U.S. patent application number 11/969728 was filed with the patent office on 2008-06-05 for modulating plant nitrogen levels.
This patent application is currently assigned to Ceres, Inc.. Invention is credited to Steven Craig Bobzin, Boris Jankowski, Emilio Margolles-Clark, Joon-Hyun Park, Richard Schneeberger.
Application Number | 20080131581 11/969728 |
Document ID | / |
Family ID | 36587364 |
Filed Date | 2008-06-05 |
United States Patent
Application |
20080131581 |
Kind Code |
A1 |
Schneeberger; Richard ; et
al. |
June 5, 2008 |
MODULATING PLANT NITROGEN LEVELS
Abstract
Methods and materials for modulating (e.g., increasing or
decreasing) nitrogen levels in plants are disclosed. For example,
nucleic acids encoding nitrogen-modulating polypeptides are
disclosed as well as methods for using such nucleic acids to
transform plant cells. Plants and plant products having increased
nitrogen levels are also disclosed.
Inventors: |
Schneeberger; Richard;
(Carlsbad, CA) ; Margolles-Clark; Emilio; (Miami,
FL) ; Park; Joon-Hyun; (Oak Park, CA) ;
Jankowski; Boris; (Santa Monica, CA) ; Bobzin; Steven
Craig; (Malibu, CA) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Ceres, Inc.
Thousand Oaks
CA
|
Family ID: |
36587364 |
Appl. No.: |
11/969728 |
Filed: |
January 4, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11292951 |
Dec 2, 2005 |
7335510 |
|
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11969728 |
|
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|
60705119 |
Aug 2, 2005 |
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60637311 |
Dec 16, 2004 |
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Current U.S.
Class: |
426/615 ;
435/419; 800/278; 800/298 |
Current CPC
Class: |
Y02A 40/146 20180101;
C12N 15/8251 20130101; C12N 15/8261 20130101 |
Class at
Publication: |
426/615 ;
800/278; 435/419; 800/298 |
International
Class: |
A23L 1/212 20060101
A23L001/212; C12N 15/87 20060101 C12N015/87; C12N 15/82 20060101
C12N015/82; A01H 1/00 20060101 A01H001/00 |
Claims
1. A method of modulating the level of nitrogen in a plant, said
method comprising introducing into a plant cell an isolated nucleic
acid comprising a nucleotide sequence encoding a polypeptide having
80 percent or greater sequence identity to an amino acid sequence
selected from the group consisting of SEQ ID NOs:4-18, wherein a
tissue of a plant produced from said plant cell has a difference in
the level of nitrogen as compared to the corresponding level in
tissue of a control plant that does not comprise said nucleic
acid.
2. The method of claim 1, wherein said difference is an increase in
the level of nitrogen.
3. The method of claim 1, wherein said isolated nucleic acid is
operably linked to a regulatory region.
4. The method of claim 3, wherein said regulatory region is a
promoter selected from the group consisting of YP0092, PT0676,
PT0708, PT0613, PT0672, PT0678, PT0688, PT0837, the napin promoter,
the Arcelin-5 promoter, the phaseolin gene promoter, the soybean
trypsin inhibitor promoter, the ACP promoter, the stearoyl-ACP
desaturase gene promoter, the soybean .alpha. subunit of
.beta.-conglycinin promoter, the oleosin promoter, the 15 kD zein
promoter, the 16 kD zein promoter, the 19 kD zein promoter, the 22
kD zein promoter, the 27 kD zein promoter, the Osgt-1 promoter, the
beta-amylase gene promoter, and the barley hordein gene
promoter.
5. A method of producing a plant tissue, said method comprising
growing a plant cell comprising an isolated nucleic acid comprising
a nucleotide sequence encoding a polypeptide having 80 percent or
greater sequence identity to an amino acid sequence selected from
the group consisting of SEQ ID NOs:4-18, wherein said tissue has a
difference in the level of nitrogen as compared to the
corresponding level in tissue of a control plant that does not
comprise said nucleic acid.
6. A plant cell comprising an isolated nucleic acid comprising a
nucleotide sequence encoding a polypeptide having 80 percent or
greater sequence identity to an amino acid sequence selected from
the group consisting of SEQ ID NOs:4-18, wherein a tissue of a
plant produced from said plant cell has a difference in the level
of nitrogen as compared to the corresponding level in tissue of a
control plant that does not comprise said nucleic acid.
7. A transgenic plant comprising the plant cell of claim 6.
8. Progeny of the plant of claim 7, wherein said progeny has a
difference in the level of nitrogen as compared to the level of
nitrogen in a corresponding control plant that does not comprise
said isolated nucleic acid.
9. Seed from a transgenic plant according to claim 7.
10. Vegetative tissue from a transgenic plant according to claim
7.
11. A food product comprising vegetative tissue from a transgenic
plant according to claim 7.
12. A feed product comprising vegetative tissue from a transgenic
plant according to claim 7.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/292,951, filed Dec. 2, 2005, which claims priority under 35
U.S.C. .sctn.119 to U.S. Provisional Application No. 60/705,119,
filed Aug. 2, 2005, and to U.S. Provisional Application No.
60/637,311, filed Dec. 16, 2004. The disclosures of the prior
applications are considered part of (and are incorporated by
reference in) the disclosure of this application.
TECHNICAL FIELD
[0002] This document provides methods and materials related to
modulating (e.g., increasing or decreasing) nitrogen levels in
plants. For example, this document provides plants having increased
nitrogen levels as well as materials and methods for making plants
and plant products having increased nitrogen levels.
BACKGROUND
[0003] The photoautotrophic production of organic nitrogenous
compounds is crucial to plant metabolism, growth, and development.
Protein and amino acid contents of harvested plant materials are of
great agronomic importance in many crop species. Light-driven
nitrogen (N) assimilation in leaves has evolved to operate
alongside and integrate with photosynthesis and respiration. The
production of reduced carbon (C) in photosynthesis and its
reoxidation in respiration are necessary to produce both the energy
and C skeletons required for the incorporation of inorganic N into
amino acids. Conversely, N assimilation is required to sustain the
output of organic C and N. This network is further complicated by
the concomitant operation of photorespiratory metabolism. Both the
rate of N assimilation and the coordination of C and N assimilation
are under multifactorial control by a repertoire of signals, which
provide information on C and N status. There is a need for
compositions and methods that can increase nitrogen content in
plants under varying nitrogen conditions.
SUMMARY
[0004] This document provides methods and materials related to
plants having modulated (e.g., increased or decreased) nitrogen
content. For example, this document provides transgenic plants and
plant cells having increased levels of nitrogen, nucleic acids used
to generate transgenic plants and plant cells having increased
levels of nitrogen, and methods for making plants and plant cells
having increased levels of nitrogen. Such plants and plant cells
can be grown to produce seeds having increased nitrogen content.
The seeds may be used to produce foodstuffs and animal feed having
increased nutritional (e.g., protein) content, which may benefit
both food producers and consumers. While not being bound to any
particular mode of action, the nitrogen-modulating polypeptides
provided herein may have activities as transport proteins. For
example, the polypeptides provided herein may be involved in
transport of nitrogen, e.g., organic nitrogen in the form of
peptides or amino acids, or inorganic nitrogen in the form of
ammonium or nitrate.
[0005] In one embodiment, a method of modulating the level of
nitrogen in a plant is provided. The method comprises introducing
into a plant cell an isolated nucleic acid comprising a nucleotide
sequence encoding a polypeptide having 80 percent or greater
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO:2, SEQ ID NOs:4-18, and the consensus
sequence set forth in FIG. 1, where a tissue of a plant produced
from the plant cell has a difference in the level of nitrogen as
compared to the corresponding level in tissue of a control plant
that does not comprise the nucleic acid.
[0006] In another embodiment, a method of modulating the level of
nitrogen in a plant is provided. The method comprises introducing
into a plant cell an isolated nucleic acid comprising a nucleotide
sequence encoding a polypeptide having 80 percent or greater
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:16,
and the consensus sequence set forth in FIG. 1, where a tissue of a
plant produced from the plant cell has a difference in the level of
nitrogen as compared to the corresponding level in tissue of a
control plant that does not comprise the nucleic acid.
[0007] In a further embodiment, a method of modulating the level of
nitrogen in a plant is provided. The method comprises introducing
into a plant cell an isolated nucleic acid comprising a nucleotide
sequence encoding a polypeptide having 80 percent or greater
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID
NO:16, where a tissue of a plant produced from the plant cell has a
difference in the level of nitrogen as compared to the
corresponding level in tissue of a control plant that does not
comprise the nucleic acid.
[0008] The sequence identity can be 85 percent or greater, 90
percent or greater, or 95 percent or greater. The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:2. The nucleotide sequence can encode a
polypeptide comprising an amino acid sequence corresponding to SEQ
ID NO:4. The nucleotide sequence can encode a polypeptide
comprising an amino acid sequence corresponding to the consensus
sequence set forth in FIG. 1. The difference can be an increase in
the level of nitrogen.
[0009] The isolated nucleic acid can be operably linked to a
regulatory region. The regulatory region can be a tissue-specific
regulatory region. The tissue-specific regulatory region can be a
promoter. The promoter can be selected from the group consisting of
YP0092, PT0676, PT0708, the napin promoter, the Arcelin-5 promoter,
the phaseolin gene promoter, the soybean trypsin inhibitor
promoter, the ACP promoter, the stearoyl-ACP desaturase gene, the
soybean .alpha. subunit of .beta.-conglycinin promoter, the oleosin
promoter, the 15 kD zein promoter, the 16 kD zein promoter, the 19
kD zein promoter, the 22 kD zein promoter, the 27 kD zein promoter,
the Osgt-1 promoter, the beta-amylase gene promoter, and the barley
hordein gene promoter. The promoter can be selected from the group
consisting of PT0613, PT0672, PT0678, PT0688, PT0837, YP0128,
YP0275, PT0625, PT0660, PT0683, and PT0758. The regulatory region
can be a broadly expressing promoter. The broadly expressing
promoter can be selected from the group consisting of p13879,
p32449, 21876, p326, YP0158, YP0214, YP0380, PT0848, PT0633,
YP0050, YP0144, and YP0190. The regulatory region can be an
inducible promoter.
[0010] The plant can be a dicot. The plant can be a member of the
genus Brassica, Glycine, Gossypiurn, Helianthus, Lactuca,
Lycopersicon, Solanum, Vitis, Pisum, Medicago, Carthamus, Arachis,
Olea, Linum, or Trifolium. The plant can be a monocot. The plant
can be a member of the genus Zea, Triticum, Hordeum, Secale, Oryza,
Triticosecale, Avena, Musa, Elaeis, Phleum, or Sorghum. The tissue
can be seed tissue.
[0011] A method of producing a plant tissue is also provided. The
method comprises growing a plant cell comprising an isolated
nucleic acid comprising a nucleotide sequence encoding a
polypeptide having 80 percent or greater sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID
NO:2, SEQ ID NOs:4-18, and the consensus sequence set forth in FIG.
1, where the tissue has a difference in the level of nitrogen as
compared to the corresponding level in tissue of a control plant
that does not comprise the nucleic acid.
[0012] In another embodiment, a method of producing a plant tissue
is provided. The method comprises growing a plant cell comprising
an isolated nucleic acid comprising a nucleotide sequence encoding
a polypeptide having 80 percent or greater sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:16, and the consensus
sequence set forth in FIG. 1, where the tissue has a difference in
the level of nitrogen as compared to the corresponding level in
tissue of a control plant that does not comprise the nucleic
acid.
[0013] In yet another embodiment, a method of producing a plant
tissue is provided. The method comprises growing a plant cell
comprising an isolated nucleic acid comprising a nucleotide
sequence encoding a polypeptide having 80 percent or greater
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID
NO:16, where the tissue has a difference in the level of nitrogen
as compared to the corresponding level in tissue of a control plant
that does not comprise the nucleic acid.
[0014] The sequence identity can be 85 percent or greater, 90
percent or greater, or 95 percent or greater. The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:2. The nucleotide sequence can encode a
polypeptide comprising an amino acid sequence corresponding to SEQ
ID NO:4. The nucleotide sequence can encode a polypeptide
comprising an amino acid sequence corresponding to the consensus
sequence set forth in FIG. 1. The difference can be an increase in
the level of nitrogen.
[0015] The isolated nucleic acid can be operably linked to a
regulatory region. The regulatory region can be a tissue-specific
regulatory region. The tissue-specific regulatory region can be a
promoter. The promoter can be selected from the group consisting of
YP0092, PT0676, PT0708, the napin promoter, the Arcelin-5 promoter,
the phaseolin gene promoter, the soybean trypsin inhibitor
promoter, the ACP promoter, the stearoyl-ACP desaturase gene, the
soybean .alpha. subunit of .beta.-conglycinin promoter, the oleosin
promoter, the 15 kD zein promoter, the 16 kD zein promoter, the 19
kD zein promoter, the 22 kD zein promoter, the 27 kD zein promoter,
the Osgt-1 promoter, the beta-amylase gene promoter, and the barley
hordein gene promoter. The promoter can be selected from the group
consisting of PT0613, PT0672, PT0678, PT0688, PT0837, YP0128,
YP0275, PT0625, PT0660, PT0683, and PT0758. The regulatory region
can be a broadly expressing promoter. The broadly expressing
promoter can be selected from the group consisting of p13879,
p32449, 21876, p326, YP0158, YP0214, YP0380, PT0848, PT0633,
YP0050, YP0144, and YP0190. The regulatory region can be an
inducible promoter.
[0016] The plant tissue can be dicotyledonous. The plant tissue can
be a member of the genus Brassica, Glycine, Gossypium, Helianthus,
Lactuca, Lycopersicon, Solanum, Vitis, Pisum, Medicago, Carthamus,
Arachis, Olea, Linum, or Trifolium. The plant tissue can be
monocotyledonous. The plant tissue can be a member of the genus
Zea, Triticum, Hordeum, Secale, Oryza, Triticosecale, Avena, Musa,
Elaeis, Phleum, or Sorghum. The tissue can be seed tissue.
[0017] A plant cell is also provided. The plant cell comprises an
isolated nucleic acid comprising a nucleotide sequence encoding a
polypeptide having 80 percent or greater sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID
NO:2, SEQ ID NOs:4-18, and the consensus sequence set forth in FIG.
1, where a tissue of a plant produced from the plant cell has a
difference in the level of nitrogen as compared to the
corresponding level in tissue of a control plant that does not
comprise the nucleic acid.
[0018] In another embodiment, a plant cell is provided. The plant
cell comprises an isolated nucleic acid comprising a nucleotide
sequence encoding a polypeptide having 80 percent or greater
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:16,
and the consensus sequence set forth in FIG. 1, where a tissue of a
plant produced from the plant cell has a difference in the level of
nitrogen as compared to the corresponding level in tissue of a
control plant that does not comprise the nucleic acid.
[0019] In yet another embodiment, a plant cell is provided. The
plant cell comprises an isolated nucleic acid comprising a
nucleotide sequence encoding a polypeptide having 80 percent or
greater sequence identity to an amino acid sequence selected from
the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and
SEQ ID NO:16, where a tissue of a plant produced from the plant
cell has a difference in the level of nitrogen as compared to the
corresponding level in tissue of a control plant that does not
comprise the nucleic acid.
[0020] The sequence identity can be 85 percent or greater, 90
percent or greater, or 95 percent or greater, The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:2. The nucleotide sequence can encode a
polypeptide comprising an amino acid sequence corresponding to SEQ
ID NO:4. The nucleotide sequence can encode a polypeptide
comprising an amino acid sequence corresponding to the consensus
sequence set forth in FIG. 1. The difference can be an increase in
the level of nitrogen.
[0021] The isolated nucleic acid can be operably linked to a
regulatory region. The regulatory region can be a tissue-specific
regulatory region. The tissue-specific regulatory region can be a
promoter. The promoter can be selected from the group consisting of
YP0092, PT0676, PT0708, the napin promoter, the Arcelin-5 promoter,
the phaseolin gene promoter, the soybean trypsin inhibitor
promoter, the ACP promoter, the stearoyl-ACP desaturase gene, the
soybean .alpha. subunit of .beta.-conglycinin promoter, the oleosin
promoter, the 15 kD zein promoter, the 16 kD zein promoter, the 19
kD zein promoter, the 22 kD zein promoter, the 27 kD zein promoter,
the Osgt-1 promoter, the beta-amylase gene promoter, and the barley
hordein gene promoter. The promoter can be selected from the group
consisting of PT0613, PT0672, PT0678, PT0688, PT0837, YP0128,
YP0275, PT0625, PT0660, PT0683, and PT0758. The regulatory region
can be a broadly expressing promoter. The broadly expressing
promoter can be selected from the group consisting of p13879,
p32449, 21876, p326, YP0158, YP0214, YP0380, PT0848, PT0633,
YP0050, YP0144, and YP0190. The regulatory region can be an
inducible promoter.
[0022] The plant can be a dicot. The plant can be a member of the
genus Brassica, Glycine, Gossypium, Helianthus, Lactuca,
Lycopersicon, Solanum, Vitis, Pisum, Medicago, Carthamus, Arachis,
Olea, Linum, or Trifolium. The plant can be a monocot. The plant
can be a member of the genus Zea, Triticum, Hordeum, Secale, Oryza,
Triticosecale, Avena, Musa, Elaeis, Phleum, or Sorghum. The tissue
can be seed tissue.
[0023] A transgenic plant is also provided. The transgenic plant
comprises any of the plant cells described above. Progeny of the
transgenic plant are also provided. The progeny have a difference
in the level of nitrogen as compared to the level of nitrogen in a
corresponding control plant that does not comprise the isolated
nucleic acid. Seed and vegetative tissue from the transgenic plant
are also provided. In addition, food products and feed products
comprising vegetative tissue from the transgenic plant are
provided.
[0024] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used to practice the invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0025] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A-1G are an alignment of SEQ ID NO:4 with orthologous
amino acid sequences SEQ ID NOs:5-7, SEQ ID NOs:9-12, SEQ ID NO:14,
and SEQ ID NOs:16-18. The consensus sequence determined by the
alignment is set forth.
DETAILED DESCRIPTION
[0027] This invention features methods and materials related to
plants, plant products, plant tissues, and plant cells having
modulated (e.g., increased or decreased) levels of nitrogen. For
example, this document provides plants and plant cells having
increased nitrogen levels as well as methods for producing such
plants and plant cells. The methods can include transforming a
plant cell with a nucleic acid encoding a nitrogen-modulating
polypeptide, wherein expression of the polypeptide results in
increased levels of nitrogen. Plants and plant cells produced using
such methods can be grown to produce seeds having increased
nitrogen levels. The seeds may be used to produce foodstuffs and
animal feed having increased nutritional (e.g., protein) content,
which may benefit both food producers and consumers.
Polypeptides
[0028] The term "polypeptide" as used herein refers to a compound
of two or more subunit amino acids, amino acid analogs, or other
peptidomimetics, regardless of post-translational modification,
e.g., phosphorylation or glycosylation. The subunits may be linked
by peptide bonds or other bonds such as, for example, ester or
ether bonds. The term "amino acid" refers to natural and/or
unnatural or synthetic amino acids, including D/L optical isomers.
Full-length proteins, analogs, mutants, and fragments thereof are
encompassed by this definition.
[0029] Described herein are nitrogen-modulating polypeptides.
Nitrogen-modulating polypeptides can be effective to modulate
nitrogen levels when expressed in a plant or plant cell. Modulation
of the level of nitrogen can be either an increase or a decrease in
the level of nitrogen relative to the corresponding level in a
control plant. A nitrogen-modulating polypeptide can be a
transporter polypeptide, such as an oligopeptide transporter
polypeptide.
[0030] A nitrogen-modulating polypeptide can be a proton-dependent
oligopeptide transport (POT) family polypeptide. POT family
polypeptides are reported to be involved in the intake of small
peptides with the concomitant uptake of a proton. SEQ ID NO:2 and
SEQ ID NO:4 set forth the amino acid sequences of Arabidopsis
clones, identified herein as Ceres cDNA ID 2998984 (SEQ ID NO:1)
and Ceres clone 117581 (SEQ ID NO:3), respectively, each of which
has a PTR2 domain characteristic of a peptide transporter
polypeptide.
[0031] A nitrogen-modulating polypeptide can comprise the amino
acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.
Alternatively, a nitrogen-modulating polypeptide can be a homolog,
ortholog, or variant of the polypeptide having the amino acid
sequence set forth in SEQ ID NO:2 or SEQ ID NO:4. For example, a
nitrogen-modulating polypeptide can have an amino acid sequence
with at least 40% sequence identity, e.g., 41%, 45%, 47%, 48%, 49%,
50%, 51%, 52%, 56%, 57%, 60%, 61%, 62%, 63%, 64%, 65%, 67%, 68%,
70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity,
to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.
In some embodiments, a nitrogen-modulating polypeptide comprises an
amino acid sequence with at least 40% sequence identity to SEQ ID
NO:2 and a chloroplast targeting signal sequence at the N-terminus
of the polypeptide.
[0032] Amino acid sequences of orthologs of the polypeptide having
the amino acid sequence set forth in SEQ ID NO:4 are provided in
FIGS. 1A-1G, along with a consensus sequence. A consensus amino
acid sequence for such orthologs was determined by aligning amino
acid sequences, e.g., amino acid sequences related to SEQ ID NO:4,
from a variety of species and determining the most common amino
acid or type of amino acid at each position. For example, the
alignment in FIG. 1 provides the amino acid sequences of Ceres
clone 117581 (SEQ ID NO:4), CeresClone:328378 (SEQ ID NO:5),
gi|2655098 (SEQ ID NO:6), gi|34895718 (SEQ ID NO:7), gi|4102839
(SEQ ID NO:9), gi|31088360 (SEQ ID NO:10), gi|6635838 (SEQ ID
NO:11), gi|56784523 (SEQ ID NO:12), gi|50059161 (SEQ ID NO:14),
CeresClone:352232 (SEQ ID NO:16), gi|33411520 (SEQ ID NO:17), and
gi|31429847 (SEQ ID NO:18). Other orthologs include gi|50933627
(SEQ ID NO:8), gi|56784524 (SEQ ID NO:13), and gi|6409176 (SEQ ID
NO:15).
[0033] In some cases, a nitrogen-modulating polypeptide can include
a polypeptide having at least 80% sequence identity (e.g., 80%,
85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity) to an amino
acid sequence corresponding to SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or the
consensus sequence set forth in FIG. 1.
[0034] It will be appreciated that a number of nucleic acids can
encode a polypeptide having a particular amino acid sequence. The
degeneracy of the genetic code is well known to the art; i.e., for
many amino acids, there is more than one nucleotide triplet that
serves as the codon for the amino acid. For example, codons in the
coding sequence for a given nitrogen-modulating polypeptide can be
modified such that optimal expression in a particular plant species
is obtained, using appropriate codon bias tables for that
species.
[0035] A nitrogen-modulating polypeptide encoded by a recombinant
nucleic acid can be a native nitrogen-modulating polypeptide, i.e.,
one or more additional copies of the coding sequence for a
nitrogen-modulating polypeptide that is naturally present in the
cell. Alternatively, a nitrogen-modulating polypeptide can be
heterologous to the cell, e.g., a transgenic Lycopersicon plant can
contain the coding sequence for a transporter polypeptide from a
Glycine plant.
[0036] A nitrogen-modulating polypeptide can include additional
amino acids that are not involved in nitrogen modulation, and thus
can be longer than would otherwise be the case. For example, a
nitrogen-modulating polypeptide can include an amino acid sequence
that functions as a reporter. Such a nitrogen-modulating
polypeptide can be a fusion protein in which a green fluorescent
protein (GFP) polypeptide is fused to SEQ ID NO:2, or in which a
yellow fluorescent protein (YFP) polypeptide is fused to SEQ ID
NO:4. In some embodiments, a nitrogen-modulating polypeptide
includes a purification tag or a leader sequence added to the amino
or carboxy terminus.
[0037] Nitrogen-modulating polypeptide candidates suitable for use
in the invention can be identified by analysis of nucleotide and
polypeptide sequence alignments. For example, performing a query on
a database of nucleotide or polypeptide sequences can identify
orthologs of nitrogen-modulating polypeptides. Sequence analysis
can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of
nonredundant databases using known nitrogen-modulating polypeptide
amino acid sequences. Those proteins in the database that have
greater than 40% sequence identity can be identified as candidates
for further evaluation for suitability as a nitrogen-modulating
polypeptide. If desired, manual inspection of such candidates can
be carried out in order to narrow the number of candidates to be
further evaluated. Manual inspection can be performed by selecting
those candidates that appear to have domains suspected of being
present in nitrogen-modulating polypeptides, e.g., conserved
functional domains.
[0038] The identification of conserved regions in a template or
subject polypeptide can facilitate production of variants of wild
type nitrogen-modulating polypeptides. Conserved regions can be
identified by locating a region within the primary amino acid
sequence of a template polypeptide that is a repeated sequence,
forms some secondary structure (e.g., helices and beta sheets),
establishes positively or negatively charged domains, or represents
a protein motif or domain. See, e.g., the Pfam web site describing
consensus sequences for a variety of protein motifs and domains at
sanger.ac.uk/Pfam and genome.wustl.edu/Pfam. A description of the
information included at the Pfam database is described in
Sonnhammer et al., 1998, Nucl. Acids Res. 26: 320-322; Sonnhammer
et al., 1997, Proteins 28:405-420; and Bateman et al., 1999, Nucl.
Acids Res. 27:260-262.
[0039] Conserved regions also can be determined by aligning
sequences of the same or related polypeptides from closely related
species. Closely related species preferably are from the same
family. In some embodiments, alignment of sequences from two
different species is adequate. For example, sequences from
Arabidopsis and Zea mays can be used to identify one or more
conserved regions.
[0040] Typically, polypeptides that exhibit at least about 40%
amino acid sequence identity are useful to identify conserved
regions. Conserved regions of related polypeptides can exhibit at
least 45% amino acid sequence identity (e.g., at least 50%, at
least 60%, at least 70%, at least 80%, or at least 90% amino acid
sequence identity). In some embodiments, a conserved region of
target and template polypeptides exhibit at least 92, 94, 96, 98,
or 99% amino acid sequence identity. Amino acid sequence identity
can be deduced from amino acid or nucleotide sequences. In certain
cases, highly conserved domains have been identified within
nitrogen-modulating polypeptides. These conserved regions can be
useful in identifying functionally similar (orthologous)
nitrogen-modulating polypeptides.
[0041] In some instances, suitable nitrogen-modulating polypeptides
can be synthesized on the basis of consensus functional domains
and/or conserved regions in polypeptides that are homologous
nitrogen-modulating polypeptides. Domains are groups of
substantially contiguous amino acids in a polypeptide that can be
used to characterize protein families and/or parts of proteins.
Such domains have a "fingerprint" or "signature" that can comprise
conserved (1) primary sequence, (2) secondary structure, and/or (3)
three-dimensional conformation. Generally, domains are correlated
with specific in vitro and/or in vivo activities. A domain can have
a length of from 10 amino acids to 400 amino acids, e.g., 10 to 50
amino acids, or 25 to 100 amino acids, or 35 to 65 amino acids, or
35 to 55 amino acids, or 45 to 60 amino acids, or 200 to 300 amino
acids, or 300 to 400 amino acids.
[0042] Consensus domains and conserved regions can be identified by
homologous polypeptide sequence analysis as described above. The
suitability of polypeptides for use as nitrogen-modulating
polypeptides can be evaluated by functional complementation
studies.
[0043] Nucleic Acids
[0044] Isolated nucleic acids are provided herein. The terms
"nucleic acid" and "polynucleotide" are used interchangeably
herein, and refer to both RNA and DNA, including cDNA, genomic DNA,
synthetic DNA, and DNA (or RNA) containing nucleic acid analogs.
Polynucleotides can have any three-dimensional structure. A nucleic
acid can be double-stranded or single-stranded (i.e., a sense
strand or an antisense strand). Non-limiting examples of
polynucleotides include genes, gene fragments, exons, introns,
messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA,
micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branched
polynucleotides, plasmids, vectors, isolated DNA of any sequence,
isolated RNA of any sequence, nucleic acid probes, and primers, as
well as nucleic acid analogs.
[0045] An isolated nucleic acid can be, for example, a
naturally-occurring DNA molecule, provided one of the nucleic acid
sequences normally found immediately flanking that DNA molecule in
a naturally-occurring genome is removed or absent. Thus, an
isolated nucleic acid includes, without limitation, a DNA molecule
that exists as a separate molecule, independent of other sequences
(e.g., a chemically synthesized nucleic acid, or a cDNA or genomic
DNA fragment produced by the polymerase chain reaction (PCR) or
restriction endonuclease treatment). An isolated nucleic acid also
refers to a DNA molecule that is incorporated into a vector, an
autonomously replicating plasmid, a virus, or into the genomic DNA
of a prokaryote or eukaryote. In addition, an isolated nucleic acid
can include an engineered nucleic acid such as a DNA molecule that
is part of a hybrid or fusion nucleic acid. A nucleic acid existing
among hundreds to millions of other nucleic acids within, for
example, cDNA libraries or genomic libraries, or gel slices
containing a genomic DNA restriction digest, is not to be
considered an isolated nucleic acid.
[0046] Isolated nucleic acid molecules can be produced by standard
techniques. For example, polymerase chain reaction (PCR) techniques
can be used to obtain an isolated nucleic acid containing a
nucleotide sequence described herein. PCR can be used to amplify
specific sequences from DNA as well as RNA, including sequences
from total genomic DNA or total cellular RNA. Various PCR methods
are described, for example, in PCR Primer: A Laboratory Manual,
Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory
Press, 1995. Generally, sequence information from the ends of the
region of interest or beyond is employed to design oligonucleotide
primers that are identical or similar in sequence to opposite
strands of the template to be amplified. Various PCR strategies
also are available by which site-specific nucleotide sequence
modifications can be introduced into a template nucleic acid.
Isolated nucleic acids also can be chemically synthesized, either
as a single nucleic acid molecule (e.g., using automated DNA
synthesis in the 3' to 5' direction using phosphoramidite
technology) or as a series of oligonucleotides. For example, one or
more pairs of long oligonucleotides (e.g., >100 nucleotides) can
be synthesized that contain the desired sequence, with each pair
containing a short segment of complementarity (e.g., about 15
nucleotides) such that a duplex is formed when the oligonucleotide
pair is annealed. DNA polymerase is used to extend the
oligonucleotides, resulting in a single, double-stranded nucleic
acid molecule per oligonucleotide pair, which then can be ligated
into a vector. Isolated nucleic acids of the invention also can be
obtained by mutagenesis of, e.g., a naturally occurring DNA.
[0047] As used herein, the term "percent sequence identity" refers
to the degree of identity between any given query sequence and a
subject sequence. A subject sequence typically has a length that is
more than 80 percent, e.g., more than 82, 85, 87, 89, 90, 93, 95,
97, 99, 100, 105, 110, 115, or 120 percent, of the length of the
query sequence. A query nucleic acid or amino acid sequence is
aligned to one or more subject nucleic acid or amino acid sequences
using the computer program ClustalW (version 1.83, default
parameters), which allows alignments of nucleic acid or protein
sequences to be carried out across their entire length (global
alignment). Chema, et al. (2003) Nucleic Acids Res 31
(13):3497-500.
[0048] ClustalW calculates the best match between a query and one
or more subject sequences, and aligns them so that identities,
similarities and differences can be determined. Gaps of one or more
residues can be inserted into a query sequence, a subject sequence,
or both, to maximize sequence alignments. For fast pairwise
alignment of nucleic acid sequences, the following default
parameters are used: word size: 2; window size: 4; scoring method:
percentage; number of top diagonals: 4; and gap penalty: 5. For
multiple alignment of nucleic acid sequences, the following
parameters are used: gap opening penalty: 10.0; gap extension
penalty: 5.0; and weight transitions: yes. For fast pairwise
alignment of protein sequences, the following parameters are used:
word size: 1; window size: 5; scoring method: percentage; number of
top diagonals: 5; gap penalty: 3. For multiple alignment of protein
sequences, the following parameters are used: weight matrix:
blosum; gap opening penalty: 10.0; gap extension penalty: 0.05;
hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn,
Asp, Gln, Glu, Arg, and Lys; residue-specific gap penalties: on.
The output is a sequence alignment that reflects the relationship
between sequences. ClustalW can be run, for example, at the Baylor
College of Medicine Search Launcher site
(searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at
the European Bioinformatics Institute site on the World Wide Web
(ebi.ac.uk/clustalw). To determine a "percent identity" between a
query sequence and a subject sequence, the number of matching bases
or amino acids in the alignment is divided by the total number of
matched and mismatched bases or amino acids, followed by
multiplying the result by 100. It is noted that the percent
identity value can be rounded to the nearest tenth. For example,
78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while
78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2. It
also is noted that the length value will always be an integer.
[0049] The term "exogenous" with respect to a nucleic acid
indicates that the nucleic acid is part of a recombinant nucleic
acid construct, or is not in its natural environment. For example,
an exogenous nucleic acid can be a sequence from one species
introduced into another species, i.e., a heterologous nucleic acid.
Typically, such an exogenous nucleic acid is introduced into the
other species via a recombinant nucleic acid construct. An
exogenous nucleic acid can also be a sequence that is native to an
organism and that has been reintroduced into cells of that
organism. An exogenous nucleic acid that includes a native sequence
can often be distinguished from the naturally occurring sequence by
the presence of non-natural sequences linked to the exogenous
nucleic acid, e.g., non-native regulatory sequences flanking a
native sequence in a recombinant nucleic acid construct. In
addition, stably transformed exogenous nucleic acids typically are
integrated at positions other than the position where the native
sequence is found. It will be appreciated that an exogenous nucleic
acid may have been introduced into a progenitor and not into the
cell under consideration. For example, a transgenic plant
containing an exogenous nucleic acid can be the progeny of a cross
between a stably transformed plant and a non-transgenic plant. Such
progeny are considered to contain the exogenous nucleic acid.
[0050] Recombinant constructs are also provided herein and can be
used to transform plants or plant cells in order to modulate
nitrogen levels. A recombinant nucleic acid construct comprises a
nucleic acid encoding a nitrogen-modulating polypeptide as
described herein, operably linked to a regulatory region suitable
for expressing the nitrogen-modulating polypeptide in the plant or
cell. Thus, a nucleic acid can comprise a nucleotide sequence that
encodes any of the nitrogen-modulating polypeptides as set forth in
SEQ ID NO:2, SEQ ID NOs:4-18, and the consensus sequence set forth
in FIG. 1.
[0051] Vectors containing nucleic acids such as those described
herein also are provided. A "vector" is a replicon, such as a
plasmid, phage, or cosmid, into which another DNA segment may be
inserted so as to bring about the replication of the inserted
segment. Generally, a vector is capable of replication when
associated with the proper control elements. Suitable vector
backbones include, for example, those routinely used in the art
such as plasmids, viruses, artificial chromosomes, BACs, YACs, or
PACs. The term "vector" includes cloning and expression vectors, as
well as viral vectors and integrating vectors. An "expression
vector" is a vector that includes a regulatory region. Suitable
expression vectors include, without limitation, plasmids and viral
vectors derived from, for example, bacteriophage, baculoviruses,
and retroviruses. Numerous vectors and expression systems are
commercially available from such corporations as Novagen (Madison,
Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.),
and Invitrogen/Life Technologies (Carlsbad, Calif.).
[0052] The vectors provided herein also can include, for example,
origins of replication, scaffold attachment regions (SARs), and/or
markers. A marker gene can confer a selectable phenotype on a plant
cell. For example, a marker can confer biocide resistance, such as
resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or
hygromycin), or an herbicide (e.g., chlorosulfuron or
phosphinothricin). In addition, an expression vector can include a
tag sequence designed to facilitate manipulation or detection
(e.g., purification or localization) of the expressed polypeptide.
Tag sequences, such as green fluorescent protein (GFP), glutathione
S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or
Flag.TM. tag (Kodak, New Haven, Conn.) sequences typically are
expressed as a fusion with the encoded polypeptide. Such tags can
be inserted anywhere within the polypeptide, including at either
the carboxyl or amino terminus.
Regulatory Regions
[0053] The term "regulatory region" refers to nucleotide sequences
that influence transcription or translation initiation and rate,
and stability and/or mobility of a transcription or translation
product. Regulatory regions include, without limitation, promoter
sequences, enhancer sequences, response elements, protein
recognition sites, inducible elements, protein binding sequences,
5' and 3' untranslated regions (UTRs), transcriptional start sites,
termination sequences, polyadenylation sequences, and introns.
[0054] As used herein, the term "operably linked" refers to
positioning of a regulatory region and a sequence to be transcribed
in a nucleic acid so as to allow or facilitate transcription of
such a sequence. For example, to bring a coding sequence under the
control of a promoter, the translation initiation site of the
translational reading frame of the polypeptide is typically
positioned between one and about fifty nucleotides downstream of
the promoter. A promoter can, however, be positioned as much as
about 5,000 nucleotides upstream of the translation initiation
site, or about 2,000 nucleotides upstream of the transcription
start site. A promoter typically comprises at least a core (basal)
promoter. A promoter also may include at least one control element,
such as an enhancer sequence, an upstream element or an upstream
activation region (UAR). For example, a suitable enhancer is a
cis-regulatory element (-212 to -154) from the upstream region of
the octopine synthase (ocs) gene. Fromm et al., The Plant Cell
1:977-984 (1989). The choice of promoters to be included depends
upon several factors, including, but not limited to, efficiency,
selectability, inducibility, desired expression level, and cell- or
tissue-preferential expression. It is a routine matter for one of
skill in the art to modulate the expression of a coding sequence by
appropriately selecting and positioning promoters and other
regulatory regions relative to the coding sequence.
[0055] Some suitable promoters initiate transcription only, or
predominantly, in certain cell types. For example, a promoter that
is active predominantly in a reproductive tissue (e.g., fruit,
ovule, pollen, pistils, female gametophyte, egg cell, central cell,
nucellus, suspensor, synergid cell, flowers, embryonic tissue,
embryo sac, embryo, zygote, endosperm, integument, or seed coat)
can be used. Thus, as used herein a cell type- or
tissue-preferential promoter is one that drives expression
preferentially in the target tissue, but may also lead to some
expression in other cell types or tissues as well. Methods for
identifying and characterizing promoter regions in plant genomic
DNA include, for example, those described in the following
references: Jordano, et al., Plant Cell, 1:855-866 (1989); Bustos,
et al., Plant Cell, 1:839-854 (1989); Green, et al., EMBO J. 7,
4035-4044 (1988); Meier, et al., Plant Cell, 3, 309-316 (1991); and
Zhang, et al., Plant Physiology 110: 1069-1079 (1996).
[0056] Examples of various classes of promoters are described
below. Some of the promoters indicated below are described in more
detail in U.S. Patent Application Ser. Nos. 60/505,689; 60/518,075;
60/544,771; 60/558,869; 60/583,691; 60/619,181; 60/637,140;
10/950,321; 10/957,569; 11/058,689; 11/172,703; 11/208,308; and
PCT/US05/23639. It will be appreciated that a promoter may meet
criteria for one classification based on its activity in one plant
species, and yet meet criteria for a different classification based
on its activity in another plant species. Nucleotide sequences of
promoters are set forth in SEQ ID NOs:19-25.
[0057] Broadly Expressing Promoters
[0058] A promoter can be said to be "broadly expressing" when it
promotes transcription in many, but not necessarily all, plant
tissues. For example, a broadly expressing promoter can promote
transcription of an operably linked sequence in one or more of the
shoot, shoot tip (apex), and leaves, but weakly or not at all in
tissues such as roots or stems. As another example, a broadly
expressing promoter can promote transcription of an operably linked
sequence in one or more of the stem, shoot, shoot tip (apex), and
leaves, but can promote transcription weakly or not at all in
tissues such as reproductive tissues of flowers and developing
seeds. Non-limiting examples of broadly expressing promoters that
can be included in the nucleic acid constructs provided herein
include the p326 (SEQ ID NO:19), YP0144 (SEQ ID NO:20), YP0190 (SEQ
ID NO:21), p13879 (SEQ ID NO:22), YP0050 (SEQ ID NO:23), p32449
(SEQ ID NO:24), 21876 (SEQ ID NO:25), YP0158, YP0214, YP0380,
PT0848, and PT0633 promoters. Additional examples include the
cauliflower mosaic virus (CaMV) 35S promoter, the mannopine
synthase (MAS) promoter, the 1' or 2' promoters derived from T-DNA
of Agrobacterium tumefaciens, the figwort mosaic virus 34S
promoter, actin promoters such as the rice actin promoter, and
ubiquitin promoters such as the maize ubiquitin-1 promoter. In some
cases, the CaMV 35S promoter is excluded from the category of
broadly expressing promoters.
[0059] Root Promoters
[0060] Root-active promoters confer transcription in root tissue,
e.g., root endodermis, root epidermis, or root vascular tissues. In
some embodiments, root-active promoters are root-preferential
promoters, i.e., confer transcription only or predominantly in root
tissue. Root-preferential promoters include the YP0128, YP0275,
PT0625, PT0660, PT0683, and PT0758 promoters. Other
root-preferential promoters include the PT0613, PT0672, PT0678,
PT0688, and PT0837 promoters, which drive transcription primarily
in root tissue and to a lesser extent in ovules and/or seeds. Other
examples of root-preferential promoters include the root-specific
subdomains of the CaMV 35S promoter (Lam et al., Proc. Natl. Acad.
Sci. USA 86:7890-7894 (1989)), root cell specific promoters
reported by Conkling et al., Plant Physiol. 93:1203-1211 (1990),
and the tobacco RD2 gene promoter.
[0061] Maturing Endosperm Promoters
[0062] In some embodiments, promoters that drive transcription in
maturing endosperm can be useful. Transcription from a maturing
endosperm promoter typically begins after fertilization and occurs
primarily in endosperm tissue during seed development and is
typically highest during the cellularization phase. Most suitable
are promoters that are active predominantly in maturing endosperm,
although promoters that are also active in other tissues can
sometimes be used. Non-limiting examples of maturing endosperm
promoters that can be included in the nucleic acid constructs
provided herein include the napin promoter, the Arcelin-5 promoter,
the phaseolin gene promoter (Bustos et al., Plant Cell 1(9):839-853
(1989)), the soybean trypsin inhibitor promoter (Riggs et al.,
Plant Cell 1(6):609-621 (1989)), the ACP promoter (Baerson et al.,
Plant Mol Biol, 22(2):255-267 (1993)), the stearoyl-ACP desaturase
gene (Slocombe et al., Plant Physiol 104(4):167-176 (1994)), the
soybean .alpha. subunit of .beta.-conglycinin promoter (Chen et
al., Proc Natl Acad Sci USA 83:8560-8564 (1986)), the oleosin
promoter (Hong et al., Plant Mol Biol 34(3):549-555 (1997)), and
zein promoters, such as the 15 kD zein promoter, the 16 kD zein
promoter, 19 kD zein promoter, 22 kD zein promoter and 27 kD zein
promoter. Also suitable are the Osgt-1 promoter from the rice
glutelin-1 gene (Zheng et al., Mol. Cell. Biol. 13:5829-5842
(1993)), the beta-amylase gene promoter, and the barley hordein
gene promoter. Other maturing endosperm promoters include the
YP0092, PT0676, and PT0708 promoters.
[0063] Ovary Tissue Promoters
[0064] Promoters that are active in ovary tissues such as the ovule
wall and mesocarp can also be useful, e.g., a polygalacturonidase
promoter, the banana TRX promoter, and the melon actin
promoter.
[0065] Embryo Sac/Early Endosperm Promoters
[0066] To achieve expression in embryo sac/early endosperm,
regulatory regions can be used that are active in polar nuclei
and/or the central cell, or in precursors to polar nuclei, but not
in egg cells or precursors to egg cells. Most suitable are
promoters that drive expression only or predominantly in polar
nuclei or precursors thereto and/or the central cell. A pattern of
transcription that extends from polar nuclei into early endosperm
development can also be found with embryo sac/early
endosperm-preferential promoters, although transcription typically
decreases significantly in later endosperm development during and
after the cellularization phase. Expression in the zygote or
developing embryo typically is not present with embryo sac/early
endosperm promoters.
[0067] Promoters that may be suitable include those derived from
the following genes; Arabidopsis viviparous-1 (see, GenBank No.
U93215); Arabidopsis atmycl (see, Urao (1996) Plant Mol. Biol.,
32:571-57; Conceicao (1994) Plant, 5:493-505); Arabidopsis F1E
(GenBank No. AF129516); Arabidopsis MEA; Arabidopsis FIS2 (GenBank
No. AF096096); and F1E 1.1 (U.S. Pat. No. 6,906,244). Other
promoters that may be suitable include those derived from the
following genes: maize MAC1 (see, Sheridan (1996) Genetics,
142:1009-1020); maize Cat3 (see, GenBank No. L05934; Abler (1993)
Plant Mol. Biol., 22:10131-1038). Other promoters include the
following Arabidopsis promoters: YP0039, YP0101, YP1002, YP0110,
YP0117, YP0119, YP0137, DME, YP0285, and YP0212. Other promoters
that may be useful include the following rice promoters: p530c10,
pOsFIE2-2, pOsMEA, pOsYp102, and pOsYp285.
[0068] Embryo Promoters
[0069] Regulatory regions that preferentially drive transcription
in zygotic cells following fertilization can provide
embryo-preferential expression. Most suitable are promoters that
preferentially drive transcription in early stage embryos prior to
the heart stage, but expression in late stage and maturing embryos
is also suitable. Embryo-preferential promoters include the barley
lipid transfer protein (Ltp1) promoter (Plant Cell Rep (2001)
20:647-654).
[0070] Photosynthetically Active Tissue Promoters
[0071] Photosynthetically active tissue promoters confer
transcription in photosynthetically active tissue such as leaves
and stems. Most suitable are promoters that drive expression only
or predominantly such tissues. Examples of such promoters include
the ribulose-1,5-bisphosphate carboxylase (RbcS) promoters such as
the RbcS promoter from eastern larch (Larix laricina), the pine
cab6 promoter (Yamamoto et al., Plant Cell Physiol. 35:773-778
(1994)), the Cab-1 gene promoter from wheat (Fejes et al., Plant
Mol. Biol. 15:921-932 (1990)), the CAB-1 promoter from spinach
(Lubberstedt et al., Plant Physiol. 104:997-1006 (1994)), the cab1R
promoter from rice (Luan et al., Plant Cell 4:971-981 (1992)), the
pyruvate orthophosphate dikinase (PPDK) promoter from corn
(Matsuoka et al., Proc Natl Acad. Sci. USA 90:9586-9590 (1993)),
the tobacco Lhcb1*2 promoter (Cerdan et al., Plant Mol. Biol.
33:245-255 (1997)), the Arabidopsis thaliana SUC2 sucrose-H+
symporter promoter (Truemit et al., Planta 196:564-570 (1995)), and
thylakoid membrane protein promoters from spinach (psaD, psaF,
psae, PC, FNR, atpC, atpD, cab, rbcS).
[0072] Inducible Promoters
[0073] Inducible promoters confer transcription in response to
external stimuli such as chemical agents or environmental stimuli.
For example, inducible promoters can confer transcription in
response to hormones such as giberellic acid or ethylene, or in
response to light or drought.
[0074] Basal Promoters
[0075] A basal promoter is the minimal sequence necessary for
assembly of a transcription complex required for transcription
initiation. Basal promoters frequently include a "TATA box" element
that may be located between about 15 and about 35 nucleotides
upstream from the site of transcription initiation. Basal promoters
also may include a "CCAAT box" element (typically the sequence
CCAAT) and/or a GGGCG sequence, which can be located between about
40 and about 200 nucleotides, typically about 60 to about 120
nucleotides, upstream from the transcription start site.
[0076] Other Promoters
[0077] Other classes of promoters include, but are not limited to,
leaf-preferential, stem/shoot-preferential, callus-preferential,
and senescence-preferential promoters. Promoters designated YP0086,
YP0188, YP0263, PT0758, PT0743, PT0829, YP0119, and YP0096, as
described in the above-referenced patent applications, may also be
useful.
[0078] Other Regulatory Regions
[0079] A 5' untranslated region (UTR) can be included in nucleic
acid constructs described herein. A 5' UTR is transcribed, but is
not translated, and lies between the start site of the transcript
and the translation initiation codon and may include the +1
nucleotide. A 3' UTR can be positioned between the translation
termination codon and the end of the transcript. UTRs can have
particular functions such as increasing mRNA stability or
attenuating translation. Examples of 3' UTRs include, but are not
limited to, polyadenylation signals and transcription termination
sequences, e.g., a nopaline synthase termination sequence.
[0080] It will be understood that more than one regulatory region
may be present in a recombinant polynucleotide, e.g., introns,
enhancers, upstream activation regions, transcription terminators,
and inducible elements. Thus, more than one regulatory region can
be operably linked to the sequence of a polynucleotide encoding a
carbon-modulating polypeptide.
Transgenic Plants and Plant Cells
[0081] The invention also features transgenic plant cells and
plants comprising at least one recombinant nucleic acid construct
described herein. A plant or plant cell can be transformed by
having a construct integrated into its genome, i.e., can be stably
transformed. Stably transformed cells typically retain the
introduced nucleic acid with each cell division. A plant or plant
cell can also be transiently transformed such that the construct is
not integrated into its genome. Transiently transformed cells
typically lose all or some portion of the introduced nucleic acid
construct with each cell division such that the introduced nucleic
acid cannot be detected in daughter cells after a sufficient number
of cell divisions. Both transiently transformed and stably
transformed transgenic plants and plant cells can be useful in the
methods described herein.
[0082] Transgenic plant cells used in methods described herein can
constitute part or all of a whole plant. Such plants can be grown
in a manner suitable for the species under consideration, either in
a growth chamber, a greenhouse, or in a field. Transgenic plants
can be bred as desired for a particular purpose, e.g., to introduce
a recombinant nucleic acid into other lines, to transfer a
recombinant nucleic acid to other species, or for further selection
of other desirable traits. Alternatively, transgenic plants can be
propagated vegetatively for those species amenable to such
techniques. Progeny include descendants of a particular plant or
plant line. Progeny of an instant plant include seeds formed on
F.sub.1, F.sub.2, F.sub.3, F.sub.4, F.sub.5, F.sub.6 and subsequent
generation plants, or seeds formed on BC.sub.1, BC.sub.2, BC.sub.3,
and subsequent generation plants, or seeds formed on
F.sub.1BC.sub.1, F.sub.1BC.sub.2, F.sub.1BC.sub.3, and subsequent
generation plants. The designation F.sub.1 refers to the progeny of
a cross between two parents that are genetically distinct. The
designations F.sub.2, F.sub.3, F.sub.4, F.sub.5 and F.sub.6 refer
to subsequent generations of self- or sib-pollinated progeny of an
F.sub.1 plant. Seeds produced by a transgenic plant can be grown
and then selfed (or outcrossed and selfed) to obtain seeds
homozygous for the nucleic acid construct.
[0083] Transgenic plants can be grown in suspension culture, or
tissue or organ culture. For the purposes of this invention, solid
and/or liquid tissue culture techniques can be used. When using
solid medium, transgenic plant cells can be placed directly onto
the medium or can be placed onto a filter that is then placed in
contact with the medium. When using liquid medium, transgenic plant
cells can be placed onto a flotation device, e.g., a porous
membrane that contacts the liquid medium. Solid medium typically is
made from liquid medium by adding agar. For example, a solid medium
can be Murashige and Skoog (MS) medium containing agar and a
suitable concentration of an auxin, e.g., 2,4-dichlorophenoxyacetic
acid (2,4-D), and a suitable concentration of a cytokinin, e.g.,
kinetin.
[0084] When transiently transformed plant cells are used, a
reporter sequence encoding a reporter polypeptide having a reporter
activity can be included in the transformation procedure and an
assay for reporter activity or expression can be performed at a
suitable time after transformation. A suitable time for conducting
the assay typically is about 1-21 days after transformation, e.g.,
about 1-14 days, about 1-7 days, or about 1-3 days. The use of
transient assays is particularly convenient for rapid analysis in
different species, or to confirm expression of a heterologous
nitrogen-modulating polypeptide whose expression has not previously
been confirmed in particular recipient cells.
[0085] Techniques for introducing nucleic acids into
monocotyledonous and dicotyledonous plants are known in the art,
and include, without limitation, Agrobacterium-mediated
transformation, viral vector-mediated transformation,
electroporation and particle gun transformation, e.g., U.S. Pat.
Nos. 5,538,880; 5,204,253; 6,329,571 and 6,013,863. If a cell or
cultured tissue is used as the recipient tissue for transformation,
plants can be regenerated from transformed cultures if desired, by
techniques known to those skilled in the art.
Plant Species
[0086] The polynucleotides and vectors described herein can be used
to transform a number of monocotyledonous and dicotyledonous plants
and plant cell systems, including dicots such as alfalfa, amaranth,
apple, beans (including kidney beans, lima beans, dry beans, green
beans), broccoli, cabbage, carrot, castor bean, cherry, chick peas,
chicory, clover, cocoa, coffee, cotton, crambe, flax, grape,
grapefruit, lemon, lentils, lettuce, linseed, mango, melon (e.g.,
watermelon, cantaloupe), mustard, orange, peach, peanut, pear,
peas, pepper, plum, potato, oilseed rape, rapeseed (high erucic
acid and canola), safflower, sesame, soybean, spinach, strawberry,
sugar beet, sunflower, sweet potatoes, tea, tomato, and yams, as
well as monocots such as banana, barley, bluegrass, date palm,
fescue, field corn, garlic, millet, oat, oil palm, onion,
pineapple, popcorn, rice, rye, ryegrass, sorghum, sudangrass,
sugarcane, sweet corn, switchgrass, timothy, and wheat. Brown
seaweeds, green seaweeds, red seaweeds, and microalgae can also be
used.
[0087] Thus, the methods and compositions described herein can be
used with dicotyledonous plants belonging, for example, to the
orders Apiales, Arecales, Aristochiales, Asterales, Batales,
Campanulales, Capparales, Caryophyllales, Casuarinales,
Celastrales, Cornales, Cucurbitales, Diapensales, Dilleniales,
Dipsacales, Ebenales, Ericales, Eucomiales, Euphorbiales, Fabales,
Fagales, Gentianales, Geraniales, Haloragales, Hamamelidales,
Illiciales, Juglandales, Lamiales, Laurales, Lecythidales,
Leitneriales, Linales, Magniolales, Malvales, Myricales, Myrtales,
Nymphaeales, Papaverales, Piperales, Plantaginales, Plumbaginales,
Podostemales, Polemoniales, Potvgalales, Polygonales, Primulales,
Proteales, Rafflesiales, Ranunculales, Rhamnales, Rosales,
Rubiales, Salicales, Santales, Sapindales, Sarraceniaceae,
Scrophulariales, Solanales, Trochodendrales, Theales, Umbellales,
Urticales, and Violales. The methods and compositions described
herein also can be utilized with monocotyledonous plants such as
those belonging to the orders Alismatales, Arales, Arecales,
Asparagales, Bromeliales, Commelinales, Cyclanthales, Cyperales,
Eriocaulales, Hydrocharitales, Juncales, Liliales, Najadales,
Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales,
Zingiberales, and with plants belonging to Gymnospermae, e.g.,
Cycadales, Ginkgoales, Gnetales, and Pinales.
[0088] The methods and compositions can be used over a broad range
of plant species, including species from the dicot genera
Amaranthus, Arachis, Brassica, Calendula, Camellia, Capsicum,
Carthamus, Cicer, Cichorium, Cinnamomum, Citrus, Citrullus, Coffea,
Crambe, Cucumis, Cucurbita, Daucus, Dioscorea, Fragaria, Glycine,
Gossypium, Helianthus, Lactuca, Lens, Linum, Lycopersicon, Malus,
Mangifera, Medicago, Mentha, Nicotiana, Ocimum, Olea, Phaseolus,
Pistacia, Pisum, Prunus, Pyrus, Rosmarinus, Salvia, Sesamum,
Solanum, Spinacia, Theobroma, Thymus, Trifolium, Vaccinium, Vigna,
and Vitis; and the monocot genera Allium, Ananas, Asparagus, Avena,
Curcuma, Elaeis, Festuca, Hordeum, Lemna, Lolium, Musa, Oryza,
Panicum, Pennisetum, Phleum, Poa, Saccharum, Secale, Sorghum,
Triticosecale, Triticum, and Zea.
[0089] The methods and compositions described herein also can be
used with brown seaweeds, e.g., Ascophyllum nodosum, Fucus
vesiculosus, Fucus serratus, Himanthalia elongata, and Undaria
pinnatifida; red seaweeds, e.g., Chondrus crispus, Cracilaria
verrucosa, Porphyra umbilicalis, and Palmaria paimata; green
seaweeds, e.g., Enteromorpha spp. and Ulva spp.; and microalgae,
e.g., Spirulina spp. (S. platensis and S. maxima) and Odontella
aurita. In addition, the methods and compositions can be used with
Crypthecodinium cohnii, Schizochytriuni spp., and Haeniatococcus
pluvialis.
[0090] In some embodiments, a plant is a member of the species
Ananus comosus, Brassica campestris, Brassica napus, Brassica
oleracea, Glycine max, Gossypium spp., Lactuca sativa, Lycopersicon
esculentum, Musa paradisiaca, Oryza sativa, Solanum tuberosum,
Triticum aestivum, Vitis vinifera, or Zea mays.
Methods of Inhibiting Expression of Nitrogen-Modulating
Polypeptides
[0091] The polynucleotides and recombinant vectors described herein
can be used to express or inhibit expression of a
nitrogen-modulating polypeptide in a plant species of interest. The
term "expression" refers to the process of converting genetic
information of a polynucleotide into RNA through transcription,
which is catalyzed by an enzyme, RNA polymerase, and into protein,
through translation of mRNA on ribosomes. "Up-regulation" or
"activation" refers to regulation that increases the production of
expression products (mRNA, polypeptide, or both) relative to basal
or native states, while "down-regulation" or "repression" refers to
regulation that decreases production of expression products (mRNA,
polypeptide, or both) relative to basal or native states.
[0092] A number of nucleic-acid based methods, including anti-sense
RNA, ribozyme directed RNA cleavage, and interfering RNA (RNAi) can
be used to inhibit protein expression in plants. Antisense
technology is one well-known method. In this method, a nucleic acid
segment from the endogenous gene is cloned and operably linked to a
promoter so that the antisense strand of RNA is transcribed. The
recombinant vector is then transformed into plants, as described
above, and the antisense strand of RNA is produced. The nucleic
acid segment need not be the entire sequence of the endogenous gene
to be repressed, but typically will be substantially identical to
at least a portion of the endogenous gene to be repressed.
Generally, higher homology can be used to compensate for the use of
a shorter sequence. Typically, a sequence of at least 30
nucleotides is used (e.g., at least 40, 50, 80, 100, 200, 500
nucleotides or more).
[0093] Thus, for example, an isolated nucleic acid provided herein
can be an antisense nucleic acid to one of the aforementioned
nucleic acids encoding a nitrogen-modulating polypeptide, e.g., SEQ
ID NO:2, SEQ ID NOs:4-18, or the consensus sequence set forth in
FIG. 1. A nucleic acid that decreases the level of a transcription
or translation product of a gene encoding a nitrogen-modulating
polypeptide is transcribed into an antisense nucleic acid similar
or identical to the sense coding sequence of the
nitrogen-modulating polypeptide. Alternatively, the transcription
product of an isolated nucleic acid can be similar or identical to
the sense coding sequence of a nitrogen-modulating polypeptide, but
is an RNA that is unpolyadenylated, lacks a 5' cap structure, or
contains an unsplicable intron.
[0094] In another method, a nucleic acid can be transcribed into a
ribozyme, or catalytic RNA, that affects expression of an mRNA.
(See, U.S. Pat. No. 6,423,885). Ribozymes can be designed to
specifically pair with virtually any target RNA and cleave the
phosphodiester backbone at a specific location, thereby
functionally inactivating the target RNA. Heterologous nucleic
acids can encode ribozymes designed to cleave particular mRNA
transcripts, thus preventing expression of a polypeptide.
Hammerhead ribozymes are useful for destroying particular mRNAs,
although various ribozymes that cleave mRNA at site-specific
recognition sequences can be used. Hammerhead ribozymes cleave
mRNAs at locations dictated by flanking regions that form
complementary base pairs with the target mRNA. The sole requirement
is that the target RNA contain a 5'-UG-3' nucleotide sequence. The
construction and production of hammerhead ribozymes is known in the
art. See, for example, U.S. Pat. No. 5,254,678 and WO 02/46449 and
references cited therein. Hammerhead ribozyme sequences can be
embedded in a stable RNA such as a transfer RNA (tRNA) to increase
cleavage efficiency in vivo. Perriman, et al., Proc. Natl. Acad.
Sci. USA, 92(13):6175-6179 (1995); de Feyter and Gaudron, Methods
in Molecular Biology, Vol. 74, Chapter 43, "Expressing Ribozymes in
Plants", Edited by Turner, P. C, Humana Press Inc., Totowa, N.J.
RNA endoribonucleases such as the one that occurs naturally in
Tetrahymena thermophila, and which have been described extensively
by Cech and collaborators can be useful. See, for example, U.S.
Pat. No. 4,987,071.
[0095] Methods based on RNA interference (RNAi) can be used. RNA
interference is a cellular mechanism to regulate the expression of
genes and the replication of viruses. This mechanism is thought to
be mediated by double-stranded small interfering RNA molecules. A
cell responds to such a double-stranded RNA by destroying
endogenous mRNA having the same sequence as the double-stranded
RNA. Methods for designing and preparing interfering RNAs are known
to those of skill in the art; see, e.g., WO 99/32619 and WO
01/75164. For example, a construct can be prepared that includes a
sequence that is transcribed into an interfering RNA. Such an RNA
can be one that can anneal to itself, e.g., a double stranded RNA
having a stem-loop structure. One strand of the stem portion of a
double stranded RNA comprises a sequence that is similar or
identical to the sense coding sequence of the polypeptide of
interest, and that is from about 10 nucleotides to about 2,500
nucleotides in length. The length of the sequence that is similar
or identical to the sense coding sequence can be from 10
nucleotides to 500 nucleotides, from 15 nucleotides to 300
nucleotides, from 20 nucleotides to 100 nucleotides, or from 25
nucleotides to 100 nucleotides. The other strand of the stem
portion of a double stranded RNA comprises an antisense sequence of
the nitrogen-modulating polypeptide of interest, and can have a
length that is shorter, the same as, or longer than the
corresponding length of the sense sequence. The loop portion of a
double stranded RNA can be from 10 nucleotides to 5,000
nucleotides, e.g., from 15 nucleotides to 1,000 nucleotides, from
20 nucleotides to 500 nucleotides, or from 25 nucleotides to 200
nucleotides. The loop portion of the RNA can include an intron.
See, e.g., WO 99/53050.
[0096] In some nucleic-acid based methods for inhibition of gene
expression in plants, a suitable nucleic acid can be a nucleic acid
analog. Nucleic acid analogs can be modified at the base moiety,
sugar moiety, or phosphate backbone to improve, for example,
stability, hybridization, or solubility of the nucleic acid.
Modifications at the base moiety include deoxyuridine for
deoxythymidine, and 5-methyl-2'-deoxycytidine and
5-bromo-2'-deoxycytidine for deoxycytidine. Modifications of the
sugar moiety include modification of the 2' hydroxyl of the ribose
sugar to form 2'-O-methyl or 2'-O-allyl sugars. The deoxyribose
phosphate backbone can be modified to produce morpholino nucleic
acids, in which each base moiety is linked to a six-membered
morpholino ring, or peptide nucleic acids, in which the
deoxyphosphate backbone is replaced by a pseudopeptide backbone and
the four bases are retained. See, for example, Summerton and
Weller, 1997, Antisense Nucleic Acid Drug Dev., 7:187-195; Hyrup et
al., 1996, Bioorgan. Med. Chem., 4: 5-23. In addition, the
deoxyphosphate backbone can be replaced with, for example, a
phosphorothioate or phosphorodithioate backbone, a
phosphoroamidite, or an alkyl phosphotriester backbone.
Transgenic Plant Phenotypes
[0097] A transformed cell, callus, tissue, or plant can be
identified and isolated by selecting or screening the engineered
plant material for particular traits or activities, e.g., those
encoded by marker genes or antibiotic resistance genes. Such
screening and selection methodologies are well known to those
having ordinary skill in the art. In addition, physical and
biochemical methods can be used to identify transformants. These
include Southern analysis or PCR amplification for detection of a
polynucleotide; Northern blots, S1 RNase protection,
primer-extension, or RT-PCR amplification for detecting RNA
transcripts; enzymatic assays for detecting enzyme or ribozyme
activity of polypeptides and polynucleotides; and protein gel
electrophoresis, Western blots, immunoprecipitation, and
enzyme-linked immunoassays to detect polypeptides. Other techniques
such as in situ hybridization, enzyme staining, and immunostaining
also can be used to detect the presence or expression of
polypeptides and/or polynucleotides. Methods for performing all of
the referenced techniques are well known.
[0098] Transgenic plants can have an altered phenotype as compared
to a corresponding control plant that either lacks the transgene or
does not express the transgene. A polypeptide can affect the
phenotype of a plant (e.g., a transgenic plant) when expressed in
the plant, e.g., at the appropriate time(s), in the appropriate
tissue(s), or at the appropriate expression levels. Phenotypic
effects can be evaluated relative to a control plant that does not
express the exogenous polynucleotide of interest, such as a
corresponding wild type plant, a corresponding plant that is not
transgenic for the exogenous polynucleotide of interest but
otherwise is of the same genetic background as the transgenic plant
of interest, or a corresponding plant of the same genetic
background in which expression of the polypeptide is suppressed,
inhibited, or not induced (e.g., where expression is under the
control of an inducible promoter). A plant can be said "not to
express" a polypeptide when the plant exhibits less than 10%, e.g.,
less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or
0.001%, of the amount of polypeptide or mRNA encoding the
polypeptide exhibited by the plant of interest. Expression can be
evaluated using methods including, for example, RT-PCR, Northern
blots, S1 RNase protection, primer extensions, Western blots,
protein gel electrophoresis, immunoprecipitation, enzyme-linked
immunoassays, chip assays, and mass spectrometry. It should be
noted that if a polypeptide is expressed under the control of a
tissue-specific or broadly expressing promoter, expression can be
evaluated in the entire plant or in a selected tissue. Similarly,
if a polypeptide is expressed at a particular time, e.g., at a
particular time in development or upon induction, expression can be
evaluated selectively at a desired time period.
[0099] In some embodiments, a plant in which expression of a
nitrogen-modulating polypeptide is modulated can have increased
levels of seed nitrogen. For example, a nitrogen-modulating
polypeptide described herein can be expressed in a transgenic
plant, resulting in increased levels of seed nitrogen. The seed
nitrogen level can be increased by at least 5 percent, e.g., 5, 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, or more than 100 percent, as compared to the seed nitrogen
level in a corresponding control plant that does not express the
transgene. In some embodiments, a plant in which expression of a
nitrogen-modulating polypeptide is modulated can have decreased
levels of seed nitrogen. The seed nitrogen level can be decreased
by at least 5 percent, e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
or more than 50 percent, as compared to the seed nitrogen level in
a corresponding control plant that does not express the
transgene.
[0100] Plants for which modulation of levels of seed nitrogen can
be useful include, without limitation, alfalfa, lettuce, carrot,
onion, broccoli, tomato, potato, sugarcane, grape, cotton, canola,
sweet corn, popcorn, field corn, peas, beans, safflower, soybean,
coffee, amaranth, rapeseed, peanut, sunflower, oil palm, wheat,
rye, barley, oat, rice, millet, strawberry, pineapple, melon,
peach, pear, apple, cherry, orange, lemon, grapefruit, plum, mango,
banana, fescue, ryegrass, bluegrass, clover, timothy, sudangrass,
switchgrass and sorghum. Increases in seed nitrogen in such plants
can provide improved nutritional content in geographic locales
where dietary intake of protein/amino acid is often insufficient.
Decreases in seed nitrogen in such plants can be useful in
situations where seeds are not the primary plant part that is
harvested for human or animal consumption.
[0101] In some embodiments, a plant in which expression of a
nitrogen-modulating polypeptide is modulated can have increased or
decreased levels of nitrogen in one or more non-seed tissues, e.g.,
leaf tissues, stem tissues, root or corm tissues, or fruit tissues
other than seed. For example, the nitrogen level can be increased
by at least 5 percent, e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100 percent,
as compared to the nitrogen level in a corresponding control plant
that does not express the transgene. In some embodiments, a plant
in which expression of a nitrogen-modulating polypeptide is
modulated can have decreased levels of nitrogen in one or more
non-seed tissues. The nitrogen level can be decreased by at least 5
percent, e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than
50 percent as compared to the nitrogen level in a corresponding
control plant that does not express the transgene.
[0102] Plants for which modulation of levels of nitrogen in
non-seed tissues can be useful include, without limitation,
alfalfa, lettuce, carrot, onion, broccoli, tomato, potato,
sugarcane, grape, sweet corn, popcorn, field corn, peas, beans,
safflower, soybean, coffee, amaranth, rapeseed, peanut, sunflower,
oil palm, wheat, rye, barley, oat, rice, millet, strawberry,
pineapple, melon, peach, pear, apple, cherry, orange, lemon,
grapefruit, plum, mango, banana, fescue, ryegrass, bluegrass,
clover, timothy, sudangrass, switchgrass and sorghum.
[0103] Increases in non-seed nitrogen in such plants can provide
improved nutritional content in edible fruits and vegetables, or
improved animal forage. Decreases in non-seed nitrogen can provide
more efficient partitioning of nitrogen to plant part(s) that are
harvested for human or animal consumption.
[0104] Typically, a difference (e.g., an increase) in the amount of
nitrogen in a transgenic plant or cell relative to a control plant
or cell is considered statistically significant at p<0.05 with
an appropriate parametric or non-parametric statistic, e.g.,
Chi-square test, Student's t-test, Mann-Whitney test, or F-test. In
some embodiments, a difference in the amount of nitrogen is
statistically significant at p<0.01, p<0.005, or p<0.001.
A statistically significant difference in, for example, the amount
of nitrogen in seeds of a transgenic plant compared to the amount
in cells of a control plant indicates that (1) the recombinant
nucleic acid present in the transgenic plant results in altered
nitrogen levels and/or (2) the recombinant nucleic acid warrants
further study as a candidate for altering the amount of nitrogen in
a plant.
Articles of Manufacture
[0105] Also provided herein are articles of manufacture that can
include, for example, a mixture of seeds (e.g., a substantially
uniform mixture of seeds) from the transgenic plants provided
herein. The seed mixture can be conditioned and packaged using
means known in the art to prepare an article of manufacture. A
package of seed can have a label e.g., a tag or label secured to
the packaging material, a label printed on the packaging material
or a label inserted within the package. The label can indicate that
plants grown from the seeds contained within the package can
produce a crop having a higher level of seed nitrogen relative to
corresponding control plants.
[0106] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Transgenic Plants
[0107] The following symbols are used in the Examples: T.sub.1:
first generation transformant; T.sub.2: second generation, progeny
of self-pollinated T.sub.1 plants; T.sub.3: third generation,
progeny of self-pollinated T.sub.2 plants; T.sub.4: fourth
generation, progeny of self-pollinated T.sub.3 plants. Independent
transformations are referred to as events.
[0108] The following nucleic acids were isolated from Arabidopsis
thaliana ecotype Wassilewskija (Ws) plants. Ceres cDNA ID 2998984
(SEQ ID NO:1) is a genomic DNA clone that is predicted to encode a
505 amino acid (SEQ ID NO:2) putative oligopeptide transporter
polypeptide. Ceres clone 117581 (cDNA ID 23364185; SEQ ID NO:3) is
a cDNA clone that is predicted to encode a 587 amino acid (SEQ ID
NO:4) putative peptide transporter polypeptide.
[0109] Each isolated polynucleotide described above was cloned into
a vector containing a phosphinothricin acetyltransferase gene,
which confers Finale.TM. resistance to transformed plants. NB42-35S
binary vectors were constructed that contained Ceres cDNA ID
2998984 or Ceres clone 117581 operably linked to the cauliflower
mosaic virus (CaMV) 35S regulatory region. The NB42-35S binary
vector is a derivative of the pMOG800 binary vector.
[0110] Wild-type Arabidopsis thaliana ecotype C24 plants were
transformed separately with each NB42-35S binary vector containing
Ceres cDNA ID 2998984 or Ceres clone 117581. The transformations
were performed essentially as described in Bechtold et al., C.R.
Acad. Sci. Paris, 316:1194-1199 (1993).
[0111] Transgenic Arabidopsis lines containing Ceres cDNA ID
2998984 or Ceres clone 117581 were designated SR00829 and SR05001,
respectively. The presence of the Ceres cDNA ID 2998984 vector in
SR00829 and the Ceres clone 117581 vector in SR05001 was confirmed
by Finale.TM. resistance, polymerase chain reaction (PCR)
amplification from green leaf tissue extract, and sequencing of PCR
products. As controls for transgenic Arabidopsis ecotype C24
plants, wild-type Arabidopsis ecotype C24 plants were transformed
with the empty vector NB42-35S.
[0112] The in planta nucleotide sequences of Ceres cDNA ID 2998984
and Ceres clone 117581 were compared to the homologous Arabidopsis
ecotype Columbia sequences. The in planta nucleotide sequence of
Ceres clone 117581 differed from the homologous Columbia sequence
by one nucleotide. The in planta nucleotide sequence of Ceres cDNA
ID 2998984 contained base pair insertions, deletions, and
substitutions compared to the homologous Columbia sequence.
[0113] To determine if the overall structure of the predicted
polypeptide encoded by Ceres cDNA ID 2998984 was similar to that of
the predicted polypeptide encoded by the homologous Columbia
nucleotide sequence, both sequences were analyzed for potential
transmembrane domains characteristic of transporter polypeptides
using the TMpred program (ch.embnet.org/software/TMPRED_form). The
analysis indicated that both predicted polypeptides had the
expected 12 transmembrane spanning regions. However, prediction of
the translation initiation sites indicated that the predicted
polypeptide encoded by Ceres cDNA ID 2998984 was 40 amino acids
shorter at the amino terminus than the predicted polypeptide
encoded by the Columbia homolog. Therefore, the predicted
polypeptide encoded by Ceres cDNA ID 2998984 lacked the secretory
signal peptide sequence contained in the first 40 amino acids of
the predicted Columbia polypeptide sequence according to the
SignalP algorithm (cbs.dtu.dk/services/SignalP/). Analysis of the
predicted polypeptide sequence encoded by Ceres cDNA ID 2998984
using the iPSORT signal sequence prediction algorithm
(hc.ims.u-tokyo.ac.jp/iPSORT/index.html#aaindex) indicated that the
first 40 amino acids contained a potential chloroplast targeting
signal sequence.
[0114] Transgenic Arabidopsis lines were screened as follows: 1)
T.sub.1 candidates in the greenhouse were screened for
morphological phenotypes, 2) T.sub.2 seeds were analyzed for carbon
and nitrogen content, 3) increased carbon and/or nitrogen content
was confirmed in T.sub.3 seeds, and 4) T.sub.2 plants were
evaluated for negative phenotypes and Finale.TM. segregation.
[0115] Five events of each of SR00829 and SR05001 were screened for
visible phenotypic alterations in the T.sub.1 generation. The
physical appearance of all of the T.sub.1 plants was identical to
that of the corresponding control plants.
Example 2
Analysis of Carbon and Nitrogen Content in Transgenic Arabidopsis
Seeds
[0116] Approximately 2.00.+-.0.15 mg of dried transgenic
Arabidopsis seeds (about 100 seeds) were weighed into a tin cup and
analyzed for total carbon and nitrogen content.
[0117] Three matched controls were prepared in a manner identical
to the experimental samples and spaced evenly throughout the batch.
The first three samples in every batch were a blank (empty tin
cup), bypass, (approximately 5 mg of aspartic acid), and a standard
(5.00.+-.0.15 mg aspartic acid), respectively. Aspartic acid was
weighed into a tin cup using an analytical balance. Blanks were
entered between every 15 experimental samples.
[0118] Analysis was completed using a FlashEA 1112 NC Analyzer
(Thermo Finnigan, San Jose, Calif.). The instrument parameters were
as follows: left furnace 900.degree. C., right furnace 840.degree.
C., oven 50.degree. C., gas flow carrier 130 mL/min., and gas flow
reference 100 mL/min. The data parameter LLOD was 0.25 mg for the
standard and different for other materials. The data parameter LLOQ
was 3 mg for the standard, 1 mg for seed tissue, and different for
other materials.
[0119] Quantification was performed using EA 1112 software. The
results were normalized and expressed in absolute percentages. Each
sample was analyzed in triplicate, and the standard deviation was
calculated. Non-transgenic controls were previously determined to
have a total carbon content of 53.3.+-.2.4% and a total nitrogen
content of 3.9.+-.0.3%. The deviation from theoretical of the
aspartic acid standard was .+-.2.0% for carbon and .about.1.0% for
nitrogen. To be declared valid, each run was required to have an
aspartic acid (standard) weight of 5 mg.+-.0.15 mg, and the
blank(s) were required to have no recorded nitrogen or carbon
content. The percent standard deviation between replicate samples
was required to be below 10%.
Example 3
Results for SR00829 Events
[0120] T.sub.2 and T.sub.3 seeds from two events of SR00829
containing Ceres cDNA ID 2998984 were analyzed for total carbon and
nitrogen content as described in Example 2.
[0121] The nitrogen content of T.sub.2 seeds from two events of
SR00829 was significantly increased compared to the nitrogen
content of corresponding control seeds. As presented in Table 1,
the nitrogen content was increased to 10% and 109% in seeds from
events -01 and -02, respectively, compared to the nitrogen content
in control seeds.
TABLE-US-00001 TABLE 1 Total nitrogen content (% control) of
T.sub.2 and T.sub.3 seeds from SR00829 events Event -01 Event -02
Control T.sub.2 110 .+-. 1 109 .+-. 2 100 .+-. 1 p-value 0.001
0.002 NA T.sub.3 111 .+-. 3 118 .+-. 5 100 .+-. 3 p-value <0.01
0.01 NA
[0122] The nitrogen content of T.sub.3 seeds from two events of
SR00829 was significantly increased compared to the nitrogen
content of corresponding control seeds. As presented in Table 1,
the nitrogen content was increased to 111% and 118% in seeds from
events -01 and -02, respectively, compared to the nitrogen content
in control seeds.
[0123] The carbon content of T.sub.2 and T.sub.3 seeds from SR00829
events was not observed to differ significantly from the carbon
content of corresponding control seeds.
[0124] T.sub.3 seeds from SR00829 events analyzed for carbon and
nitrogen content were collected from one T.sub.2 plant from each
event.
[0125] The segregation of Finale.TM. resistance in T.sub.2 plants
from events -01 and -02 of SR00829 was a 3:1 ratio of resistant to
sensitive.
[0126] There were no observable or statistically significant
differences between T.sub.2 SR00829 and control plants in
germination, onset of flowering, rosette area, fertility, plant
height, and general morphology/architecture.
Example 4
Results for SR05001 Events
[0127] T.sub.2 and T.sub.3 seeds from two events of SR05001
containing Ceres clone 117581 were analyzed for total carbon and
nitrogen content as described in Example 2.
[0128] The carbon content of T.sub.2 seeds from one event of
SR05001 was significantly decreased compared to the carbon content
of corresponding control seeds. As presented in Table 2, the carbon
content was decreased to 97% in seeds from event -02 compared to
the carbon content in control seeds.
TABLE-US-00002 TABLE 2 Total carbon content (% control) of T.sub.2
and T.sub.3 seeds from SR05001 events Event -02 Event -03 Control
T.sub.2 97 .+-. 2 102 .+-. 2 100 .+-. 1 p-value 0.03 0.14 NA
T.sub.3 106 .+-. 1 107 .+-. 2 100 .+-. 2 p-value 0.01 0.01 NA
[0129] The nitrogen content of T.sub.2 seeds from two events of
SR05001 was significantly increased compared to the nitrogen
content of corresponding control seeds. As presented in Table 3,
the nitrogen content was increased to 112% and 115% in seeds from
events -02 and -03, respectively, compared to the nitrogen content
in control seeds.
TABLE-US-00003 TABLE 3 Total nitrogen content (% control) of
T.sub.2 and T.sub.3 seeds from SR05001 events Event -02 Event -03
Control T.sub.2 112 .+-. 3 115 .+-. 1 100 .+-. 4 p-value <0.01
<0.01 NA T.sub.3 109 .+-. 1 106 .+-. 2 100 .+-. 2 p-value
<0.01 0.02 NA
[0130] The carbon content of T.sub.3 seeds from two events of
SR05001 was significantly increased compared to the carbon content
of corresponding control seeds. As presented in Table 2, the carbon
content was increased to 106% and 107% in seeds from events -02 and
-03, respectively, compared to the carbon content in control
seeds.
[0131] The nitrogen content of T.sub.3 seeds from two events of
SR05001 was significantly increased compared to the nitrogen
content of corresponding control seeds. As presented in Table 3,
the nitrogen content was increased to 109% and 106% in seeds from
events -02 and -03, respectively, compared to the nitrogen content
in control seeds.
[0132] T.sub.3 seeds from SR05001 events analyzed for carbon and
nitrogen content were collected from one T.sub.2 plant from each
event.
[0133] The segregation of Finale.TM. resistance in T.sub.2 plants
from events -02 and -03 of SR05001 was a 3:1 ratio of resistant to
sensitive.
[0134] There were no observable or statistically significant
differences between T.sub.2 SR05001 and control plants in
germination, onset of flowering, rosette area, fertility, seed
size, and general morphology/architecture.
Example 5
Determination of Functional Homolog and/or Ortholog Sequences
[0135] A subject sequence was considered a functional homolog or
ortholog of a query sequence if the subject and query sequences
encoded proteins having a similar function and/or activity. A
process known as Reciprocal BLAST (Rivera et al., Proc. Natl. Acad.
Sci. USA, 95:6239-6244 (1998)) was used to identify potential
functional homolog and/or ortholog sequences from databases
consisting of all available public and proprietary peptide
sequences, including NR from NCBI and peptide translations from
Ceres clones.
[0136] Before starting a Reciprocal BLAST process, a specific query
polypeptide was searched against all peptides from its source
species using BLAST in order to identify polypeptides having
sequence identity of 80% or greater to the query polypeptide and an
alignment length of 85% or greater along the shorter sequence in
the alignment. The query polypeptide and any of the aforementioned
identified polypeptides were designated as a cluster.
[0137] The main Reciprocal BLAST process consists of two rounds of
BLAST searches; forward search and reverse search. In the forward
search step, a query polypeptide sequence, "polypeptide A," from
source species SA was BLASTed against all protein sequences from a
species of interest. Top hits were determined using an E-value
cutoff of 10-5 and an identity cutoff of 35%. Among the top hits,
the sequence having the lowest E-value was designated as the best
hit, and considered a potential functional homolog or ortholog. Any
other top hit that had a sequence identity of 80% or greater to the
best hit or to the original query polypeptide was considered a
potential functional homolog or ortholog as well. This process was
repeated for all species of interest. In the reverse search round,
the top hits identified in the forward search from all species were
BLASTed against all protein sequences from the source species SA. A
top hit from the forward search that returned a polypeptide from
the aforementioned cluster as its best hit was also considered as a
potential functional homolog or ortholog.
[0138] Functional homologs and/or orthologs were identified by
manual inspection of potential functional homolog and/or ortholog
sequences. Representative functional orthologs for SEQ ID NO:4 are
shown in FIG. 1. The percent identities of functional orthologs to
SEQ ID NO:4 are shown below in Table 4.
TABLE-US-00004 TABLE 4 Percent identity to Ceres clone 117581 (SEQ
ID NO: 4) SEQ ID % Designation Species NO: Identity e-value
CeresClone: 328378 Zea mays 5 70.4 0 gi|2655098 Hordeum vulgare 6
68.2 0 subsp. vulgare gi|34895718 Oryza sativa subsp. 7 68 0
japonica gi|50933627 Oryza sativa subsp. 8 64 0 japonica gi|4102839
Lycopersicon 9 63 0 esculentum gi|31088360 Vicia faba 10 62.9 0
gi|6635838 Prunus dulcis 11 61.4 0 gi|56784523 Oryza sativa subsp.
12 56.7 0 japonica gi|56784524 Oryza sativa subsp. 13 51.6 0
japonica gi|6409176 Oryza sativa 15 49.5 0 gi|50059161 Triticum
aestivum 14 49.5 0 CeresClone: 352232 Zea mays 16 48.5 0
gi|33411520 Prunus persica 17 47.1 0 gi|31429847 Oryza sativa 18
47.1 0
OTHER EMBODIMENTS
[0139] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
2511446DNAArabidopsis thalianamisc_feature(1)..(1446)Ceres CDNA ID
no. 2998984 1atggatcaag aagctcttct cgtcggaaga actcttctga agcgcggtat
tccgaccatt 60cctttcattc tagcaagtca ggctttggag aaacttgcat attttggttt
ggtaccgaac 120atgatactct tcttgacggt ggaatacggc atgggaacag
cggaagcggc caacatcctt 180ttcctctggt ctgccgccac caatttcttc
cctcttgttg gtgcttttat cgctgattct 240tacaccggtc ggtttcctct
gatcgggttt ggatcctcca tcagccttac gggaatggtt 300ttgttatggc
tgacaacaat aattcgacca gaatgcgata aattaacaaa cgtgtgccaa
360cccaccacac tgctcaaaag tgttcttcta tattcctttt ttgccctcac
cgccattggt 420gccggcggcg ttagatcctc ctgcttagcc ttcgctgccg
accagctcca acctaatcag 480acatcacgtg tcaccacatc atccctagaa
acgctcttca actggtacta cttctccgtc 540atggtcgcat gctttctttc
tcagtctttg ctcgtcttcg ttcagacaac gtatggttgg 600cagatcggtt
ttggagtttc tgtcgctgcc atggctctat cggtcgcttt gttcttcgcg
660gcgtctccgt actatgtaag gtttcagaaa ccgacacgga actcaaggaa
tccatggaag 720ctatgtaggg tgcaacaagt agaagatctt aaatctctca
tcaatgtcat accaatttgg 780tcaacaggga tcatcttgtc acttgtcacg
gcttgccaag tctccttcat agtccttcaa 840gctaagacca tggatcgcca
caccttcatt cagggtttcg agattcctcc aggctcttac 900ggcattttct
tggtcatctc ctttttgctc ttccttggtc tttacgatct tgttatcgtc
960ccattacttt cttgggctct aagagaaccc tttcgattgg gagttatggt
gagaatgtgg 1020gctgggtatg taatatcggt tttgtgcatc tccgctcttg
cggctacgga gtacgcgagg 1080agaaaaacag cgagagacga gagtggtacc
aagttgtcgg cgatgtggct attaccgtac 1140atgatattag gaggcattgc
ggaagcactt aatacaatag cacagaacga gttcttctac 1200tcagaacttc
ccaaaaccat gtcaagcgtc gccaccacac tctccagtct caacatggcc
1260gcagcaagcc tcatctcctc ctggatcatc accatcgttg acgttaccac
ttacgggagc 1320tggatcacag agaatataga cgagggacac ttggactatt
actactggct cttggtggga 1380ttatctttgc tgaatgtttt gtattttgtg
tggtgtaaga aatcttatgg taaatgtagt 1440atataa 14462505PRTArabidopsis
thalianamisc_feature(50)..(443)Pfam Name PTR2; Pfam Description POT
family 2Met Ile Leu Phe Leu Thr Val Glu Tyr Gly Met Gly Thr Ala Glu
Ala1 5 10 15Ala Asn Ile Leu Phe Leu Trp Ser Ala Ala Thr Asn Phe Phe
Pro Leu 20 25 30Val Gly Ala Phe Ile Ala Asp Ser Tyr Thr Gly Arg Phe
Pro Leu Thr 35 40 45Gly Phe Gly Ser Ser Ile Ser Leu Thr Gly Met Val
Leu Leu Trp Leu 50 55 60Thr Thr Ile Asn Arg Pro Glu Cys Asp Lys Leu
Thr Asn Val Cys Gln65 70 75 80Pro Thr Thr Leu Leu Lys Ser Val Leu
Leu Tyr Ser Phe Phe Ala Leu 85 90 95Thr Ala Ile Gly Ala Gly Gly Val
Arg Ser Ser Cys Leu Ala Phe Ala 100 105 110Ala Asp Gln Leu Gln Pro
Asn Gln Lys Ser Arg Val Thr Thr Ser Ser 115 120 125Val Glu Thr Leu
Phe Asn Trp Tyr Tyr Phe Ser Val Met Val Ala Cys 130 135 140Phe Leu
Ser Gln Ser Leu Leu Val Phe Val Gln Thr Thr Tyr Gly Trp145 150 155
160Gln Ile Gly Phe Gly Val Ser Val Ala Ala Met Ala Leu Ser Val Ala
165 170 175Leu Phe Phe Ala Ala Ser Pro Tyr Tyr Val Arg Phe Lys Cys
Glu Ser 180 185 190Gly Leu Val Thr Gly Leu Phe Gln Val Leu Val Ala
Ala Phe Arg Asn 195 200 205Arg His Val Asp Leu Ser Ser Glu Glu His
Ile Ile Ser Tyr His His 210 215 220Glu Thr Gly Ser Ser Phe Ser Ile
Pro Ser Gln Lys Leu Arg Tyr Leu225 230 235 240Asn Lys Ala Cys Val
Thr Asn Asn Ser Lys Gln Asp Leu Ala Leu Thr 245 250 255Gly Asn Ser
Arg Asn Pro Trp Lys Leu Cys Arg Val Gln Gln Val Glu 260 265 270Asp
Leu Lys Ser Leu Ile Asn Val Ile Pro Ile Trp Ser Thr Gly Ile 275 280
285Ile Leu Ser Leu Val Thr Ala Cys Gln Val Ser Phe Ile Val Leu Gln
290 295 300Ala Lys Thr Met Asp Arg His Thr Phe Ile Gln Gly Phe Glu
Ile Pro305 310 315 320Pro Gly Ser Tyr Gly Ile Phe Leu Val Ile Ser
Phe Leu Leu Phe Leu 325 330 335Gly Leu Tyr Asp Leu Val Ile Val Pro
Leu Leu Ser Trp Ala Leu Arg 340 345 350Ala Pro Phe Arg Leu Gly Val
Met Val Arg Met Trp Ala Gly Tyr Val 355 360 365Ile Ser Val Leu Cys
Ile Ser Ala Leu Ala Ala Thr Glu Tyr Ala Arg 370 375 380Arg Lys Thr
Ala Arg Asp Glu Ser Gly Thr Lys Leu Ser Ala Met Trp385 390 395
400Leu Leu Pro Tyr Met Ile Leu Gly Gly Ile Ala Glu Ala Leu Asn Thr
405 410 415Ile Ala Gln Asn Glu Phe Phe Tyr Ser Glu Leu Pro Lys Thr
Met Ser 420 425 430Ser Val Ala Thr Thr Leu Ser Ser Leu Asn Met Ala
Ala Ala Ser Leu 435 440 445Ile Ser Ser Trp Ile Ile Thr Ile Val Asp
Val Thr Thr Tyr Gly Ser 450 455 460Trp Ile Thr Glu Asn Ile Asp Glu
Gly His Leu Asp Tyr Tyr Tyr Trp465 470 475 480Leu Leu Val Gly Leu
Ser Leu Leu Asn Val Leu Tyr Phe Val Trp Cys 485 490 495Lys Lys Ser
Tyr Gly Lys Cys Ser Ile 500 50531764DNAArabidopsis
thalianamisc_feature(1)..(1764)Ceres CLONE ID no. 117581
3atggaagaaa aagatgtgta tacgcaagat ggaactgttg atattcacaa caatcctgca
60aacaaggaga aaaccggaaa ttggaaagct tgccgcttca ttctcggaaa tgagtgctgt
120gaaagattgg cctactatgg catgggcact aaccttgtga attatcttga
gagccgtctg 180aatcaaggca atgctacggc tgcaaataac gtcacgaatt
ggtctggaac atgttatata 240actcctttga ttggagcctt tatagctgat
gcttaccttg gacgatattg gactattgca 300acttttgttt tcatctatgt
ctccggtatg actcttttga cattatcagc ttcagttcct 360ggacttaaac
caggtaactg caatgctgat acttgtcatc caaattctag tcagactgct
420gttttctttg tcgcgcttta tatgattgct cttggaactg gcggtataaa
gccgtgtgtt 480tcgtcctttg gagctgatca gtttgatgag aatgatgaga
atgagaagat caagaaaagt 540tctttcttca actggtttta cttctccatt
aatgttggag ctctcattgc tgcaactgtt 600ctcgtctgga tacaaatgaa
tgttggttgg ggatggggtt tcggtgttcc aacagtcgcg 660atggttatcg
cggtttgctt tttcttcttc ggaagccgtt tttacagact tcagagacct
720ggagggagtc cacttactag gatctttcag gttatagtag cggcttttcg
gaagataagt 780gttaaggttc cagaggacaa gtctctgctc tttgaaactg
cagatgatga gagtaacatc 840aaaggtagcc ggaaacttgt gcacacagat
aacttaaagt tttttgacaa ggcagcggtt 900aagagtcaat ctgatagcat
caaagacggg gaagtcaatc catggagact atgttctgtt 960actcaagttg
aagaacttaa gtcaataatc acacttcttc cagtttgggc cacaggaata
1020gtcttcgcca cagtgtacag ccaaatgagc acaatgtttg tgttacaagg
aaacacaatg 1080gaccaacaca tgggaaaaaa ctttgaaatc ccatcagctt
cactctcact tttcgacact 1140gtcagtgtac tcttctggac tcctgtctat
gaccagttca ttatcccgct ggcaagaaag 1200ttcacacgca atgaacgagg
cttcactcag cttcaacgta tgggtatagg tcttgtggtc 1260tccatctttg
ccatgatcac tgcaggagtc ttggaggttg tcaggcttga ttatgtcaaa
1320actcacaatg catatgacca aaaacagatc catatgtcga tattctggca
gataccgcag 1380tatttactta tcggttgtgc agaagttttc acctttatag
gtcagcttga gtttttctat 1440gatcaggctc ctgatgccat gagaagtctc
tgctctgctt tgtcgttgac cacggttgcg 1500ttggggaact atttgagcac
agttcttgtg acggttgtga tgaagataac gaagaagaac 1560ggtaaaccgg
gttggatacc ggataacttg aaccgaggcc atcttgatta ctttttctac
1620ttgttggcaa ctctcagttt cctcaacttc ttagtgtacc tctggatttc
aaaacgctac 1680aaatacaaga aagctgttgg tcgagcacat aaatgctgca
gtctcgagcc gatcgttcaa 1740acatttggca ataaagtttc ttaa
17644587PRTArabidopsis thalianamisc_feature(96)..(499)Pfam Name
PTR2; Pfam Description POT family 4Met Glu Glu Lys Asp Val Tyr Thr
Gln Asp Gly Thr Val Asp Ile His1 5 10 15Asn Asn Pro Ala Asn Lys Glu
Lys Thr Gly Asn Trp Lys Ala Cys Arg 20 25 30Phe Ile Leu Gly Asn Glu
Cys Cys Glu Arg Leu Ala Tyr Tyr Gly Met 35 40 45Gly Thr Asn Leu Val
Asn Tyr Leu Glu Ser Arg Leu Asn Gln Gly Asn 50 55 60Ala Thr Ala Ala
Asn Asn Val Thr Asn Trp Ser Gly Thr Cys Tyr Ile65 70 75 80Thr Pro
Leu Ile Gly Ala Phe Ile Ala Asp Ala Tyr Leu Gly Arg Tyr 85 90 95Trp
Thr Ile Ala Thr Phe Val Phe Ile Tyr Val Ser Gly Met Thr Leu 100 105
110Leu Thr Leu Ser Ala Ser Val Pro Gly Leu Lys Pro Gly Asn Cys Asn
115 120 125Ala Asp Thr Cys His Pro Asn Ser Ser Gln Thr Ala Val Phe
Phe Val 130 135 140Ala Leu Tyr Met Ile Ala Leu Gly Thr Gly Gly Ile
Lys Pro Cys Val145 150 155 160Ser Ser Phe Gly Ala Asp Gln Phe Asp
Glu Asn Asp Glu Asn Glu Lys 165 170 175Ile Lys Lys Ser Ser Phe Phe
Asn Trp Phe Tyr Phe Ser Ile Asn Val 180 185 190Gly Ala Leu Ile Ala
Ala Thr Val Leu Val Trp Ile Gln Met Asn Val 195 200 205Gly Trp Gly
Trp Gly Phe Gly Val Pro Thr Val Ala Met Val Ile Ala 210 215 220Val
Cys Phe Phe Phe Phe Gly Ser Arg Phe Tyr Arg Leu Gln Arg Pro225 230
235 240Gly Gly Ser Pro Leu Thr Arg Ile Phe Gln Val Ile Val Ala Ala
Phe 245 250 255Arg Lys Ile Ser Val Lys Val Pro Glu Asp Lys Ser Leu
Leu Phe Glu 260 265 270Thr Ala Asp Asp Glu Ser Asn Ile Lys Gly Ser
Arg Lys Leu Val His 275 280 285Thr Asp Asn Leu Lys Phe Phe Asp Lys
Ala Ala Val Lys Ser Gln Ser 290 295 300Asp Ser Ile Lys Asp Gly Glu
Val Asn Pro Trp Arg Leu Cys Ser Val305 310 315 320Thr Gln Val Glu
Glu Leu Lys Ser Ile Ile Thr Leu Leu Pro Val Trp 325 330 335Ala Thr
Gly Ile Val Phe Ala Thr Val Tyr Ser Gln Met Ser Thr Met 340 345
350Phe Val Leu Gln Gly Asn Thr Met Asp Gln His Met Gly Lys Asn Phe
355 360 365Glu Ile Pro Ser Ala Ser Leu Ser Leu Phe Asp Thr Val Ser
Val Leu 370 375 380Phe Trp Thr Pro Val Tyr Asp Gln Phe Ile Ile Pro
Leu Ala Arg Lys385 390 395 400Phe Thr Arg Asn Glu Arg Gly Phe Thr
Gln Leu Gln Arg Met Gly Ile 405 410 415Gly Leu Val Val Ser Ile Phe
Ala Met Ile Thr Ala Gly Val Leu Glu 420 425 430Val Val Arg Leu Asp
Tyr Val Lys Thr His Asn Ala Tyr Asp Gln Lys 435 440 445Gln Ile His
Met Ser Ile Phe Trp Gln Ile Pro Gln Tyr Leu Leu Ile 450 455 460Gly
Cys Ala Glu Val Phe Thr Phe Ile Gly Gln Leu Glu Phe Phe Tyr465 470
475 480Asp Gln Ala Pro Asp Ala Met Arg Ser Leu Cys Ser Ala Leu Ser
Leu 485 490 495Thr Thr Val Ala Leu Gly Asn Tyr Leu Ser Thr Val Leu
Val Thr Val 500 505 510Val Met Lys Ile Thr Lys Lys Asn Gly Lys Pro
Gly Trp Ile Pro Asp 515 520 525Asn Leu Asn Arg Gly His Leu Asp Tyr
Phe Phe Tyr Leu Leu Ala Thr 530 535 540Leu Ser Phe Leu Asn Phe Leu
Val Tyr Leu Trp Ile Ser Lys Arg Tyr545 550 555 560Lys Tyr Lys Lys
Ala Val Gly Arg Ala His Lys Cys Cys Ser Leu Glu 565 570 575Pro Ile
Val Gln Thr Phe Gly Asn Lys Val Ser 580 5855580PRTZea
maysmisc_feature(1)..(580)Ceres CLONE ID no. 328378 5Met Gly Glu
Val Glu Asp Met Tyr Thr Gln Asp Gly Thr Val Asp Met1 5 10 15Lys Gly
Asn Pro Ala Val Lys Lys Gly Thr Gly Asn Trp Arg Ala Cys 20 25 30Pro
Tyr Ile Leu Ala Asn Glu Cys Cys Glu Arg Leu Ala Tyr Tyr Gly 35 40
45Met Ser Thr Asn Leu Val Asn Tyr Met Lys Thr Arg Leu Gly Gln Val
50 55 60Asn Ser Val Ala Ser Asn Asn Val Thr Asn Trp Gln Gly Thr Cys
Tyr65 70 75 80Ile Thr Pro Leu Ile Gly Ala Phe Phe Ala Asp Ala Tyr
Met Gly Arg 85 90 95Phe Trp Thr Ile Ala Ile Phe Met Ile Ile Tyr Ile
Phe Gly Leu Ala 100 105 110Leu Leu Thr Met Ala Ser Ser Val Lys Gly
Leu Val Pro Thr Ser Cys 115 120 125Gly Asp Lys Asp Val Cys His Pro
Thr Asp Ala Gln Ala Ala Val Val 130 135 140Phe Val Ala Leu Tyr Leu
Ile Ala Leu Gly Thr Gly Gly Ile Lys Pro145 150 155 160Cys Val Ser
Ser Phe Gly Ala Asp Gln Phe Asp Glu Asn Asp Glu Arg 165 170 175Glu
Lys Lys Ser Lys Ser Ser Phe Phe Asn Trp Phe Tyr Phe Ser Ile 180 185
190Asn Ile Gly Ala Leu Val Ala Ser Thr Val Leu Val Tyr Val Gln Thr
195 200 205His Val Gly Trp Gly Trp Gly Phe Gly Ile Pro Ala Val Val
Met Ala 210 215 220Ile Ala Val Gly Ser Phe Phe Val Gly Thr Pro Leu
Tyr Arg His Gln225 230 235 240Lys Pro Gly Gly Ser Pro Leu Thr Arg
Ile Ala Gln Val Leu Val Ala 245 250 255Cys Ala Arg Lys Trp Asn Val
Ala Val Pro Ala Asp Lys Ser Arg Leu 260 265 270His Glu Thr Val Asp
Gly Glu Ser Gly Ile Glu Gly Ser Arg Lys Leu 275 280 285Glu His Ser
Glu Gln Leu Ala Cys Leu Asp Arg Ala Ala Val Val Thr 290 295 300Ala
Glu Asp Gly Ala Glu Ala Ser Pro Trp Arg Leu Cys Ser Val Thr305 310
315 320Gln Val Glu Glu Leu Lys Ser Val Ile Arg Leu Leu Pro Ile Trp
Ala 325 330 335Ser Gly Ile Val Phe Ala Ala Val Tyr Ser Gln Met Ser
Thr Met Phe 340 345 350Val Leu Gln Gly Asn Thr Leu Asp Gln Ser Met
Gly Pro Arg Phe Lys 355 360 365Ile Pro Ser Ala Thr Leu Ser Met Val
Asp Thr Ile Ser Val Ile Val 370 375 380Trp Val Pro Val Tyr Asp Arg
Ala Ile Val Pro Leu Val Arg Ser Tyr385 390 395 400Thr Gly Arg Pro
Arg Gly Phe Thr Gln Leu Gln Arg Met Gly Ile Gly 405 410 415Leu Val
Val Ser Ile Phe Ser Met Val Ala Ala Gly Val Leu Asp Ile 420 425
430Val Arg Leu Arg Ala Ile Ala Arg His Gly Leu Tyr Gly Glu Asp Asp
435 440 445Ile Val Pro Ile Ser Ile Phe Trp Gln Ile Pro Gln Tyr Phe
Ile Ile 450 455 460Gly Cys Ala Glu Val Phe Thr Phe Val Gly Gln Leu
Glu Phe Phe Tyr465 470 475 480Asp Gln Ala Pro Asp Ala Met Arg Ser
Met Cys Ser Ala Leu Ser Leu 485 490 495Thr Thr Val Ala Leu Gly Asn
Tyr Leu Ser Thr Val Leu Val Thr Ile 500 505 510Val Thr His Ile Thr
Thr Arg His Gly Arg Ile Gly Trp Ile Pro Glu 515 520 525Asn Leu Asn
Arg Gly His Leu Asp Tyr Phe Phe Trp Leu Leu Ala Val 530 535 540Leu
Ser Leu Leu Asn Phe Leu Ala Tyr Leu Val Ile Ala Ser Trp Tyr545 550
555 560Lys Tyr Lys Lys Thr Ala Asp Asp Tyr Pro Gly Ala Lys Gly Glu
His 565 570 575Gly Thr Glu His 5806579PRTHordeum
vulgaremisc_feature(1)..(579)Public GI no. 2655098 6Met Gly Glu Val
Ala Ala Glu Met Tyr Thr Gln Asp Gly Thr Val Asp1 5 10 15Ile Lys Gly
Asn Pro Ala Leu Lys Lys Asp Thr Gly Asn Trp Arg Ala 20 25 30Cys Pro
Tyr Ile Leu Ala Asn Glu Cys Cys Glu Arg Leu Ala Tyr Tyr 35 40 45Gly
Met Ser Thr Asn Leu Val Asn Phe Met Lys Asp Arg Met Gly Met 50 55
60Ala Asn Ala Ala Ala Ala Asn Asn Val Thr Asn Trp Gly Gly Thr Cys65
70 75 80Tyr Ile Thr Pro Leu Ile Gly Ala Phe Leu Ala Asp Ala Tyr Leu
Gly 85 90 95Arg Phe Trp Thr Ile Ala Ser Phe Met Ile Ile Tyr Ile Phe
Gly Leu 100 105 110Gly Leu Leu Thr Met Ala Thr Ser Val His Gly Leu
Val Pro Ala Cys 115 120 125Ala Ser Lys Gly Val Cys Asp Pro Thr Pro
Gly Gln Ser Ala Ala Val 130 135 140Phe Ile Ala Leu Tyr Leu Ile Ala
Leu Gly Thr Gly Gly Ile Lys Pro145 150 155 160Cys Val Ser Ser Phe
Gly Ala Asp Gln Phe Asp Glu His Asp Asp Val 165 170 175Glu Arg Lys
Ser Lys Ser Ser Phe Phe Asn Trp Phe Tyr Phe Ser Ile 180 185
190Asn Ile Gly Ala Leu Val Ala Ser Ser Val Leu Val Tyr Val Gln Thr
195 200 205His Val Gly Trp Ser Trp Gly Phe Gly Ile Pro Ala Val Val
Met Ala 210 215 220Ile Ala Val Gly Ser Phe Phe Val Gly Thr Ser Leu
Tyr Arg His Gln225 230 235 240Arg Pro Gly Gly Ser Pro Leu Thr Arg
Ile Ala Gln Val Leu Val Ala 245 250 255Ala Thr Arg Lys Leu Gly Val
Ala Val Asp Gly Ser Ala Leu Tyr Glu 260 265 270Thr Ala Asp Lys Glu
Ser Gly Ile Glu Gly Ser Arg Lys Leu Glu His 275 280 285Thr Arg Gln
Phe Arg Phe Leu Asp Lys Ala Ala Val Glu Thr His Ala 290 295 300Asp
Arg Thr Ala Ala Ala Pro Ser Pro Trp Arg Leu Cys Thr Val Thr305 310
315 320Gln Val Glu Glu Leu Lys Ser Val Val Arg Leu Leu Pro Ile Trp
Ala 325 330 335Ser Gly Ile Val Phe Ala Thr Val Tyr Gly Gln Met Ser
Thr Met Phe 340 345 350Val Leu Gln Gly Asn Thr Leu Asp Ala Ser Met
Gly Pro Lys Phe Lys 355 360 365Ile Pro Ser Ala Ser Leu Ser Ile Phe
Asp Thr Leu Ser Val Ile Ala 370 375 380Trp Val Pro Val Tyr Asp Arg
Ile Leu Val Pro Ala Val Arg Ser Val385 390 395 400Thr Gly Arg Pro
Arg Gly Phe Thr Gln Leu Gln Arg Met Gly Ile Gly 405 410 415Leu Val
Val Ser Met Phe Ala Met Leu Ala Ala Gly Val Leu Glu Leu 420 425
430Val Arg Leu Arg Thr Ile Ala Gln His Gly Leu Tyr Gly Glu Lys Asp
435 440 445Val Val Pro Ile Ser Ile Phe Trp Gln Val Pro Gln Tyr Phe
Ile Ile 450 455 460Gly Cys Ala Glu Val Phe Thr Phe Val Gly Gln Leu
Glu Phe Phe Tyr465 470 475 480Asp Gln Ala Pro Asp Ala Met Arg Ser
Met Cys Ser Ala Leu Ser Leu 485 490 495Thr Thr Val Ala Leu Gly Asn
Tyr Leu Ser Thr Leu Leu Val Thr Val 500 505 510Val Ala Lys Val Thr
Thr Arg Gly Gly Lys Gln Gly Trp Ile Pro Asp 515 520 525Asn Leu Asn
Val Gly His Leu Asp Tyr Phe Phe Trp Leu Leu Ala Ala 530 535 540Leu
Ser Leu Val Asn Phe Ala Val Tyr Leu Leu Ile Ala Ser Trp Tyr545 550
555 560Thr Tyr Lys Lys Thr Ala Gly Asp Ser Pro Asp Ala Lys Gly Gly
Ala 565 570 575His Asp Gln7580PRTOryza sativa (japonica
cultivar-group)misc_feature(1)..(580)Public GI no. 34895718 7Met
Gly Glu Val Ala Glu Asp Ile Tyr Thr Gln Asp Gly Thr Val Asp1 5 10
15Val Lys Gly Asn Pro Ala Thr Lys Lys Asn Thr Gly Asn Trp Arg Ala
20 25 30Cys Pro Tyr Ile Leu Ala Asn Glu Cys Cys Glu Arg Leu Ala Tyr
Tyr 35 40 45Gly Met Ser Thr Asn Leu Val Asn Tyr Met Lys Thr Arg Leu
Gly Gln 50 55 60Glu Ser Ala Ile Ala Ala Asn Asn Val Thr Asn Trp Ser
Gly Thr Cys65 70 75 80Tyr Ile Thr Pro Leu Leu Gly Ala Phe Leu Ala
Asp Ala Tyr Met Gly 85 90 95Arg Phe Trp Thr Ile Ala Ser Phe Met Ile
Ile Tyr Ile Leu Gly Leu 100 105 110Ala Leu Leu Thr Met Ala Ser Ser
Val Lys Gly Leu Val Pro Ala Cys 115 120 125Asp Gly Gly Ala Cys His
Pro Thr Glu Ala Gln Thr Gly Val Val Phe 130 135 140Leu Ala Leu Tyr
Leu Ile Ala Leu Gly Thr Gly Gly Ile Lys Pro Cys145 150 155 160Val
Ser Ser Phe Gly Ala Asp Gln Phe Asp Glu Asn Asp Glu Gly Glu 165 170
175Lys Arg Ser Lys Ser Ser Phe Phe Asn Trp Phe Tyr Phe Ser Ile Asn
180 185 190Ile Gly Ala Leu Val Ala Ser Ser Val Leu Val Tyr Val Gln
Thr His 195 200 205Val Gly Trp Gly Trp Gly Phe Gly Ile Pro Ala Val
Val Met Ala Val 210 215 220Ala Val Ala Ser Phe Phe Val Gly Thr Pro
Leu Tyr Arg His Gln Arg225 230 235 240Pro Gly Gly Ser Pro Leu Thr
Arg Ile Ala Gln Val Leu Val Ala Ser 245 250 255Ala Arg Lys Trp Gly
Val Glu Val Pro Ala Asp Gly Ser Arg Leu His 260 265 270Glu Thr Leu
Asp Arg Glu Ser Gly Ile Glu Gly Ser Arg Lys Leu Glu 275 280 285His
Thr Gly Gln Phe Ala Cys Leu Asp Arg Ala Ala Val Glu Thr Pro 290 295
300Glu Asp Arg Ser Ala Ala Asn Ala Ser Ala Trp Arg Leu Cys Thr
Val305 310 315 320Thr Gln Val Glu Glu Leu Lys Ser Val Val Arg Leu
Leu Pro Ile Trp 325 330 335Ala Ser Gly Ile Val Phe Ala Thr Val Tyr
Gly Gln Met Ser Thr Met 340 345 350Phe Val Leu Gln Gly Asn Thr Leu
Asp Ala Ser Met Gly Pro His Phe 355 360 365Ser Ile Pro Ala Ala Ser
Leu Ser Ile Phe Asp Thr Leu Ser Val Ile 370 375 380Val Trp Val Pro
Val Tyr Asp Arg Leu Ile Val Pro Ala Val Arg Ala385 390 395 400Val
Thr Gly Arg Pro Arg Gly Phe Thr Gln Leu Gln Arg Met Gly Ile 405 410
415Gly Leu Val Ile Ser Val Phe Ser Met Leu Ala Ala Gly Val Leu Asp
420 425 430Val Val Arg Leu Arg Ala Ile Ala Arg His Gly Leu Tyr Gly
Asp Lys 435 440 445Asp Val Val Pro Ile Ser Ile Phe Trp Gln Val Pro
Gln Tyr Phe Ile 450 455 460Ile Gly Ala Ala Glu Val Phe Thr Phe Val
Gly Gln Leu Glu Phe Phe465 470 475 480Tyr Asp Gln Ala Pro Asp Ala
Met Arg Ser Met Cys Ser Ala Leu Ser 485 490 495Leu Thr Thr Val Ala
Leu Gly Asn Tyr Leu Ser Thr Leu Leu Val Thr 500 505 510Ile Val Thr
His Val Thr Thr Arg Asn Gly Ala Val Gly Trp Ile Pro 515 520 525Asp
Asn Leu Asn Arg Gly His Leu Asp Tyr Phe Phe Trp Leu Leu Ala 530 535
540Val Leu Ser Leu Ile Asn Phe Gly Val Tyr Leu Val Ile Ala Ser
Trp545 550 555 560Tyr Thr Tyr Lys Lys Thr Ala Asp Ser Pro Asp Asp
Lys Ala Glu His 565 570 575Ala Gly Ala Asn 5808572PRTOryza sativa
(japonica cultivar-group)misc_feature(1)..(572)Public GI no.
50933627 8Met Glu Ala Thr Thr Thr Asp Gly Thr Thr Asp His Ala Gly
Lys Pro1 5 10 15Ala Val Arg Ser Lys Ser Gly Thr Trp Arg Ala Cys Pro
Phe Ile Leu 20 25 30Gly Asn Glu Cys Cys Glu Arg Leu Ala Tyr Tyr Gly
Met Ser Ala Asn 35 40 45Leu Val Asn Tyr Met Val Asp Arg Leu Arg Gln
Gly Asn Ala Gly Ala 50 55 60Ala Ala Ser Val Asn Asn Trp Ser Gly Thr
Cys Tyr Val Met Pro Leu65 70 75 80Val Gly Ala Phe Leu Ala Asp Ala
Tyr Leu Gly Arg Tyr Arg Thr Ile 85 90 95Ala Ala Phe Met Ala Leu Tyr
Ile Val Gly Leu Ala Leu Leu Thr Met 100 105 110Ser Ala Ser Val Pro
Gly Met Lys Pro Pro Asn Cys Ala Thr Ile Ser 115 120 125Ala Ser Ser
Cys Gly Pro Ser Pro Gly Gln Ser Ala Ala Phe Phe Val 130 135 140Ala
Leu Tyr Leu Ile Ala Leu Gly Thr Gly Gly Ile Lys Pro Cys Val145 150
155 160Ser Ser Phe Gly Ala Asp Gln Phe Asp Asp Ala Asp Pro Arg Glu
His 165 170 175Arg Ser Lys Ala Ser Phe Phe Asn Trp Phe Tyr Met Ser
Ile Asn Val 180 185 190Gly Ala Leu Val Ala Ser Ser Val Leu Val Trp
Val Gln Met Asn Val 195 200 205Gly Trp Gly Trp Gly Phe Gly Ile Pro
Ala Val Ala Met Ala Val Ala 210 215 220Val Ala Ser Phe Leu Met Gly
Ser Ser Leu Tyr Arg His Gln Lys Pro225 230 235 240Gly Gly Ser Pro
Leu Thr Arg Met Leu Gln Val Val Val Ala Ala Ala 245 250 255Arg Lys
Ser Arg Val Ala Leu Pro Ala Asp Ala Ala Ala Leu Leu Tyr 260 265
270Glu Gly Asp Lys Leu Ala Cys Gly Thr Arg Arg Leu Ala His Thr Glu
275 280 285Gln Phe Arg Trp Leu Asp Arg Ala Ala Val Val Thr Pro Thr
Thr Asp 290 295 300Lys Asp Asp Asp Thr Gly Ser Arg Trp Arg Leu Cys
Pro Val Thr Gln305 310 315 320Val Glu Glu Leu Lys Ala Val Val Arg
Leu Leu Pro Val Trp Ala Ser 325 330 335Gly Ile Val Met Ser Ala Val
Tyr Gly Gln Met Ser Thr Met Phe Val 340 345 350Leu Gln Gly Asn Thr
Leu Asp Pro Arg Met Gly Ala Thr Phe Lys Ile 355 360 365Pro Ser Ala
Ser Leu Ser Ile Phe Asp Thr Leu Ala Val Leu Ala Trp 370 375 380Val
Pro Val Tyr Asp Arg Leu Ile Val Pro Ala Ala Arg Arg Phe Thr385 390
395 400Gly His Pro Arg Gly Phe Thr Gln Leu Gln Arg Met Gly Ile Gly
Leu 405 410 415Leu Ile Ser Val Phe Ser Met Val Ala Ala Gly Val Leu
Glu Val Val 420 425 430Arg Leu Arg Val Ala Ala Ala His Gly Met Leu
Asp Ser Thr Ser Tyr 435 440 445Leu Pro Ile Ser Ile Phe Trp Gln Val
Gln Tyr Phe Ile Ile Gly Ala 450 455 460Ala Glu Val Phe Ala Phe Ile
Gly Gln Ile Asp Phe Phe Tyr Asp Gln465 470 475 480Ala Pro Asp Asp
Met Arg Ser Thr Cys Thr Ala Leu Ser Leu Thr Ser 485 490 495Ser Ala
Leu Gly Asn Tyr Leu Ser Thr Leu Leu Val Val Ile Val Thr 500 505
510Ala Ala Ser Thr Arg Gly Gly Gly Leu Gly Trp Ile Pro Asp Asn Leu
515 520 525Asn Arg Gly His Leu Asp Tyr Phe Phe Trp Leu Leu Ala Ala
Leu Ser 530 535 540Ala Val Asn Phe Leu Val Tyr Leu Trp Ile Ala Asn
Trp Tyr Arg Cys545 550 555 560Lys Thr Ile Thr Thr Thr Glu Ala Ala
Ala Gln Thr 565 5709580PRTLycopersicon
esculentummisc_feature(1)..(580)Public GI no. 4102839 9Met Lys Tyr
Leu Phe Ser Lys Asn Gly Gly Leu Leu Glu Asp Glu Asn1 5 10 15Ser Gly
Leu Tyr Thr Arg Asp Gly Ser Val Asp Ile Lys Gly Asn Pro 20 25 30Val
Leu Lys Ser Glu Thr Gly Asn Trp Arg Ala Cys Pro Phe Ile Leu 35 40
45Gly Asn Glu Cys Cys Glu Arg Leu Ala Tyr Tyr Gly Ile Ala Ala Asn
50 55 60Leu Val Thr Tyr Leu Thr Lys Lys Leu His Glu Gly Asn Val Ser
Ala65 70 75 80Ala Arg Asn Val Thr Thr Trp Gln Gly Thr Cys Tyr Ile
Thr Pro Leu 85 90 95Ile Gly Ala Val Leu Ala Asp Ala Tyr Trp Gly Arg
Tyr Trp Thr Ile 100 105 110Ala Thr Phe Ser Thr Ile Tyr Phe Ile Gly
Met Cys Thr Leu Thr Leu 115 120 125Ser Ala Ser Val Pro Ala Phe Lys
Pro Pro Gln Cys Val Gly Ser Val 130 135 140Cys Pro Ser Ala Ser Pro
Ala Gln Tyr Ala Ile Phe Phe Phe Gly Leu145 150 155 160Tyr Leu Ile
Ala Leu Gly Thr Gly Gly Ile Lys Pro Cys Val Ser Ser 165 170 175Phe
Gly Ala Asp Gln Phe Asp Asp Thr Asp Pro Lys Glu Arg Val Lys 180 185
190Lys Gly Ser Phe Phe Asn Trp Phe Tyr Phe Ser Ile Asn Ile Gly Ala
195 200 205Leu Ile Ser Ser Ser Leu Ile Val Trp Ile Gln Glu Asn Ala
Gly Trp 210 215 220Gly Leu Gly Phe Gly Ile Pro Ala Val Phe Met Gly
Ile Ala Ile Ala225 230 235 240Ser Phe Phe Phe Gly Thr Pro Leu Tyr
Arg Phe Gln Lys Pro Gly Gly 245 250 255Ser Pro Leu Thr Arg Met Cys
Gln Val Leu Val Ala Val Phe His Lys 260 265 270Trp Asn Leu Ser Val
Pro Asp Asp Ser Thr Leu Leu Tyr Glu Thr Pro 275 280 285Asp Lys Ser
Ser Ala Ile Glu Gly Ser Arg Lys Leu Leu His Thr Asp 290 295 300Glu
Leu Arg Cys Leu Asp Lys Ala Ala Val Val Ser Asp Asn Glu Leu305 310
315 320Thr Thr Gly Asp Tyr Ser Asn Ala Trp Arg Leu Cys Thr Val Thr
Gln 325 330 335Val Glu Glu Leu Lys Ile Leu Ile Arg Met Phe Pro Ile
Trp Ala Thr 340 345 350Gly Ile Val Phe Ser Ala Val Tyr Ala Gln Met
Ser Thr Met Phe Val 355 360 365Glu Gln Gly Met Val Met Asp Thr Ala
Val Gly Ser Phe Lys Ile Pro 370 375 380Ala Ala Ser Leu Ser Thr Phe
Asp Thr Ile Ser Val Ile Val Trp Val385 390 395 400Pro Val Tyr Asp
Lys Ile Leu Val Pro Ile Ala Arg Arg Phe Thr Gly 405 410 415Ile Glu
Arg Gly Phe Ser Glu Leu Gln Arg Met Gly Ile Gly Leu Phe 420 425
430Leu Ser Met Leu Cys Met Ser Ala Ala Ala Ile Val Glu Ile Arg Arg
435 440 445Leu Gln Leu Ala Arg Asp Leu Gly Leu Val Asp Glu Ala Val
Ser Val 450 455 460Pro Leu Ser Ile Phe Trp Gln Ile Pro Gln Tyr Phe
Ile Leu Gly Ala465 470 475 480Ala Glu Ile Phe Thr Phe Ile Gly Gln
Leu Glu Phe Phe Tyr Asp Gln 485 490 495Ser Pro Asp Ala Met Arg Ser
Leu Cys Ser Ala Leu Ser Leu Leu Thr 500 505 510Thr Ala Leu Gly Asn
Tyr Leu Ser Ser Phe Ile Leu Thr Val Val Thr 515 520 525Ser Ile Thr
Thr Arg Gly Gly Lys Pro Gly Trp Ile Pro Asn Asn Leu 530 535 540Asn
Gly Gly His Leu Asp Tyr Phe Phe Trp Leu Leu Ala Ala Leu Ser545 550
555 560Phe Phe Asn Leu Val Ile Tyr Val Phe Leu Cys Gln Met Tyr Lys
Ser 565 570 575Lys Lys Ala Ser 58010584PRTVicia
fabamisc_feature(1)..(584)Public GI no. 31088360 10Met Gly Ser Val
Glu Asp Asp Ser Ser Arg Leu Glu Glu Ala Leu Ile1 5 10 15Gln Asp Glu
Glu Ser Lys Leu Tyr Thr Gly Asp Gly Ser Val Asp Phe 20 25 30Lys Gly
Arg Pro Val Leu Lys Lys Asn Thr Gly Asn Trp Lys Ala Cys 35 40 45Pro
Phe Ile Leu Gly Asn Glu Cys Cys Glu Arg Leu Ala Tyr Tyr Gly 50 55
60Ile Ala Thr Asn Leu Val Lys Pro Ile Leu Leu Ala Lys Leu His Glu65
70 75 80Gly Asn Val Ser Ala Ala Arg Asn Val Thr Thr Trp Gln Gly Thr
Cys 85 90 95Tyr Leu Ala Pro Leu Ile Gly Ala Val Leu Ala Asp Ser Tyr
Trp Gly 100 105 110Arg Tyr Trp Thr Ile Ala Ile Phe Ser Met Ile Tyr
Phe Ile Gly Met 115 120 125Gly Thr Leu Thr Leu Ser Ala Ser Ile Pro
Ala Leu Lys Pro Ala Glu 130 135 140Cys Leu Gly Ala Val Cys Pro Pro
Ala Thr Pro Ala Gln Tyr Ala Val145 150 155 160Phe Phe Ile Gly Leu
Tyr Leu Ile Ala Leu Gly Thr Gly Gly Ile Lys 165 170 175Pro Cys Val
Ser Ser Phe Gly Ala Asp Gln Phe Asp Asp Thr Asp Ser 180 185 190Arg
Glu Arg Val Lys Lys Gly Ser Phe Phe Asn Trp Phe Tyr Phe Ser 195 200
205Ile Asn Ile Gly Ala Leu Ile Ser Ser Ser Phe Ile Val Trp Ile Gln
210 215 220Glu Asn Ala Gly Trp Gly Leu Gly Phe Gly Ile Pro Ala Leu
Phe Met225 230 235 240Gly Leu Ala Ile Gly Ser Phe Phe Leu Gly Thr
Pro Leu Tyr Arg Phe 245 250 255Gln Lys Pro Gly Gly Ser Pro Leu Thr
Arg Met Cys Gln Val Val Ala 260 265 270Ala Ser Phe Arg Lys Arg Asn
Leu Thr Val Pro Glu Asp Ser Ser Leu 275 280 285Leu Tyr Glu Thr Pro
Asp Lys Ser Ser Ala Ile Glu Gly Ser Arg Lys 290 295 300Leu Gln His
Ser Asp Glu Leu Arg Cys Leu Asp
Arg Ala Ala Val Ile305 310 315 320Ser Asp Asp Glu Arg Lys Arg Gly
Asp Tyr Ser Asn Leu Trp Arg Leu 325 330 335Cys Thr Val Thr Gln Val
Glu Glu Leu Lys Ile Leu Ile Arg Met Phe 340 345 350Pro Val Trp Ala
Thr Gly Ile Val Phe Ser Ala Val Tyr Ala Gln Met 355 360 365Ser Thr
Met Phe Val Glu Gln Gly Thr Met Met Asp Thr Ser Val Gly 370 375
380Ser Phe Lys Ile Pro Ala Ala Ser Leu Ser Thr Phe Asp Val Ile
Ser385 390 395 400Val Ile Phe Trp Val Pro Val Tyr Asp Arg Phe Ile
Val Pro Ile Ala 405 410 415Arg Lys Phe Thr Gly Lys Glu Arg Gly Phe
Ser Glu Leu Gln Arg Met 420 425 430Gly Ile Gly Leu Phe Ile Ser Val
Leu Cys Met Ser Ala Ala Ala Ile 435 440 445Val Glu Ile Lys Arg Leu
Gln Leu Ala Lys Glu Leu Asp Leu Val Asp 450 455 460Lys Ala Val Pro
Val Pro Leu Thr Ile Phe Leu Gln Ile Pro Gln Tyr465 470 475 480Phe
Leu Leu Gly Ala Ala Glu Val Phe Thr Phe Val Gly Gln Leu Glu 485 490
495Phe Phe Tyr Asp Gln Ser Pro Asp Ala Met Arg Ser Leu Cys Ser Ala
500 505 510Leu Ser Leu Leu Thr Thr Ser Leu Gly Asn Tyr Leu Ser Ser
Phe Ile 515 520 525Leu Thr Val Val Leu Tyr Phe Thr Thr Arg Gly Gly
Asn Pro Gly Trp 530 535 540Ile Pro Asp Asn Leu Asn Lys Gly His Leu
Asp Tyr Phe Ser Gly Leu545 550 555 560Ala Gly Leu Ser Phe Leu Asn
Met Phe Leu Tyr Ile Val Ala Ala Lys 565 570 575Arg Tyr Lys Ser Lys
Lys Ala Ser 58011559PRTPrunus dulcismisc_feature(1)..(559)Public GI
no. 6635838 11Met Gly Ser Leu Glu Glu Glu Arg Ser Leu Leu Glu Asp
Gly Leu Ile1 5 10 15Gln Asp Glu Thr Asn Gly Leu Tyr Thr Gly Asp Gly
Ser Val Asp Ile 20 25 30Thr Gly Lys Pro Val Leu Lys Gln Ser Thr Gly
Asn Trp Xaa Ala Cys 35 40 45Pro Phe Ile Leu Gly Thr Glu Cys Cys Glu
Arg Leu Ala Phe Tyr Gly 50 55 60Ile Ser Thr Asn Leu Val Thr Tyr Leu
Thr His Lys Leu His Glu Gly65 70 75 80Asn Val Ser Ala Ala Arg Asn
Val Thr Thr Trp Ser Gly Thr Cys Tyr 85 90 95Leu Thr Pro Leu Ile Gly
Ala Val Leu Ala Asp Ala Tyr Trp Gly Arg 100 105 110Tyr Trp Thr Ile
Ala Ile Phe Ser Thr Ile Tyr Phe Ile Gly Met Cys 115 120 125Thr Leu
Thr Ile Ser Ala Ser Val Pro Ala Leu Lys Pro Pro Gln Cys 130 135
140Val Asp Ser Val Cys Pro Ser Ala Ser Pro Ala Gln Tyr Gly Val
Phe145 150 155 160Phe Phe Gly Leu Tyr Leu Ile Ala Leu Arg Thr Gly
Gly Ile Lys Pro 165 170 175Cys Val Ser Ser Phe Gly Ala Asp Gln Phe
Asp Asp Thr Asp Ser Arg 180 185 190Glu Arg Val Lys Lys Gly Ser Phe
Phe Asn Trp Phe Tyr Phe Ser Ile 195 200 205Asn Ile Gly Ala Leu Val
Ser Ser Thr Leu Ile Val Trp Val Gln Asp 210 215 220Asn Ala Gly Trp
Gly Leu Gly Phe Gly Ile Pro Ala Leu Phe Met Gly225 230 235 240Ile
Ala Ile Val Ser Phe Phe Ser Gly Thr Pro Leu Tyr Arg Phe Gln 245 250
255Lys Pro Gly Gly Ser Pro Leu Thr Arg Met Cys Gln Val Leu Val Ala
260 265 270Ser Phe Arg Lys Trp Asn Leu Asp Val Pro Arg Asp Ser Ser
Leu Leu 275 280 285Tyr Glu Thr Gln Asp Lys Gly Ser Ala Ile Lys Gly
Ser Arg Lys Leu 290 295 300Glu His Ser Asp Glu Leu Asn Cys Leu Asp
Lys Ala Ala Val Ile Ser305 310 315 320Glu Thr Glu Thr Lys Thr Gly
Asp Phe Ser Asn Pro Trp Arg Ile Cys 325 330 335Thr Val Thr Gln Val
Glu Glu Leu Lys Ile Leu Ile Arg Met Phe Pro 340 345 350Ile Trp Ala
Thr Gly Ile Val Phe Ser Ala Val Tyr Ala Gln Met Ala 355 360 365Thr
Met Phe Val Glu Gln Gly Met Met Met Asp Thr Ser Val Gly Ser 370 375
380Phe Thr Ile Pro Pro Ala Ser Leu Ser Ser Phe Asp Val Ile Ser
Val385 390 395 400Ile Phe Trp Val Pro Ile Tyr Asp Arg Phe Ile Val
Pro Ile Ala Arg 405 410 415Lys Phe Thr Gly Lys Glu Arg Gly Phe Ser
Glu Leu Gln Arg Met Gly 420 425 430Ile Gly Leu Phe Leu Ser Val Leu
Cys Met Ser Ala Ala Ala Val Val 435 440 445Glu Met Lys Arg Leu Gln
Leu Ala Thr Glu Leu Gly Leu Val Asp Lys 450 455 460Glu Val Ala Val
Pro Leu Ser Ile Phe Trp Gln Ile Pro Gln Tyr Phe465 470 475 480Leu
Leu Gly Ala Ala Glu Ile Phe Thr Phe Ile Gly Gln Leu Glu Phe 485 490
495Phe Tyr Asp Gln Ser Ser Asp Ala Met Arg Ser Leu Cys Ser Ala Leu
500 505 510Ser Ala Ser Asp Asp Phe Ile Gly Lys Leu Ser Glu Leu Phe
Asp Ser 515 520 525Asp Ile Val Thr Tyr Phe Thr Thr Gln Gly Gly Lys
Ala Gly Trp Ile 530 535 540Pro Asp Asn Leu Asn Asp Gly His Leu Asp
Tyr Phe Ser Gly Ser545 550 55512557PRTOryza sativa (japonica
cultivar-group)misc_feature(1)..(557)Public GI no. 56784523 12Met
Glu Gly Val Glu Ser Cys Asn Gly Arg His Ala Asp Ala Asp Asp1 5 10
15Arg Arg Thr Ser Lys Lys Asp Arg Arg Thr Thr Trp Ala Ser Ala Phe
20 25 30Ile Leu Val Asn Asn Phe Met Gln Tyr Thr Ala Tyr Phe Gly Val
Ser 35 40 45Thr Asn Leu Val Asn Tyr Leu Lys Tyr Arg Leu His Glu Gly
Ser Lys 50 55 60Ser Ala Ala Asn Asp Val Thr Asn Trp Gln Gly Thr Gly
Ser Ile Thr65 70 75 80Pro Leu Val Ala Ala Tyr Leu Ala Asp Ala Phe
Leu Gly Arg Tyr Trp 85 90 95Thr Ile Leu Leu Phe Met Ala Ile Ser Val
Leu Gly Tyr Gly Val Leu 100 105 110Ala Ala Ser Ala Ala Ala Pro Ala
Leu Leu His Gly Gly Ala Ala Ala 115 120 125Phe Tyr Ala Gly Leu Tyr
Leu Val Ala Leu Gly Ser Gly Leu Leu Val 130 135 140Val Met Ala Pro
Phe Gly Ala Gly Gln Phe Asp Glu Ala Asp Glu Gly145 150 155 160Glu
Arg Arg Arg Gln Ser Ser Phe Phe Asn Trp Phe Tyr Leu Ser Leu 165 170
175Asn Phe Gly Ser Leu Val Gly Gly Thr Val Leu Val Trp Val Gln Thr
180 185 190Ser Val Gly Trp Gly Ile Gly Tyr Gly Val Pro Ala Ile Phe
Ser Ala 195 200 205Leu Ser Val Ala Val Phe Leu Ala Gly Thr Ala Ala
Tyr Arg Arg Cys 210 215 220Gln Pro Pro Gly Gly Ser Pro Leu Thr Arg
Ile Ala Gln Val Val Val225 230 235 240Ala Ala Ala Arg Lys His Asp
Val Glu Val Pro Ala Asp Ala Ser Leu 245 250 255Leu His Glu Cys Cys
Asp Ala Val Asp Gly Met Ser Ala Ile Gln Gly 260 265 270Ser Arg Arg
Leu Val His Thr Asp Gln Phe Arg Phe Leu Asp Lys Ala 275 280 285Ala
Val Glu Thr Ala Gly Asp Lys Ala Glu Pro Ser Pro Trp Arg Leu 290 295
300Cys Thr Val Thr Gln Val Glu Glu Leu Lys Cys Val Leu Arg Leu
Leu305 310 315 320Pro Val Trp Ala Ser Gly Ile Ile Phe Ala Ala Ala
Tyr Thr Gln Met 325 330 335Thr Thr Thr Phe Val Leu Gln Gly Asp Thr
Leu Asp Pro Arg Ile Gly 340 345 350Gly Phe Lys Val Pro Ala Ala Val
Leu Ser Val Phe Asp Thr Leu Ser 355 360 365Val Met Leu Trp Val Pro
Leu Tyr Asp Arg Ala Ile Val Pro Leu Ala 370 375 380Arg Arg Val Thr
Gly His Asp Arg Gly Phe Thr Gln Leu Ala Arg Met385 390 395 400Gly
Val Gly Leu Val Ile Leu Thr Val Ala Met Leu Val Ala Gly Thr 405 410
415Leu Glu Val Ala Arg Arg Arg Val Ile Ala Arg His Gly Leu Tyr Gly
420 425 430Asp Asp Gly Asp Gly Gly Tyr Leu Pro Leu Ser Ile Phe Trp
Gln Val 435 440 445Pro Gln Tyr Val Val Val Gly Ala Ser Glu Val Phe
Thr Phe Ile Gly 450 455 460Gln Met Glu Phe Phe Tyr Asp Gln Ala Pro
Asp Ala Met Arg Ser Leu465 470 475 480Cys Ser Gly Leu Ser Ser Thr
Ser Phe Ala Leu Gly Asn Tyr Ala Ser 485 490 495Ser Ala Ile Val Val
Val Val Ala Arg Ala Thr Ala Arg Gly Gly Arg 500 505 510Leu Gly Trp
Ile Pro Asp Asn Ile Asn Arg Gly His Leu Asp Asp Phe 515 520 525Phe
Trp Leu Leu Ala Val Leu Cys Val Ala Asn Phe Ala Ala Tyr Leu 530 535
540Leu Ile Ala Arg Trp Tyr Thr Tyr Lys Lys Thr Val Asp545 550
55513545PRTOryza sativa (japonica
cultivar-group)misc_feature(1)..(545)Public GI no. 56784524 13Met
Glu Gly Val Glu Ser Asn Asp Arg His Gly Gly Ala Ala Ala Asp1 5 10
15Arg Arg Lys Ser Asn Arg Arg Asn Arg Trp Ala Cys Thr Phe Ile Leu
20 25 30Ala Asn Asn Phe Phe Gln Asn Met Ala Tyr Phe Gly Val Ser Thr
Asn 35 40 45Leu Val Asn Tyr Leu Lys Tyr Arg Leu His Glu Gly Ser Lys
Ser Ala 50 55 60Ala Asn Asn Val Thr Asn Trp Glu Gly Thr Gly Ser Ile
Ala Pro Leu65 70 75 80Val Ala Gly Tyr Leu Ala Asp Ala Phe Leu Gly
Arg Tyr Trp Thr Ile 85 90 95Val Leu Ser Met Val Ile Ser Ala Val Val
Arg Ser Ser Pro Pro Pro 100 105 110Ala Met Gln Gly Tyr Gly Val Leu
Ala Ala Ser Ala Ser Val Ile Arg 115 120 125Leu Glu Ser Ala Ala Leu
Tyr Ala Gly Met Tyr Leu Val Ala Leu Gly 130 135 140Gly Val Leu Glu
Pro Ile Met Ala Pro Phe Gly Ala Asp Gln Phe Asp145 150 155 160Asp
Gly Glu Asp Asp Gln Arg Gly Arg Arg Gln Ser Ser Phe Phe Asn 165 170
175Trp Phe Tyr Leu Ser Leu Asn Cys Gly Ser Leu Val Gly Gly Thr Val
180 185 190Leu Val Trp Val Gln Thr Ser Val Gly Trp Gly Val Gly Tyr
Gly Val 195 200 205Pro Ala Ile Phe Ser Ala Leu Ser Val Ala Val Phe
Leu Ala Gly Thr 210 215 220Ala Thr Tyr Arg Arg Asp Gln Pro Pro Gly
Gly Ser Pro Leu Thr Arg225 230 235 240Ile Ala Gln Val Val Val Ala
Ala Val Arg Lys Phe Asp Val Glu Ile 245 250 255Pro Ser Asp Ser Ser
Met Leu Tyr Glu Ser Asp Ala Val Asp Gly Met 260 265 270Pro Ala Ile
His Gly Arg Arg Arg Leu Leu His Thr Gly Gln Phe Arg 275 280 285Phe
Leu Asp Arg Ala Thr Val Lys Thr Ala Gly Glu Lys Ala Ala Gln 290 295
300Ser Pro Trp Arg Leu Cys Thr Val Thr Gln Val Glu Glu Leu Lys
Cys305 310 315 320Val Leu Arg Leu Leu Pro Val Trp Ala Thr Gly Ile
Ile Tyr Ala Ala 325 330 335Ala Tyr Thr Gln Val Thr Thr Thr Phe Ile
Leu Gln Gly Asp Thr Leu 340 345 350Asp Arg Ser Leu Gly Arg Phe Lys
Val Pro Ala Ala Ala Leu Ser Ile 355 360 365Phe His Thr Leu Ser Val
Ile Leu Trp Val Ala Leu Tyr Asp Arg Ala 370 375 380Ile Val Pro Leu
Ala Arg Arg Val Thr Arg His Asp Gly Gly Phe Thr385 390 395 400Gln
Leu Ala Arg Met Gly Val Gly Leu Val Ile Leu Thr Val Ala Met 405 410
415Ala Ala Ala Gly Ala Leu Glu Ala Ala Arg Arg Arg Leu Ile Ala Arg
420 425 430Pro Ser Val Phe Trp Gln Val Pro Gln Tyr Ala Val Val Gly
Ala Ser 435 440 445Glu Val Phe Thr Leu Ile Gly Gln Met Glu Phe Phe
Tyr Asp Gln Ala 450 455 460Pro Asp Ala Met Arg Ser Leu Cys Ser Ala
Leu Ser Ser Thr Ser Phe465 470 475 480Ala Leu Gly Asp Tyr Ala Ser
Ser Ala Leu Val Val Val Ala Ala Arg 485 490 495Arg Gly Gly Ala Pro
Gly Trp Ile Pro Asp Asp Ile Asn Arg Gly His 500 505 510Leu Asp Tyr
Phe Phe Trp Leu Leu Thr Ala Leu Cys Val Ala Asn Phe 515 520 525Ala
Ala Tyr Leu Leu Ile Ala Arg Trp Tyr Thr Tyr Lys Lys Thr Val 530 535
540Asp54514586PRTTriticum aestivummisc_feature(1)..(586)Public GI
no. 50059161 14Met Asp Ser Thr Asp Gln Phe Asp Asn Ser Pro Leu Leu
Asp Gly Asp1 5 10 15Gly Ser Ser Gln Glu Asn Thr Thr Glu Tyr Thr Gly
Asp Gly Ser Val 20 25 30Cys Ile Ser Gly His Pro Ala Ser Arg Lys His
Thr Gly Asn Trp Lys 35 40 45Ala Ser Phe Leu Ile Ile Val Cys Ser Phe
Cys Cys Tyr Leu Ala Tyr 50 55 60Ser Ser Ile Gly Lys Asn Leu Val Ser
Tyr Leu Thr Lys Val Leu His65 70 75 80Glu Thr Asn Leu Asp Ala Ala
Arg His Val Ala Thr Trp Gln Gly Thr 85 90 95Ser Tyr Leu Ala Pro Leu
Val Gly Ala Phe Val Ala Asp Ser Tyr Leu 100 105 110Gly Lys Tyr Arg
Thr Ala Leu Ile Ala Cys Lys Ile Phe Ile Ile Gly 115 120 125Met Met
Met Leu Leu Leu Ser Ala Ala Leu Gln Leu Ile Ser Ala Gly 130 135
140Pro His Ala Trp Thr Val Trp Val His Leu Val Ser Ser Gln Tyr
Thr145 150 155 160Ile Phe Leu Ile Gly Leu Tyr Met Val Gly Leu Gly
Tyr Gly Ala Gln 165 170 175Arg Pro Cys Val Thr Ser Phe Gly Ala Asp
Gln Phe Asp Asp Thr Asp 180 185 190Tyr Val Glu Lys Thr Arg Lys Ser
Ser Phe Phe Asn Trp His Tyr Phe 195 200 205Ala Ile Asn Ala Gly Ser
Leu Ile Ala Gly Thr Val Ile Val Trp Val 210 215 220Gln Glu His Glu
Gly Trp Leu Trp Gly Phe Thr Ile Ser Thr Leu Phe225 230 235 240Val
Thr Leu Gly Val Cys Ile Phe Phe Leu Gly Ser Ile Val Tyr Arg 245 250
255Phe Gln Lys Pro Arg Gly Ser Pro Leu Thr Arg Leu Cys Gln Val Val
260 265 270Ile Ala Ala Thr Arg Asn Phe Asp Lys Val Leu Pro Cys Asp
Ser Ser 275 280 285Ala Leu Tyr Glu Phe Met Gly Gln Gly Ser Ala Ile
Glu Gly Arg Arg 290 295 300Lys Leu Glu His Thr Thr Gly Leu Gly Phe
Phe Asp Lys Ala Ala Ile305 310 315 320Val Thr Leu Pro Asp Cys Glu
Ser Pro Gly Gln His Asn Lys Trp Lys 325 330 335Ile Cys Thr Val Thr
Gln Val Glu Glu Leu Lys Ile Leu Ile Arg Met 340 345 350Phe Pro Ile
Trp Ser Ala Met Ile Leu Phe Ala Ala Val Gln Glu Gln 355 360 365Met
Ser Ser Thr Phe Val Glu Gln Gly Met Ala Met Asp Lys His Ile 370 375
380Gly Ser Phe Glu Ile Pro Ser Ala Ser Phe Gln Cys Val Asp Thr
Ile385 390 395 400Thr Val Ile Val Leu Val Pro Ile Tyr Glu Arg Leu
Ile Val Pro Val 405 410 415Ile Arg Lys Phe Thr Gly Arg Ala Asn Gly
Ile Thr Ser Pro Gln Arg 420 425 430Ile Gly Ile Gly Leu Cys Phe Ser
Met Phe Ser Met Val Ser Ala Ala 435 440 445Leu Val Glu Gly Asn Arg
Leu Gln Ile Ala Gln Ala Glu Gly Leu Val 450 455 460His Arg Lys Val
Ala Val Pro Met Ser Ile Met Trp Gln Gly Pro Gln465 470 475 480Tyr
Phe Leu Leu Gly Val Ala Glu Val Phe Ser Asn Ile Gly Leu Thr
485 490 495Glu Ala Phe Tyr Asp Glu Ser Pro Asp Gly Met Arg Ser Leu
Cys Met 500 505 510Ala Phe Ser Leu Val Asn Met Ser Ala Gly Asn Tyr
Leu Ser Ser Leu 515 520 525Ile Leu Ser Leu Val Pro Val Phe Thr Ala
Arg Gly Gly Ser Pro Gly 530 535 540Trp Ile Pro Asp Asn Leu Asn Glu
Gly His Leu Asp Arg Phe Tyr Leu545 550 555 560Met Met Ala Gly Leu
Ser Phe Phe Asn Ile Val Val Phe Val Phe Cys 565 570 575Ala Met Arg
Tyr Lys Cys Lys Lys Ala Ser 580 58515584PRTOryza
sativamisc_feature(1)..(584)Public GI no. 6409176 15Met Asp Ser Ser
Tyr Gln His Asp Lys Pro Leu Leu Asp Glu Glu Asn1 5 10 15Ser Ser Gln
Val Thr Leu Glu Tyr Thr Gly Asp Gly Ser Val Cys Ile 20 25 30Arg Gly
His Pro Ala Leu Arg Lys His Thr Gly Asn Trp Lys Gly Ser 35 40 45Ser
Leu Ala Ile Val Phe Ser Phe Cys Ser Tyr Leu Ala Phe Thr Ser 50 55
60Ile Val Lys Asn Leu Val Ser Tyr Leu Thr Lys Val Leu His Glu Thr65
70 75 80Asn Val Ala Ala Ala Arg Asp Val Ala Thr Trp Ser Gly Thr Ser
Tyr 85 90 95Leu Ala Pro Leu Val Gly Ala Phe Leu Ala Asp Ser Tyr Leu
Gly Lys 100 105 110Tyr Cys Thr Ile Leu Ile Phe Cys Thr Ile Phe Ile
Ile Gly Leu Met 115 120 125Met Leu Leu Leu Ser Ala Ala Val Pro Leu
Ile Ser Thr Gly Pro His 130 135 140Ser Trp Ile Ile Trp Thr Asp Pro
Val Ser Ser Gln Asn Ile Ile Phe145 150 155 160Phe Val Gly Leu Tyr
Met Val Ala Leu Gly Tyr Gly Ala Gln Cys Pro 165 170 175Cys Ile Ser
Ser Phe Gly Ala Asp Gln Phe Asp Asp Thr Asp Glu Asn 180 185 190Glu
Arg Thr Lys Lys Ser Ser Phe Phe Asn Trp Thr Tyr Phe Val Ala 195 200
205Asn Ala Gly Ser Leu Ile Ser Gly Thr Val Ile Val Trp Val Gln Asp
210 215 220His Lys Gly Trp Ile Trp Gly Phe Thr Ile Ser Ala Leu Phe
Val Tyr225 230 235 240Leu Gly Phe Gly Thr Phe Ile Phe Gly Ser Ser
Met Tyr Asp Phe Arg 245 250 255Asn Leu Glu Glu Ala Pro Leu Ala Arg
Ile Cys Gln Val Val Val Ala 260 265 270Ala Ile His Lys Arg Asp Lys
Asp Leu Pro Cys Asp Ser Ser Val Leu 275 280 285Tyr Glu Phe Leu Gly
Gln Ser Ser Ala Ile Glu Gly Ser Arg Lys Leu 290 295 300Glu His Thr
Thr Gly Leu Lys Phe Phe Asp Arg Ala Ala Met Val Thr305 310 315
320Pro Ser Asp Phe Glu Ser Asp Gly Leu Leu Asn Thr Trp Lys Ile Cys
325 330 335Thr Val Thr Gln Val Glu Glu Leu Lys Ile Leu Ile Arg Met
Phe Pro 340 345 350Val Trp Ala Thr Met Ile Leu Phe Ala Ala Val Leu
Asp Asn Met Phe 355 360 365Ser Thr Phe Ile Glu Gln Gly Met Val Met
Glu Lys His Ile Gly Ser 370 375 380Phe Glu Ile Pro Ala Ala Ser Phe
Gln Ser Ile Asp Val Ile Ala Val385 390 395 400Leu Ile Leu Val Pro
Val Tyr Glu Arg Val Leu Val Pro Val Phe Arg 405 410 415Lys Phe Thr
Gly Arg Ala Asn Gly Ile Thr Pro Leu Gln Arg Met Gly 420 425 430Ile
Gly Leu Phe Phe Ser Met Leu Ser Met Val Ser Ala Ala Leu Val 435 440
445Glu Ser Asn Arg Leu Arg Ile Ala Gln Asp Glu Gly Leu Val His Arg
450 455 460Lys Val Ala Val Pro Met Ser Ile Leu Trp Gln Gly Pro Gln
Tyr Phe465 470 475 480Leu Ile Gly Val Gly Glu Val Phe Ser Asn Ile
Gly Leu Thr Glu Phe 485 490 495Phe Tyr Gln Glu Ser Pro Asp Ala Met
Arg Ser Leu Cys Leu Ala Phe 500 505 510Ser Leu Ala Asn Val Ser Ala
Gly Ser Tyr Leu Ser Ser Phe Ile Val 515 520 525Ser Leu Val Pro Val
Phe Thr Ala Arg Glu Gly Ser Pro Gly Trp Ile 530 535 540Pro Asp Asn
Leu Asn Glu Gly His Leu Asp Arg Phe Phe Trp Met Met545 550 555
560Ala Gly Leu Cys Phe Leu Asn Met Leu Ala Phe Val Phe Cys Ala Met
565 570 575Arg Tyr Lys Cys Lys Lys Ala Ser 58016584PRTZea
maysmisc_feature(1)..(584)Ceres CLONE ID no. 352232 16Met Asp Ala
Gln Asp Asp Asp Glu Arg Pro Leu Ile Ile His Arg Leu1 5 10 15Pro Leu
Leu Gln Asp Glu Ser Thr Ser Gly Phe Thr Ser Asp Gly Thr 20 25 30Val
Asp Leu Arg Asn Gln Pro Ala Arg Lys Gln Arg Thr Gly Asn Trp 35 40
45Arg Ala Cys Phe Phe Ile Leu Gly Ala Glu Phe Ala Glu Cys Val Ala
50 55 60Phe Phe Ala Ile Ser Lys Asn Leu Val Thr Tyr Leu Thr Gly Val
Leu65 70 75 80His Glu Ser Asn Val Asp Ala Ala Thr Thr Val Ser Thr
Trp Ile Gly 85 90 95Thr Ser Phe Phe Thr Pro Leu Val Gly Ala Phe Leu
Ala Asp Thr Phe 100 105 110Trp Gly Arg Tyr Trp Thr Ile Leu Ala Phe
Leu Ser Val Tyr Val Thr 115 120 125Gly Met Thr Val Leu Thr Ala Ser
Ala Leu Leu Pro Leu Leu Met Gly 130 135 140Ala Ser Tyr Ser Arg Ser
Ala His Arg Leu Ser Ala Tyr Leu Gly Leu145 150 155 160Tyr Leu Ala
Ala Leu Gly Thr Gly Gly Ile Lys Pro Cys Val Cys Ala 165 170 175Leu
Gly Ala Asp Gln Phe Asp Ala Ser Asp Pro Val Glu Arg Arg Ala 180 185
190Lys Gly Ser Phe Phe Asn Trp Tyr Tyr Phe Ser Ile Asn Ile Gly Ser
195 200 205Leu Leu Ser Ala Thr Val Val Val Trp Val Gln Asp Asn Val
Gly Trp 210 215 220Gly Val Gly Phe Ala Ile Pro Thr Leu Leu Met Leu
Ser Gly Leu Val225 230 235 240Leu Phe Val Ala Gly Arg Lys Val Tyr
Arg Tyr Gln Arg Val Gly Gly 245 250 255Ser Pro Leu Thr Arg Ala Ser
Gln Val Val Val Ala Ala Val Arg Asn 260 265 270Tyr Arg Leu Val Leu
Pro Glu Pro Asp Asp Ser Ser Ala Ala Leu Leu 275 280 285His Gln Ala
Pro Pro Gly Thr Thr Glu Gly Asn Tyr Ser Thr Met Gln 290 295 300His
Thr Ser Gln Phe Arg Phe Leu Asp Lys Ala Ala Ile Val Ala Ala305 310
315 320Ser Ser Gly Glu Lys Gly Ala Thr Ala Ser Pro Trp Arg Leu Cys
Thr 325 330 335Val Ser Gln Val Glu Glu Leu Lys Thr Val Leu Arg Met
Phe Pro Val 340 345 350Trp Val Ser Met Val Leu Phe Phe Ala Ala Thr
Ala Gln Met Ser Ser 355 360 365Thr Phe Ile Glu Gln Gly Glu Thr Ile
Asp Asn Arg Val Gly Pro Phe 370 375 380Thr Val Pro Pro Ala Ser Leu
Ser Thr Phe Asp Val Ile Ser Val Met385 390 395 400Val Cys Ile Pro
Ile Tyr Asp Lys Ala Leu Val Pro Leu Ala Arg Arg 405 410 415Ala Thr
Gly Lys Glu Arg Gly Leu Ser Gln Leu Gln Arg Leu Gly Val 420 425
430Gly Leu Ala Leu Ser Val Ala Gly Met Val Tyr Ala Ala Leu Leu Glu
435 440 445Ala Arg Arg Leu Ser Leu Ala Arg Ala Ala Ala Gly Gly Arg
Pro Pro 450 455 460Met Ser Ile Met Trp Gln Ala Pro Ala Phe Ala Val
Leu Gly Ala Gly465 470 475 480Glu Val Phe Ala Thr Ile Gly Ile Leu
Glu Phe Phe Tyr Asp Gln Ser 485 490 495Pro Asp Gly Met Lys Ser Leu
Gly Thr Ala Leu Ala Gln Leu Ala Val 500 505 510Ala Ala Gly Asn Tyr
Phe Asn Ser Ala Val Leu Xaa Ala Val Ala Ala 515 520 525Val Thr Thr
Arg Asn Gly Glu Ala Gly Trp Ile Pro Asp Asp Leu Asp 530 535 540Lys
Gly His Leu Asp Tyr Phe Phe Trp Phe Met Ala Val Leu Gly Val545 550
555 560Val Asn Leu Leu His Phe Leu His Cys Ser Val Arg Tyr Arg Gly
Ser 565 570 575Ser Asn Asn Ser Thr Tyr Ser Ser 58017596PRTPrunus
persicamisc_feature(1)..(596)Public GI no. 33411520 17Met Ser Asn
Cys Thr Leu Pro Glu Thr Gln Glu Lys Leu Thr Leu Pro1 5 10 15Asp Ala
Trp Asp Phe Lys Gly Arg Pro Ala Glu Arg Ser Lys Thr Gly 20 25 30Gly
Trp Thr Ala Ala Ala Met Ile Leu Gly Gly Glu Ala Cys Glu Arg 35 40
45Leu Thr Thr Leu Gly Ile Ala Val Asn Leu Val Thr Tyr Leu Thr Gly
50 55 60Thr Met His Leu Gly Asn Ala Thr Ser Ala Asn Thr Val Thr Asn
Phe65 70 75 80Leu Gly Thr Ser Phe Met Leu Cys Leu Leu Gly Gly Phe
Val Ala Asp 85 90 95Thr Phe Leu Gly Arg Tyr Leu Thr Ile Ala Ile Phe
Ala Thr Phe Gln 100 105 110Ala Met Gly Val Thr Ile Leu Thr Ile Ser
Thr Thr Ile Pro Ser Leu 115 120 125Arg Pro Pro Lys Cys Thr Ser Asp
Thr Ser Thr Pro Cys Ile Pro Ala 130 135 140Ser Gly Lys Gln Leu Met
Val Leu Tyr Ile Ala Leu Tyr Leu Thr Ala145 150 155 160Leu Gly Thr
Gly Gly Leu Lys Ser Ser Val Ser Gly Phe Gly Ser Asp 165 170 175Gln
Phe Asp Glu Ser Asp Lys Gln Glu Arg Arg Gln Met Thr Asn Phe 180 185
190Phe Asn Trp Phe Phe Phe Phe Ile Ser Ile Gly Ser Leu Ala Ala Val
195 200 205Thr Val Leu Val Tyr Ile Gln Asp Asn Leu Gly Arg Gln Trp
Gly Tyr 210 215 220Gly Ile Cys Val Cys Ala Ile Val Leu Gly Leu Ile
Val Phe Leu Ser225 230 235 240Gly Thr Arg Arg Tyr Arg Phe Lys Lys
Leu Val Gly Ser Pro Leu Thr 245 250 255Gln Ile Ser Gly Val Cys Val
Ala Ala Trp Arg Lys Arg Asn Met Glu 260 265 270Leu Pro Ser Asp Met
Ser Phe Leu Tyr Asn Val Asp Asp Ile Asp Asp 275 280 285Gly Leu Lys
Lys Lys Lys Lys Gln Lys Leu Pro His Ser Lys Gln Phe 290 295 300Arg
Phe Leu Asp Lys Ala Ala Ile Lys Glu Pro Lys Thr Thr Ser Gly305 310
315 320Thr Ala Met Ile Ile Asn Lys Trp Ser Leu Ser Thr Leu Thr Asp
Val 325 330 335Glu Glu Val Lys Leu Ile Ile Arg Met Leu Pro Ile Trp
Ala Thr Thr 340 345 350Ile Met Phe Trp Thr Val Tyr Ala Gln Met Thr
Thr Phe Ser Val Ser 355 360 365Gln Ala Thr Ser Met Asp Arg His Ile
Gly Lys Ser Phe Gln Ile Pro 370 375 380Pro Ala Ser Leu Thr Ala Phe
Phe Val Gly Ser Ile Leu Leu Thr Val385 390 395 400Pro Val Tyr Asp
Arg Leu Ile Val Pro Met Ala Arg Lys Ala Leu Glu 405 410 415Asn Pro
Gln Gly Leu Thr Pro Leu Gln Arg Met Gly Val Gly Leu Val 420 425
430Phe Ser Ile Phe Ala Met Val Ala Ala Ala Leu Thr Glu Val Lys Arg
435 440 445Leu Asn Ile Ala Arg Ser His Gly Leu Thr Asp Asn Pro Thr
Ala Glu 450 455 460Ile Pro Leu Ser Val Phe Trp Leu Val Pro Gln Phe
Phe Phe Val Gly465 470 475 480Ser Gly Glu Ala Phe Thr Tyr Ile Gly
Gln Leu Asp Phe Phe Leu Arg 485 490 495Glu Cys Pro Lys Gly Met Lys
Thr Met Ser Thr Gly Leu Phe Leu Ser 500 505 510Thr Leu Ser Leu Gly
Phe Phe Phe Ser Ser Leu Leu Val Thr Ile Val 515 520 525His Lys Thr
Thr Gly His Asn Lys Pro Trp Leu Ala Asp Asn Leu Asn 530 535 540Gln
Gly Lys Leu Tyr Asp Phe Tyr Trp Leu Leu Ala Leu Leu Ser Ala545 550
555 560Leu Asn Leu Val Ile Tyr Leu Phe Cys Ala Asn Trp Tyr Val Tyr
Lys 565 570 575Asp Lys Arg Leu Ala Glu Glu Gly Ile Glu Leu Glu Glu
Pro Glu Ile 580 585 590Cys Ala His Ala 59518576PRTOryza sativa
(japonica cultivar-group)misc_feature(1)..(576)Public GI no.
31429847 18Met Ala Ala Ile Glu Glu Glu Arg Pro Leu Leu Pro Leu Gln
Ser Gln1 5 10 15Asp Val Gly Ser Glu Tyr Thr Arg Asp Gly Ser Val Asp
Ile Asn Lys 20 25 30Glu Pro Ala Leu Lys His Ser Thr Gly Asn Trp Arg
Ala Cys Phe Leu 35 40 45Ile Leu Gly Val Glu Phe Cys Glu Asn Met Thr
Tyr Phe Val Ile Ser 50 55 60Arg Asn Leu Val Thr Phe Leu Thr Thr Val
Leu His Glu Ser Lys Val65 70 75 80Asp Ala Ala Arg Asn Val Ser Ala
Trp Val Gly Ala Cys Phe Leu Thr 85 90 95Pro Val Val Gly Ala Phe Leu
Ala Asp Thr Tyr Trp Gly Arg Tyr Trp 100 105 110Thr Ile Val Val Phe
Leu Pro Val Tyr Ile Thr Gly Met Leu Ile Val 115 120 125Thr Val Ser
Ala Ser Leu Pro Met Phe Leu Thr Ser Ser Glu His Gly 130 135 140Asn
Val His Arg Ser Val Val Tyr Leu Gly Leu Tyr Leu Ala Ala Leu145 150
155 160Gly Ser Gly Ala Met Lys Pro Cys Thr Ser Ser Phe Gly Ala Asp
Gln 165 170 175Phe Asp Ser Thr Asp Leu Glu Glu Leu Pro Lys Lys Ala
Ser Phe Phe 180 185 190Ser Trp Ser Phe Tyr Met Thr Thr Val Ser Thr
Leu Leu Ser Ser Thr 195 200 205Val Leu Val Trp Leu Gln Asp Asn Val
Gly Trp Gly Val Gly Cys Ala 210 215 220Ile Pro Thr Val Phe Met Ile
Ile Ser Phe Pro Val Phe Ile Ala Gly225 230 235 240Ser Arg Val Tyr
Arg Phe Arg Asn Leu Gly Phe Ser Pro Leu Lys Ser 245 250 255Leu Cys
Gln Val Ile Val Ala Ala Val Arg Lys Cys His Leu Gln Leu 260 265
270Pro Glu Asn Lys Ser Leu Leu Tyr Glu Pro Ser Asn Ser Ser Ser Thr
275 280 285Thr Glu Ala Ser His Lys Ile Gln Pro Thr Asn Gln Phe Arg
Phe Leu 290 295 300Asp Lys Ala Ala Ile Val Leu Pro Pro Ser Asp Glu
Thr Cys Ile Lys305 310 315 320Pro Met Ser Ser Trp Ser Leu Cys Thr
Val Thr Gln Val Glu Glu Leu 325 330 335Lys Met Leu Leu Arg Met Phe
Pro Thr Trp Ala Ser Phe Val Ile Phe 340 345 350Phe Ala Val Asn Gly
Gln Met Ser Ser Thr Phe Ile Glu Gln Gly Met 355 360 365Ala Met Asp
Asn His Val Gly Ser Phe Ala Ile Pro Pro Ala Ser Leu 370 375 380Thr
Ile Ile Ala Val Leu Ser Val Leu Val Leu Val Pro Val Tyr Glu385 390
395 400Ile Ile Ser Val Pro Leu Val Lys His Phe Thr Gly Gln Asp Lys
Gly 405 410 415Phe Ser His Ala Gln Arg Ile Gly Ile Gly Leu Ser Leu
Ser Met Ile 420 425 430Met Met Val Tyr Ala Ala Leu Leu Glu Met Lys
Arg Leu Ala Ile Val 435 440 445Gln Ser Ser Gly Leu Ala Asp His Asn
Val Ala Ala Pro Met Ser Ile 450 455 460Leu Trp Gln Thr Pro Ala Tyr
Phe Leu Gln Gly Val Ser Glu Ile Phe465 470 475 480Ser Cys Ile Gly
Met Ser Gln Phe Phe Tyr Asp Gln Ala Pro Asp Ser 485 490 495Met Lys
Ser Val Cys Ala Ala Leu Gly Gln Leu Ala Ile Ala Ser Gly 500 505
510Ala Tyr Phe Asn Thr Phe Val Leu Gly Ala Val Ala Val Ile Thr Thr
515 520 525Ser Ser Gly Ala Pro Gly Trp Ile Pro Asp Asn Leu Asn Glu
Gly His 530 535 540Leu Asp Tyr Phe Phe Trp Met Met Ala Thr Leu Ser
Leu Leu Asn Leu545 550 555 560Ala Met Phe Val Tyr Ser Ser Thr Arg
His Arg Glu Asn Thr Ala Ser 565 570
575191954DNAArabidopsis thalianamisc_feature(1)..(1954)Ceres
Promoter p326 19gtgggtaaaa gtatccttct ttgtgcattt ggtattttta
agcatgtaat aagaaaaacc 60aaaatagacg gctggtattt aataaaagga gactaatgta
tgtatagtat atgatttgtg 120tggaatataa taaagttgta aaatatagat
gtgaagcgag tatctatctt ttgactttca 180aaggtgatcg atcgtgttct
ttgtgatagt tttggtcgtc ggtctacaag tcaacaacca 240ccttgaagtt
ttcgcgtctc ggtttcctct tcgcatctgg tatccaatag catacatata
300ccagtgcgga aaatggcgaa gactagtggg cttgaaccat aaggtttggc
cccaatacgg 360attccaaaca acaagcctag cgcagtcttt tgggatgcat
aagactaaac tgtcgcagtg 420atagacgtaa gatatatcga cttgattgga
atcgtctaag ctaataagtt taccttgacc 480gtttatagtt gcgtcaacgt
ccttatggag attgatgccc atcaaataaa cctgaaaatc 540catcaccatg
accaccataa actcccttgc tgccgctgct ttggcttgag caaggtgttt
600ccttgtaaag ctccgatctt tggataaagt gttccacttt ttgcaagtag
ctctgacccc 660tctcagagat gtcaccggaa tcttagacag aacctcctct
gccaaatcac ttggaagatc 720ggacaatgtc atcatttttg caggtaattt
ctccttcgtt gctgctttgg cttgagcacg 780gtgcttcttt gtaaagctcc
gatctttgga taagagcgga tcggaatcct ctaggaggtg 840ccagtccctt
gacctattaa tttatagaag gttttagtgt attttgttcc aatttcttct
900ctaacttaac aaataacaac tgcctcatag tcatgggctt caaattttat
cgcttggtgt 960atttcgttat ttgcaaggcc ttggcccatt ttgagcccaa
taactaaatc tagccttttc 1020agaccggaca tgaacttcgc atattggcgt
aactgtgcag ttttaccttt ttcggatcag 1080acaagatcag atttagacca
cccaacaata gtcagtcata tttgacaacc taagctagcc 1140gacactacta
aaaagcaaac aaaagaagaa ttctatgttg tcattttacc ggtggcaagt
1200ggacccttct ataaaagagt aaagagacag cctgtgtgtg tataatctct
aattatgttc 1260accgacacaa tcacacaaac ccttctctaa tcacacaact
tcttcatgat ttacgacatt 1320aattatcatt aactctttaa attcacttta
catgctcaaa aatatctaat ttgcagcatt 1380aatttgagta ccgataacta
ttattataat cgtcgtgatt cgcaatcttc ttcattagat 1440gctgtcaagt
tgtactcgca cgcggtggtc cagtgaagca aatccaacgg tttaaaacct
1500tcttacattt ctagatctaa tctgaaccgt cagatatcta gatctcattg
tctgaacaca 1560gttagatgaa actgggaatg aatctggacg aaattacgat
cttacaccaa ccccctcgac 1620gagctcgtat atataaagct tatacgctcc
tccttcacct tcgtactact actaccacca 1680catttcttta gctcaacctt
cattactaat ctccttttaa ggtatgttca cttttcttcg 1740attcatactt
tctcaagatt cctgcatttc tgtagaattt gaaccaagtg tcgatttttg
1800tttgagagaa gtgttgattt atagatctgg ttattgaatc tagattccaa
tttttaattg 1860attcgagttt gttatgtgtg tttatactac ttctcattga
tcttgtttga tttctctgct 1920ctgtattagg tttctttcgt gaatcagatc ggaa
195420881DNAArabidopsis thalianamisc_feature(1)..(881)Ceres
Promoter YP0144 20ggaaagaagc ggtttatgcg ctgcgcataa cactattatg
tctcgggaga acaaagatgg 60aagcaagagc ggtttgattg gaccgggact ctttagtggc
cttgtttttg gctctacttc 120tgatcattct cagtctggag ctagcgctgt
ctctgattgt actgattctg ttgaacgaat 180acagtttgag aataggcaga
agaacaagaa gatgatgata ccgatgcagg ttctagtacc 240ttcatcaatg
aaatctccaa gtaattcaca tgaaggagaa acaaacatct atgacttcat
300ggttccggag gagagagttc acggcggtgg gctagtaatg tctttacttg
gtggctccat 360tgatcgaaac tgaaagccat ttatggtaaa agtgtcacat
tctcagcaaa aacctgtgta 420aagctgtaaa atgtgtggga atctccgaat
ctgtttgtag ccggttacgt tatgctggat 480caaaaactca agatttgttg
gatattgtta tgctggatcg gtggtgaaac cacttcccgg 540ttgctaaata
aataaacgtt tttgttttat aatctttttc actaaacggc agtatgggcc
600tttagtgggc ttcctttaag cgaccaatac aatcgtcgca ccggaatcta
ctaccattta 660taggtttatt catgtaaaac ctcggaaaat ttgagagcca
caacggtcaa gagacaaaaa 720caacttgaag ataaagggat aaggaaggct
tcctacatga tggacaacat ttctttccac 780acaaattctc ataataaaaa
tcttataata caaatactta cgtcataatc attcaatcta 840gtccccatgt
tttaaggtcc tgtttcttgt ctgatacaaa t 881211002DNAArabidopsis
thalianamisc_feature(1)..(1002)Ceres Promoter YP0190 21taaatagtga
cattggtaag aagaaaaaaa acactattaa atagtgaaaa aatggtttat 60aactctctta
attaacatta cttattattg ctagcaccta aaatctccca caaaatattt
120gttgtaaaac acaaatttac aaaatgattt tgtttttaaa ttagtaacac
atgttcatat 180atacgttaat aagaacatac cctatatgat tttatataaa
aaaatttctt tgagacgtct 240tattcttttt tctttaataa tatgcaattg
tgagagtttg gatttgaatg gtagcattag 300aagcaaactt gaaccaaaca
tatttcatga agtcaaactt gaaccaatgt gatcactaat 360cacagtgttc
gcagtgtaag gcatcagaaa atagaagaag ggacatagct atgaatcata
420taatcttgac acatgtttta taggttttag gtgtgtatgc taacaaaaaa
tgagacagct 480ttcttctaat agacttaata tttgggctaa atgtaccaca
gttgtgaatt tcttacaaaa 540atgggccgag ctacaaaaaa ctacaggccc
actctcaact cttatcaaac gacagcgttt 600tactttttta aaagcacaca
ctttttgttt ggtgtcggtg acggtgagtt tcgtccgctc 660ttcctttaaa
ttgaagcaac ggttttgatc cgatcaaatc caacggtgct gattacacaa
720agcccgagac gaaaacgttg actattaagt taggttttaa tctcagccgt
taatctacaa 780atcaacggtt ccctgtaaaa cgaatcttcc ttccttcttc
acttccgcgt cttctctctc 840aatcacctca aaaaaatcga tttcatcaaa
atattcaccc gcccgaattt gactctccga 900tcatcgtctc cgaatctaga
tcgacgagat caaaacccta gaaatctaaa tcggaatgag 960aaattgattt
tgatacgaat tagggatctg tgtgttgagg ac 1002221514DNAArabidopsis
thalianamisc_feature(1)..(1514)Ceres Promoter p13879 22tttcgatcct
cttctttttt aggtttcttg atttgatgat cgccgccagt agagccgtcg 60tcggaagttt
cagagattaa aaccatcacc gtgtgagttg gtagcgaatt aacggaaagt
120ctaagtcaag attttttaaa aagaaattta tgtgtgaaaa gaagccgttg
tgtatattta 180tataatttag aaaatgtttc atcattttaa ttaaaaaatt
aataatttgt agaagaaaga 240agcatttttt atacataaat catttacctt
ctttactgtg tttttcttca cttacttcat 300ttttactttt ttacaaaaaa
gtgaaaagta aattacgtaa ttggtaacat aaattcactt 360taaatttgca
tatgttttgt tttcttcgga aactatatcg aaaagcaaac ggaaagaact
420tcacaaaaaa ccctagctaa ctaaagacgc atgtgttctt cttattcttc
atatatcctc 480tgtttcttgt gttctgtttt gagtcttaca ttttcaatat
ctgactctga ttactatatc 540taaaagggaa catgaagaac ttgagaccat
gttaaactgt acaatgcctt caaacatggc 600taactaaaga tacattagat
ggctttacag tgtgtaatgc ttattatctt taggtttttt 660aaatcccttg
tattaagtta tttaccaaat tatgttcttg tactgcttat tggcttggtt
720gttgtgtgct ttgtaaacaa cacctttggc tttatttcat cctttgtaaa
cctactggtc 780tttgttcagc tcctcttgga agtgagtttg tatgcctgga
acgggtttta atggagtgtt 840tatcgacaaa aaaaaaatgt agcttttgaa
atcacagaga gtagttttat attcaaatta 900catgcatgca actaagtagc
aacaaagttg atatggccga gttggtctaa ggcgccagat 960taaggttctg
gtccgaaagg gcgtgggttc aaatcccact gtcaacattc tctttttctc
1020aaattaatat ttttctgcct caatggttca ggcccaatta tactagacta
ctatcgcgac 1080taaaataggg actagccgaa ttgatccggc ccagtatcag
ttgtgtatca ccacgttatt 1140tcaaatttca aactaaggga taaagatgtc
atttgacata tgagatattt ttttgctcca 1200ctgagatatt tttctttgtc
ccaagataaa atatcttttc tcgcatcgtc gtctttccat 1260ttgcgcatta
aaccaaaaag tgtcacgtga tatgtcccca accactacga attttaacta
1320cagatttaac catggttaaa ccagaattca cgtaaaccga ctctaaacct
agaaaatatc 1380taaaccttgg ttaatatctc agccccctta taaataacga
gacttcgtct acatcgttct 1440acacatctca ctgctcacta ctctcactgt
aatcccttag atcttctttt caaatttcac 1500cattgcactg gatg
1514231026DNAArabidopsis thalianamisc_feature(1)..(1026)Ceres
Promoter YP0050 23aatctgatct ctagtccagt cgattggtac ttgagggaaa
catcatattt ttaaaccttg 60tctcagtaag ctaacacaca ccccttgtga ttacttatcc
atgtttatcc acaagaatgc 120agttggattg agatattttc ttctttgttg
aaatcaggcc tcaaggtgtt catgtggtct 180gcaaaaaaat tcccaaaaat
aaagatagtg acatctgaaa tcgataatgg attagacgaa 240gagtttcgtg
ttattccttg gtatgggcgg gtttggggac agatattttg gcacagacga
300ggactaggcc actgtggtcc tgcagcatta ggtgtccctt ccatgtcctg
cattacattt 360tattgatgga ttcatcaccc tatctactac aacggctaca
caaactatga agagttttgt 420ttactaataa atgcccaagt gaggggtcga
tcgaacccgg gacacgtttt tcagtttacc 480atatagaatt atccttggaa
cccttgatac tccatagaac atcaccacct ctgttgtcat 540ctcaggaatc
caggttcaaa cctagtctct ctctccctag tgggaggtat atggccactg
600ggccaatgat gacaaaatgc aaaaaaaata aaatacattt gggttcatta
tctaaaatat 660ctcttgtgtt tgtaagtttt ggttgcacac tcgtgtggtt
gaagtgtgtg tgagaggtac 720tatacaatac actctgcttt tgttttgtac
ctatctcttt ctcttctcca catatccaag 780actttgggga taaagctgag
atcattggtt gccatttggt tgtgtagaag caatcaccca 840tttgctttat
ccgaggttga taaatttcct cgggttctcc ttctgacacg tatgacaaat
900tctaatagta tattcctcgt agatattacc tatatattct caatagttgc
aggtacttaa 960ggctttgtct tggcatcctc gtcctcttca gcaaaactcg
tctctcttgc actccaaaaa 1020gcaacc 1026242016DNAArabidopsis
thalianamisc_feature(1)..(2016)Ceres Promoter p32449 24gatcggcctt
cttcaggtct tctctgtagc tctgttactt ctatcacagt tatcgggtat 60ttgagaaaaa
agagttagct aaaatgaatt tctccatata atcatggttt actacaggtt
120tacttgattc gcgttagctt tatctgcatc caaagttttt tccatgatgt
tatgtcatat 180gtgataccgt tactatgttt ataactttat acagtctggt
tcactggagt ttctgtgatt 240atgttgagta catactcatt catcctttgg
taactctcaa gtttaggttg tttgaattgc 300ctctgttgtg atacttattg
tctattgcat caatcttcta atgcaccacc ctagactatt 360tgaacaaaga
gctgtttcat tcttaaacct ctgtgtctcc ttgctaaatg gtcatgcttt
420aatgtcttca cctgtctttc tcttctatag atatgtagtc ttgctagata
gttagttcta 480cagctctctt ttgtagtctt gttagagagt tagttgagat
attacctctt aaaagtatcc 540ttgaacgctt tccggttatg accaatttgt
tgtagctcct tgtaagtaga acttactggg 600accagcgaga cagtttatgt
gaatgttcat gcttaagtgt cgaacgtatc tatctctact 660atagctctgt
agtcttgtta gacagttagt tttatatctc catttttttg tagtcttgct
720agttgagata ttacctcttc tcttcaaagt atccttgaac gctcaccggt
tatgaaatct 780ctacactata gctctgtagt cttgctagat agttagttct
ttagctctct ttttgtagcc 840tagttcttta gctctccttt tgtagccttg
ctacagagta agatgggata ttacctcctt 900gaacgctctc cggttatgac
caatttgttg tagctccttg taagtagaac ttaggataga 960gtgagtcaac
tttaagaaag aacctagtat gtggcataac cagattgcag gctctgtctc
1020ggctacagta acgtaactct atagctcttt gttttgttca gaaagaacca
gtgattggat 1080gattcgtcct tagaaactgg acctaacaac agtcattggc
tttgaaatca agccacaaca 1140atgcctatat gaaccgtcca tttcatttat
ccgtttcaaa ccagcccatt acatttcgtc 1200ccattgataa ccaaaagcgg
ttcaatcaga ttatgtttta attttaccaa attctttatg 1260aagtttaaat
tatactcaca ttaaaaggat tattggataa tgtaaaaatt ctgaacaatt
1320actgattttg gaaaattaac aaatattctt tgaaatagaa gaaaaagcct
ttttcctttt 1380gacaacaaca tataaaatca tactcccatt aaaaagattt
taatgtaaaa ttctgaatat 1440aagatatttt ttacaacaac aaccaaaaat
atttattttt ttcctttttt acagcaacaa 1500gaaggaaaaa cttttttttt
tgtcaagaaa aggggagatt atgtaaacag ataaaacagg 1560gaaaataact
aaccgaactc tcttaattaa catcttcaaa taaggaaaat tatgatccgc
1620atatttagga agatcaatgc attaaaacaa cttgcacgtg gaaagagaga
ctatacgctc 1680cacacaagtt gcactaatgg tacctctcac aaaccaatca
aaatactgaa taatgccaac 1740gtgtacaaat tagggtttta cctcacaacc
atcgaacatt ctcgaaacat tttaaacagc 1800ctggcgccat agatctaaac
tctcatcgac caatttttga ccgtccgatg gaaactctag 1860cctcaaccca
aaactctata taaagaaatc ttttccttcg ttattgctta ccaaatacaa
1920accctagccg ccttattcgt cttcttcgtt ctctagtttt ttcctcagtc
tctgttctta 1980gatcccttgt agtttccaaa tcttccgata aggcct
2016251823DNAArabidopsis thalianamisc_feature(1)..(1823)Ceres
Promoter 21876 25gtctcttaaa aaggatgaac aaacacgaaa ctggtggatt
atacaaatgt cgccttatac 60atatatcggt tattggccaa aagagctatt ttaccttatg
gataatggtg ctactatggt 120tggagttgga ggtgtagttc aggcttcacc
ttctggttta agccctccaa tgggtaatgg 180taaatttccg gcaaaaggtc
ctttgagatc agccatgttt tccaatgttg aggtcttata 240ttccaagtat
gagaaaggta aaataaatgc gtttcctata gtggagttgc tagatagtag
300tagatgttat gggctacgaa ttggtaagag agttcgattt tggactagtc
cactcggata 360ctttttcaat tatggtggtc ctggaggaat ctcttgtgga
gtttgatatt tgcgagtata 420atctttgaac ttgtgtagat tgtacccaaa
accgaaaaca tatcctatat aaatttcatt 480atgagagtaa aattgtttgt
tttatgtatc atttctcaac tgtgattgag ttgactattg 540aaaacatatc
ttagataagt ttcgttatga gagttaatga tgattgatga catacacact
600cctttatgat ggtgattcaa cgttttggag aaaatttatt tataatctct
cataaattct 660ccgttattag ttgaataaaa tcttaaatgt ctcctttaac
catagcaaac caacttaaaa 720atttagattt taaagttaag atggatattg
tgattcaacg attaattatc gtaatgcata 780ttgattatgt aaaataaaat
ctaactaccg gaatttattc aataactcca ttgtgtgact 840gcatttaaat
atatgtttta tgtcccatta attaggctgt aatttcgatt tatcaattta
900tatactagta ttaatttaat tccatagatt tatcaaagcc aactcatgac
ggctagggtt 960ttccgtcacc ttttcgatca tcaagagagt ttttttataa
aaaaatttat acaattatac 1020aatttcttaa ccaaacaaca cataattata
agctatttaa catttcaaat tgaaaaaaaa 1080aatgtatgag aattttgtgg
atccattttt gtaattcttt gttgggtaaa ttcacaacca 1140aaaaaataga
aaggcccaaa acgcgtaagg gcaaattagt aaaagtagaa ccacaaagag
1200aaagcgaaaa ccctagacac ctcgtagcta taagtaccct cgagtcgacc
aggattaggg 1260tgcgctctca tatttctcac attttcgtag ccgcaagact
cctttcagat tcttacttgc 1320aggttagata ttttctctct ttagtgtctc
cgatcttcat cttcttatga ttattgtagc 1380tgtttagggt ttagattctt
agttttagct ctatattgac tgtgattatc gcttattctt 1440tgctgttgtt
atactgcttt tgattctcta gctttagatc cgtttactcg tcgatcaata
1500ttgttcctat tgagtctgat gtataatcct ctgattaatt gatagcgttt
agttttgata 1560tcgtcttcgc atgtttttta tcatgtcgat ctgtatctgc
tctggttata gttgattctg 1620atgtatttgg ttggtgatgt tccttagatt
tgatatacct gttgtctcgt ggtttgatat 1680gatagctcaa ctggtgatat
gtggttttgt ttcagtggat ctgtgtttga ttatattgtt 1740gacgttttgg
ttgttgtatg gttgatggtt gatgtatttt tgttgattct gatgtttcga
1800tttttgtttt tgttttgaca gct 1823
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