glu 35 40 45 Leu Arg Glu Ile Thr Ala Lys His Pro Trp Asn Leu
Met Thr Thr Ser 50 55 60 Ala Asp Glu Gly Gln Phe Leu Asn Met Leu
Leu Lys Leu Ile Asn Ala 65 70 75 80 Lys Asn Thr Met Glu Ile Gly Val
Tyr Thr Gly Tyr Ser Leu Leu Ala 85 90 95 Thr Ala Leu Ala Leu Pro
Asp Asp Gly Lys Ile Leu Ala Met Asp Ile 100 105 110 Asn Arg Glu Asn
Phe Glu Ile Gly Leu Pro Val Ile Glu Lys Ala Gly 115 120 125 Leu Ala
His Lys Ile Asp Phe Arg Glu Gly Pro Ala Leu Pro Leu Leu 130 135 140
Asp Gln Leu Val Gln Asp Glu Lys Asn His Gly Thr Tyr Asp Phe Ile 145
150 155 160 Phe Val Asp Ala Asp Lys Asp Asn Tyr Ile Asn Tyr His Lys
Arg Leu 165 170 175 Ile Asp Leu Val Lys Val Gly Gly Leu Ile Gly Tyr
Asp Asn Thr Leu 180 185 190 Trp Asn Gly Ser Val Val Ala Pro Ala Asp
Ala Pro Leu Arg Lys Tyr 195 200 205 Val Arg Tyr Tyr Arg Asp Phe Val
Leu Glu Leu Asn Lys Ala Leu Ala 210 215 220 Val Asp Pro Arg Val Glu
Ile Cys Met Leu Pro Val Gly Asp Gly Ile 225 230 235 240 Thr Leu Cys
Arg Arg Val Ser 245 105247PRTPopulus species 105Met Ala Thr Asn Gly
Glu Glu Gln Gln Ser Gln Ala Gly Arg His Gln 1 5 10 15 Glu Val Gly
His Lys Ser Leu Leu Gln Ser Asp Ala Leu Tyr Gln Tyr 20 25 30 Ile
Leu Glu Thr Ser Val Tyr Pro Arg Glu Pro Glu Cys Met Lys Glu 35 40
45 Leu Arg Glu Val Thr Ala Lys His Pro Trp Asn Ile Met Thr Thr Ser
50 55 60 Ala Asp Glu Gly Gln Phe Leu Asn Met Leu Leu Lys Leu Val
Asn Ala 65 70 75 80 Lys Asn Thr Met Glu Ile Gly Val Tyr Thr Gly Tyr
Ser Leu Leu Ala 85 90 95 Thr Ala Leu Ala Ile Pro Glu Asp Gly Lys
Ile Leu Ala Met Asp Ile 100 105 110 Asn Arg Glu Asn Tyr Glu Leu Gly
Leu Pro Val Ile Gln Lys Ala Gly 115 120 125 Val Ala His Lys Ile Asp
Phe Lys Glu Gly Pro Ala Leu Pro Val Leu 130 135 140 Asp Gln Met Ile
Glu Asp Gly Lys Cys His Gly Ser Phe Asp Phe Ile 145 150 155 160 Phe
Val Asp Ala Asp Lys Asp Asn Tyr Ile Asn Tyr His Lys Arg Leu 165 170
175 Ile Glu Leu Val Lys Val Gly Gly Leu Ile Gly Tyr Asp Asn Thr Leu
180 185 190 Trp Asn Gly Ser Val Val Ala Pro Pro Asp Ala Pro Met Arg
Lys Tyr 195 200 205 Val Arg Tyr Tyr Arg Asp Phe Val Leu Glu Leu Asn
Lys Ala Leu Ala 210 215 220 Ala Asp Pro Arg Ile Glu Ile Cys Met Leu
Pro Val Gly Asp Gly Ile 225 230 235 240 Thr Leu Cys Arg Arg Ile Gln
245 106258PRTZea mays 106Met Ala Thr Thr Ala Thr Glu Ala Ala Pro
Ala Gln Glu Gln Gln Ala 1 5 10 15 Asn Gly Asn Gly Glu Gln Lys Thr
Arg His Ser Glu Val Gly His Lys 20 25 30 Ser Leu Leu Lys Ser Asp
Asp Leu Tyr Gln Tyr Ile Leu Asp Thr Ser 35 40 45 Val Tyr Pro Arg
Glu Pro Glu Ser Met Lys Glu Leu Arg Glu Val Thr 50 55 60 Ala Lys
His Pro Trp Asn Leu Met Thr Thr Ser Ala Asp Glu Gly Gln 65 70 75 80
Phe Leu Asn Met Leu Ile Lys Leu Ile Gly Ala Lys Lys Thr Met Glu 85
90 95 Ile Gly Val Tyr Thr Gly Tyr Ser Leu Leu Ala Thr Ala Leu Ala
Leu 100 105 110 Pro Glu Asp Gly Thr Ile Leu Ala Met Asp Ile Asn Arg
Glu Asn Tyr 115 120 125 Glu Leu Gly Leu Pro Cys Ile Glu Lys Ala Gly
Val Ala His Lys Ile 130 135 140 Asp Phe Arg Glu Gly Pro Ala Leu Pro
Val Leu Asp Asp Leu Ile Ala 145 150 155 160 Glu Glu Lys Asn His Gly
Ser Phe Asp Phe Val Phe Val Asp Ala Asp 165 170 175 Lys Asp Asn Tyr
Leu Asn Tyr His Glu Arg Leu Leu Lys Leu Val Lys 180 185 190 Leu Gly
Gly Leu Ile Gly Tyr Asp Asn Thr Leu Trp Asn Gly Ser Val 195 200 205
Val Leu Pro Asp Asp Ala Pro Met Arg Lys Tyr Ile Arg Phe Tyr Arg 210
215 220 Asp Phe Val Leu Val Leu Asn Lys Ala Leu Ala Ala Asp Asp Arg
Val 225 230 235 240 Glu Ile Cys Gln Leu Pro Val Gly Asp Gly Val Thr
Leu Cys Arg Arg 245 250 255 Val Lys 107264PRTZea mays 107Met Ala
Thr Thr Ala Thr Glu Ala Thr Lys Thr Thr Ala Pro Ala Gln 1 5 10 15
Glu Gln Gln Ala Asn Gly Asn Gly Asn Gly Glu Gln Lys Thr Arg His 20
25 30 Ser Glu Val Gly His Lys Ser Leu Leu Lys Ser Asp Asp Leu Tyr
Gln 35 40 45 Tyr Ile Leu Asp Thr Ser Val Tyr Pro Arg Glu Pro Glu
Ser Met Lys 50 55 60 Glu Leu Arg Glu Ile Thr Ala Lys His Pro Trp
Asn Leu Met Thr Thr 65 70 75 80 Ser Ala Asp Glu Gly Gln Phe Leu Asn
Met Leu Ile Lys Leu Ile Gly 85 90 95 Ala Lys Lys Thr Met Glu Ile
Gly Val Tyr Thr Gly Tyr Ser Leu Leu 100 105 110 Ala Thr Ala Leu Ala
Leu Pro Glu Asp Gly Thr Ile Leu Ala Met Asp 115 120 125 Ile Asn Arg
Glu Asn Tyr Glu Leu Gly Leu Pro Cys Ile Asn Lys Ala 130 135 140 Gly
Val Gly His Lys Ile Asp Phe Arg Glu Gly Pro Ala Leu Pro Val 145 150
155 160 Leu Asp Asp Leu Val Ala Asp Lys Glu Gln His Gly Ser Phe Asp
Phe 165 170 175 Ala Phe Val Asp Ala Asp Lys Asp Asn Tyr Leu Asn Tyr
His Glu Arg 180 185 190 Leu Leu Lys Leu Val Arg Pro Gly Gly Leu Ile
Gly Tyr Asp Asn Thr 195 200 205 Leu Trp Asn Gly Ser Val Val Leu Pro
Asp Asp Ala Pro Met Arg Lys 210 215 220 Tyr Ile Arg Phe Tyr Arg Asp
Phe Val Leu Ala Leu Asn Ser Ala Leu 225 230 235 240 Ala Ala Asp Asp
Arg Val Glu Ile Cys Gln Leu Pro Val Gly Asp Gly 245 250 255 Val Thr
Leu Cys Arg Arg Val Lys 260
* * * * *
References