U.S. patent application number 13/465846 was filed with the patent office on 2012-08-23 for nucleotide sequences and polypeptides encoded thereby useful for increasing plant size and increasing the number and size of leaves.
This patent application is currently assigned to CERES, INC.. Invention is credited to David Vandinh Dang, Kenneth FELDMANN, Shing Kwok, Roger Pennell, Hongyu Zhang.
Application Number | 20120216317 13/465846 |
Document ID | / |
Family ID | 34215338 |
Filed Date | 2012-08-23 |
United States Patent
Application |
20120216317 |
Kind Code |
A1 |
FELDMANN; Kenneth ; et
al. |
August 23, 2012 |
NUCLEOTIDE SEQUENCES AND POLYPEPTIDES ENCODED THEREBY USEFUL FOR
INCREASING PLANT SIZE AND INCREASING THE NUMBER AND SIZE OF
LEAVES
Abstract
Isolated polynucleotides and polypeptides encoded thereby are
described, together with the use of those products for making
transgenic plants that are characterized by increased size, have an
increased number and size of rosette leaves and are
late-flowering.
Inventors: |
FELDMANN; Kenneth; (Tucson,
AZ) ; Pennell; Roger; (Malibu, CA) ; Kwok;
Shing; (Woodland Hills, CA) ; Dang; David
Vandinh; (San Diego, CA) ; Zhang; Hongyu;
(Thousand Oaks, CA) |
Assignee: |
CERES, INC.
Thousand Oaks
CA
|
Family ID: |
34215338 |
Appl. No.: |
13/465846 |
Filed: |
May 7, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10572827 |
Mar 7, 2007 |
8193409 |
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PCT/US2003/025997 |
Aug 18, 2003 |
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13465846 |
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Current U.S.
Class: |
800/290 ;
435/320.1; 435/419; 435/468; 435/6.12; 506/9; 530/376; 536/23.6;
800/278; 800/298 |
Current CPC
Class: |
C12N 15/8261 20130101;
Y02A 40/146 20180101; C07K 14/415 20130101 |
Class at
Publication: |
800/290 ;
536/23.6; 530/376; 435/320.1; 800/278; 800/298; 435/419; 435/468;
506/9; 435/6.12 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C07K 14/415 20060101 C07K014/415; C12Q 1/68 20060101
C12Q001/68; A01H 5/10 20060101 A01H005/10; C12N 5/10 20060101
C12N005/10; C40B 30/04 20060101 C40B030/04; C12N 15/29 20060101
C12N015/29; A01H 5/00 20060101 A01H005/00 |
Claims
1. An isolated nucleic acid molecule comprising: a) a nucleic acid
having a nucleotide sequence which encodes an amino acid sequence
exhibiting at least 85% sequence identity to an amino acid sequence
according to any one of SEQ ID NOS. 3, 5, 7, 10, 12, 14, 17, 19,
21, 24, 26, 28, 31, 34, 36, 38, 41, 43, 45, 48 and 49; b) a nucleic
acid which is a complement of a nucleotide sequence according to
paragraph (a); c) a nucleic acid which is the reverse of the
nucleotide sequence according to subparagraph (a), such that the
reverse nucleotide sequence has a sequence order which is the
reverse of the sequence order of the nucleotide sequence according
to subparagraph (a); or d) a nucleic acid capable of hybridising to
a nucleic acid according to any one of paragraphs (a)-(c), under
conditions that permit formation of a nucleic acid duplex at a
temperature from about 40.degree. C. and 48.degree. C. below the
melting temperature of the nucleic acid duplex.
2. The isolated nucleic acid molecule according to claim 1, which
has the sequence according to any one of SEQ ID NOS. 1, 2, 4, 6, 8,
9, 11, 13, 15, 16, 18, 20, 22, 23, 35, 27, 29, 30, 32, 33, 35, 37,
39, 40, 42, 44 and 46.
3. The isolated nucleic acid molecule according to claim 1, wherein
said amino acid sequence comprises the sequence according to SEQ ID
NOS. 3, 5, 7, 10, 12, 14, 17, 19, 21, 24, 26, 28, 31, 34, 36, 38,
41, 43, 45, 48 and 49.
4. The isolated nucleic acid molecule of claim 1, wherein said
amino acid has the structure according to SEQ ID NO. 48 or 49.
5. A vector construct comprising: a) a first nucleic acid having a
regulatory sequence capable of causing transcription and/or
translation in a plant; and b) a second nucleic acid having the
sequence of the isolated nucleic acid molecule according to claim
1; wherein said first and second nucleic acids are operably linked
and wherein said second nucleic acid is heterologous to any element
in said vector construct.
6. The vector construct according to claim 5, wherein said first
nucleic acid is native to said second nucleic acid.
7. The vector construct according to claim 5, wherein said first
nucleic acid is heterologous to said second nucleic acid.
8. A host cell comprising an isolated nucleic acid molecule
according to claim 1, wherein said nucleic acid molecule is flanked
by exogenous sequence.
9. A host cell comprising a vector construct according to claim
5.
10. An isolated polypeptide comprising an amino acid sequence
exhibiting at least 85% sequence identity of an amino acid sequence
according to any one of SEQ ID Nos. 3, 5, 7, 10, 12, 14, 17, 19,
21, 24, 26, 28, 31, 34, 36, 38, 41, 43, 45, 48 and 49, and capable
of causing a plant to have an increased size or an increased number
and size of rosette leaves as compared to a wild type-plant.
11. A method of introducing an isolated nucleic acid into a host
cell comprising: a) providing an isolated nucleic acid molecule
according to claim 1; and b) contacting said isolated nucleic with
said host cell under conditions that permit insertion of said
nucleic acid into said host cell.
12. A method of transforming a host cell which comprises contacting
a host cell with a vector construct according to claim 5.
13. A method of modulating the flowering time or size of a plant,
or the size or number of rosette leaves of a plant comprising
transforming said plant with a nucleic acid molecule according to
claim 1 or a vector according to claim 5.
14. A method of increasing the size of a plant comprising
transforming said plant with a nucleic acid molecule according to
claim 1 or a vector according to claim 5.
15. A method of increasing the size or number of rosette leaves of
a plant comprising transforming said plant with a nucleic acid
molecule according to claim 1 or a vector according to claim 5.
16. A method for increasing the size of a plant, or the size or
number of rosette leaves, comprising transforming a plant with a
nucleic acid molecule that codes for a polypeptide according to SEQ
ID NO. 48 or 49.
17. A method for detecting a nucleic acid in a sample which
comprises: a) providing an isolated nucleic acid molecule according
to claim 1; b) contacting said isolated nucleic acid molecule with
a sample under conditions which permit a comparison of the sequence
of said isolated nucleic acid molecule with the sequence of DNA in
said sample; and c) analyzing the result of said comparison.
18. A plant, plant cell, plant material or seed of a plant which
comprises a nucleic acid molecule according to claim 1 which is
exogenous or heterologous to said plant or plant cell.
19. A plant, plant cell, plant material or seed of a plant which
comprises a vector construct according to claim 5.
20. A plant which has been regenerated from a plant cell or seed
according to claim 18.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to isolated polynucleotides,
polypeptides encoded thereby, and the use of those products for
making transgenic plants that are characterized by increased size,
have an increased number and size of rosette leaves and are
late-flowering.
BACKGROUND OF THE INVENTION
[0002] There are more than 300,000 species of plants. They show a
wide diversity of forms, ranging from delicate liverworts, adapted
for life in a damp habitat, to cacti, capable of surviving in the
desert. The plant kingdom includes herbaceous plants, such as corn,
whose life cycle is measured in months, to the giant redwood tree,
which can live for thousands of years. This diversity reflects the
adaptations of plants to survive in a wide range of habitats. This
is seen most clearly in the flowering plants (phylum
Angiospermophyta), which are the most numerous, with over 250,000
species. They are also the most widespread, being found from the
tropics to the arctic.
[0003] The process of plant breeding involving man's intervention
in natural breeding and selection is some 20,000 years old. It has
produced remarkable advances in adapting existing species to serve
new purposes. The world's economics was largely based on the
successes of agriculture for most of these 20,000 years.
[0004] Plant breeding involves choosing parents, making crosses to
allow recombination of gene (alleles) and searching for and
selecting improved forms. Success depends on the genes/alleles
available, the combinations required and the ability to create and
find the correct combinations necessary to give the desired
properties to the plant. Molecular genetics technologies are now
capable of providing new genes, new alleles and the means of
creating and selecting plants with the new, desired
characteristics.
[0005] Great agronomic value can result from modulating the size of
a plant as a whole or of any of its organs. For example, the green
revolution came about as a result of creating dwarf wheat plants,
which produced a higher seed yield than taller plants because they
could withstand higher levels and inputs of fertilizer and water.
Modulation of the size and stature of an entire plant or a
particular portion of a plant allows productions of plants
specifically improved for agriculture, horticulture and other
industries. For example, reductions in height of specific
ornamentals, crops and tree species can be beneficial, while
increasing height of others may be beneficial.
[0006] Increasing the length of the floral stems of cut flowers in
some species would also be useful, while increasing leaf size in
others would be economically attractive. Enhancing the size of
specific plant parts, such as seeds and fruit, to enhance yields by
specifically stimulating hormone (e.g. Brassinolide) synthesis in
these cells is beneficial. Another application is to stimulate
early flowering by altering levels of gibberellic acid in specific
cells. Changes in organ size and biomass also results in changes in
the mass of constituent molecules.
[0007] To summarize, molecular genetic technologies provide the
ability to modulate and manipulate plant size and stature of the
entire plant as well as at the cell, tissue and organ levels. Thus,
plant morphology can be altered to maximize the desired plant
trait.
SUMMARY OF THE INVENTION
[0008] The present invention, therefore, relates to isolated
polynucleotides, polypeptides encoded thereby, and the use of those
products for making transgenic plants that are characterized by
increased size, have an increased number and size of rosette leaves
and are late-flowering, as compared to the non-transformed,
wild-type plant.
[0009] The present invention also relates to processes for
increasing the yield in plants, recombinant nucleic acid molecules
and polypeptides used for these processes, their uses as well as to
plants with an increased yield.
[0010] In the field of agriculture and forestry constantly efforts
are being made to produce plants with an increased yield, in
particular in order to guarantee the supply of the constantly
increasing world population with food and to guarantee the supply
of reproducible raw materials. Conventionally, it is tried to
obtain plants with an increased yield by breeding, which is,
however time-consuming and labor-intensive. Furthermore,
appropriate breeding programs have to be performed for each
relevant plant species.
[0011] Progress has partly been made by the genetic manipulation of
plants, that is by introducing into and expressing recombinant
nucleic acid molecules in plants. Such approaches have the
advantage of usually not being limited to one plant species but
being transferable to other plant species. In EP-A 0 511 979, e.g.,
it was described that the expression of a prokaryotic asparagine
synthetase in plant cells inter alia leads to an increased biomass
production. In WO 96/21737, e.g., the production of plants with an
increased yield by the expression of deregulated or unregulated
fructose-1,6-bisphosphatase due to the increase of the
photosynthesis rate is described. Nevertheless, there still is a
need of generally applicable processes for improving the yield in
plants interesting for agriculture or forestry. Therefore, the
present invention relates to a process for increasing the yield in
plants, characterized in that recombinant DNA molecules stably
integrated into the genome of plants are expressed.
[0012] It was surprisingly found that the expression of the
proteins according to the invention specifically leads to an
increase in yield.
[0013] The term "increase in yield" preferably relates to an
increase of the biomass production, in particular when determined
as the fresh weight of the plant. Such an increase in yield
preferably refers to the so-called "sink" organs of the plant,
which are the organs that take up the photoassimilates produced
during photosynthesis. Particularly preferred are parts of plants
which can be harvested, such as seeds, fruits, storage roots,
roots, tubers, flowers, buds, shoots, stems or wood. The increase
in yield according to the invention is at least 3% with regard to
the biomass in comparison to non-transformed plants of the same
genotype when cultivated under the same conditions, preferably at
least 10% and particularly preferred at least 20%.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 is a map of the DNA vector CRS 338 utilized in the
transformation procedures described herein.
BRIEF DESCRIPTION OF THE INDIVIDUAL TABLES
Table--Reference Tables
[0015] The sequences of the instant invention are described in the
Sequence Listing and the Reference Table (sometimes referred to as
the REF Table. The Reference Table refers to a number of "Maximum
Length Sequences" or "MLS." Each MLS corresponds to the longest
cDNA and is described in the Av subsection of the Reference
Table.
[0016] The Reference Table includes the following information
relating to each MLS:
[0017] I. cDNA Sequence [0018] A. 5' UTR [0019] B. Coding Sequence
[0020] C. 3' UTR
[0021] II. Genomic Sequence [0022] A. Exons [0023] B. Introns
[0024] C. Promoters
[0025] III. Link of cDNA Sequences to Clone IDs
[0026] IV. Multiple Transcription Start Sites
[0027] V. Polypeptide Sequences [0028] A. Signal Peptide [0029] B.
Domains [0030] C. Related Polypeptides
[0031] VI. Related Polynucleotide Sequences
[0032] I. cDNA Sequence
[0033] The Reference Table indicates which sequence in the Sequence
Table represents the sequence of each MLS. The MLS sequence can
comprise 5' and 3' UTR as well as coding sequences. In addition,
specific cDNA clone numbers also are included in the Reference
Table when the MLS sequence relates to a specific cDNA clone.
[0034] A. 5' UTR
[0035] The location of the 5' UTR can be determined by comparing
the most 5' MLS sequence with the corresponding genomic sequence as
indicated in the Reference Table. The sequence that matches,
beginning at any of the transcriptional start sites and ending at
the last nucleotide before any of the translational start sites
corresponds to the 5' UTR.
[0036] B. Coding Region
[0037] The coding region is the sequence in any open reading frame
found in the MLS. Coding regions of interest are indicated in the
PolyP SEQ subsection of the Reference Table.
[0038] C. 3' UTR
[0039] The location of the 3' UTR can be determined by comparing
the most 3' MLS sequence with the corresponding genomic sequence as
indicated in the Reference Table. The sequence that matches,
beginning at the translational stop site and ending at the last
nucleotide of the MLS corresponds to the 3' UTR.
[0040] II. Genomic Sequence
[0041] Further, the Reference Table indicates the specific "gi"
number of the genomic sequence if the sequence resides in a public
databank. For each genomic sequence, Reference tables indicate
which regions are included in the MLS. These regions can include
the 5' and 3' UTRs as well as the coding sequence of the MLS. See,
for example, the scheme below:
##STR00001##
[0042] The Reference Table reports the first and last base of each
region that are included in an MLS sequence. An example is shown
below:
[0043] gi No. 47000:
[0044] 37102 . . . 37497
[0045] 37593 . . . 37925
[0046] The numbers indicate that the MLS contains the following
sequences from two regions of gi No. 47000; a first region
including bases 37102-37497, and a second region including bases
37593-37925.
[0047] A. Exon Sequences
[0048] The location of the exons can be determined by comparing the
sequence of the regions from the genomic sequences with the
corresponding MLS sequence as indicated by the Reference Table.
[0049] i. Initial Exon
[0050] To determine the location of the initial exon, information
from the
[0051] (1) polypeptide sequence section;
[0052] (2) cDNA polynucleotide section; and
[0053] (3) the genomic sequence section
[0054] of the Reference Table is used. First, the polypeptide
section will indicate where the translational start site is located
in the MLS sequence. The MLS sequence can be matched to the genomic
sequence that corresponds to the MLS. Based on the match between
the MLS and corresponding genomic sequences, the location of the
translational start site can be determined in one of the regions of
the genomic sequence. The location of this translational start site
is the start of the first oxen.
[0055] Generally, the last base of the exon of the corresponding
genomic region, in which the translational start site was located,
will represent the end of the initial exon. In some cases, the
initial exon will end with a stop codon, when the initial exon is
the only exon.
[0056] In the case when sequences representing the MLS are in the
positive strand of the corresponding genomic sequence, the last
base will be a larger number than the first base. When the
sequences representing the MLS are in the negative strand of the
corresponding genomic sequence, then the last base will be a
smaller number than the first base.
[0057] ii. Internal Exons
[0058] Except for the regions that comprise the 5' and 3' UTRs,
initial exon, and terminal exon, the remaining genomic regions that
match the MLS sequence are the internal exons. Specifically, the
bases defining the boundaries of the remaining regions also define
the intron/exon junctions of the internal exons,
[0059] iii. Terminal Exon
[0060] As with the initial exon, the location of the terminal exon
is determined with information from the
[0061] (1) polypeptide sequence section;
[0062] (2) cDNA polynucleotide section; and
[0063] (3) the genomic sequence section
[0064] of the Reference Table. The polypeptide section will
indicate where the stop codon is located in the MLS sequence. The
MLS sequence can be matched to the corresponding genomic sequence.
Based on the match between MLS and corresponding genomic sequences,
the location of the stop codon can be determined in one of the
regions of the genomic sequence. The location of this stop codon is
the end of the terminal exon. Generally, the first base of the exon
of the corresponding genomic region that matches the cDNA sequence,
in which the stop codon was located, will represent the beginning
of the terminal exon. In some cases, the translational start site
will represent the start of the terminal exon, which will be the
only exon.
[0065] In the case when the MLS sequences are in the positive
strand of the corresponding genomic sequence, the last base will be
a larger number than the first base. When the MLS sequences are in
the negative strand of the corresponding genomic sequence, then the
last base will be a smaller number than the first base.
[0066] B. Intron Sequences
[0067] In addition, the introns corresponding to the MLS are
defined by identifying the genomic sequence located between the
regions where the genomic sequence comprises exons. Thus, introns
are defined as starting one base downstream of a genomic region
comprising an exon, and end one base upstream from a genomic region
comprising an exon.
[0068] C. Promoter Sequences
[0069] As indicated below, promoter sequences corresponding to the
MLS are defined as sequences upstream of the first exon; more
usually, as sequences upstream of the first of multiple
transcription start sites; even more usually as sequences about
2,000 nucleotides upstream of the first of multiple transcription
start sites.
[0070] III. Link of cDNA Sequences to Clone IDs
[0071] As noted above, the Reference Table identifies the cDNA
clone(s) that relate to each MLS. The MLS sequence can be longer
than the sequences included in the cDNA clones. In such a case, the
Reference Table indicates the region of the MLS that is included in
the clone. If either the 5' or 3' termini of the cDNA clone
sequence is the same as the MLS sequence, no mention will be
made.
[0072] IV. Multiple Transcription Start Sites
[0073] Initiation of transcription can occur at a number of sites
of the gene. The Reference Table indicates the possible multiple
transcription sites for each gene. In the Reference Table, the
location of the transcription start sites can be either a positive
or negative number.
[0074] The positions indicated by positive numbers refer to the
transcription start sites as located in the MLS sequence. The
negative numbers indicate the transcription start site within the
genomic sequence that corresponds to the MLS.
[0075] To determine the location of the transcription start sites
with the negative numbers, the MLS sequence is aligned with the
corresponding genomic sequence. In the instances when a public
genomic sequence is referenced, the relevant corresponding genomic
sequence can be found by direct reference to the nucleotide
sequence indicated by the "gi" number shown in the public genomic
DNA section of the Reference Table. When the position is a negative
number, the transcription start site is located in the
corresponding genomic sequence upstream of the base that matches
the beginning of the MLS sequence in the alignment. The negative
number is relative to the first base of the MLS sequence which
matches the genomic sequence corresponding to the relevant "gi"
number.
[0076] In the instances when no public genomic DNA is referenced,
the relevant nucleotide sequence for alignment is the nucleotide
sequence associated with the amino acid sequence designated by "gi"
number of the later PolyP SEQ subsection.
[0077] V. Polypeptide Sequences
[0078] The PolyP SEQ subsection lists SEQ ID NOS. and Ceres SEQ ID
NO for polypeptide sequences corresponding to the coding sequence
of the MLS sequence and the location of the translational start
site with the coding sequence of the MLS sequence.
[0079] The MLS sequence can have multiple translational start sites
and can be capable of producing more than one polypeptide
sequence.
[0080] Subsection (Dp) provides (where present) information
concerning amino acid sequences that are found to be related and
have some percentage of sequence identity to the polypeptide
sequences of the Reference and Sequence Tables. These related
sequences are identified by a "gi" number.
Tables 3 and 4--Protein Group Matrix Table
[0081] In addition to each consensus sequence of the invention (see
below), Applicants have generated a scoring matrix to provide
further description of the consensus sequence. The first row of
each matrix indicates the residue position in the consensus
sequence. The matrix reports the number of occurrences of all the
amino acids that were found in the group members for every residue
position of the signature sequence. The matrix also indicates for
each residue position, how many different organisms were found to
have a polypeptide in the group that included a residue at the
relevant position. The last line of the matrix indicates all the
amino acids that were found at each position of the consensus.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0082] The following terms are utilized throughout this
application:
Allelic variant: An "allelic variant" is an alternative form of the
same SDF, which resides at the same chromosomal locus in the
organism. Allelic variations can occur in any portion of the gene
sequence, including regulatory regions. Allelic variants can arise
by normal genetic variation in a population. Allelic variants can
also be produced by genetic engineering methods. An allelic variant
can be one that is found in a naturally occurring plant, including
a cultivar or ecotype. An allelic variant may or may not give rise
to a phenotypic change, and may or may not be expressed. An allele
can result in a detectable change in the phenotype of the trait
represented by the locus. A phenotypically silent allele can give
rise to a product. Chimeric: The term "chimeric" is used to
describe genes, as defined supra, or constructs wherein at least
two of the elements of the gene or construct, such as the promoter
and the coding sequence and/or other regulatory sequences and/or
filler sequences and/or complements thereof, are heterologous to
each other. Constitutive Promoter: Promoters referred to herein as
"constitutive promoters" actively promote transcription under most,
but not necessarily all, environmental conditions and states of
development or cell differentiation. Examples of constitutive
promoters include the cauliflower mosaic virus (CaMV) 35S
transcript initiation region and the 1' or 2' promoter derived from
T-DNA of Agrobacterium tumefaciens, and other transcription
initiation regions from various plant genes, such as the maize
ubiquitin-1 promoter, known to those of skill. Coordinately
Expressed: The term "coordinately expressed," as used in the
current invention, refers to genes that are expressed at the same
or a similar time and/or stage and/or under the same or similar
environmental conditions. Domain: Domains are fingerprints or
signatures that can be used to characterize protein families and/or
parts of proteins. Such fingerprints or signatures can comprise
conserved (1) primary sequence, (2) secondary structure, and/or (3)
three-dimensional conformation. Generally, each domain has been
associated with either a family of proteins or motifs. Typically,
these families and/or motifs have been correlated with specific
in-vitro and/or in-vivo activities. A domain can be any length,
including the entirety of the sequence of a protein. Detailed
descriptions of the domains, associated families and motifs, and
correlated activities of the polypeptides of the instant invention
are described below. Usually, the polypeptides with designated
domain(s) can exhibit at least one activity that is exhibited by
any polypeptide that comprises the same domain(s). Endogenous: The
term "endogenous," within the context of the current invention
refers to any polynucleotide, polypeptide or protein sequence which
is a natural part of a cell or organisms regenerated from said
cell. Exogenous: "Exogenous," as referred to within, is any
polynucleotide, polypeptide or protein sequence, whether chimeric
or not, that is initially or subsequently introduced into the
genome of an individual host cell or the organism regenerated from
said host cell by any means other than by a sexual cross. Examples
of means by which this can be accomplished are described below, and
include Agrobacterium-mediated transformation (of dicots--e.g.
Salomon et al. EMBO J. 3:141 (1984); Herrera-Estrella et al. EMBO
J. 2:987 (1983); of monocots, representative papers are those by
Escudero et al., Plant J. 10:355 (1996), Ishida et al., Nature
Biotechnology 14:745 (1996), May et al., Bio/Technology 13:486
(1995)), biolistic methods (Armaleo et al., Current Genetics 17:97
1990)), electroporation, in planta techniques, and the like. Such a
plant containing the exogenous nucleic acid is referred to here as
a T.sub.0 for the primary transgenic plant and T.sub.1 for the
first generation. The term "exogenous" as used herein is also
intended to encompass inserting a naturally found element into a
non-naturally found location. Gene: The term "gene," as used in the
context of the current invention, encompasses all regulatory and
coding sequence contiguously associated with a single hereditary
unit with a genetic function. Genes can include non-coding
sequences that modulate the genetic function that include, but are
not limited to, those that specify polyadenylation, transcriptional
regulation, DNA conformation, chromatin conformation, extent and
position of base methylation and binding sites of proteins that
control all of these. Genes comprised of "exons" (coding
sequences), which may be interrupted by "introns" (non-coding
sequences), encode proteins. A gene's genetic function may require
only RNA expression or protein production, or may only require
binding of proteins and/or nucleic acids without associated
expression. In certain cases, genes adjacent to one another may
share sequence in such a way that one gene will overlap the other.
A gene can be found within the genome of an organism, artificial
chromosome, plasmid, vector, etc., or as a separate isolated
entity. Heterologous sequences: "Heterologous sequences" are those
that are not operatively linked or are not contiguous to each other
in nature. For example, a promoter from corn is considered
heterologous to an Arabidopsis coding region sequence. Also, a
promoter from a gene encoding a growth factor from corn is
considered heterologous to a sequence encoding the corn receptor
for the growth factor. Regulatory element sequences, such as UTRs
or 3' end termination sequences that do not originate in nature
from the same gene as the coding sequence originates from, are
considered heterologous to said coding sequence. Elements
operatively linked in nature and contiguous to each other are not
heterologous to each other. On the other hand, these same elements
remain operatively linked but become heterologous if other filler
sequence is placed between them. Thus, the promoter and coding
sequences of a corn gene expressing an amino acid transporter are
not heterologous to each other, but the promoter and coding
sequence of a corn gene operatively linked in a novel manner are
heterologous. Homologous gene: In the current invention,
"homologous gene" refers to a gene that shares sequence similarity
with the gene of interest. This similarity may be in only a
fragment of the sequence and often represents a functional domain
such as, examples including without limitation a DNA binding
domain, a domain with tyrosine kinase activity, or the like. The
functional activities of homologous genes are not necessarily the
same. Inducible Promoter: An "inducible promoter" in the context of
the current invention refers to a promoter which is regulated under
certain conditions, such as light, chemical concentration, protein
concentration, conditions in an organism, cell, or organelle, etc.
A typical example of an inducible promoter, which can be utilized
with the polynucleotides of the present invention, is PARSK1, the
promoter from the Arabidopsis gene encoding a serine-threonine
kinase enzyme, and which promoter is induced by dehydration,
abscissic acid and sodium chloride (Wang and Goodman, Plant J. 8:37
(1995)). Examples of environmental conditions that may affect
transcription by inducible promoters include anaerobic conditions,
elevated temperature, or the presence of light. Orthologous Gene:
In the current invention "orthologous gene" refers to a second gene
that encodes a gene product that performs a similar function as the
product of a first gene. The orthologous gene may also have a
degree of sequence similarity to the first gene. The orthologous
gene may encode a polypeptide that exhibits a degree of sequence
similarity to a polypeptide corresponding to a first gene. The
sequence similarity can be found within a functional domain or
along the entire length of the coding sequence of the genes and/or
their corresponding polypeptides. Percentage of sequence identity:
"Percentage of sequence identity," as used herein, is determined by
comparing two optimally aligned sequences over a comparison window,
where the fragment of the polynucleotide or amino acid sequence in
the comparison window may comprise additions or deletions (e.g.,
gaps or overhangs) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison and
multiplying the result by 100 to yield the percentage of sequence
identity. Optimal alignment of sequences for comparison may be
conducted by the local homology algorithm of Smith and Waterman
Add. APL. Math. 2:482 (1981), by the homology alignment algorithm
of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search
for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci.
(USA) 85: 2444 (1988), by computerized implementations of these
algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group (GCG), 575
Science Dr., Madison, Wis.), or by inspection. Given that two
sequences have been identified for comparison, GAP and BESTFIT are
preferably employed to determine their optimal alignment.
Typically, the default values of 5.00 for gap weight and 0.30 for
gap weight length are used. The term "substantial sequence
identity" between polynucleotide or polypeptide sequences refers to
polynucleotide or polypeptide comprising a sequence that has at
least 80% sequence identity, preferably at least 85%, more
preferably at least 90% and most preferably at least 95%, even more
preferably, at least 96%, 97%, 98% or 99% sequence identity
compared to a reference sequence using the programs. Plant
Promoter: A "plant promoter" is a promoter capable of initiating
transcription in plant cells and can drive or facilitate
transcription of a fragment of the SDF of the instant invention or
a coding sequence of the SDF of the instant invention. Such
promoters need not be of plant origin. For example, promoters
derived from plant viruses, such as the CaMV35S promoter or from
Agrobacterium tumefaciens such as the T-DNA promoters, can be plant
promoters. A typical example of a plant promoter of plant origin is
the maize ubiquitin-1 (ubi-1) promoter known to those of skill.
Promoter: The term "promoter," as used herein, refers to a region
of sequence determinants located upstream from the start of
transcription of a gene and which are involved in recognition and
binding of RNA polymerase and other proteins to initiate and
modulate transcription. 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 usually located between 15 and 35 nucleotides
upstream from the site of initiation of transcription. Basal
promoters also sometimes include a "CCAAT box" element (typically a
sequence CCAAT) and/or a GGGCG sequence, usually located between 40
and 200 nucleotides, preferably 60 to 120 nucleotides, upstream
from the start site of transcription. Regulatory Sequence: The term
"regulatory sequence," as used in the current invention, refers to
any nucleotide sequence that influences transcription or
translation initiation and rat; and stability and/or mobility of
the transcript or polypeptide product. Regulatory sequences
include, but are not limited to, promoters, promoter control
elements, protein binding sequences, 5' and 3' UTRs,
transcriptional start site, termination sequence, polyadenylation
sequence, introns, certain sequences within a coding sequence, etc.
Signal Peptide: A "signal peptide" as used in the current invention
is an amino acid sequence that targets the protein for secretion,
for transport to an intracellular compartment or organelle or for
incorporation into a membrane. Signal peptides are indicated in the
tables and a more detailed description located below. Specific
Promoter: In the context of the current invention, "specific
promoters" refers to a subset of inducible promoters that have a
high preference for being induced in a specific tissue or cell
and/or at a specific time during development of an organism. By
"high preference" is meant at least 3-fold, preferably 5-fold, more
preferably at least 10-fold still more preferably at least 20-fold,
50-fold or 100-fold increase in transcription in the desired tissue
over the transcription in any other tissue. Typical examples of
temporal and/or tissue specific promoters of plant origin that can
be used with the polynucleotides of the present invention, are:
PTA29, a promoter which is capable of driving gene transcription
specifically in tapetum and only during anther development
(Koltonow et al., Plant Cell 2:1201 (1990); RCc2 and RCc3,
promoters that direct root-specific gene transcription in rice (Xu
et al., Plant Mol. 27:237 (1995); TobRB27, a root-specific promoter
from tobacco (Yamamoto et al., Plant Cell 3:371 (1991)). Examples
of tissue-specific promoters under developmental control include
promoters that initiate transcription only in certain tissues or
organs, such as root, ovule, fruit, seeds, or flowers. Other
suitable promoters include those from genes encoding storage
proteins or the lipid body membrane protein, oleosin. A few
root-specific promoters are noted above. Stringency: "Stringency"
as used herein is a function of probe length, probe composition
(G+C content), and salt concentration, organic solvent
concentration, and temperature of hybridization or wash conditions.
Stringency is typically compared by the parameter T.sub.m, which is
the temperature at which 50% of the complementary molecules in the
hybridization are hybridized, in terms of a temperature
differential from T.sub.m. High stringency conditions are those
providing a condition of T.sub.m-5.degree. C. to T.sub.m-10.degree.
C. Medium or moderate stringency conditions are those providing
T.sub.m-20.degree. C. to T.sub.m-29.degree. C. Low stringency
conditions are those providing a condition of T.sub.m-40.degree. C.
to T.sub.m-48.degree. C. The relationship of hybridization
conditions to T.sub.m (in .degree. C.) is expressed in the
mathematical equation
T.sub.m=81.5-16.6(log.sub.10[Na.sup.+])+0.41(% G+C)-(600/N) (1)
where N is the length of the probe. This equation works well for
probes 14 to 70 nucleotides in length that are identical to the
target sequence. The equation below for T.sub.m of DNA-DNA hybrids
is useful for probes in the range of 50 to greater than 500
nucleotides, and for conditions that include an organic solvent
(formamide).
T.sub.m=81.5+16.6 log {[Na.sup.+]/(1+0.7[Na.sup.+])}+0.41(%
G+C)-500/L0.63(% formamide) (2)
where L is the length of the probe in the hybrid. (P. Tijessen,
"Hybridization with Nucleic Acid Probes" in Laboratory Techniques
in Biochemistry and Molecular Biology, P. C. vand der Vliet, ed.,
c. 1993 by Elsevier, Amsterdam.) The T.sub.m of equation (2) is
affected by the nature of the hybrid; for DNA-RNA hybrids T.sub.m
is 10-15.degree. C. higher than calculated, for RNA-RNA hybrids
T.sub.m is 20-25.degree. C. higher. Because the T.sub.m decreases
about 1.degree. C. for each 1% decrease in homology when a long
probe is used (Bonner et al., J. Mol. Biol. 81:123 (1973)),
stringency conditions can be adjusted to favor detection of
identical genes or related family members.
[0083] Equation (2) is derived assuming equilibrium and therefore,
hybridizations according to the present invention are most
preferably performed under conditions of probe excess and for
sufficient time to achieve equilibrium. The time required to reach
equilibrium can be shortened by inclusion of a hybridization
accelerator such as dextran sulfate or another high volume polymer
in the hybridization buffer.
[0084] Stringency can be controlled during the hybridization
reaction or after hybridization has occurred by altering the salt
and temperature conditions of the wash solutions used. The formulas
shown above are equally valid when used to compute the stringency
of a wash solution. Preferred wash solution stringencies lie within
the ranges stated above; high stringency is 5-8.degree. C. below
T.sub.m, medium or moderate stringency is 26-29.degree. C. below
T.sub.m and low stringency is 45-48.degree. C. below T.sub.m.
Substantially free of: A composition containing A is "substantially
free of" B when at least 85% by weight of the total A+B in the
composition is A. Preferably, A comprises at least about 90% by
weight of the total of A+B in the composition, more preferably at
least about 95% or even 99% by weight. For example, a plant gene or
DNA sequence can be considered substantially free of other plant
genes or DNA sequences. Translational start site: In the context of
the current invention, a "translational start site" is usually an
ATG in the cDNA transcript, more usually the first ATG. A single
cDNA, however, may have multiple translational start sites.
Transcription start site: "Transcription start site" is used in the
current invention to describe the point at which transcription is
initiated. This point is typically located about 25 nucleotides
downstream from a TFIID binding site, such as a TATA box.
Transcription can initiate at one or more sites within the gene,
and a single gene may have multiple transcriptional start sites,
some of which may be specific for transcription in a particular
cell-type or tissue. Untranslated region (UTR): A "UTR" is any
contiguous series of nucleotide bases that is transcribed, but is
not translated. These untranslated regions may be associated with
particular functions such as increasing mRNA message stability.
Examples of UTRs include, but are not limited to polyadenylation
signals, terminations sequences, sequences located between the
transcriptional start site and the first exon (5' UTR) and
sequences located between the last exon and the end of the mRNA (3'
UTR). Variant: The term "variant" is used herein to denote a
polypeptide or protein or polynucleotide molecule that differs from
others of its kind in some way. For example, polypeptide and
protein variants can consist of changes in amino acid sequence
and/or charge and/or post-translational modifications (such as
glycosylation, etc),
2. Important Characteristics of the Polynucleotides of the
Invention
[0085] The genes and polynucleotides of the present invention are
of interest because when they are misexpressed (i.e. when expressed
at a non-material location or in an increased amount) they produce
plants with increased height, increased primary inflorescence
thickness, an increase in the number and size of leaves,
particularly rosette leaves, and a delay in flowering time without
reduction in fertility. These traits can be used to exploit or
maximize plant products. For example, an increase in plant height
is beneficial in species grown or harvested for their main stem or
trunk, such as ornamental cut flowers, fiber crops (e.g. flax,
kenaf, hesperaloe, hemp) and wood producing trees. Increase in
inflorescence thickness is also desirable for some ornamentals,
while increases in the number and size of leaves can lead to
increased production/harvest from leaf crops such as lettuce,
spinach, cabbage and tobacco. The genes of the invention can also
be used to increase the size of particular
tissues/organs/organelles by placing the gene(s) under the control
of a tissue/organ/organelle-specific promoter, to thereby increase
particularly the size of the plant fruit and seed.
3. The Genes of the Invention
[0086] The sequences of the invention were isolated from
Arabidopsis (polynucleotide and polypeptide SEQ ID NOS. 29-47),
Maize (polynucleotide and polypeptide SEQ NOS. 1-14) and Brassica
(polynucleotide and polypeptide SEQ ID NOS. 15-28), and are
considered orthologous genes because the polypeptides perform
similar functions in a transgenic plant.
[0087] Based upon the orthologous sequences, Applicants have
determined that plants having the desired characteristics discussed
above can be obtained by transformation of a plant or plant cell
with a polynucleotide (stably integrated into the plant genome)
that codes for a polypeptide that comprises one of the following
consensus sequences:
TABLE-US-00001 (SEQ ID NO. 49)
(S,E)t<8>(E,G)<2-5>t<11-14>WT(N,D)E+H<2>Ya<1>-
;(S,Y)aEtSFV<1>Q(L,S)<8-
83>(P,E)r<2-4>+<9-89>E<2>(D,G)QNF<2>n
(SEQ ID NO. 48)
V(E,K)tE(T,P)Ttt(M,G)(Y,I)t(A,K)G(K,N)(E,R)(Y,V)a<1>t<1-
4>WT(N,D)E+H<1>(L,S)Ya(K,S)SMEASFVnQL<0-
30>K(V,A)a<2>(G,E)<2>(Q,E)<9-19>(H,C)<1>(F,V)(L-
,P)<1>(S,N)PW<0-
2>a<1>+r+P<0-8>tD<2>(E,N)<8>(G,D)<0-6>S(G-
,P)t<1>t<2>+<6- 17>(Q,K)a<3>(E,S)<1-
3>EVtDQNF<2>n(G,E)(I,A)<1>t(E,S)(N,T)(G,E)t<1>K<2&-
gt;K<1>(V,R)(M,R)aS (E,R)t
[0088] The consensus sequence contains both lower-case and
upper-case letters. The upper-case letters represent the standard
one-letter amino acid abbreviations. The lower case letters
represent classes of amino acids: [0089] "t" refers to tiny amino
acids, which are specifically alanine, glycine, serine and
threonine. [0090] "p" refers to polar amino acids, which are
specifically, asparagine and glutamine [0091] "n" refers to
negatively charged amino acids, which are specifically, aspartic
acid and glutamic acid [0092] "+" refers to positively charged
residues, which are specifically, lysine, arginine, and histidine
[0093] "r" refers to aromatic residues, which are specifically,
phenylalanine, tyrosine, and tryptophan, [0094] "a" refers to
aliphatic residues, which are specifically, isoleucine, valine,
leucine, and methonine [0095] "< >" refers to the number of
residues present. For example, A <8>S indicates that eight
residues separate the alanine residue from the serine residue.
"A<8>S" is equivalent to "A XXXXX XXXS." Likewise
"A<1-3>S" indicates that at least one, but as many as three
residues separate alanine from serine.
[0096] In addition to the sequences of SEQ ID NOS. 1-49, the
invention also encompasses variants, fragments or fusions of the
polypeptides that produce the same phenotypic effect after
transformation into a host plant.
[0097] A type of variant of the polypeptides comprises amino acid
substitutions. Conservative substitutions are preferred to maintain
the function or activity of the polypeptide. Such substitutions
include conservation of charge, polarity, hydrophobicity, size,
etc. For example, one or more amino acid residues within the
sequence can be substituted with another amino acid of similar
polarity that acts as a functional equivalent, for example
providing a hydrogen bond in an enzymatic catalysis. Substitutes
for an amino acid within an exemplified sequence are preferably
made among the members of the class to which the amino acid
belongs. For example, the nonpolar (hydrophobic) amino acids
include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan and methionine. The polar neutral amino
acids include glycine, serine, threonine, cysteine, tyrosine,
asparagine, and glutamine. The positively charged (basic) amino
acids include arginine, lysine and histidine. The negatively
charged (acidic) amino acids include aspartic acid and glutamic
acid.
[0098] The variants include those that have a percentage of
sequence identity to SEQ NOS. 1-49 with the range of at least 80%,
or preferably at least 85, 90, 95, 96, 97, 98 or 99%. Within that
scope of percentage of sequence identity, a polypeptide of the
invention may have additional individual amino acids or amino acid
sequences inserted into the polypeptide in the middle thereof
and/or at the N-terminal and/or C-terminal ends thereof, Likewise,
some of the amino acids or amino acid sequences may be deleted from
the polypeptide. Amino acid substitutions may also be made in the
sequences; conservative substitutions being preferred.
[0099] One preferred class of variants are those that comprise (1)
the domain of an encoded polypeptide and/or (2) residues conserved
between the encoded polypeptide and related polypeptides. For this
class of variants, the encoded polypeptide sequence is changed by
insertion, deletion, or substitution at positions flanking the
domain and/or conserved residues. Another class of variants
includes those that comprise an encoded polypeptide sequence that
is changed in the domain or conserved residues by a conservative
substitution.
4. Use of the Genes to Make Transgenic Plants
[0100] To use the sequences of the present invention or a
combination of them or parts and/or mutants and/or fusions and/or
variants of them, recombinant DNA constructs are prepared which
comprise the polynucleotide sequences of the invention inserted
into a vector, and which are suitable for transformation of plant
cells. The construct can be made using standard recombinant DNA
techniques (Sambrook et al. 1989) and can be introduced to the
species of interest by Agrobacterium-mediated transformation or by
other means of transformation as referenced below.
[0101] The vector backbone can be any of those typical in the art
such as plasmids, viruses, artificial chromosomes, BACs, YACs and
PACs and vectors of the sort described by [0102] (a) BAC: Shizuya
et al., Proc. Natl. Acad. Sci. USA 89: 8794-8797 (1992); Hamilton
et al., Proc. Natl. Acad. Sci. USA 93: 9975-9979 (1996); [0103] (b)
YAC: Burke et al., Science 236:806-812 (1987); [0104] (c) PAC:
Sternberg N. et al., Proc Natl Acad Sci USA. January; 87(1):103-7
(1990); [0105] (d) Bacteria-Yeast Shuttle Vectors: Bradshaw et al.,
Nucl Acids Res 23: 4850-4856 (1995); [0106] (e) Lambda Phage
Vectors: Replacement Vector, e.g., Frischauf et al., J. Mol. Biol
170: 827-842 (1983); or Insertion vector, e.g., Huynh et al., In:
Glover NM (ed) DNA Cloning: A practical Approach, Vol. 1 Oxford:
IRL Press (1985); T-DNA gene fusion vectors: Walden et al., Mol
Cell Biol 1: 175-194 (1990); and [0107] (g) Plasmid vectors:
Sambrook et al., infra.
[0108] Typically, the construct will comprise a vector containing a
sequence of the present invention with any desired transcriptional
and/or translational regulatory sequences, such as promoters, UTRs,
and 3' end termination sequences. Vectors can also include origins
of replication, scaffold attachment regions (SARs), markers,
homologous sequences, introns, etc. The vector may also comprise a
marker gene that confers a selectable phenotype on plant cells. The
marker may encode biocide resistance, particularly antibiotic
resistance, such as resistance to kanamycin, G418, bleomycin,
hygromycin, or herbicide resistance, such as resistance to
chlorosulfuron or phosphinotricin.
[0109] A plant promoter fragment may be used that directs
transcription of the gene in all tissues of a regenerated plant and
may be a constitutive promoter, such as 355. Alternatively, the
plant promoter may direct transcription of a sequence of the
invention in a specific tissue (tissue-specific promoters) or may
be otherwise under more precise environmental control (inducible
promoters).
[0110] If proper polypeptide production is desired, a
polyadenylation region at the 3'-end of the coding region is
typically included. The polyadenylation region can be derived from
the natural gene, from a variety of other plant genes, or from
T-DNA.
Knock-In Constructs
[0111] Ectopic expression of the sequences of the invention can
also be accomplished using a "knock-in" approach. Here, the first
component, an "activator line," is created by generating a
transgenic plant comprising a transcriptional activator operatively
linked to a promoter. The second component comprises the desired
cDNA sequence operatively linked to the target binding
sequence/region of the transcriptional activator. The second
component can be transformed into the "activator line" or be used
to transform a host plant to produce a "target" line that can be
crossed with the "activator line" by ordinary breeding methods. In
either case, the result is the same. That is, the promoter drives
production of the transcriptional activator protein that then binds
to the target binding region to facilitate expression of the
desired cDNA.
[0112] Any promoter that functions in plants can be used in the
first component, such as the 35S Cauliflower Mosaic Virus promoter
or a tissue or organ specific promoter. Suitable transcriptional
activator polypeptides include, but are not limited to, those
encoding HAP1 and GAL4. The binding sequence recognized and
targeted by the selected transcriptional activator protein is used
in the second component.
Transformation
[0113] Techniques for transforming a wide variety of higher plant
species are well known and described in the technical and
scientific literature. See, e.g. Weising et al., Ann. Rev. Genet.
22:421 (1988); and Christou, Euphytica, v. 85, n. 1-3:13-27,
(1995).
[0114] Processes for the transformation of monocotyledonous and
dicotyledonous plants are known to the person skilled in the art.
For the introduction of DNA into a plant host cell a variety of
techniques is available. These techniques comprise the
transformation of plant cells with T-DNA using Agrobacterium
tumefaciens or Agrobacterium rhizogenes as transformation means,
the fusion of protoplasts, the injection, the electroporation of
DNA, the introduction of DNA by means of the biolistic method as
well as further possibilities.
[0115] For the injection and electroporation of DNA in plant cells
the plasmids do not have to fulfill specific requirements. Simple
plasmids such as pUC derivatives can be used.
[0116] The use of agrobacteria for the transformation of plant
cells has extensively been examined and sufficiently disclosed in
the specification of EP-A 120 516, in Hoekema (In: The Binary Plant
Vector System Offsetdrukkerij Kanters B. V., Alblasserdam (1985),
Chapter V), Fraley et al. (Crit. Rev. Plant. Sci. 4, 1-46) and An
et al. (EMBO J. 4 (1985), 277-287).
[0117] For the transfer of the DNA to the plant cell plant explants
can be co-cultivated with Agrobacterium tumefaciens or
Agrobacterium rhizogenes. From the infected plant material (for
example leaf explants, segments of stems, roots but also
protoplasts or suspension cultivated plant cells) whole plants can
be regenerated in a suitable medium which may contain antibiotics
or biozides for the selection of transformed cells. The plants
obtained that way can then be examined for the presence of the
introduced DNA. Other possibilities for the introduction of foreign
DNA using the biolistic method or by protoplast transformation are
known (cf., e.g., Willmitzer, L., 1993 Transgenic plants. In:
Biotechnology, A Multi-Volume Comprehensive Treatise (H. J. Rehm,
G. Reed, A. Pithier, P. Stadler, eds.), Vol. 2, 627-659, VCH
Weinheim-New York-Basel-Cambridge).
[0118] The transformation of dicotyledonous plants via
Ti-plasmid-vector systems with the help of Agrobacterium
tumefaciens is well-established. Recent studies have indicated that
also monocotyledonous plants can be transformed by means of vectors
based on Agrobacterium (Chan et al., Plant Mol. Biol. 22 (1993),
491-506; Hiei et al., Plant J. 6 (1994), 271-282; Deng et al.,
Science in China 33 (1990), 28-34; Wilmink et al., Plant Cell
Reports 11 (1992), 76-80; May et al., Bio/Technology 13 (1995),
486.492; Conner and Domisse; Int. J. Plant Sci. 153 (1992),
550-555; Ritchie et al., Transgenic Res. 2 (1993), 252-265).
[0119] Alternative systems for the transformation of
monocotyledonous plants are the transformation by means of the
biolistic method (Wan and Lemaux, Plant Physiol. 104 (1994), 37-48;
Vasil et al., Bio/Technology 11 (1993), 1553-1558; Ritala et al.,
Plant Mol. Biol. 24 (1994), 317-325; Spencer et al., Theor. Appl.
Genet. 79 (1990), 625-631), the protoplast transformation, the
electroporation of partially permeabilized cells, as well as the
introduction of DNA by means of glass fibers.
[0120] In particular the transformation of maize is described in
the literature several times (cf., e.g., WO95/06128, EP 0 513 849;
EP 0 465 875; Fromm et al., Biotechnology 8 (1990), 833-844;
Gordon-Kamm et al., Plant Cell 2 (1990), 603-618; Koziel et al.,
Biotechnology 11 (1993), 194-200). In EP 292 435 and in Shillito et
al. (Bio/Technology 7 (1989), 581) a process is described with the
help of which and starting from a mucus-free, soft (friable) maize
callus fertile plants can be obtained. Prioli and Sondahl
(Bio/Technology 7 (1989), 589) describe the regenerating and
obtaining of fertile plants from maize protoplasts of the Cateto
maize inbred line Cat 100-1.
[0121] The successful transformation of other cereal species has
also been described, for example for barley (Wan and Lemaux, see
above; Ritala et al., see above) and for wheat (Nehra et al., Plant
J. 5 (1994), 285-297).
[0122] Once the introduced DNA has been integrated into the genome
of the plant cell, it usually is stable there and is also contained
in the progenies of the originally transformed cell. It usually
contains a selection marker which makes the transformed plant cells
resistant to a biozide or an antibiotic such as kanamycin, G 418,
bleomycin, hygromycin or phosphinotricin and others. Therefore, the
individually chosen marker should allow the selection of
transformed cells from cells lacking the introduced DNA.
[0123] The transformed cells grow within the plant in the usual way
(see also McCormick et al., Plant Cell Reports 5 (1986), 81-84).
The resulting plants can be cultured normally. Seeds can be
obtained from the plants.
[0124] Two or more generations should be cultivated to make sure
that the phenotypic feature is maintained stably and is
transmitted. Seeds should be harvested to make sure that the
corresponding phenotype or other properties are maintained.
[0125] DNA constructs of the invention may be introduced into the
genome of the desired plant host by a variety of conventional
techniques. For example, the DNA construct may be introduced
directly into the genomic DNA of the plant cell using techniques
such as electroporation and microinjection of plant cell
protoplasts, or the DNA constructs can be introduced directly to
plant tissue using ballistic methods, such as DNA particle
bombardment. Alternatively, the DNA constructs may be combined with
suitable T-DNA flanking regions and introduced into a conventional
Agrobacterium tumefaciens host vector. The virulence functions of
the Agrobacterium tumefaciens host will direct the insertion of the
construct and adjacent marker into the plant cell DNA when the cell
is infected by the bacteria (McCormac et al., Mol. Biotechnol.
8:199 (1997); Hamilton, Gene 200:107 (1997)); Salomon et al. EMBO
J. 3:141 (1984); Herrera-Estrella et al. EMBO J. 2:987 (1983).
[0126] Microinjection techniques are known in the art and well
described in the scientific and patent literature. The introduction
of DNA constructs using polyethylene glycol precipitation is
described in Paszkowski et al. EMBO J. 3:2717 (1984).
Electroporation techniques are described in Fromm et al. Proc. Natl
Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniques
are described in Klein et al. Nature 327:773 (1987). Agrobacterium
tumefaciens-mediated transformation techniques, including disarming
and use of binary or co-integrate vectors, are well described in
the scientific literature. See, for example Hamilton, C M., Gene
200:107 (1997); Muller et al. Mol. Gen. Genet. 207:171 (1987);
Komari et al. Plant J. 10:165 (1996); Venkateswarlu et al.
Biotechnology 9:1103 (1991) and Gleave, A P., Plant Mol. Biol.
20:1203 (1992); Graves and Goldman, Plant Mol. Biol. 7:34 (1986)
and Gould et al., Plant Physiology 95:426 (1991).
[0127] Transformed plant cells that have been obtained by any of
the above transformation techniques can be cultured to regenerate a
whole plant that possesses the transformed genotype and thus the
desired phenotype. Such regeneration techniques rely on
manipulation of certain phytohormones in a tissue culture growth
medium, typically relying on a biocide and/or herbicide marker that
has been introduced together with the desired nucleotide sequences.
Plant regeneration from cultured protoplasts is described in Evans
et al., Protoplasts Isolation and Culture in "Handbook of Plant
Cell Culture," pp. 124-176, MacMillan Publishing Company, New York,
1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp.
21-73, CRC Press, Boca Raton, 1988. Regeneration can also be
obtained from plant callus, explants, organs, or parts thereof.
Such regeneration techniques are described generally in Klee et al.
Ann. Rev. of Plant Phys, 38:467 (1987). Regeneration of monocots
(rice) is described by Hosoyama et al. (Biosci. Biotechnol.
Biochem. 58:1500 (1994)) and by Ghosh et al. (J. Biotechnol. 32:1
(1994)). The nucleic acids of the invention can be used to confer
the trait of increased height, increased primary inflorescence
thickness, an increase in the number and size of leaves and a delay
in flowering time, without reduction in fertility, on essentially
any plant.
[0128] The nucleotide sequences according to the invention can
generally encode any appropriate proteins from any organism, in
particular from plants, fungi, bacteria or animals. The sequences
preferably encode proteins from plants or fungi. Preferably, the
plants are higher plants, in particular starch or oil storing
useful plants, for example potato or cereals such as rice, maize,
wheat, barley, rye, triticale, oat, millet, etc., as well as
spinach, tobacco, sugar beet, soya, cotton etc.
[0129] The process according to the invention can in principle be
applied to any plant. Therefore, monocotyledonous as well as
dicotyledonous plant species are particularly suitable. The process
is preferably used with plants that are interesting for
agriculture, horticulture and/or forestry.
[0130] Examples thereof are vegetable plants such as, for example,
cucumber, melon, pumpkin, eggplant, zucchini, tomato, spinach,
cabbage species, peas, beans, etc., as well as fruits such as, for
example, pears, apples, etc.
[0131] Thus, the invention has use over a broad range of plants,
including species from the genera Anacardium, Arachis, Asparagus,
Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus,
Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria,
Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus,
Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Mahe, Manihot,
Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannesetum,
Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus,
Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus,
Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.
[0132] One of skill will recognize that after the expression
cassette is stably incorporated in transgenic plants and confirmed
to be operable, it can be introduced into other plants by sexual
crossing. Any of a number of standard breeding techniques can be
used, depending upon the species to be crossed.
5. Phenotype Studies
[0133] The genes of the invention were utilized to transform plants
(specifically Arabidopsis as a model species) and the results show
the improved phenotype characteristics of the transgenic
plants.
[0134] 5.1. Phenotype Experiments for Clone 8490
[0135] Ectopic expression of cDNA 12337825 (clone 8490--SEQ ID No.
39) under the control of the 35S promoter results in plants having
a number of phenotypes including: [0136] Taller plants [0137]
Thicker inflorescences [0138] Larger rosettes [0139] Increased
rosette leaf number [0140] Slightly delayed flowering
[0141] As a result, misexpression of cDNA 12337825 (SEQ ID No. 39)
is useful to increase overall plant size/biomass. A gene with a
direct role in controlling the size of an endosperm is also
potentially advantageous for seed size and, if misexpressed with an
appropriate promoter, for plant growth and development.
[0142] Clone 8490 contains cDNA 12337825, which when analyzed in
transcript profiling (Txp) experiments (discussed below) was
down-regulated in the root meristematic region of the plant
relative to root cell elongation zone and up-regulated in an
interploidy cross that stimulates endosperm (a paternal tetraploid
gives rise to large endosperm and large seed).
Materials and Methods:
[0143] Generation and Phenotypic Evaluation of T.sub.1 and T.sub.2
Lines Containing 35S::cDNA 12337825.
[0144] Wild-type Arabidopsis Wassilewskija (WS) plants were
transformed with a Ti plasmid containing cDNA 12337825 in the sense
orientation relative to the 35S constitutive promoter. The Ti
plasmid vector used for this construct, CRS 338 (FIG. 1), contains
a plant selectable marker gene phosphinothricin acetyltransferase
(PAT) that confers herbicide resistance to transformed plants. The
transformation is conducted as follows:
Procedure: Agrobacterium-Mediated Transformation of Arabidopsis
Materials:
0.2% Phytagar
[0145] 2 g Phytagar
[0146] 1 L nanopure water
YEB (for 1 L)
[0147] 5 g extract of meat
[0148] 5 g Bacto peptone
[0149] 1 g yeast extract
[0150] 5 g sucrose
[0151] 0.24 g magnesium sulfate
Infiltration Medium (IM) (for 1 L)
[0152] 2.2 g MS salts
[0153] 50 g sucrose
[0154] 5 ul BAP solution (stock is 2 mg/ml)
Methods:
1. Stratification of WS-2 Seed.
[0155] Add 0.5 ml WS-2 (CS2360) seed to 50 ml of 0.2% Phytagar in a
50 ml Corning tube and vortex until seeds and Phytagar form a
homogenous mixture. [0156] Cover tube with foil and stratify at
4.degree. C. for 3 days.
2. Preparation of Seed Mixture.
[0156] [0157] Obtain stratified seed from cooler. [0158] Add seed
mixture to a 1000 ml beaker. [0159] Add an additional 950 ml of
0.2% Phytagar and mix to homogenize.
3. Preparation of Soil Mixture.
[0159] [0160] Mix 24 L SunshineMix #5 soil with 16 L Therm-O-Rock
vermiculite in cement mixer to make a 60:40 soil mixture. [0161]
Amend soil mixture by adding 2 Tbsp Marathon and 3 Tbsp Osmocote
and mix contents thoroughly. [0162] Add 1 Tbsp Peters fertilizer to
3 gallons of water and add to soil mixture and mix thoroughly.
[0163] Fill 4-inch pots with soil mixture and round the surface to
create a slight dome. [0164] Cover pots with 8-inch squares of
nylon netting and fasten using rubber bands. [0165] Place 14 4-inch
pots into each no-hole utility flat.
4. Planting.
[0165] [0166] Using a 60 ml syringe, aspirate 35 ml of the seed
mixture. [0167] Exude 25 drops of the seed mixture onto each pot.
[0168] Repeat until all pots have been seeded. [0169] Place flats
on greenhouse bench, cover flat with clear propagation domes, place
55% shade cloth on top of flats and subirrigate by adding 1 inch of
water to bottom of each flat.
5. Plant Maintenance.
[0169] [0170] 3 to 4 days after planting, remove clear lids and
shade cloth. [0171] Subirrigate flats with water as needed. [0172]
After 7-10 days, thin pots to 20 plants per pot using forceps.
[0173] After 2 weeks, subirrigate all plants with Peters fertilizer
at a rate of 1 Tsp per gallon water. [0174] When bolts are about
5-10 cm long, clip them between the first node and the base of stem
to induce secondary bolts. [0175] 6 to 7 days after clipping,
perform dipping infiltration.
6. Preparation of Agrobacterium.
[0175] [0176] Add 150 ml fresh YEB to 250 ml centrifuge bottles and
cap each with a foam plug (Identi-Plug). [0177] Autoclave for 40
min at 121.degree. C. [0178] After cooling to room temperature,
uncap and add 0.1 ml each of carbenicillin, spectinomycin and
rifampicin stock solutions to each culture vessel. [0179] Obtain
Agrobacterium starter block (96-well block with Agrobacterium
cultures grown to an OD.sub.600 of approximately 1.0) and inoculate
one culture vessel per construct by transferring 1 ml from
appropriate well in the starter block. [0180] Cap culture vessels
and place on Lab-Line incubator shaker set at 27.degree. C. and 250
RPM. [0181] Remove after Agrobacterium cultures reach an OD.sub.600
of approximately 1.0 (about 24 hours), cap culture vessels with
plastic caps, place in Sorvall SLA 1500 rotor and centrifuge at
8000 RPM for 8 min at 4.degree. C. [0182] Pour out supernatant and
put bottles on ice until ready to use. [0183] Add 200 ml
Infiltration Media (IM) to each bottle, resuspend Agrobacterium
pellets and store on ice.
7. Dipping Infiltration.
[0183] [0184] Pour resuspended Agrobacterium into 16 oz
polypropylene containers. [0185] Invert 4-inch pots and submerge
the aerial portion of the plants into the Agrobacterium suspension
and let stand for 5 min. [0186] Pour out Agrobacterium suspension
into waste bucket while keeping polypropylene container in place
and return the plants to the upright position. [0187] Place 10
covered pots per flat. [0188] Fill each flat with 1-inch of water
and cover with shade cloth. [0189] Keep covered for 24 hr and then
remove shade cloth and polypropylene containers. [0190] Resume
normal plant maintenance. [0191] When plants have finished
flowering cover each pot with a ciber plant sleeve. [0192] After
plants are completely dry, collect seed and place into 2.0 ml micro
tubes and store in 100-place cryogenic boxes.
[0193] Ten independently transformed events were selected and
evaluated for their qualitative phenotype in the T.sub.1 generation
as follows:
Procedure: High Throughput Phenotypic Screening of Misexpression
Mutants-T.sub.1 Generation
[0194] 1. Soil Preparation. Wear gloves at all times. [0195] In a
large container, mix 60% autoclaved SunshineMix #5 with 40%
vermiculite. [0196] Add 2.5 Tbsp of Osmocote, and 2.5 Tbsp of 1%
granular Marathon per 25 L of soil. [0197] Mix thoroughly.
2. Fill Com-Packs With Soil.
[0197] [0198] Loosely fill D601 Corn-Packs level to the rim with
the prepared soil. [0199] Place filled pot into utility flat with
holes, within a no-hole utility flat. [0200] Repeat as necessary
for planting. One flat set should contain 6 pots.
3. Saturate Soil.
[0200] [0201] Evenly water all pots until the soil is saturated and
water is collecting in the bottom of the flats. [0202] After the
soil is completely saturated, dump out the excess water.
4. Plant the Seed.
5. Stratify the Seeds.
[0202] [0203] After sowing the seed for all the flats, place them
into a dark 4.degree. C. cooler. [0204] Keep the flats in the
cooler for 2 nights for WS seed. Other ecotypes may take longer.
This cold treatment will help promote uniform germination of the
seed. 6. Remove Flats From Cooler and Cover With Shade Cloth.
(Shade cloth is only needed in the greenhouse) [0205] After the
appropriate time, remove the flats from the cooler and place onto
growth racks or benches. [0206] Cover the entire set of flats with
55% shade cloth. The cloth is necessary to cut down the light
intensity during the delicate germination period. [0207] The cloth
and domes should remain on the flats until the cotyledons have
fully expanded. This usually takes about 4-5 days under standard
greenhouse conditions.
7. Remove 55% Shade Cloth and Propagation Domes.
[0207] [0208] After the cotyledons have fully expanded, remove both
the 55% shade cloth and propagation domes. 8. Spray Plants With
Finale Mixture. Wear gloves and protective clothing at all times.
[0209] Prepare working Finale mixture by mixing 3 ml concentrated
Finale in 48 oz of water in the Poly-TEK sprayer. [0210] Completely
and evenly spray plants with a fine mist of the Finale mixture.
[0211] Repeat Finale spraying every 3-4 days until only
transformants remain. (Approximately 3 applications are necessary.)
[0212] When satisfied that only transformants remain, discontinue
Finale spraying.
9. Weed Out Excess Transformants.
[0212] [0213] Weed out excess transformants such that a maximum
number of five plants per pot exist evenly spaced throughout the
pot. 10. Label Individual plants.
11. Screen Each Pot For Phenotypes.
[0213] [0214] When a phenotype is observed, label a tag describing
the phenotype. [0215] Repeat screening process at 4 development
stages: Seedling, Rosette, Flowering, and Senescence. [0216]
Seedling--the time after the cotyledons have emerged, but before
the 3.sup.rd true leaf begins to form. [0217] Rosette--the time
from the emergence of the 3.sup.rd true leaf through just before
the primary bolt begins to elongate. [0218] Flowering--the time
from the emergence of the primary bolt to the onset of senescence
(with the exception of noting the flowering time itself, most
observations should be made at the stage where approximately 50% of
the flowers have opened). [0219] Senescence--the time following the
onset of senescence (with the exception of "delayed senescence",
most observations should be made after the plant has completely
dried).
12. Quality Control for T1 Overexpressers-Misexpression Lines.
13. Individual Plant Staking.
[0219] [0220] During the flowering stage of development, it is
necessary to separate individual plants so that they do not entwine
themselves, causing cross-contamination and making seed collection
very difficult. [0221] Place a Hyacinth stake in the soil next to
the rosette, being careful not to damage the plant. [0222]
Carefully wrap the primary and secondary bolts around the stake.
[0223] Very loosely wrap a single plastic coated twist tie around
the stake and the plant to hold it in place.
14. Seed Collection Preparation.
[0223] [0224] When senescence begins and flowers stop forming, stop
watering. This will allow the plant to dry properly for seed
collection. 15. Collect Seed from Plants
[0225] Two events showing the most advantageous T.sub.1 phenotypes
(large, late-flowering) were chosen for evaluation in the T.sub.2
generation. The T.sub.2 growth conditions follow the above T.sub.1
protocol. The experimental design differs from the T.sub.1 planting
in that each T.sub.2 plant is contained within its own pot, and no
herbicide selection is used. All pots for each T.sub.2 event are
contained within the same flat and the plants are randomly
distributed within each flat. The controls for each set of
measurements are the segregating progeny of the given T.sub.1 event
which do not contain the T-DNA (internal controls). All analyses
are done via soil-based experiments under long day light conditions
(16 hours) in the Ceres greenhouse.
[0226] T2 measurements being taken are as follows: [0227] Days to
bolt=number of days between sowing of seed and emergence of first
inflorescence. [0228] Number of Leaves=number of rosette leaves
present at date of first bolt. [0229] Rosette Area=Area of rosette
at time of initial bolt emergence, using ((L.times.W)*3.14)/4,
[0230] Primary Inflorescence Thickness=diameter of primary
inflorescence 2.5 cm up from base. This measurement was taken at
the termination of flowering/onset of senescence. [0231]
Height=length of longest inflorescence from base to apex. This
measurement was taken at the termination of flowering/onset of
senescence.
[0232] PCR was used to amplify the cDNA insert in one randomly
chosen T.sub.1 plant. This PCR product was then sequenced to
confirm that the correct insert was contained in the plants. The
quality control process was performed as per standard protocol.
[0233] In the T.sub.2 generation, PCR was used to confirm the
presence or absence of the insert in each plant. To confirm that
genomic DNA was present in the reaction mixture, a second set of
reactions was run for each sample using primers that amplify a
sequence from the RAP2.7 gene. Each sample template yielding a PCR
product for RAP2.7 was deemed of adequate template quality.
Results:
Qualitative Analysis of the T.sub.1 Plants:
[0234] All ten events were late flowering, produced larger rosettes
with more leaves and tall, thick inflorescences compared to the
controls (see results in Table 5). The transgenic "control" was a
set of different 35S::cDNA expressing plants which were
indistinguishable from the untransformed WS wild type.
TABLE-US-00002 TABLE 5 Qualitative phenotypes observed in 35S::cDNA
12337825 T.sub.1 events Increased Rosette Size Increased Rosette
Late Tall & Event Leaf Number Flowering Thick ME03459-01 x x x
ME03459-02 x x x ME03459-03 x x x ME03459-04 x x x ME03459-05 x x x
ME03459-06 x x x ME04358-01 x x x ME04358-02 x x x ME04358-03 x x x
ME04358-04 x x x
Quantitative Analysis of the T.sub.2 Plants:
[0235] Events ME03459-01 and ME03459-04 were evaluated in greater
detail in the T.sub.2 generation. Seventeen individuals were sown
and observed for event 01, whereas 18 individuals were sown and
observed for event 04. The transgenic plants for both events showed
increased height, increased primary inflorescence thickness,
increased number of rosette leaves, a larger rosette, and delay of
flowering time to a 0.05 level of statistical significance (Table
6). Both events had normal fertility. All plants noted in the table
as ME03459-01 or ME03459-04 were segregating progeny of the T.sub.1
event which we had confirmed to contain the transgene under test.
All plants noted in the table as -01 Control or -04 Control were
T.sub.2 segregating progeny which did not contain the transgene
under test (internal controls).
[0236] Both events produce significantly more seeds than the
control, as would be expected for a typical, fertile, late
flowering plant.
[0237] Event ME03459-01 is the strongest expresser as noted in
Table 5. The rosette area, number of leaves, thickness of the
inflorescence and days to bolt are all greater than event -04.
[0238] Segregation frequencies of the transgene under test suggest
that each event contains a single insert, as calculated by a
Chi-square test. The T.sub.2 seeds segregate 3R:1S for both events
(data not shown).
TABLE-US-00003 TABLE 6 Quantitative phenotypes observed in
35S::cDNA 12337825 T.sub.2 events Number Primary of Rosette Number
Inflorescence Days Event/ Obser- Area of Height Thickness to
Control vations (mm.sup.2) Leaves (cm) (inches) Bolt ME03459-01 14
7023.0* 11.0* 75.6* 0.068* 21.9* -01 Control 3 2348.5 8.0 52.2
0.050 19.0 ME03459-04 9 4977.7* 9.4* 68.9* 0.055* 20.8* -04 Control
5 2521.1 7.5 54.0 0.051 18.1 *significantly different from control
at 0.05 level, via t-test
Summary of Results
[0239] The ectopic expression of cDNA 12337825 with a strong
constitutive promoter (35S) results in taller plants, with thicker
inflorescences, a larger rosette, and more rosette leaves. 12337825
is normally regulated in shoot and root apices, suggesting that the
encoded protein may help to regulate meristem function. The
increase in plant size observed by this expression is accompanied
by a delay in flowering time, but no reduction in fertility. It may
also be a useful gene to increase root growth, given the similar
expression pattern in shoot meristems and root tip cells.
[0240] Assuming conservation of process controlling vegetative
growth across species, this gene and protein is likely to function
similarly in other species. Increased vegetative biomass should
give an improved source:sink ratio and improved fixation of carbon
to sucrose and starch. It may in and of itself play into improved
yield. Taller inflorescences give the opportunity for more flowers
and therefore more seeds. The combination of improved biomass and
inflorescence stature may give a significant improvement in yield.
Thicker inflorescences may prevent against "snap" against wind,
rain or drought. Biomass advantage and presumed photosynthesis
advantage should be useful in corn and soybean.
[0241] Therefore, this gene/protein is especially useful for
controlling the number/rate of cell division in meristems without
disturbing overall plant morphology. It could be developed in crops
with an appropriate promoter to regulate size and growth rate of
many individual organs. The use of a tissue-specific promoter may
be particularly desirable. For example, if an increase of leaf size
is desired without an increase in root size, the coding sequences
of the invention can be operably linked to a leaf specific promoter
for this purpose. Alternatively, if an increase in plant size is
desired with no change in flowering time, the coding sequences of
the invention can be modulated with a leaf specific promoter that
does not direct expression in the floral meristem.
[0242] The protein is useful for creating sturdier stems in corn
and preventing against "snap".
[0243] 5.2. Phenotype Experiments for Clone 8161--cDNA 5662747
[0244] Ectopic expression of Ceres cDNA 5662747(SEQ ID No. 29)
under the control of the 35S promoter results in plants having a
number of phenotypes including: [0245] Taller plants [0246] Thicker
inflorescences [0247] Qualitatively larger rosettes [0248]
Qualitatively increased rosette leaf number [0249] Delayed
flowering
[0250] As a result, misexpression of Ceres cDNA 5662747 (SEQ ID No.
29) is useful to increase overall plant size/biomass.
[0251] Clone 8161 contains cDNA 5662747, which when analyzed in
transcript profiling experiments (discussed below) was
down-regulated in both the shoot and root tips of the plant
relative to whole plant mRNA extracts suggesting a function in
meristem activity.
Materials and Methods:
[0252] Generation and Phenotypic Evaluation of T.sub.1 and T.sub.2
Lines Containing 35S::cDNA 5662747.
[0253] Wild-type Arabidopsis Wassilewskija (WS) plants were
transformed with a Ti plasmid containing cDNA 5662747 in the sense
orientation relative to the 35S constitutive promoter as per
standard protocol (See "Ceres Protocol-Agrobacterium-Mediated
Transformation of Arabidopsis"). The Ti plasmid vector used for
this construct, CRS 311, contains a plant selectable marker gene
phosphinothricin acetyltransferase (PAT) that confers herbicide
resistance to transformed plants.
[0254] Ten independently transformed events were selected and
evaluated for their qualitative phenotype in the T.sub.1 generation
as per standard protocol. Three events showing the strongest
T.sub.1 phenotypes were chosen for evaluation in the T.sub.2
generation. The T.sub.2 growth conditions followed the above
T.sub.1 protocol. The experimental design differed from the T.sub.1
planting in that each T.sub.2 plant was contained with its own pot,
and no herbicide selection was used. All the pots for each T.sub.2
event were contained within the same flat and the plants were
randomly distributed within each flat. The controls for each set of
measurements were the segregating progeny of other T.sub.1 events
which did not contain this gene (internal controls). All analyses
were done via soil-based experiments under long day light
conditions (16 hours) in the Ceres greenhouse.
[0255] T2 measurements were taken as follows: [0256] Height=length
of longest inflorescence from base to apex. This measurement was
taken at the termination of flowering/onset of senescence. [0257]
Primary Inflorescence Thickness=diameter of primary inflorescence
2.5 cm up from base. This measurement was taken at the termination
of flowering/onset of senescence. [0258] Days to bolt=number of
days between sowing of seed and eruption of first
infloreseence.
[0259] PCR was used to amplify the cDNA insert in one randomly
chosen T.sub.1 plant. This PCR product was then sequenced to
confirm that the correct insert was contained in the plants. The
quality control process was performed as per standard protocol.
[0260] In the T.sub.2 generation, PCR was used to confirm the
presence or absence of the insert in each plant. To confirm that
genomic DNA was present in the reaction mixture, a second set of
reactions was run for each sample using primers that amplify a
sequence from the RAP2.7 gene. Each sample template yielded a PCR
product for RAP2.7, so all DNA samples were deemed of adequate
template quality.
Results:
Qualitative Analysis of the T.sub.1 Plants:
[0261] All ten events showed a variety of phenotypes different from
wild-type transgenic controls (Table 7); obvious differences from
the controls were noted. The transgenic "control" was a set of
different 35S::cDNA expressing plants which were indistinguishable
from the untransformed WS wildtype. The most pronounced variant
phenotype was that of reduced secondary inflorescence formation,
slightly delayed flowering time, larger rosettes with more leaves,
and tall, thick inflorescences. This pot of plants was used only to
provide a size comparison.
TABLE-US-00004 TABLE 7 Qualitative phenotypes observed in 35S::cDNA
5662747 T.sub.1 events Increased Rosette Reduced Size Increased
Late Secondary Rosette Leaf Flower- Inflor. Tall & Fertility
Event Number ing Formation Thick Defects ME01795-01 x x x x
ME01795-02 x x x ME01795-03 x ME01795-04 x x x x ME01795-05 x
ME01795-06 x x x x ME01795-07 x x x ME01795-08 x ME01795-09 x
ME01795-10 x x x x
Quantitative Analysis of the T.sub.2 Plants:
[0262] Events 01, 04, and 10 were evaluated in greater detail in
the T.sub.2 generation. Fourteen individuals were sown for each
event. The transgenic plants of all 3 events showed increased
height, primary inflorescence thickness, and delay of flowering
time to a 0.01 level of statistical significance (Table 8). These
plants also had qualitatively larger rosettes which contained more
leaves (data not shown). All plants, noted in the table as
ME01795-01, ME01795-04, or ME01795-10, were segregating progeny of
the T.sub.1 event which we had confirmed to contain the transgene
under test. All plants noted in the table as -01 Control, -04
Control, or -10 Controls were T.sub.2 segregating progeny which did
not contain the transgene under test (internal controls).
[0263] One item of note in the T.sub.y analysis is that the reduced
secondary inflorescence formation observed in T.sub.1 plants is no
longer present in T.sub.2 plants. In addition, the delay in
flowering time appears to have increased in severity from the
T.sub.1 to T.sub.2 generation.
[0264] Segregation frequencies of the transgene under test suggest
that each event contains a single insert, as shown by a Chi-square
test (Table 8 and data not shown).
TABLE-US-00005 TABLE 8 Quantitative phenotypes seen in 35S::cDNA
5662747 T.sub.2 events Primary Number of Height Inflorescence Days
to Event/Control Observations (cm) Thickness (mm) Bolt ME01795-01 8
64.3* 1.062* 29.8* -01 Control 6 48.3 1.048 24.5 ME01795-04 9 70.9*
1.065* 35.8* -04 Control 5 42.4 1.047 25.8 ME01795-10 8 67.9*
1.069* 31.3* -10 Control 6 43.3 1.049 25.3 *significantly different
from control at 0.01 level, via t-test
Expression: Ceres clone 8161 is down-regulated in both the shoot
apical meristem and root tips of the plant relative to whole plant
mRNA extracts.
Summary of Results
[0265] The ectopic expression of cDNA 5662747 with a strong
constitutive promoter (35S) results in taller plants, with thicker
inflorescences, a larger rosette, and more rosette leaves. cDNA
5662747 is normally regulated in shoot and root apices, suggesting
that the encoded protein may help to regulate meristem function.
The increase in plant size seen by this expression is accompanied
by a delay in flowering time, but no reduction in fertility. As the
T.sub.1 plants had a much less severe delay in flowering than the
T.sub.2 plants, but still produced the large-plant phenotype, it
may be possible to use a promoter of different strength or with a
different spatial expression pattern with the cDNA to maintain an
increase in plant height and stem/inflorescence thickness without
any increase in flowering time. Alternatively, it might be possible
to co-express an early flowering gene (e.g., LEAFY) to thereby
alleviate/counter balance any late flowering effects. In addition,
the gene of the invention (cDNA 5662747) can be utilize to
transform a plant line known to have an early flowering
characteristic, to thereby create a transformed line with normal
flowering time. It may also be a useful gene to increase root
growth, given the similar expression pattern in shoot meristems and
root tip cells.
[0266] Assuming conservation of process controlling vegetative
growth across species, this gene and protein is likely to function
similarly in other species. Increased vegetative biomass should
give an improved source:sink ratio and improved fixation of carbon
to sucrose and starch. It may in and of itself play into improved
yield. Taller inflorescences give the opportunity for more flowers
and therefore more seeds. The combination of improved biomass and
inflorescence stature may give a significant improvement in yield.
Thicker inflorescences may prevent against "snap" against wind,
rain or drought, Biomass advantage and presumed photosynthesis
advantage should be useful in corn and soybean.
[0267] Therefore this gene/protein is especially useful for
controlling the number/rate of cell division in meristems without
disturbing overall plant morphology. It could be developed in crops
with an appropriate promoter to regulate size and growth rate of
many individual organs. The use of a tissue-specific promoter may
be particularly desirable. For example, if an increase of leaf size
is desired without an increase in root size, the coding sequences
of the invention can be operably linked to a leaf specific promoter
for this purpose. Alternatively, if an increase in plant size is
desired with no change in flowering time, the coding sequences of
the invention can be modulated with a leaf specific promoter that
does not direct expression in the floral meristem.
Microarray Analysis
[0268] A major way that a cell controls its response to internal or
external stimuli is by regulating the rate of transcription of
specific genes. For example, the differentiation of cells during
organogenensis into forms characteristic of the organ is associated
with the selective activation and repression of large numbers of
genes. Thus, specific organs, tissues and cells are functionally
distinct due to the different populations of mRNAs and protein
products they possess. Internal signals program the selective
activation and repression programs. For example, internally
synthesized hormones produce such signals. The level of hormone can
be raised by increasing the level of transcription of genes
encoding proteins concerned with hormone synthesis.
[0269] To measure how a cell reacts to internal and/or external
stimuli, individual mRNA levels can be measured and used as an
indicator for the extent of transcription of the gene, Cells can be
exposed to a stimulus, and mRNA can be isolated and assayed at
different time points after stimulation. The mRNA from the
stimulated cells can be compared to control cells that were not
stimulated. The mRNA levels that are higher in the stimulated cell
versus the control indicate a stimulus-specific response of the
cell. The same is true of mRNA levels that are lower in stimulated
cells versus the control condition.
[0270] Similar studies can be performed with cells taken from an
organism with a defined mutation in their genome as compared with
cells without the mutation. Altered mRNA levels in the mutated
cells indicate how the mutation causes transcriptional changes.
These transcriptional'changes are associated with the phenotype
that the mutated cells exhibit that is different from the phenotype
exhibited by the control cells.
[0271] Applicants have utilized microarray techniques to measure
the levels of mRNAs in cells from plants transformed with the
polynucleotides of the invention. In general, transformants with
the genes of the invention were grown to an appropriate stage, and
tissue samples were prepared for the microarray differential
expression analysis.
Microarray Experimental Procedures and Results
Procedures
[0272] A summary of the parameters utilized for each of the
differential expression analysis experiments is provided in TABLE
9.
1. Sample Tissue Preparation
[0273] Tissue samples for each of the expression analysis
experiments were prepared as follows:
[0274] (a) Roots
[0275] Seeds of Arabidopsis thaliana (Ws) were sterilized in full
strength bleach for less than 5 min., washed more than 3 times in
sterile distilled deionized water and plated on MS agar plates. The
plates were placed at 4.degree. C. for 3 nights and then placed
vertically into a growth chamber having 16 hr light/8 hr dark
cycles, 23.degree. C., 70% relative humidity and .about.11,000 LUX.
After 2 weeks, the roots were cut from the agar, flash frozen in
liquid nitrogen and stored at -80.degree. C.
[0276] (b) Rosette Leaves, Stems, and Siliques
[0277] Arabidopsis thaliana (Ws) seed was vernalized at 4.degree.
C. for 3 days before sowing in Metro-mix soil type 350. Flats were
placed in a growth chamber having 16 hr light/8 hr dark, 80%
relative humidity, 23.degree. C. and 13,000 LUX for germination and
growth. After 3 weeks, rosette leaves, stems, and siliques were
harvested, flash frozen in liquid nitrogen and stored at
-80.degree. C. until use. After 4 weeks, siliques (<5 mm, 5-10
mm and >10 mm) were harvested, flash frozen in liquid nitrogen
and stored at -80.degree. C. until use. 5 week old whole plants
(used as controls) were harvested, flash frozen in liquid nitrogen
and kept at -80.degree. C. until RNA was isolated.
[0278] (c) Germination
[0279] Arabidopsis thaliana seeds (ecotype Ws) were sterilized in
bleach and rinsed with sterile water. The seeds were placed in 100
mm petri plates containing soaked autoclaved filter paper. Plates
were foil-wrapped and left at 4.degree. C. for 3 nights to
vernalize. After cold treatment, the foil was removed and plates
were placed into a growth chamber having 16 hr light/8 hr dark
cycles, 23.degree. C., 70% relative humidity and .about.11,000 lux.
Seeds were collected 1 d, 2 d, 3 d and 4 d later, flash frozen in
liquid nitrogen and stored at -80.degree. C. until RNA was
isolated.
[0280] (d) Abscissic Acid (ABA)
[0281] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in trays and left at 4.degree. C. for 4 days to vernalize.
They were then transferred to a growth chamber having grown 16 hr
light/8 hr dark, 13,000 LUX, 70% humidity, and 20.degree. C. and
watered twice a week with 1 L of 1.times. Hoagland's solution.
Approximately 1,000 14 day old plants were spayed with 200-250 Mk
of 100 .mu.M ABA in a 0.02% solution of the detergent Silwet L-77.
Whole seedlings, including roots, were harvested within a 15 to 20
minute time period at 1 hr and 6 hr after treatment, flash-frozen
in liquid nitrogen and stored at -80.degree. C.
[0282] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in 1-liter beakers with 100 .mu.M ABA for
treatment. Control plants were treated with water. After 6 hr and
24 hr, aerial and root tissues were separated and flash frozen in
liquid nitrogen prior to storage at -80.degree. C.
[0283] (e) Brassinosteroid Responsive
[0284] Two separate experiments were performed, one with
epi-brassinolide and one with the brassinosteroid biosynthetic
inhibitor brassinazole. In the epi-brassinolide experiments, seeds
of wild-type Arabidopsis thaliana (ecotype Wassilewskija) and the
brassinosteroid biosynthetic mutant dwf4-1 were sown in trays and
left at 4.degree. C. for 4 days to vernalize. They were then
transferred to a growth chamber having 16 hr light/8 hr dark,
11,000 LUX, 70% humidity and 22.degree. C. temperature. Four week
old plants were spayed with a 1 .mu.M solution of epi-brassinolide
and shoot parts (unopened floral primordia and shoot apical
meristems) harvested three hours later. Tissue was flash-frozen in
liquid nitrogen and stored at -80.degree. C. In the brassinazole
experiments, seeds of wild-type Arabidopsis thaliana (ecotype
Wassilewskija) were grown as described above. Four week old plants
were spayed with a 1 .mu.M solution of brassinazole and shoot parts
(unopened floral primordia and shoot apical meristems) harvested
three hours later. Tissue was flash-frozen in liquid nitrogen and
stored at -80.degree. C.
[0285] In addition to the spray experiments, tissue was prepared
from two different mutants; (1) a dwf4-1 knock out mutant and (2) a
mutant overexpressing the dwf4-1 gene.
[0286] Seeds of wild-type Arabidopsis thaliana (ecotype
Wassilewskija) and of the dwf4-1 knock out and overexpressor
mutants were sown in trays and left at 4.degree. C. for 4 days to
vernalize. They were then transferred to a growth chamber having 16
hr light/8 hr dark, 11,000 LUX, 70% humidity and 22.degree. C.
temperature. Tissue from shoot parts (unopened floral primordia and
shoot apical meristems) was flash-frozen in liquid nitrogen and
stored at -80.degree. C.
[0287] Another experiment was completed with seeds of Arabidopsis
thaliana (ecotype Wassilewskija) were sown in trays and left at
4.degree. C. for 4 days to vernalize. They were then transferred to
a growth chamber. Plants were grown under long-day (16 hr light: 8
hr. dark) conditions, 13,000 LUX light intensity, 70% humidity,
20.degree. C. temperature and watered twice a week with 1 L
1.times. Hoagland's solution (recipe recited in Feldmann et al.,
(1987) Mol. Gen. Genet, 208: 1-9 and described as complete nutrient
solution). Approximately 1,000 14 day old plants were spayed with
200-250 mls of 0.1 .mu.M Epi-Brassinolite in 0.02% solution of the
detergent Silwet L-77. At 1 hr. and 6 hrs, after treatment aerial
tissues were harvested within a 15 to 20 minute time period and
flash-frozen in liquid nitrogen.
[0288] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX, Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in 1-liter beakers with 0.1 .mu.M
epi-brassinolide for treatment. Control plants were treated with
distilled deionized water. After 24 hr, aerial and root tissues
were separated and flash frozen in liquid nitrogen prior to storage
at -80.degree. C.
[0289] (f) Nitrogen: High to Low
[0290] Wild type Arabidopsis thaliana seeds (ecotpye Ws) were
surface sterilized with 30% Clorox, 0.1% Triton X-100 for 5
minutes. Seeds were then rinsed with 4-5 exchanges of sterile
double distilled deionized water. Seeds were vernalized at
4.degree. C. for 2-4 days in darkness. After cold treatment, seeds
were plated on modified 1.times. MS media (without NH.sub.4NO.sub.3
or KNO.sub.3), 0.5% sucrose, 0.5 g/L MES pH5.7, 1% phytagar and
supplemented with KNO.sub.3 to a final concentration of 60 mM (high
nitrate modified 1.times. MS media). Plates were then grown for 7
days in a Percival growth chamber at 22.degree. C. with 16 hr.
light/8 hr dark.
[0291] Germinated seedlings were then transferred to a sterile
flask containing 50 mL of high nitrate modified 1.times.MS liquid
media. Seedlings were grown with mild shaking for 3 additional days
at 22.degree. C. in 16 hr. light/8 hr dark (in a Percival growth
chamber) on the high nitrate modified 1.times. MS liquid media.
[0292] After three days of growth on high nitrate modified
1.times.MS liquid media, seedlings were transferred either to a new
sterile flask containing 50 mL of high nitrate modified 1.times. MS
liquid media or to low nitrate modified 1.times. MS liquid media
(containing 20 .quadrature.M KNO.sub.3). Seedlings were grown in
these media conditions with mild shaking at 22.degree. C. in 16 hr
light/8 hr dark for the appropriate time points and whole seedlings
harvested for total RNA isolation via the Trizol method
(LifeTech.). The time points used for the microarray experiments
were 10 min. and 1 hour time points for both the high and low
nitrate modified 1.times.MS media.
[0293] Alternatively, seeds that were surface sterilized in 30%
bleach containing 0.1% Triton X-100 and further rinsed in sterile
water, were planted on MS agar, (0.5% sucrose) plates containing 50
mM KNO.sub.3 (potassium nitrate). The seedlings were grown under
constant light (3500 LUX) at 22.degree. C. After 12 days, seedlings
were transferred to MS agar plates containing either 1 mM KNO.sub.3
or 50 mM KNO.sub.3. Seedlings transferred to agar plates containing
50 mM KNO.sub.3 were treated as controls in the experiment.
Seedlings transferred to plates with 1 mM KNO.sub.3 were rinsed
thoroughly with sterile MS solution containing 1 mM KNO.sub.3.
There were ten plates per transfer. Root tissue was collected and
frozen in 15 mL Falcon tubes at various time points which included
1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 9 hours, 12 hours, 16
hours, and 24 hours.
[0294] Maize 35A19 Pioneer hybrid seeds were sown on flats
containing sand and grown in a Conviron growth chamber at
25.degree. C., 16 hr light/8 hr dark, 13,000 LUX and 80% relative
humidity. Plants were watered every three days with double
distilled deionized water. Germinated seedlings are allowed to grow
for 10 days and were watered with high nitrate modified 1.times. MS
liquid media (see above). On day 11, young corn seedlings were
removed from the sand (with their roots intact) and rinsed briefly
in high nitrate modified 1.times.MS liquid media. The equivalent of
half a flat of seedlings were then submerged (up to their roots) in
a beaker containing either 500 mL of high or low nitrate modified
1.times.MS liquid media (see above for details).
[0295] At appropriate time points, seedlings were removed from
their respective liquid media, the roots separated from the shoots
and each tissue type flash frozen in liquid nitrogen and stored at
-80.degree. C. This was repeated for each time point. Total RNA was
isolated using the Trizol method (see above) with root tissues
only.
[0296] Corn root tissues isolated at the 4 hr and 16 hr time points
were used for the microarray experiments. Both the high and low
nitrate modified 1.times.MS media were used.
[0297] (g) Nitrogen: Low to High
[0298] Arabidopsis thaliana ecotype Ws seeds were sown on flats
containing 4 L of a 1:2 mixture of Grace Zonolite vermiculite and
soil. Flats were watered with 3 L of water and vernalized at
4.degree. C. for five days. Flats were placed in a Conviron growth
chamber having 16 hr light/8 hr dark at 20.degree. C., 80% humidity
and 17,450 LUX. Flats were watered with approximately 1.5 L of
water every four days. Mature, bolting plants (24 days after
germination) were bottom treated with 2 L of either a control (100
mM mannitol pH 5.5) or an experimental (50 mM ammonium nitrate, pH
5.5) solution, Roots, leaves and siliques were harvested separately
30, 120 and 240 minutes after treatment, flash frozen in liquid
nitrogen and stored at -80.degree. C.
[0299] Hybrid maize seed (Pioneer hybrid 35A19) were aerated
overnight in deionized water. Thirty seeds were plated in each
flat, which contained 4 liters of Grace zonolite vermiculite. Two
liters of water were bottom fed and flats were kept in a Conviron
growth chamber with 16 hr light/8 hr dark at 20.degree. C. and 80%
humidity. Flats were watered with 1 L of tap water every three
days. Five day old seedlings were treated as described above with 2
L of either a control (100 mM mannitol pH 6.5) solution or 1 L of
an experimental (50 mM ammonium nitrate, pH 6.8) solution. Fifteen
shoots per time point per treatment were harvested 10, 90 and 180
minutes after treatment, flash frozen in liquid nitrogen and stored
at -80.degree. C.
[0300] Alternatively, seeds of Arabidopsis thaliana (ecotype
Wassilewskija) were left at 4.degree. C. for 3 days to vernalize.
They were then sown on vermiculite in a growth chamber having 16
hours light/8 hours dark, 12,000-14,000 LUX, 70% humidity, and
20.degree. C. They were bottom-watered with tap water, twice
weekly. Twenty-four days old plants were sprayed with either water
(control) or 0.6% ammonium nitrate at 4 .mu.L/cm.sup.2 of tray
surface. Total shoots and some primary roots were cleaned of
vermiculite, flash-frozen in liquid nitrogen and stored at
-80.degree. C.
[0301] (h) Methyl Jasmonate
[0302] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in trays and left at 4.degree. C. for 4 days to vernalize
before being transferred to a growth chamber having 16 hr light/8
hr. dark, 13,000 LUX, 70% humidity, 20.degree. C. temperature and
watered twice a week with 1 L of a 1.times. Hoagland's solution.
Approximately 1,000 14 day old plants were spayed with 200-250 mls
of 0.001% methyl jasmonate in a 0.02% solution of the detergent
Silwet L-77. At 1 hr and 6 hrs after treatment, whole seedlings,
including roots, were harvested within a 15 to 20 minute time
period, flash-frozen in liquid nitrogen and stored at -80.degree.
C.
[0303] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in 1-liter beakers with 0.001% methyl jasmonate
for treatment. Control plants were treated with water. After 24 hr,
aerial and root tissues were separated and flash frozen in liquid
nitrogen prior to storage at -80.degree. C.
[0304] (i) Salicylic Acid
[0305] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in trays and left at 4.degree. C. for 4 days to vernalize
before being transferred to a growth chamber having 16 hr light/8
hr. dark, 13,000 LUX, 70% humidity, 20.degree. C. temperature and
watered twice a week with 1 L of a 1.times. Hoagland's solution.
Approximately 1,000 14 day old plants were spayed with 200-250 mls
of 5 mM salicylic acid (solubilized in 70% ethanol) in a 0.02%
solution of the detergent Silwet L-77. At 1 hr and 6 hrs after
treatment, whole seedlings, including roots, were harvested within
a 15 to 20 minute time period flash-frozen in liquid nitrogen and
stored at -80.degree. C.
[0306] Alternatively, seeds of wild-type Arabidopsis thaliana
(ecotype Columbia) and mutant CS3726 were sown in soil type 200
mixed with osmocote fertilizer and Marathon insecticide and left at
4.degree. C. for 3 days to vernalize. Flats were incubated at room
temperature with continuous light. Sixteen days post germination
plants were sprayed with 2 mM SA, 0.02% SilwettL-77 or control
solution (0.02% SilwettL-77. Aerial parts or flowers were harvested
1 hr, 4 hr, 6 hr, 24 hr and 3 weeks post-treatment flash frozen and
stored at -80.degree. C.
[0307] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in 1-liter beakers with 2 mM SA for treatment.
Control plants were treated with water. After 12 hr and 24 hr,
aerial and root tissues were separated and flash frozen in liquid
nitrogen prior to storage at -80.degree. C.
[0308] (j) Drought Stress
[0309] Seeds of Arabidopsis thaliana (Wassilewskija) were sown in
pots and left at 4.degree. C. for three days to vernalize before
being transferred to a growth chamber having 16 hr light/8 hr dark,
150,000-160,000 LUX, 20.degree. C. and 70% humidity. After 14 days,
aerial tissues were cut and left to dry on 3mM Whatman paper in a
petri-plate for 1 hour and 6 hours. Aerial tissues exposed for 1
hour and 6 hours to 3 mM Whatman paper wetted with 1.times.
Hoagland's solution served as controls. Tissues were harvested,
flash-frozen in liquid nitrogen and stored at -80.degree. C.
[0310] Alternatively, Arabidopsis thaliana (Ws) seed was vernalized
at 4.degree. C. for 3 days before sowing in Metromix soil type 350.
Flats were placed in a growth chamber with 23.degree. C., 16 hr
light/8 hr. dark, 80% relative humidity, .about.13,000 LUX for
germination and growth. Plants were watered with 1-1.5 L of water
every four days. Watering was stopped 16 days after germination for
the treated samples, but continued for the control samples. Rosette
leaves and stems, flowers and siliques were harvested 2 d, 3 d, 4
d, 5 d, 6 d and 7 d after watering was stopped. Tissue was flash
frozen in liquid nitrogen and kept at -80.degree. C. until RNA was
isolated. Flowers and siliques were also harvested on day 8 from
plants that had undergone a 7 d drought treatment followed by 1 day
of watering. Control plants (whole plants) were harvested after 5
weeks, flash frozen in liquid nitrogen and stored as above.
[0311] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX, Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in empty 1-liter beakers at room temperature
for treatment. Control plants were placed in water. After 1 hr, 6
hr, 12 hr and 24 hr aerial and root tissues were separated and
flash frozen in liquid nitrogen prior to storage at -80.degree.
C.
[0312] (k) Osmotic Stress
[0313] Seeds of Arabidopsis thaliana (Wassilewskija) were sown in
trays and left at 4.degree. C. for three days to vernalize before
being transferred to a growth chamber having 16 hr light/8 hr dark,
12,000-14,000 LUX, 20.degree. C., and 70% humidity. After 14 days,
the aerial tissues were cut and placed on 3 MM Whatman paper in a
petri-plate wetted with 20% PEG (polyethylene glycol-M.sub.r 8,000)
in 1.times. Hoagland's solution. Aerial tissues on 3 MM Whatman
paper containing 1.times. Hoagland's solution alone served as the
control. Aerial tissues were harvested at 1 hour and 6 hours after
treatment, flash-frozen in liquid nitrogen and stored at
-80.degree. C.
[0314] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX, Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in 1-liter beakers with 10% PEG (polyethylene
glycol-M.sub.r 8,000) for treatment. Control plants were treated
with water. After 1 hr and 6 hr aerial and root tissues were
separated and flash frozen in liquid nitrogen prior to storage at
-80.degree. C.
[0315] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in 1-liter beakers with 150 mM NaCl for
treatment. Control plants were treated with water. After 1 hr, 6
hr, and 24 hr aerial and root tissues were separated and flash
frozen in liquid nitrogen prior to storage at -80.degree. C.
[0316] (l) Heat Shock Treatment
[0317] Seeds of Arabidopsis Thaliana (Wassilewskija) were sown in
trays and left at 4.degree. C. for three days to vernalize before
being transferred to a growth chamber with 16 hr light/8 hr dark,
12,000-14,000 Lux, 70% humidity and 20.degree. C., fourteen day old
plants were transferred to a 42.degree. C. growth chamber and
aerial tissues were harvested 1 hr and 6 hr after transfer. Control
plants were left at 20.degree. C. and aerial tissues were
harvested, Tissues were flash-frozen in liquid nitrogen and stored
at -80.degree. C.
[0318] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in 1-liter beakers containing 42.degree. C.
water for treatment. Control plants were treated with water at
25.degree. C. After 1 hr and 6 hr aerial and root tissues were
separated and flash frozen in liquid nitrogen prior to storage at
-80.degree. C.
[0319] (m) Cold Shock Treatment
[0320] Seeds of Arabidopsis thaliana (Wassilewskija) were sown in
trays and left at 4.degree. C. for three days to vernalize before
being transferred to a growth chamber having 16 hr light/8 hr dark,
12,000-14,000 LUX, 20.degree. C. and 70% humidity. Fourteen day old
plants were transferred to a 4.degree. C. dark growth chamber and
aerial tissues were harvested 1 hour and 6 hours later. Control
plants were maintained at 20.degree. C. and covered with foil to
avoid exposure to light. Tissues were flash-frozen in liquid
nitrogen and stored at -80.degree. C.
[0321] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 7 days. Seedlings were carefully removed from
the sand and placed in 1-liter beakers containing 4.degree. C.
water for treatment. Control plants were treated with water at
25.degree. C. After 1 hr and 6 hr aerial and root tissues were
separated and flash frozen in liquid nitrogen prior to storage at
-80.degree. C.
[0322] (n) Arabidopsis Seeds
[0323] Fruits (pod+seed) 0-5 mm
[0324] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in pots and left at 4.degree. C. for two to three days to
vernalize. They were then transferred to a growth chamber. Plants
were grown under long-day (16 hr light: 8 hr dark) conditions,
7000-8000 LUX light intensity, 70% humidity, and 22.degree. C.
temperature. 3-4 siliques (fruits) bearing developing seeds were
selected from at least 3 plants and were hand-dissected to
determine what developmental stage(s) were represented by the
enclosed embryos. Description of the stages of Arabidopsis
embryogenesis used in this determination were summarized by Bowman
(1994). Silique lengths were then determined and used as an
approximate determinant for embryonic stage, Siliques 0-5 mm in
length containing post fertilization through pre-heart stage [0-72
hours after fertilization (HAF)] embryos were harvested and flash
frozen in liquid nitrogen.
[0325] Fruits (pod+seed) 5-10 mm
[0326] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in pots and left at 4.degree. C. for two to three days to
vernalize. They were then transferred to a growth chamber. Plants
were grown under long-day (16 hr light: 8 hr dark) conditions,
7000-8000 LUX light intensity, 70% humidity, and 22.degree. C.
temperature. 3-4 siliques (fruits) bearing developing seeds were
selected from at least 3 plants and were hand-dissected to
determine what developmental stage(s) were represented by the
enclosed embryos. Description of the stages of Arabidopsis
embryogenesis used in this determination were summarized by Bowman
(1994). Silique lengths were then determined and used as an
approximate determinant for embryonic stage. Siliques 5-10 mm in
length containing heart-through early upturned-U-stage [72-120
hours after fertilization (HAF)] embryos were harvested and flash
frozen in liquid nitrogen.
[0327] Fruits (pod+seed)>10 mm
[0328] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in pots and left at 4.degree. C. for two to three days to
vernalize. They were then transferred to a growth chamber. Plants
were grown under long-day (16 hr light: 8 hr dark) conditions,
7000-8000 LUX light intensity, 70% humidity, and 22.degree. C.
temperature. 3-4 siliques (fruits) bearing developing seeds were
selected from at least 3 plants and were hand-dissected to
determine what developmental stage(s) were represented by the
enclosed embryos. Description of the stages of Arabidopsis
embryogenesis used in this determination were summarized by Bowman
(1994). Silique lengths were then determined and used as an
approximate determinant for embryonic stage. Siliques >10 mm in
length containing green, late upturned-U-stage [>120 hours after
fertilization (HAF)-9 days after flowering (DAF)] embryos were
harvested and flash frozen in liquid nitrogen.
[0329] Green Pods 5-10 mm (Control Tissue for Samples 72-74)
[0330] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in pots and left at 4.degree. C. for two to three days to
vernalize. They were then transferred to a growth chamber. Plants
were grown under long-day (16 hr light: 8 hr dark) conditions,
7000-8000 LUX light intensity, 70% humidity, and 22.degree. C.
temperature. 3-4 siliques (fruits) bearing developing seeds were
selected from at least 3 plants and were hand-dissected to
determine what developmental stage(s) were represented by the
enclosed embryos. Description of the stages of Arabidopsis
embryogenesis used in this determination were summarized by Bowman
(1994). Silique lengths were then determined and used as an
approximate determinant for embryonic stage. Green siliques 5-10 mm
in length containing developing seeds 72-120 hours after
fertilization (HAF)] were opened and the seeds removed. The
remaining tissues (green pods minus seed) were harvested and flash
frozen in liquid nitrogen.
[0331] Green Seeds from Fruits >10 mm
[0332] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in pots and left at 4.degree. C. for two to three days to
vernalize. They were then transferred to a growth chamber. Plants
were grown under long-day (16 hr light: 8 hr dark) conditions,
7000-8000 LUX light intensity, 70% humidity, and 22.degree. C.
temperature. 3-4 siliques (fruits) bearing developing seeds were
selected from at least 3 plants and were hand-dissected to
determine what developmental stage(s) were represented by the
enclosed embryos. Description of the stages of Arabidopsis
embryogenesis used in this determination were summarized by Bowman
(1994). Silique lengths were then determined and used as an
approximate determinant for embryonic stage. Green siliques >10
mm in length containing developing seeds up to 9 days after
flowering (DAF)] were opened and the seeds removed and harvested
and flash frozen in liquid nitrogen.
[0333] Brown Seeds from Fruits >10 mm
[0334] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in pots and left at 4.degree. C. for two to three days to
vernalize. They were then transferred to a growth chamber. Plants
were grown under long-day (16 hr light: 8 hr dark) conditions,
7000-8000 LUX light intensity, 70% humidity, and 22.degree. C.
temperature. 3-4 siliques (fruits) bearing developing seeds were
selected from at least 3 plants and were hand-dissected to
determine what developmental stage(s) were represented by the
enclosed embryos. Description of the stages of Arabidopsis
embryogenesis used in this determination were summarized by Bowman
(1994). Silique lengths were then determined and used as an
approximate determinant for embryonic stage. Yellowing siliques
>10 mm in length containing brown, dessicating seeds >11 days
after flowering (DAF)] were opened and the seeds removed and
harvested and flash frozen in liquid nitrogen.
[0335] Green/Brown Seeds from Fruits >10 mm
[0336] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in pots and left at 4.degree. C. for two to three days to
vernalize. They were then transferred to a growth chamber. Plants
were grown under long-day (16 hr light: 8 hr dark) conditions,
7000-8000 LUX light intensity, 70% humidity, and 22.degree. C.
temperature. 3-4 siliques (fruits) bearing developing seeds were
selected from at least 3 plants and were hand-dissected to
determine what developmental stage(s) were represented by the
enclosed embryos. Description of the stages of Arabidopsis
embryogenesis used in this determination were summarized by Bowman
(1994). Silique lengths were then determined and used as an
approximate determinant for embryonic stage. Green siliques >10
mm in length containing both green and brown seeds >9 days after
flowering (DAF)] were opened and the seeds removed and harvested
and flash frozen in liquid nitrogen.
[0337] Mature Seeds (24 Hours after Imbibition)
[0338] Mature dry seeds of Arabidopsis thaliana (ecotype
Wassilewskija) were sown onto moistened filter paper and left at
4.degree. C. for two to three days to vernalize. Imbibed seeds were
then transferred to a growth chamber [16 hr light: 8 hr dark
conditions, 7000-8000 LUX light intensity, 70% humidity, and
22.degree. C. temperature], the emerging seedlings harvested after
48 hours and flash frozen in liquid nitrogen.
[0339] Mature Seeds (Dry)
[0340] Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were
sown in pots and left at 4.degree. C. for two to three days to
vernalize. They were then transferred to a growth chamber. Plants
were grown under long-day (16 hr light: 8 hr dark) conditions,
7000-8000 LUX light intensity, 70% humidity, and 22.degree. C.
temperature and taken to maturity. Mature dry seeds are collected,
dried for one week at 28.degree. C., and vernalized for one week at
4.degree. C. before used as a source of RNA.
[0341] (o) Herbicide Treatment
[0342] Arabidopsis thaliana (Ws) seeds were sterilized for 5 min.
with 30% bleach, 50 .mu.l Triton in a total volume of 50 ml. Seeds
were vernalized at 4.degree. C. for 3 days before being plated onto
GM agar plates at a density of about 144 seeds per plate. Plates
were incubated in a Percival growth chamber having 16 hr light/8 hr
dark, 80% relative humidity, 22.degree. C. and 11,000 LUX for 14
days.
[0343] Plates were sprayed (.about.0.5 mls/plate) with water,
Finale (1.128 g/L), Glean (1.88 g/L), RoundUp (0.01 g/L) or Trimec
(0.08 g/L). Tissue was collected and flash frozen in liquid
nitrogen at the following time points: 0, 1, 2, 4, 8, 12 and 24
hours. Frozen tissue was stored at -80.degree. C. prior to RNA
isolation.
[0344] (p) Root Tips
[0345] Seeds of Arabidopsis thaliana (ecotye Ws) were placed on MS
plates and vernalized at 4.degree. C. for 3 days before being
placed in a 25.degree. C. growth chamber having 16 hr light/8 hr
dark, 70% relative humidity and about 3 W/m.sup.2. After 6 days,
young seedlings were transferred to flasks containing B5 liquid
medium, 1% sucrose and 0.05 mg/l indole-3-butyric acid. Flasks were
incubated at room temperature with 100 rpm agitation. Media was
replaced weekly. After three weeks, roots were harvested and
incubated for 1 hr with 2% pectinase, 0.2% cellulase, pH 7 before
straining through a #80 (Sigma) sieve. The root body material
remaining on the sieve (used as the control) was flash frozen and
stored at -80.degree. C. until use. The material that passed
through the #80 sieve was strained through a #200 (Sigma) sieve and
the material remaining on the sieve (root tips) was flash frozen
and stored at -80.degree. C. until use. Approximately 10 mg of root
tips were collected from one flask of root culture.
[0346] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in flats (10 rows, 5-6 seed/row) and covered
with clear, plastic lids before being placed in a growth chamber
having 16 hr light (25.degree. C.)/8 hr dark (20.degree. C.), 75%
relative humidity and 13,000-14,000 LUX. Covered flats were watered
every three days for 8 days. Seedlings were carefully removed from
the sand and the root tips (-2 mm long) were removed and flash
frozen in liquid nitrogen prior to storage at -80.degree. C. The
tissues above the root tips (.about.1 cm long) were cut, treated as
above and used as control tissue.
[0347] (q) Imbibed Seed
[0348] Seeds of maize hybrid 35A (Pioneer) were sown in
water-moistened sand in covered flats (10 rows, 5-6 seed/row) and
covered with clear, plastic lids before being placed in a growth
chamber having 16 hr light (25.degree. C.)/8 hr dark (20.degree.
C.), 75% relative humidity and 13,000-14,000 LUX. One day after
sowing, whole seeds were flash frozen in liquid nitrogen prior to
storage at -80.degree. C. Two days after sowing, embryos and
endosperm were isolated and flash frozen in liquid nitrogen prior
to storage at -80.degree. C. On days 3-6, aerial tissues, roots and
endosperm were isolated and flash frozen in liquid nitrogen prior
to storage at -80.degree. C.
[0349] (r) Flowers (Green, White or Buds)
[0350] Approximately 10 .quadrature.l of Arabidopsis thaliana seeds
(ecotype Ws) were sown on 350 soil (containing 0.03% marathon) and
vernalized at 4 C for 3 days. Plants were then grown at room
temperature under fluorescent lighting until flowering. Flowers
were harvested after 28 days in three different categories. Buds
that had not opened at all and were completely green were
categorized as "flower buds" (also referred to as green buds by the
investigator). Buds that had started to open, with white petals
emerging slightly were categorized as "green flowers" (also
referred to as white buds by the investigator). Flowers that had
opened mostly (with no silique elongation) with white petals
completely visible were categorized as "white flowers" (also
referred to as open flowers by the investigator). Buds and flowers
were harvested with forceps, flash frozen in liquid nitrogen and
stored at -80 C until RNA was isolated.
2. Microarray Hybridization Procedures
[0351] Microarray technology provides the ability to monitor mRNA
transcript levels of thousands of genes in a single experiment.
These experiments simultaneously hybridize two differentially
labeled fluorescent cDNA pools to glass slides that have been
previously spotted with cDNA clones of the same species. Each
arrayed cDNA spot will have a corresponding ratio of fluorescence
that represents the level of disparity between the respective mRNA
species in the two sample pools. Thousands of polynucleotides can
be spotted on one slide, and each experiment generates a global
expression pattern.
Coating Slides
[0352] The microarray consists of a chemically coated microscope
slide, referred herein as a "chip" with numerous polynucleotide
samples arrayed at a high density. The poly-L-lysine coating allows
for this spotting at high density by providing a hydrophobic
surface, reducing the spreading of spots of DNA solution arrayed on
the slides, Glass microscope slides (Gold Seal #3010 manufactured
by Gold Seal Products, Portsmouth, N.H., USA) were coated with a
0.1% W/V solution of Poly-L-lysine (Sigma, St. Louis, Mo.) using
the following protocol: [0353] 1. Slides were placed in slide racks
(Shandon Lipshaw #121). The racks were then put in chambers
(Shandon Lipshaw #121). [0354] 2. Cleaning solution was prepared:
[0355] 70 g NaOH was dissolved in 280 mL ddH2O. [0356] 420 mL 95%
ethanol was added. The total volume was 700 mL (=2.times.350 mL);
it was stirred until completely mixed. If the solution remained
cloudy, ddH.sub.2O was added until clear. [0357] 3. The solution
was poured into chambers with slides; the chambers were covered
with glass lids. The solution was mixed on an orbital shaker for 2
hr. [0358] 4. The racks were quickly transferred to fresh chambers
filled with ddH.sub.2O. They were rinsed vigorously by plunging
racks up and down. Rinses were repeated 4.times. with fresh
ddH.sub.2O each time, to remove all traces of NaOH-ethanol. [0359]
5. Polylysine solution was prepared: [0360] 0 mL poly-L-lysine+70
mL tissue culture PBS in 560 mL water, using plastic graduated
cylinder and beaker. [0361] 6. Slides were transferred to
polylysine solution and shaken for 1 hr. [0362] 7. The rack was
transferred to a fresh chambers filled with ddH.sub.2O. It was
plunged up and down 5.times. to rinse. [0363] 8. The slides were
centrifuged on microtiter plate carriers (paper towels were placed
below the rack to absorb liquid) for 5 min. @ 500 rpm. The slide
racks were transferred to empty chambers with covers. [0364] 9.
Slide racks were dried in a 45 C oven for 10 min. [0365] 10. The
slides were stored in a closed plastic slide box. [0366] 11.
Normally, the surface of lysine coated slides was not very
hydrophobic immediately after this process, but became increasingly
hydrophobic with storage. A hydrophobic surface helped ensure that
spots didn't run together while printing at high densities. After
they aged for 10 days to a month the slides were ready to use.
However, coated slides that have been sitting around for long
periods of time were usually too old to be used. This was because
they developed opaque patches, visible when held to the light, and
these resulted in high background hybridization from the
fluorescent probe. Alternatively, pre-coated glass slides were
purchased from TeleChem International, Inc, (Sunnyvale, Calif.,
94089; catalog number SMM-25, Superamine substrates). PCR
Amplification of cDNA Clone Inserts
[0367] Polynucleotides were amplified from Arabidopsis cDNA clones
using insert specific probes. The resulting 100 uL PCR reactions
were purified with Qiaquick 96 PCR purification columns (Qiagen,
Valencia, Calif., USA) and eluted in 30 uL of 5 mM Tris. 8.5 uL of
the elution were mixed with 1.5 uL of 20.times.SSC to give a final
spotting solution of DNA in 3.times.SSC. The concentrations of DNA
generated from each clone varied between 10-100 ng/ul, but were
usually about 50 ng/ul.
Arraying of PCR Products on Glass Slides
[0368] PCR products from cDNA clones were spotted onto the
poly-L-Lysine coated glass slides using an arrangement of quill-tip
pins (ChipMaker 3 spotting pins; Telechem, International, Inc.,
Sunnyvale, Calif., USA) and a robotic arrayer (PixSys 3500,
Cartesian Technologies, Irvine, Calif., USA). Around 0.5 nl of a
prepared PCR product was spotted at each location to produce spots
with approximately 100 um diameters. Spot center-to-center spacing
was from 180 um to 210 um depending on the array, Printing was
conducted in a chamber with relative humidity set at 50%.
[0369] Slides containing maize sequences were purchased from
Agilent Technology (Palo Alto, Calif. 94304).
Post-Processing of Slides
[0370] After arraying, slides were processed through a series of
steps--rehydration, UV cross-linking, blocking and
denaturation--required prior to hybridization. Slides were
rehydrated by placing them over a beaker of warm water (DNA face
down), for 2-3 sec, to distribute the DNA more evenly within the
spots, and then snap dried on a hot plate (DNA side, face up). The
DNA was then cross-linked to the slides by UV irradiation (60-55
mJ; 2400 Stratalinker, Stratagene, La Jolla, Calif., USA).
[0371] Following this a blocking step was performed to modify
remaining free lysine groups, and hence minimize their ability to
bind labeled probe DNA. To achieve this the arrays were placed in a
slide rack. An empty slide chamber was left ready on an orbital
shaker. The rack was bent slightly inwards in the middle, to ensure
the slides would not run into each other while shaking. The
blocking solution was prepared as follows:
3.times.350-ml glass chambers (with metal tops) were set to one
side, and a large round Pyrex dish with dH.sub.2O was placed ready
in the microwave. At this time, 15 ml sodium borate was prepared in
a 50 ml conical tube.
[0372] 6-g succinic anhydride was dissolved in approx. 325-350 mL
1-methyl-2-pyrrolidinone. Rapid addition of reagent was
crucial.
[0373] a. Immediately after the last flake of the succinic
anhydride dissolved, the 15-mL sodium borate was added.
[0374] b. Immediately after the sodium borate solution mixed in,
the solution was poured into an empty slide chamber.
[0375] c. The slide rack was plunged rapidly and evenly in the
solution. It was vigorously shaken up and down for a few seconds,
making sure slides never left the solution.
[0376] d. It was mixed on an orbital shaker for 15-20 min.
Meanwhile, the water in the Pyrex dish (enough to cover slide rack)
was heated to boiling.
[0377] Following this, the slide rack was gently plunge in the 95 C
water (just stopped boiling) for 2 min. Then the slide rack was
plunged 5.times.in 95% ethanol. The slides and rack were
centrifuged for 5 min. @ 500 rpm. The slides were loaded quickly
and evenly onto the carriers to avoid streaking. The arrays were
used immediately or store in slide box.
[0378] The Hybridization process began with the isolation of mRNA
from the two tissues (see "Isolation of total RNA" and "Isolation
of mRNA"; below) in question followed by their conversion to single
stranded cDNA (see "Generation of probes for hybridization",
below). The cDNA from each tissue was independently labeled with a
different fluorescent dye and then both samples were pooled
together. This final differentially labeled cDNA pool was then
placed on a processed microarray and allowed to hybridize (see
"Hybridization and wash conditions", below).
Isolation of Total RNA
[0379] Approximately 1 g of plant tissue was ground in liquid
nitrogen to a fine powder and transferred into a 50-ml centrifuge
tube containing 10 ml of Trizol reagent. The tube was vigorously
vortexed for 1 min and then incubated at room temperature for 10-20
min. on an orbital shaker at 220 rpm. Two ml of chloroform was
added to the tube and the solution vortexed vigorously for at least
30-sec before again incubating at room temperature with shaking.
The sample was then centrifuged at 12,000.times.g (10,000 rpm) for
15-20 min at 4.degree. C. The aqueous layer was removed and mixed
by inversion with 2.5 ml of 1.2 M NaCl/0.8 M Sodium Citrate and 2.5
ml of isopropyl alcohol added. After a 10 min, incubation at room
temperature, the sample was centrifuged at 12,000.times.g (10,000
rpm) for 15 min at 4.degree. C. The pellet was washed with 70%
ethanol, re-centrifuged at 8,000 rpm for 5 min and then air dried
at room temperature for 10 min. The resulting total RNA was
dissolved in either TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) or DEPC
(diethylpyrocarbonate) treated deionized water (RNAse-free water).
For subsequent isolation of mRNA using the Qiagen kit, the total
RNA pellet was dissolved in RNAse-free water.
Isolation of mRNA
[0380] mRNA was isolated using the Qiagen Oligotex mRNA Spin-Column
protocol (Qiagen, Valencia, Calif.). Briefly, 500 .mu.l OBB buffer
(20 mM Tris-Cl, pH 7.5, 1 M NaCl, 2 mM EDTA, 0.2% SDS) was added to
500 .mu.l of total RNA (0.5-0.75 mg) and mixed thoroughly. The
sample was first incubated at 70.degree. C. for 3 min, then at room
temperature for 10 minutes and finally centrifuged for 2 min at
14,000-18,000.times.g. The pellet was resuspended in 400 .mu.l OW2
buffer (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA) by
vortexing, the resulting solution placed on a small spin column in
a 1.5 ml RNase-free microcentrifuge tube and centrifuged for 1 min
at 14,000-18,000.times.g. The spin column was transferred to a new
1.5 ml RNase-free microcentrifuge tube and washed with 400 .mu.l of
OW2 buffer. To release the isolated mRNA from the resin, the spin
column was again transferred to a new RNase-free 1.5 ml
microcentrifuge tube, 20-100 .mu.l 70.degree. C. OEB buffer (5 mM
Tris-Cl, pH 7.5) added and the resin resuspended in the resulting
solution via pipeting. The mRNA solution was collected after
centrifuging for 1 min at 14,000-18,000.times.g.
[0381] Alternatively, mRNA was isolated using the Stratagene
Poly(A) Quik mRNA Isolation Kit (Startagene, La Jolla, Calif.).
Here, up to 0.5 mg of total RNA (maximum volume of 1 ml) was
incubated at 65.degree. C. for 5 minutes, snap cooled on ice and
0.1.times. volumes of 10.times. sample buffer (10 mM Tris-HCl (pH
7.5), 1 mM EDTA (pH 8.0) 5 M NaCl) added. The RNA sample was
applied to a prepared push column and passed through the column at
a rate of .about.1 drop every 2 sec. The solution collected was
reapplied to the column and collected as above. 200 .mu.l of high
salt buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 NaCl) was
applied to the column and passed through the column at a rate of
.about.1 drop every 2 sec. This step was repeated and followed by
three low salt buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 M
NaCl) washes preformed in a similar manner. mRNA was eluted by
applying to the column four separate 200 .mu.l aliquots of elution
buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA) preheated to 65.degree.
C. Here, the elution buffer was passed through the column at a rate
of 1 drop/Sec. The resulting mRNA solution was precipitated by
adding 0.1.times. volumes of 10.times. sample buffer, 2.5 volumes
of ice-cold 100% ethanol, incubating overnight at -20.degree. C.
and centrifuging at 14,000-18,000.times.g for 20-30 min at
4.degree. C. The pellet was washed with 70% ethanol and air dried
for 10 min. at room temperature before resuspension in RNase-free
deionized water.
Preparation of Yeast Controls
[0382] Plasmid DNA was isolated from the following yeast clones
using Qiagen filtered maxiprep kits (Qiagen, Valencia., Calif.):
YAL022c(Fun26), YAL031c(Fun21), YBRO32w, YDL131w, YDL182w, YDL194w,
YDL196w, YDR050c and YDR116c. Plasmid DNA was linearized with
either BsrB1 (YAL022c(Fun26), YAL031c(Fun21), YDL131w, YDL182w,
YDL194w, YDL196w, YDR050c) or AflIII (YBR032w, YDR116c) and
isolated.
In Vitro Transcription of Yeast Clones
[0383] The following solution was incubated at 37.degree. C. for 2
hours: 17 .mu.l of isolated yeast insert DNA (1 .mu.g), 20 .mu.l
5.times. buffer, 10 .mu.l 100 mM DTT, 2.5 .mu.l (100 U) RNasin, 20
.mu.l 2.5 mM (ea.) rNTPs, 2.7 (40U) SP6 polymerase and 27.8 .mu.l
RNase-free deionized water. 2 .mu.l (2 U) Ampli DNase I was added
and the incubation continued for another 15 min, 10 .mu.l 5M
NH.sub.4OAC and 100 .mu.l phenol:chloroform:isoamyl alcohol
(25:24:1) were added, the solution vortexed and then centrifuged to
separate the phases. To precipitate the RNA, 250 .mu.l ethanol was
added and the solution incubated at -20.degree. C. for at least one
hour. The sample was then centrifuged for 20 min at 4.degree. C. at
14,000-18,000.times.g, the pellet washed with 500 .mu.l of 70%
ethanol, air dried at room temperature for 10 min and resuspended
in 100 .mu.l of RNase-free deionized water. The precipitation
procedure was then repeated.
[0384] Alternatively, after the two-hour incubation, the solution
was extracted with phenol/chloroform once before adding 0.1 volume
3M sodium acetate and 2.5 volumes of 100% ethanol. The solution was
centrifuged at 15,000 rpm, 4.degree. C. for 20 minutes and the
pellet resuspended in RNase-free deionized water. The DNase I
treatment was carried out at 37.degree. C. for 30 minutes using 2 U
of Ampli DNase I in the following reaction condition: 50 mM
Tris-HCl (pH 7.5), 10 mM MgCl.sub.2. The DNase I reaction was then
stopped with the addition of NH.sub.4OAC and
phenol:chloroform:isoamyl alcohol (25:24:1), and RNA isolated as
described above.
[0385] 0.15-2.5 ng of the in vitro transcript RNA from each yeast
clone were added to each plant mRNA sample prior to labeling to
serve as positive (internal) probe controls.
Generation of Probes for Hybridization
[0386] Generation of Labeled Probes for Hybridization from
First-Strand cDNA
[0387] Hybridization probes were generated from isolated mRNA using
an Atlas.TM. Glass Fluorescent Labeling Kit (Clontech Laboratories,
Inc., Palo Alto, Calif., USA). This entails a two step labeling
procedure that first incorporates primary aliphatic amino groups
during cDNA synthesis and then couples fluorescent dye to the cDNA
by reaction with the amino functional groups. Briefly, 5 .mu.g of
oligo(dT).sub.18 primer d(TTTTTTTTTTTTTTTTTTV) was mixed with Poly
A+ mRNA (1.5-2 .mu.g mRNA isolated using the Qiagen Oligotex mRNA
Spin-Column protocol or the Stratagene Poly(A) Quik mRNA Isolation
protocol (Stratagene, La Jolla, Calif., USA)) in a total volume of
25 .mu.l. The sample was incubated in a thermocycler at 70.degree.
C. for 5 min, cooled to 48.degree. C. and 10 .mu.l of 5.times. cDNA
Synthesis Buffer (kit supplied), 5 .mu.l X dNTP mix (dATP, dCTP,
dCTP, dTTP and aminoallyl-dUTP; kit supplied), 7.5 .mu.l deionized
water and 2.5 .mu.l MMLV Reverse Transcriptase (500U) added. The
reaction was then incubated at 48.degree. C. for 30 minutes,
followed by 1 hr incubation at 42.degree. C. At the end of the
incubation the reaction was heated to 70.degree. C. for 10 min,
cooled to 37.degree. C. and 0.5 .mu.l (5 U) RNase H added, before
incubating for 15 min at 37.degree. C. The solution was vortexed
for 1 min after the addition of 0.5 .mu.l 10.5 M EDTA and 5 .mu.l
of QuickClean Resin (kit supplied) then centrifuged at
14,000-18,000.times.g for 1 min. After removing the supernatant to
a 0.45 .mu.m spin filter (kit supplied), the sample was again
centrifuged at 14,000-18,000.times.g for 1 min, and 5.5 .mu.l 3 M
sodium acetate and 137.5 .mu.l of 100% ethanol added to the sample
before incubating at -20.degree. C. for at least 1 hr. The sample
was then centrifuged at 14,000-18,000.times.g at 4.degree. C. for
20 min, the resulting pellet washed with 500 .mu.l 70% ethanol,
air-dried at room temperature for 10 min and resuspended in 10
.mu.l of 2.times. fluorescent labeling buffer (kit provided). 10
.mu.l each of the fluorescent dyes Cy3 and Cy5 (Amersham Pharmacia
(Piscataway, N.J., USA); prepared according to Atlas.TM. kit
directions of Clontech) were added and the sample incubated in the
dark at room temperature for 30 min.
[0388] The fluorescently labeled first strand cDNA was precipitated
by adding 2 .mu.l 3M sodium acetate and 50 .mu.l 100% ethanol,
incubated at -20.degree. C. for at least 2 hrs, centrifuged at
14,000-18,000.times.g for 20 min, washed with 70% ethanol,
air-dried for 10 min and dissolved in 100 .mu.l of water.
[0389] Alternatively, 3-4 .mu.g mRNA, 2.5 (.about.8.9 ng of in
vitro translated mRNA) .mu.l yeast control and 3 .mu.g oligo dTV
(TTTTTTTTTTTTTTTTTT(A/C/G) were mixed in a total volume of 24.7
.mu.l. The sample was incubated in a thermocycler at 70.degree. C.
for 10 min. before chilling on ice. To this, 8 .mu.l of 5.times.
first strand buffer (SuperScript II RNase H--Reverse Transcriptase
kit from Invitrogen (Carlsbad, Calif. 92008); cat no, 18064022),
0.8.degree. C. of aa-dUTP/dNTP mix (50.times.; 25 mM dATP, 25 mM
dGTP, 25 mM dCTP, 15 mM dTTP, 10 mM aminoallyl-dUTP), 4 .mu.l of
0.1 M DTT and 2.5 .mu.l (500 units) of Superscript R.T.II enzyme
(Stratagene) were added. The sample was incubated at 42.degree. C.
for 2 hours before a mixture of 10.degree. C. of 1M NaOH and
10.degree. C. of 0.5 M EDTA were added. After a 15 minute
incubation at 65.degree. C., 25 .mu.l of 1 M Tris pH 7.4 was added.
This was mixed with 450 .mu.l of water in a Microcon 30 column
before centrifugation at 11,000.times.g for 12 min. The column was
washed twice with 450 .mu.l (centrifugation at 11,000 g, 12 min.)
before eluting the sample by inverting the Microcon column and
centrifuging at 11,000.times.g for 20 seconds. Sample was
dehydrated by centrifugation under vacuum and stored at -20.degree.
C.
[0390] Each reaction pellet was dissolved in 9 .mu.l of 0.1 M
carbonate buffer (0.1M sodium carbonate and sodium bicarbonate,
pH=8.5-9) and 4.5 .mu.l of this placed in two microfuge tubes. 4.5
.mu.l of each dye (in DMSO) were added and the mixture incubated in
the dark for 1 hour. 4.5 .mu.l of 4 M hydroxylamine was added and
again incubated in the dark for 15 minutes.
[0391] Regardless of the method used for probe generation, the
probe was purified using a Qiagen PCR cleanup kit (Qiagen,
Valencia, Calif., USA), and eluted with 100 ul ED (kit provided).
The sample was loaded on a Microcon YM-30 (Millipore, Bedford,
Mass., USA) spin column and concentrated to 4-5 ul in volume.
[0392] Probes for the maize microarrays were generated using the
Fluorescent Linear Amplification Kit (cat. No. G2556A) from Agilent
Technologies (Palo Alto, Calif.).
Hybridization and Wash Conditions
[0393] The following Hybridization and Washing Condition were
developed:
Hybridization Conditions:
[0394] Labeled probe was heated at 95.degree. C. for 3 min and
chilled on ice. Then 25 .quadrature.L of the hybridization buffer
which was warmed at 42 C was added to the probe, mixing by
pipeting, to give a final concentration of:
50% formamide
[0395] 4.times.SSC
[0396] 0.03% SDS
5.times.Denhardt's solution 0.1 .mu.g/ml single-stranded salmon
sperm DNA
[0397] The probe was kept at 42 C. Prior to the hybridization, the
probe was heated for 1 more min., added to the array, and then
covered with a glass cover slip. Slides were placed in
hybridization chambers (Telechem, Sunnyvale, Calif.) and incubated
at 42.degree. C. overnight.
Washing Conditions:
[0398] A. Slides were washed in 1.times.SSC 0.03% SDS solution at
room temperature for 5 minutes, [0399] B. Slides were washed in
0.2.times.SSC at room temperature for 5 minutes, [0400] C. Slides
were washed in 0.05.times.SSC at room temperature for 5
minutes.
[0401] After A, B, and C, slides were spun at 800.times.g for 2
min, to dry. They were then scanned.
[0402] Maize microarrays were hybridized according to the
instructions included Fluorescent Linear Amplification Kit (cat.
No. 02556A) from Agilent Technologies (Palo Alto, Calif.).
Scanning of Slides
[0403] The chips were scanned using a ScanArray 3000 or 5000
(General Scanning, Watertown, Mass., USA). The chips were scanned
at 543 and 633 nm, at 10 um resolution to measure the intensity of
the two fluorescent dyes incorporated into the samples hybridized
to the chips.
Data Extraction and Analysis
[0404] The images generated by scanning slides consisted of two
16-bit TIFF images representing the fluorescent emissions of the
two samples at each arrayed spot. These images were then quantified
and processed for expression analysis using the data extraction
software Imagene (Biodiscovery, Los Angeles, Calif., USA). Imagene
output was subsequently analyzed using the analysis program
Genespring (Silicon Genetics, San Carlos, Calif., USA). In
Genespring, the data was imported using median pixel intensity
measurements derived from Imagene output. Background subtraction,
ratio calculation and normalization were all conducted in
Genespring. Normalization was achieved by breaking the data in to
32 groups, each of which represented one of the 32 pin printing
regions on the microarray. Groups consist of 360 to 550 spots. Each
group was independently normalized by setting the median of ratios
to one and multiplying ratios by the appropriate factor.
Results
[0405] The MA_diff Table (TABLE 10) presents the results of the
differential expression experiments for the mRNAs, as reported by
their corresponding cDNA ID number, that were differentially
transcribed under a particular set of conditions as compared to a
control sample. The cDNA ID numbers correspond to those utilized in
the Reference and Sequence Tables. Increases in mRNA abundance
levels in experimental plants versus the controls are denoted with
the plus sign (+). Likewise, reductions in mRNA abundance levels in
the experimental plants are denoted with the minus (-) sign.
[0406] The Table is organized according to the clone number with
each set of experimental conditions being denoted by the term "Expt
Rep ID:" followed by a "short name". Table 9 links each Expt Rep ID
with a short description of the experiment and the parameters. The
experiment numbers are referenced in the appropriate
utility/functions sections herein.
[0407] The sequences showing differential expression in a
particular experiment (denoted by either a "+" or "-" in the Table)
thereby shows utility for a function in a plant, and these
functions/utilities are described in detail below, where the title
of each section (i.e. a "utility section") is correlated with the
particular differential expression experiment in TABLE 9.
Organ-Affecting Genes, Gene Components, Products (Including
Differentiation and Function)
Root Genes
[0408] The economic values of roots arise not only from harvested
adventitious roots or tubers, but also from the ability of roots to
funnel nutrients to support growth of all plants and increase their
vegetative material, seeds, fruits, etc. Roots have four main
functions. First, they anchor the plant in the soil. Second, they
facilitate and regulate the molecular signals and molecular traffic
between the plant, soil, and soil fauna. Third, the root provides a
plant with nutrients gained from the soil or growth medium. Fourth,
they condition local soil chemical and physical properties.
[0409] Root genes are active or potentially active to a greater
extent in roots than in most other organs of the plant. These genes
and gene products can regulate many plant traits from yield to
stress tolerance. Root genes can be used to modulate root growth
and development.
[0410] Differential Expression of the Sequences in Roots
[0411] The relative levels of mRNA product in the root versus the
aerial portion of the plant was measured. Specifically, mRNA was
isolated from roots and root tips of Arabidopsis plants and
compared to mRNA isolated from the aerial portion of the plants
utilizing microarray procedures. Results are presented in TABLE
10.
Reproduction Genes, Gene Components and Products
[0412] Reproduction genes are defined as genes or components of
genes capable of modulating any aspect of sexual reproduction from
flowering time and inflorescence development to fertilization and
finally seed and fruit development. These genes are of great
economic interest as well as biological importance. The fruit and
vegeTable industry grosses over $1 billion USD a year. The seed
market, valued at approximately $15 billion USD annually, is even
more lucrative.
Inflorescence and Floral Development Genes, Gene Components and
Products
[0413] During reproductive growth the plant enters a program of
floral development that culminates in fertilization, followed by
the production of seeds. Senescence may or may not follow. The
flower formation is a precondition for the sexual propagation of
plants and is therefore essential for the propagation of plants
that cannot be propagated vegetatively as well as for the formation
of seeds and fruits. The point of time at which the merely
vegetative growth of plants changes into flower formation is of
vital importance for example in agriculture, horticulture and plant
breeding. Also the number of flowers is often of economic
importance, for example in the case of various useful plants
(tomato, cucumber, zucchini, cotton etc.) with which an increased
number of flowers may lead to an increased yield, or in the case of
growing ornamental plants and cut flowers.
[0414] Flowering plants exhibit one of two types of inflorescence
architecture: indeterminate, in which the inflorescence grows
indefinitely, or determinate, in which a terminal flower is
produced. Adult organs of flowering plants develop from groups of
stem cells called meristems. The identity of a meristem is inferred
from structures it produces: vegetative meristems give rise to
roots and leaves, inflorescence meristems give rise to flower
meristems, and flower meristems give rise to floral organs such as
sepals and petals. Not only are meristems capable of generating new
meristems of different identity, but their own identity can change
during development. For example, a vegetative shoot meristem can be
transformed into an inflorescence meristem upon floral induction,
and in some species, the inflorescence meristem itself will
eventually become a flower meristem. Despite the importance of
meristem transitions in plant development, little is known about
the underlying mechanisms.
[0415] Following germination, the shoot meristem produces a series
of leaf meristems on its flanks. However, once floral induction has
occurred, the shoot meristem switches to the production of flower
meristems. Flower meristems produce floral organ primordia, which
develop individually into sepals, petals, stamens or carpels. Thus,
flower formation can be thought of as a series of distinct
developmental steps, i.e. floral induction, the formation of flower
primordia and the production of flower organs. Mutations disrupting
each of the steps have been isolated in a variety of species,
suggesting that a genetic hierarchy directs the flowering process
(see for review, Weigel and Meyerowitz, In Molecular Basis of
Morphogenesis (ed. M. Bernfield). 51st Annual Symposium of the
Society for Developmental Biology, pp. 93-107, New York, 1993).
[0416] Expression of many reproduction genes and gene products is
orchestrated by internal programs or the surrounding environment of
a plant. These genes can be used to modulate traits such as fruit
and seed yield
Seed and Fruit Development Genes, Gene Components and Products
[0417] The ovule is the primary female sexual reproductive organ of
flowering plants. At maturity it contains the egg cell and one
large central cell containing two polar nuclei encased by two
integuments that, after fertilization, develops into the embryo,
endosperm, and seed coat of the mature seed, respectively. As the
ovule develops into the seed, the ovary matures into the fruit or
silique. As such, seed and fruit development requires the
orchestrated transcription of numerous polynucleotides, some of
which are ubiquitous, others that are embryo-specific and still
others that are expressed only in the endosperm, seed coat, or
fruit. Such genes are termed fruit development responsive genes and
can be used to modulate seed and fruit growth and development such
as seed size, seed yield, seed composition and seed dormancy.
[0418] Differential Expression of the Sequences in Siliques,
Inflorescences and Flowers
The relative levels of mRNA product in the siliques relative to the
plant as a whole was measured. The results are presented in TABLE
10.
[0419] Differential Expression of the Sequences in Hybrid Seed
Development
[0420] The levels of mRNA product in the seeds relative to those in
a leaf and floral stems was measured. The results are presented
TABLE 10,
Development Genes, Gene Components and Products
Imbibition and Germination Responsive Genes, Gene Components and
Products
[0421] Seeds are a vital component of the world's diet. Cereal
grains alone, which comprise .about.90% of all cultivated seeds,
contribute up to half of the global per capita energy intake. The
primary organ system for seed production in flowering plants is the
ovule. At maturity, the ovule consists of a haploid female
gametophyte or embryo sac surrounded by several layers of maternal
tissue including the nucleus and the integuments. The embryo sac
typically contains seven cells including the egg cell, two
synergids, a large central cell containing two polar nuclei, and
three antipodal cells. That pollination results in the
fertilization of both egg and central cell. The fertilized egg
develops into the embryo. The fertilized central cell develops into
the endosperm. And the integuments mature into the seed coat. As
the ovule develops into the seed, the ovary matures into the fruit
or silique. Late in development, the developing seed ends a period
of extensive biosynthetic and cellular activity and begins to
desiccate to complete its development and enter a dormant,
metabolically quiescent state. Seed dormancy is generally an
undesirable characteristic in agricultural crops, where rapid
germination and growth are required. However, some degree of
dormancy is advantageous, at least during seed development. This is
particularly true for cereal crops because it prevents germination
of grains while still on the ear of the parent plant (preharvest
sprouting), a phenomenon that results in major losses to the
agricultural industry. Extensive domestication and breeding of crop
species have ostensibly reduced the level of dormancy mechanisms
present in the seeds of their wild ancestors, although under some
adverse environmental conditions, dormancy may reappear. By
contrast, weed seeds frequently mature with inherent dormancy
mechanisms that allow some seeds to persist in the soil for many
years before completing germination.
[0422] Germination commences with imbibition, the uptake of water
by the dry seed, and the activation of the quiescent embryo and
endosperm. The result is a burst of intense metabolic activity. At
the cellular level, the genome is transformed from an inactive
state to one of intense transcriptional activity. Stored lipids,
carbohydrates and proteins are catabolized fueling seedling growth
and development. DNA and organelles are repaired, replicated and
begin functioning. Cell expansion and cell division are triggered.
The shoot and root apical meristem are activated and begin growth
and organogenesis. Schematic 4 summarizes some of the metabolic and
cellular processes that occur during imbibition. Germination is
complete when a part of the embryo, the radicle, extends to
penetrate the structures that surround it. In Arabidopsis, seed
germination takes place within twenty-four (24) hours after
imbibition, As such, germination requires the rapid and
orchestrated transcription of numerous polynucleotides. Germination
is followed by expansion of the hypocotyl and opening of the
cotyledons. Meristem development continues to promote root growth
and shoot growth, which is followed by early leaf formation.
Imbibition and Germination Genes
[0423] Imbibition and germination includes those events that
commence with the uptake of water by the quiescent dry seed and
terminate with the expansion and elongation of the shoots and
roots. The germination period exists from imbibition to when part
of the embryo, usually the radicle, extends to penetrate the seed
coat that surrounds it. Imbibition and germination genes are
defined as genes, gene components and products capable of
modulating one or more processes of imbibition and germination
described above. They are useful to modulate many plant traits from
early vigor to yield to stress tolerance.
[0424] Differential Expression of the Sequences in Germinating
Seeds and Imbibed Embryos
[0425] The levels of mRNA product in the seeds versus the plant as
a whole was measured. The results are presented in TABLE 10.
Hormone Responsive Genes, Gene Components and Products
Abscissic Acid Responsive Genes, Gene Components and Products
[0426] Plant hormones are naturally occurring substances, effective
in very small amounts, which act as signals to stimulate or inhibit
growth or regulate developmental processes in plants. Abscisic acid
(ABA) is a ubiquitous hormone in vascular plants that has been
detected in every major organ or living tissue from the root to the
apical bud. The major physiological responses affected by ABA are
dormancy, stress stomatal closure, water uptake, abscission and
senescence. In contrast to Auxins, cytokinins and gibberellins,
which are principally growth promoters, ABA primarily acts as an
inhibitor of growth and metabolic processes.
[0427] Changes in ABA concentration internally or in the
surrounding environment in contact with a plant results in
modulation of many genes and gene products. These genes and/or
products are responsible for effects on traits such as plant vigor
and seed yield.
While ABA responsive polynucleotides and gene products can act
alone, combinations of these polynucleotides also affect growth and
development. Useful combinations include different ABA responsive
polynucleotides and/or gene products that have similar
transcription profiles or similar biological activities, and
members of the same or similar biochemical pathways. Whole pathways
or segments of pathways are controlled by transcription factor
proteins and proteins controlling the activity of signal
transduction pathways. Therefore, manipulation of such protein
levels is especially useful for altering phenotypes and biochemical
activities of plants. In addition, the combination of an ABA
responsive polynucleotide and/or gene product with another
environmentally responsive polynucleotide is also useful because of
the interactions that exist between hormone-regulated pathways,
stress and defence induced pathways, nutritional pathways and
development.
[0428] Differential Expression of the Sequences in ABA Treated
Plants
[0429] The relative levels of mRNA product in plants treated with
ABA versus controls treated with water were measured. Results are
presented in TABLE 10.
Brassinosteroid Responsive Genes, Gene Components and Products
[0430] Plant hormones are naturally occuring substances, effective
in very small amounts, which act as signals to stimulate or inhibit
growth or regulate developmental processes in plants.
Brassinosteroids (BRs) are the most recently discovered, and least
studied, class of plant hormones. The major physiological response
affected by BRs is the longitudinal growth of young tissue via cell
elongation and possibly cell division. Consequently, disruptions in
BR metabolism, perception and activity frequently result in a dwarf
phenotype. In addition, because BRs are derived from the sterol
metabolic pathway, any perturbations to the sterol pathway can
affect the BR pathway. In the same way, perturbations in the BR
pathway can have effects on the later part of the sterol pathway
and thus the sterol composition of membranes.
[0431] Changes in BR concentration in the surrounding environment
or in contact with a plant result in modulation of many genes and
gene products. These genes and/or products are responsible for
effects on traits such as plant biomass and seed yield. These genes
were discovered and characterized from a much larger set of genes
by experiments designed to find genes whose mRNA abundance changed
in response to application of BRs to plants.
[0432] While BR responsive polynucleotides and gene products can
act alone, combinations of these polynucleotides also affect growth
and development. Useful combinations include different BR
responsive polynucleotides and/or gene products that have similar
transcription profiles or similar biological activities, and
members of the same or functionally related biochemical pathways.
Whole pathways or segments of pathways are controlled by
transcription factors and proteins controlling the activity of
signal transduction pathways. Therefore, manipulation of such
protein levels is especially useful for altering phenotypes and
biochemical activities of plants. In addition, the combination of a
BR responsive polynucleotide and/or gene product with another
environmentally responsive polynucleotide is useful because of the
interactions that exist between hormone-regulated pathways, stress
pathways, nutritional pathways and development. Here, in addition
to polynucleotides having similar transcription profiles and/or
biological activities, useful combinations include polynucleotides
that may have different transcription profiles but which
participate in common or overlapping pathways.
[0433] Differential Expression of the Sequences in Epi-Brassinolide
or Brassinozole Plants
[0434] The relative levels of mRNA product in plants treated with
either epi-brassinolide or brassinozole were measured. Results are
presented in TABLE 10.
Metabolism Affecting Genes, Gene Components and Products
Nitrogen Responsive Genes, Gene Components and Products
[0435] Nitrogen is often the rate-limiting element in plant growth,
and all field crops have a fundamental dependence on exogenous
nitrogen sources. Nitrogenous fertilizer, which is usually supplied
as ammonium nitrate, potassium nitrate, or urea, typically accounts
for 40% of the costs associated with crops, such as corn and wheat
in intensive agriculture. Increased efficiency of nitrogen use by
plants should enable the production of higher yields with existing
fertilizer inputs and/or enable existing yields of crops to be
obtained with lower fertilizer input, or better yields on soils of
poorer quality. Also, higher amounts of proteins in the crops could
also be produced more cost-effectively. "Nitrogen responsive" genes
and gene products can be used to alter or modulate plant growth and
development.
[0436] Differential Expression of the Sequences in Whole Seedlings,
Shoots and Roots
[0437] The relative levels of mRNA product in whole seedlings,
shoots and roots treated with either high or low nitrogen media
were compared to controls. Results are presented in TABLE 10.
Viability Genes, Gene Components and Products
[0438] Plants contain many proteins and pathways that when blocked
or induced lead to cell, organ or whole plant death. Gene variants
that influence these pathways can have profound effects on plant
survival, vigor and performance. The critical pathways include
those concerned with metabolism and development or protection
against stresses, diseases and pests. They also include those
involved in apoptosis and necrosis. Viability genes can be
modulated to affect cell or plant death. Herbicides are, by
definition, chemicals that cause death of tissues, organs and whole
plants. The genes and pathways that are activated or inactivated by
herbicides include those that cause cell death as well as those
that function to provide protection.
[0439] Differential Expression of the Sequences in Herbicide
Treated Plants and Herbicide Resistant Mutants
[0440] The relative levels of mRNA product in plants treated with
heribicide and mutants resistant to heribicides were compared to
control plants. Results are presented in TABLE 10.
Stress Responsive Genes, Gene Components and Products
Cold Responsive Genes, Gene Components and Products
[0441] The ability to endure low temperatures and freezing is a
major determinant of the geographical distribution and productivity
of agricultural crops. Even in areas considered suiTable for the
cultivation of a given species or cultivar, can give rise to yield
decreases and crop failures as a result of aberrant, freezing
temperatures. Even modest increases (1-2.degree. C.) in the
freezing tolerance of certain crop species would have a dramatic
impact on agricultural productivity in some areas. The development
of genotypes with increased freezing tolerance would provide a more
reliable means to minimize crop losses and diminish the use of
energy-costly practices to modify the microclimate.
[0442] Sudden cold temperatures result in modulation of many genes
and gene products, including promoters. These genes and/or products
are responsible for effects on traits such as plant vigor and seed
yield.
[0443] Manipulation of one or more cold responsive gene activities
is useful to modulate growth and development.
[0444] Differential Expression of the Sequences in Cold Treated
Plants
[0445] The relative levels of mRNA product in cold treated plants
were compared to control plants. Results are presented in TABLE
10.
Heat Responsive Genes, Gene Components and Products
[0446] The ability to endure high temperatures is a major
determinant of the geographical distribution and productivity of
agricultural crops. Decreases in yield and crop failure frequently
occur as a result of aberrant, hot conditions even in areas
considered suiTable for the cultivation of a given species or
cultivar. Only modest increases in the heat tolerance of crop
species would have a dramatic impact on agricultural productivity.
The development of genotypes with increased heat tolerance would
provide a more reliable means to minimize crop losses and diminish
the use of energy-costly practices to modify the microclimate.
[0447] Changes in temperature in the surrounding environment or in
a plant microclimate results in modulation of many genes and gene
products.
[0448] Differential Expression of the Sequences in Heat Treated
Plants
[0449] The relative levels of mRNA product in heat treated plants
were compared to control plants. Results are presented in TABLE
10.
Drought Responsive Genes, Gene Components and Products
[0450] The ability to endure drought conditions is a major
determinant of the geographical distribution and productivity of
agricultural crops. Decreases in yield and crop failure frequently
occur as a result of aberrant, drought conditions even in areas
considered suiTable for the cultivation of a given species or
cultivar. Only modest increases in the drought tolerance of crop
species would have a dramatic impact on agricultural productivity.
The development of genotypes with increased drought tolerance would
provide a more reliable means to minimize crop losses and diminish
the use of energy-costly practices to modify the microclimate.
[0451] Drought conditions in the surrounding environment or within
a plant, results in modulation of many genes and gene products.
[0452] Differential Expression of the Sequences in Drought Treated
Plants and Drought Mutants
[0453] The relative levels of mRNA product in drought treated
plants and drought mutants were compared to control plants. Results
are presented in TABLE 10.
Methyl Jasmonate (Jasmonate) Responsive Genes, Gene Components and
Products
[0454] Jasmonic acid and its derivatives, collectively referred to
as jasmonates, are naturally occurring derivatives of plant lipids.
These substances are synthesized from linolenic acid in a
lipoxygenase-dependent biosynthetic pathway. Jasmonates are
signalling molecules which have been shown to be growth regulators
as well as regulators of defense and stress responses. As such,
jasmonates represent a separate class of plant hormones. Jasmonate
responsive genes can be used to modulate plant growth and
development.
[0455] Differential Expression of the Sequences in Methyl Jasmonate
Treated Plants
[0456] The relative levels of mRNA product in methyl jasmonate
treated plants were compared to control plants. Results are
presented in TABLE 10.
SalicyliC Acid Responsive Genes, Gene Components and Products
[0457] Plant defense responses can be divided into two groups:
constitutive and induced. Salicylic acid (SA) is a signaling
molecule necessary for activation of the plant induced defense
system known as systemic acquired resistance or SAR. This response,
which is triggered by prior exposure to avirulent pathogens, is
long lasting and provides protection against a broad spectrum of
pathogens. Another induced defense system is the hypersensitive
response (HR). HR is far more rapid, occurs at the sites of
pathogen (avirulent pathogens) entry and precedes SAR. SA is also
the key signaling molecule for this defense pathway.
[0458] Differential Expression of the Sequences in Salicylic Acid
Treated Plants
[0459] The relative levels of mRNA product in salicylic acid
treated plants were compared to control plants. Results are
presented in TABLE 10.
Osmotic Stress Responsive Genes, Gene Components and Products
[0460] The ability to endure and recover from osmotic and salt
related stress is a major determinant of the geographical
distribution and productivity of agricultural crops. Osmotic stress
is a major component of stress imposed by saline soil and water
deficit. Decreases in yield and crop failure frequently occur as a
result of aberrant or transient environmental stress conditions
even in areas considered suitable for the cultivation of a given
species or cultivar. Only modest increases in the osmotic and salt
tolerance of a crop species would have a dramatic impact on
agricultural productivity. The development of genotypes with
increased osmotic tolerance would provide a more reliable means to
minimize crop losses and diminish the use of energy-costly
practices to modify the soil environment. Thus, osmotic stress
responsive genes can be used to modulate plant growth and
development.
[0461] Differential Expression of the Sequences in PEG Treated
Plants
[0462] The relative levels of mRNA product in PEG treated plants
were compared to control plants. Results are presented in TABLE
10.
Shade Responsive Genes, Gene Components and Products
[0463] Plants sense the ratio of Red (R):Far Red (FR) light in
their environment and respond differently to particular ratios. A
low R:FR ratio, for example, enhances cell elongation and favors
flowering over leaf production. The changes in R:FR ratios mimic
and cause the shading response effects in plants. The response of a
plant to shade in the canopy structures of agricultural crop fields
influences crop yields significantly. Therefore manipulation of
genes regulating the shade avoidance responses can improve crop
yields. While phytochromes mediate the shade avoidance response,
the down-stream factors participating in this pathway are largely
unknown. One potential downstream participant, ATHB-2, is a member
of the RD-Zip class of transcription factors and shows a strong and
rapid response to changes in the R:FR ratio. ATHB-2 overexpressors
have a thinner root mass, smaller and fewer leaves and longer
hypocotyls and petioles. This elongation arises from longer
epidermal and cortical cells, and a decrease in secondary vascular
tissues, paralleling the changes observed in wild-type seedlings
grown under conditions simulating canopy shade. On the other hand,
plants with reduced ATHB-2 expression have a thick root mass and
many larger leaves and shorter hypocotyls and petioles. Here, the
changes in the hypocotyl result from shorter epidermal and cortical
cells and increased proliferation of vascular tissue.
Interestingly, application of Auxin is able to reverse the root
phenotypic consequences of high ATHB-2 levels, restoring the
wild-type phenotype. Consequently, given that ATHB-2 is tightly
regulated by phytochrome, these data suggest that ATHB-2 may link
the Auxin and phytochrome pathways in the shade avoidance response
pathway.
[0464] Shade responsive genes can be used to modulate plant growth
and development.
[0465] Differential Expression of the Sequences in Far-Red Light
Treated Plants
[0466] The relative levels of mRNA product in far-red light treated
plants were compared to control plants. Results are presented in
TABLE 10.
Viability Genes, Gene Components and Products
[0467] Plants contain many proteins and pathways that when blocked
or induced lead to cell, organ or whole plant death. Gene variants
that influence these pathways can have profound effects on plant
survival, vigor and performance. The critical pathways include
those concerned with metabolism and development or protection
against stresses, diseases and pests. They also include those
involved in apoptosis and necrosis. The applicants have elucidated
many such genes and pathways by discovering genes that when
inactivated lead to cell or plant death.
[0468] Herbicides are, by definition, chemicals that cause death of
tissues, organs and whole plants. The genes and pathways that are
activated or inactivated by herbicides include those that cause
cell death as well as those that function to provide protection.
The applicants have elucidated these genes.
[0469] The genes defined in this section have many uses including
manipulating which cells, tissues and organs are selectively
killed, which are protected, making plants resistant to herbicides,
discovering new herbicides and making plants resistant to various
stresses.
[0470] Viability genes were also identified from a much larger set
of genes by experiments designed to find genes whose mRNA products
changed in concentration in response to applications of different
herbicides to plants. Viability genes are characteristically
differentially transcribed in response to fluctuating herbicide
levels or concentrations, whether internal or external to an
organism or cell, The MA_diff Table reports the changes in
transcript levels of various viability genes.
Early Seedling-Phase Specific Responsive Genes, Gene Components and
Products
[0471] One of the more active stages of the plant life cycle is a
few days after germination is complete, also referred to as the
early seedling phase. During this period the plant begins
development and growth of the first leaves, roots, and other organs
not found in the embryo. Generally this stage begins when
germination ends. The first sign that germination has been
completed is usually that there is an increase in length and fresh
weight of the radicle. Such genes and gene products can regulate a
number of plant traits to modulate yield. For example, these genes
are active or potentially active to a greater extent in developing
and rapidly growing cells, tissues and organs, as exemplified by
development and growth of a seedling 3 or 4 days after planting a
seed.
[0472] Rapid, efficient establishment of a seedling is very
important in commercial agriculture and horticulture. It is also
vital that resources are approximately partitioned between shoot
and root to facilitate adaptive growth, Phototropism and geotropism
need to be established. All these require post-germination process
to be sustained to ensure that vigorous seedlings are produced.
Early seedling phase genes, gene components and products are useful
to manipulate these and other processes.
Guard Cell Genes, Gene Components and Products
[0473] Scattered throughout the epidermis of the shoot are minute
pores called stomata. Each stomal pore is surrounded by two guard
cells. The guard cells control the size of the stomal pore, which
is critical since the stomata control the exchange of carbon
dioxide, oxygen, and water vapor between the interior of the plant
and the outside atmosphere. Stomata open and close through turgor
changes driven by ion fluxes, which occur mainly through the guard
cell plasma membrane and tonoplast. Guard cells are known to
respond to a number of external stimuli such as changes in light
intensity, carbon dioxide and water vapor, for example. Guard cells
can also sense and rapidly respond to internal stimuli including
changes in ABA, auxin and calcium ion flux.
[0474] Thus, genes, gene products, and fragments thereof
differentially transcribed and/or translated in guard cells can be
useful to modulate ABA responses, drought tolerance, respiration,
water potential, and water management as examples. All of which can
in turn affect plant yield including seed yield, harvest index,
fruit yield, etc.
[0475] To identify such guard cell genes, gene products, and
fragments thereof, Applicants have performed a microarray
experiment comparing the transcript levels of genes in guard cells
versus leaves. Experimental data is shown below.
Nitric Oxide Responsive Genes, Gene Components and Products
[0476] The rate-limiting element in plant growth and yield is often
its ability to tolerate suboptimal or stress conditions, including
pathogen attack conditions, wounding and the presence of various
other factors. To combat such conditions, plant cells deploy a
battery of inducible defense responses, including synergistic
interactions between nitric oxide (NO), reactive oxygen
intermediates (ROS), and salicylic acid (SA). NO has been shown to
play a critical role in the activation of innate immune and
inflammatory responses in animals. At least part of this mammalian
signaling pathway is present in plants, where NO is known to
potentiate the hypersensitive response (HR). In addition, NO is a
stimulator molecule in plant photomorphogenesis.
[0477] Changes in nitric oxide concentration in the internal or
surrounding environment, or in contact with a plant, results in
modulation of many genes and gene products.
[0478] In addition, the combination of a nitric oxide responsive
polynucleotide and/or gene product with other environmentally
responsive polynucleotides is also useful because of the
interactions that exist between hormone regulated pathways, stress
pathways, pathogen stimulated pathways, nutritional pathways and
development.
[0479] Nitric oxide responsive genes and gene products can function
either to increase or dampen the above phenotypes or activities
either in response to changes in nitric oxide concentration or in
the absence of nitric oxide fluctuations. More specifically, these
genes and gene products can modulate stress responses in an
organism. In plants, these genes and gene products are useful for
modulating yield under stress conditions. Measurements of yield
include seed yield, seed size, fruit yield, fruit size, etc.
Shoot-Apical Meristem Genes, Gene Components and Products
[0480] New organs, stems, leaves, branches and inflorescences
develop from the stem apical meristem (SAM). The growth structure
and architecture of the plant therefore depends on the behavior of
SAMs. Shoot apical meristems (SAMs) are comprised of a number of
morphologically undifferentiated, dividing cells located at the
tips of shoots. SAM genes elucidated here are capable of modifying
the activity of SAMs and thereby many traits of economic interest
from ornamental leaf shape to organ number to responses to plant
density.
[0481] In addition, a key attribute of the SAM is its capacity for
self-renewal. Thus, SAM genes of the instant invention are useful
for modulating one or more processes of SAM structure and/or
function including (I) cell size and division; (II) cell
differentiation and organ primordia. The genes and gene components
of this invention are useful for modulating any one or all of these
cell division processes generally, as in timing and rate, for
example. In addition, the polynucleotides and polypeptides of the
invention can control the response of these processes to the
internal plant programs associated with embryogenesis, and hormone
responses, for example.
[0482] Because SAMs determine the architecture of the plant,
modified plants will be useful in many agricultural, horticultural,
forestry and other industrial sectors. Plants with a different
shape, numbers of flowers and seed and fruits will have altered
yields of plant parts. For example, plants with more branches can
produce more flowers, seed or fruits. Trees without lateral
branches will produce long lengths of clean timber. Plants with
greater yields of specific plant parts will be useful sources of
constituent chemicals.
[0483] The invention being thus described, it will be apparent to
one of ordinary skill in the art that various modifications of the
materials and methods for practicing the invention can be made.
Such modifications are to be considered within the scope of the
invention as defined by the following claims.
[0484] Each of the references from the patent and periodical
literature cited herein is hereby expressly incorporated in its
entirety by such citation.
TABLE-US-00006 TABLE I REFERENCE TABLE Max Len. Seq.: rel to: Clone
IDs: 1093453 (Ac) cDNA SEQ - Pat. Appln. SEQ ID NO: 1 - Ceres SEQ
ID NO: 4788142 PolyP SEQ - Pat. Appln. SEQ ID NO 2 - Ceres SEQ ID
NO 4788143 - Loc. SEQ ID NO 1: @ 89 nt. (C) Pred. PP Nom. &
Annot. (Dp) Rel. AA SEQ - Align. NO 1 - gi No 30694168 - Desp. :
expressed protein [Arabidopsis thaliana] - % Idnt. : 63.8 - Align.
Len.: 105 - Loc. SEQ ID NO 2: 1 -> 92 aa. PolyP SEQ - Pat.
Appln. SEQ ID NO 3 - Ceres SEQ ID NO 4788144 - Loc. SEQ ID NO 1: @
167 nt. (C) Pred. PP Nom. & Annot. (Dp) Rel. AA SEQ - Align. NO
2 - gi No 30694168 - Desp. : expressed protein [Arabidopsis
thaliana] - % Idnt. : 63.8 - Align. Len.: 105 - Loc. SEQ ID NO 3: 1
-> 66 aa. PolyP SEQ - Pat. Appln. SEQ ID NO 4 - Ceres SEQ ID NO
4788145 - Loc. SEQ ID NO 1: @ 183 nt. (C) Pred. PP Nom. &
Annot. (Dp) Rel. AA SEQ Max Len. Seq. : rel to: Clone IDs: 1079596
(Ac) cDNA SEQ - Pat. Appln. SEQ ID NO: 5 - Ceres SEQ ID NO: 4796909
PolyP SEQ - Pat. Appln. SEQ ID NO 6 - Ceres SEQ ID NO 4796910 -
Loc. SEQ ID NO 5: @ 94 nt. (C) Pred. PP Nom. & Annot. (Dp) Rel.
AA SEQ - Align. NO 3 - gi No 30694168 - Desp. : expressed protein
[Arabidopsis thaliana] - % Idnt. : 63.9 - Align. Len.: 147 - Loc.
SEQ ID NO 6: 1 -> 128 aa. Polyp SEQ - Pat. Appln. SEQ ID NO 7 -
Ceres SEQ ID NO 4796911 - Loc. SEQ ID NO 5: @ 172 nt. (C) Pred. PP
Nom. & Annot. (Dp) Rel. AA SEQ - Align. NO 4 - gi No 30694168 -
Desp. : expressed protein [Arabidopsis thaliana] - % Idnt. : 63.9 -
Align. Len.: 147 - Loc. SEQ ID NO 7: 1 -> 102 aa. PolyP SEQ -
Pat. Appln. SEQ ID NO 8 - Ceres SEQ ID NO 4796912 - Loc. SEQ ID NO
5: @ 244 nt. (C) Pred. PP Nom. & Annot. (Dp) Rel. AA SEQ -
Align. NO 5 - gi No 30694168 - Desp. : expressed protein
[Arabidopsis thaliana] - % Idnt. : 63.9 - Align. Len.: 147 - Loc.
SEQ ID NO 8: 1 -> 78 aa. END_OF_FILE Max Len. Seq. : rel to:
Clone IDs: 8161 Pub gDNA: gi No: 22329272 Gen. seq. in cDNA: 129945
. . . 129790 OCKHAM3-CDS 129087 . . . 128929 OCKHAM3-CDS 128845 . .
. 128653 OCKHAM3-CDS 128277 . . . 128165 OCKHAM3-CDS 128081 . . .
128046 OCKHAM3-CDS (Ac) cDNA SEQ - Pat. Appln. SEQ ID NO: 9 - Ceres
SEQ ID NO: 12321174 PolyP SEQ - Pat. Appln. SEQ ID NO 10 - Ceres
SEQ ID NO 12321175 - Loc. SEQ ID NO 9: @ 113 nt. (C) Pred. PP Nom.
& Annot. (Dp) Rel. AA SEQ - Align. NO 6 - gi No 30694168 -
Desp. : expressed protein [Arabidopsis thaliana] - % Idnt. : 41.2 -
Align. Len.: 102 - Loc. SEQ ID NO 10: 22 -> 118 aa. Max Len.
Seq. : rel to: Clone IDs: 96 (Ac) cDNA SEQ - Pat. Appln. SEQ ID NO:
11 - Ceres SEQ ID NO: 12323601 - SEQ 11 w. TSS: 36 PolyP SEQ - Pat.
Appln. SEQ ID NO 12 - Ceres SEQ ID NO 12323602 - Loc. SEQ ID NO 11:
@ 2 nt. - Loc. Sig. P. SEQ ID NO 12: @ 22 aa. (C) Pred. PP Nom.
& Annot. (Dp) Rel. AA SEQ - Align. NO 7 - gi No 30694168 -
Desp. : expressed protein [Arabidopsis thaliana] - % Idnt. : 99.6 -
Align. Len.: 246 - Loc. SEQ ID NO 12: 28 -> 273 aa. PolyP SEQ -
Pat. Appln. SEQ ID NO 13 - Ceres SEQ ID NO 12323603 - log. SEQ ID
NO 11: @ 83 nt. (C) Pred. PP Nom. & Annot. (Dp) Rel. AA SEQ -
Align. NO 8 - gi No 30694168 - Desp. : expressed protein
[Arabidopsis thaliana] - % Idnt. : 99.6 - Align. Len.: 246 - Loc.
SEQ ID NO 13: 1 -> 246 aa. PolyP SEQ - Pat. Appln. SEQ ID NO 14
- Ceres SEQ ID NO 12323604 - Loc. SEQ ID NO 11: @ 188 nt. (C) Pred.
PP Nom. & Annot. (Dp) Rel. AA SEQ - Align. NO 9 - gi No
30694168 - Desp. : expressed protein [Arabidopsis thaliana] - %
Idnt. : 99.6 - Align. Len.: 246 - Loc. SEQ ID NO 14: 1 -> 211
aa. Max Len. Seq. : rel to: Clone IDs: 8490 Pub gDNA: gi No:
22323163 Gen. seq. in cDNA: 147882 . . . 147775 OCKHAM3-CDS 147419
. . . 147237 OCKBAM3-CDS 147148 . . . 146863 OCKHAM3-CDS 146779 . .
. 146673 OCKHAM3-CDS 146592 . . . 146536 OCKHAM3-CDS gi No:
22328163 Gen. seq. in cDNA: 7882 . . . 7775 OCKHAM3-CDS 7419 . . .
7237 OCKHAM3-CDS 7148 . . . 6863 OCKHAM3-CDS 6779 . . . 6673
OCKHAM3-CDS 6592 . . . 6536 OCKHAM3-CDS (Ac) cDNA SEQ - Pat. Appln.
SEQ ID NO: 15 - Ceres SEQ ID NO: 13491409 PolyP SEQ - Pat. Appln.
SEQ ID NO 16 - Ceres SEQ ID NO 13491410 - Loc. SEQ ID NO 15: @ 2
nt. - Loc. Sig. P. SEQ ID NO 16: @ 21 aa. (C) Pred. PP Nom. &
Annot. (Dp) Rel. AA SEQ - Align. NO 10 - gi No 30694168 - Desp. :
expressed protein [Arabidopsis thaliana] - % Idnt. : 99.6 - Align.
Len.: 246 - Loc. SEQ ID NO 16: 27 -> 272 aa. Polyp SEQ - Pat.
Appln. SEQ ID NO 17 - Ceres SEQ ID NO 13491411 - Loc. SEQ ID NO 15:
@ 80 nt. (C) Pred. PP Nom. & Annot. (Dp) Rel. AA SEQ - Align.
NO 11 - gi No 30694168 - Desp. : expressed protein [Arabidopsis
thaliana] - % Idnt. : 99.6 - Align. Len.: 246 - Loc. SEQ ID NO 17:
1 -> 246 aa. PolyP SEQ - Pat. Appln. SEQ ID NO 18 - Ceres SEQ ID
NO 13491412 - Loc. SEQ ID NO 15: @ 185 nt. (C) Pred. PP Nom. &
Annot. (Dp) Rel. AA SEQ - Align. NO 12 - gi No 30694168 - Desp. :
expressed protein [Arabidopsis thaliana] - % Idnt. : 99.6 - Align.
Len.: 245 - Loc. SEQ ID NO 18: 1 -> 211 aa. END_OF_FILE Max Len.
Seq. : rel to: Clone IDs: 305463 (Ac) cDNA SEQ - Pat. Appln. SEQ ID
NO: 1 - Ceres SEQ ID NO: 12355477 - SEQ 1 w. TSS: 27 Polyp SEQ -
Pat. Appln. SEQ ID NO 2 - Ceres SEQ ID NO 12355478 - Loc. SEQ ID NO
1: @ 462 nt. (C) Pred. PP Nom. & Annot. (Dp) Rel. AA SEQ PolyP
SEQ - Pat. Appln. SEQ ID NO 3 - Ceres SEQ ID NO 12355479 - Loc. SEQ
ID NO 1: @ 549 nt. (C) Pred. PP Nom. & Annot. (Dp) Rel. AA SEQ
Polyp SEQ - Pat. Appln. SEQ ID NO 4 - Ceres SEQ ID NO 12355480 -
Loc. SEQ ID NO 1: @ 597 nt. (C) Pred. PP Nom. & Annot. (Dp)
Rel. AA SEQ Max Len. Seq. : rel to: Clone IDs: 258437
(Ac) cDNA SEQ - Pat. Appln. SEQ ID NO: 5 - Ceres SEQ ID NO:
12410516 - SEQ 5 w. TSS: 22,79,83,85 Polyp SEQ - Pat. Appln. SEQ ID
NO 6 - Ceres SEQ ID NO 12410517 - Loc. SEQ ID NO 5: @ 553 nt. (C)
Pred. PP Nom. & Annot. (Dp) Rel. AA SEQ PolyP SEQ - Pat. Appln.
SEQ ID NO 7 - Ceres SEQ ID NO 12410518 - Loc. SEQ ID NO 5: @ 637
nt. (C) Pred. PP Nom. & Annot. (Dp) Rel. AA SEQ Polyp SEQ -
Pat. Appln. SEQ ID NO 8 - Ceres SEQ ID NO 12410519 - Loc. SEQ ID NO
5: @ 667 nt. (C) Pred. PP Nom. & Annot. (Dp) Rel. AA SEQ
END_OF_FILE
TABLE-US-00007 TABLE 3 Matrix com 28 29 30 31 32 33 34 35 36 37 38
39 40 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 1 2 1 2 2 2 2 2 2 1 v ek ga e
tp t sa sg mg yi sa ak g 41 42 43 44 45 46 47 48 49 50 51 52 53 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 3 1 1 2 1 2 1 kn er yv ml eds ta
emg w t nd e kr h 54 55 56 57 58 59 60 61 62 63 64 65 66 2 2 2 2 2
2 2 2 2 2 2 2 2 3 2 1 2 2 1 1 1 1 1 1 1 2 smr ls y li ks s m e a s
f v de 67 68 69 70 71 72 73 74 75 76 77 78 79 2 2 2 2 2 2 2 2 2 2 2
2 2 1 1 1 1 2 1 1 3 3 2 3 2 2 q l y n sh l g ans lhr gp khr nd ea
80 81 82 83 84 85 86 87 88 89 90 91 92 2 2 2 2 2 2 2 2 2 2 2 2 2 1
2 2 1 1 1 1 2 1 2 1 1 1 n vg st e s t r fs g sa g r k 93 94 95 96
97 98 99 100 101 102 103 104 105 2 2 21 2 2 2 21 2 2 2 2 2 2 1 1 1
1 2 1 1 2 2 3 3 2 4 p s q e qa f k va li hrq dre ge fvyl 106 107
108 109 110 111 112 113 114 115 116 117 118 2 2 2 2 2 2 2 2 2 2 2 2
2 3 2 3 3 4 4 4 4 3 4 2 2 2 wvc qe kys iem nekr vyti kedp qkar ptd
esvd ha rp iv 119 120 121 122 123 124 125 126 127 128 129 130 131 2
2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 2 2 2 3 3 2 4 2 2 3 n g r hr gs ga
nak skc hc ecgd fv lp rae 132 133 134 135 136 137 138 139 140 141
142 143 144 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 3 3 2 2 2 1 2 2 2 3 sn
p w imv krq hr yf kr p lr vd kc tgr 145 146 147 148 149 150 151 152
153 154 155 156 157 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 1 3 4 2 3 4
3 3 qsg inv pah vqh tsg d eag pvgm en nav qsae vrp vgl 158 159 160
161 162 163 164 165 166 167 168 169 170 2 2 2 2 2 2 2 2 2 2 2 2 2 3
4 4 4 2 3 3 2 3 3 3 1 2 sdv shgd selg nsgy gd kts kqv ga isd crl
ske s gp 171 172 173 174 175 176 177 178 179 180 181 182 183 2 2 2
2 2 2 2 2 2 2 2 2 2 2 4 3 4 3 2 3 2 2 2 1 3 3 st aved sat shna lgr
kr qes lr sg sa h scg rkp 184 185 186 187 188 189 190 191 192 193
194 195 196 2 2 2 2 2 2 2 2 2 2 2 2 2 3 4 3 2 2 3 3 3 2 3 1 1 2 dge
herp dpr qk il sla vhk gea es atr e v st 197 198 199 200 201 202
203 204 205 206 207 208 209 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 3 3 2
2 2 3 3 2 2 d q n f vap nde ed ge ia keq gas es nt 210 211 212 213
214 215 216 217 218 219 220 221 222 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3
1 3 4 1 3 2 2 2 1 2 ge sa smc k kar mcyq k tks vr mr ml s er 223 2
3 sat
TABLE-US-00008 TABLE 4 matrix_e17 21 22 23 24 25 26 27 28 29 30 31
32 33 34 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 5 4 5 3 3 3 5 2 3 3 2 se
st dst dgsel astl sqtyn sfg vly egd geqsh eg tae tlv sa 61 62 63 64
65 66 67 68 69 70 71 72 73 74 2 2 21 2 2 2 2 2 2 2 2 2 2 2 1 2 1 1
1 5 1 2 2 3 2 3 4 2 e as s f v dnser q ls yh nde sh lsm gnds al 121
122 123 124 125 126 127 128 129 130 131 132 133 134 2 2 2 2 2 2 2 2
2 2 2 2 2 2 4 4 3 6 6 2 6 7 6 5 5 3 2 2 rskt hsrk gia granke ngrkqe
sc hdcrat edgrvaiflvise lqptf rsaet sdn pe wf 181 182 183 184 185
186 187 188 189 190 191 192 193 194 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 5
4 6 5 6 4 6 6 7 5 4 5 1 hnse sgryp rlvp dyecsphrdep drpkqs qdke
ismlyt svliak vlghyclgseda ensd aeitr e 35 36 37 38 39 40 41 42 43
44 45 46 47 48 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 4 4 5 6 7 7 6 7 8 7 5
4 1 srqt mlqs yedm sfgka aeqpsdgevrmkkntliqe edagtl ydptsikmktlse
epsaqr tapsg egqa w 75 76 77 78 79 80 81 82 83 84 85 86 87 88 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 5 6 9 7 6 6 6 5 5 6 9 7 lq gs ksryg
nkqesgednwm ntksae vptckr sgptkl epnysk shdrl trspm rqhapk
fkdtnengdatvql 135 136 137 138 139 140 141 142 143 144 145 146 147
148 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 4 2 3 4 3 6 7 4 6 7 9 6 8 lmvr
krqf hr yfr knrt psn lsrega vprdsg krcv tsgqah qlsrahnitdnspnpdaisr
vgqarli 195 196 197 198 199 200 201 202 203 2 2 2 2 2 2 2 2 2 3 3 2
1 1 1 6 3 2 vma stm dg q n f vmaipr nde ed 49 50 51 52 53 54 55 56
57 58 59 60 2 2 2 2 2 2 2 2 2 2 2 2 1 2 1 2 1 4 3 1 2 4 2 3 t nd e
kr h smrn lms y li knsd sy mil 89 90 91 92 93 94 95 96 97 98 99 100
101 102 2 2 2 2 2 2 2 2 2 2 2 2 2 2 5 4 7 5 5 2 4 4 3 2 2 2 4 4
sdcnh gnca rgtipnskcarh pitsk sp qesa egad qge fy kt va lhfi hrkql
149 150 151 152 153 154 155 156 157 158 159 160 161 162 2 2 2 2 2 2
2 2 2 2 2 2 2 2 8 4 8 5 3 6 5 5 4 4 7 5 7 4 tslvqngdspe efasgc
plvga eqn neaqgyqnsat varmt vagt sdtl sthgvarselkv nksgfp gdsn 103
104 105 106 107 108 109 110 111 112 113 114 115 116 2 2 2 2 2 2 2 2
2 2 2 2 2 2 5 2 7 2 4 4 4 5 5 7 4 6 6 7 dgren ge fsvcynlwc qekt
kygs imkv neskr vcyfi krvempqrky patved esnkmdhyaptsl 163 164 165
166 167 168 169 170 171 172 173 174 175 176 2 2 2 2 2 2 2 2 2 2 2 2
2 2 6 7 6 8 6 7 5 5 5 7 6 5 4 4 kltqsd kvqrihsgakvepivsgdmcrsvli
sckped sgcfd gpaks sdtay asvgeitsthaqe snftg lrst kqld 117 118 119
120 2 2 2 2 5 7 3 4 rpqdk ilvtamdnsp gkvs 177 178 179 180 2 2 2 2 5
4 3 4 qfhst lita scl swer indicates data missing or illegible when
filed
TABLE-US-00009 TABLE 9 PARAMETERS FOR DIFFERENTIAL ANALYSIS Expt
Utility Section Rep ID Short_Name Parameter Value Viability 107881
At_Herbicide_v2_cDNA_P Timepoint (hr) 4 107881
At_Herbicide_v2_cDNA_P Treatment Glean vs. No Treatment 107891
At_Herbicide_v2_cDNA_P Timepoint (hr) 12 107891
At_Herbicide_v2_cDNA_P Treatment Trimec vs. No Treatment Root
108429 At_Tissue_Specific_Expression_cDNA_P Probe Amount 50 108429
At_Tissue_Specific_Expression_cDNA_P Probe Method operon 108429
At_Tissue_Specific_Expression_cDNA_P Tissue Green Flower vs. Whole
Plant Root 108434 At_Root_Tips_cDNA_P Tissue Root Tips Shoot
Meristem 108435 At_stm_Mutants_cDNA_P Rant Line wt Landsburg vs stm
108435 At_stm_Mutants_cDKA_P Tissue Shoot Apical Meristem Region
Reproductive and Seed & 108437
At_Tissue_Specific_Expression_cDNA_P Probe Amount 33 Fruit
Development 108437 At_Tissue_Specific_Expression_cDNA_P Probe
Method operon 108437 At_Tissue_Specific_Expression_cDNA_P Tissue
<5 mm Siliques vs. Whole Plant Reproductive and Seed &
108438 At_Tissue_Specific_Expression_cDNA_P Probe Amount 33 Fruit
Development 108438 At_Tissue_Specific_Expression_cDNA_P Probe
Method operon 108438 At_Tissue_Specific_Expression_cDNA_P Tissue 5
wk Siliques vs. Whole Plant Root 108439
At_Tissue_Specific_Expression_cDNA_P Probe Amount 33 108439
At_Tissue_Specific_Expression_cDNA_P Probe Method operon 108439
At_Tissue_Specific_Expression_cDNA_P Tissue Roots (2 wk) vs. Whole
Plant Imbibition & Germination 108461
At_Germinating_Seeds_cDNA_P Age 1 vs. 0 108461
At_Germinating_Seeds_cDNA_P Tissue Germinating Seeds Imbibition
& Germination 108462 At_Germinating_Seeds_cDNA_P Age 2 vs. 0
108462 At_Germinating_Seeds_cDNA_P Tissue Greminating Seeds Early
Seedling Phase 108463 At_Germinating_Seeds_cDNA_P Age 3 vs. 0
108463 At_Germinating_Seeds_cDNA_P Tissue Germinating Seeds Early
Seedling Phase 108464 At_Germinating_Seeds_cDNA_P Age 4 vs. 0
108464 At_Germinating_Seeds_cDNA_P Tissue Germinating Seeds
Viability 108465 At_Herbicide_v3_1_cDNA_P Timepoint (hr) 12 108465
At_Herbicide_v3_1_cDNA_P Treatment Roundup vs. No Treatment Drought
and Reproductive 108473 At_Drought_Flowers_cDNA_P Timepoint (hr) 7
d 108473 At_Drought_Flowers_cDNA_P Tissue Flowers 108473
At_Drought_Flowers_cDNA_P Treatment Drought vs. No Drought Shoot
Meristem 108480 At_Shoot_Apices_cDNA_P Plant Line Ws-2 108480
At_Shoot_Apices_cDNA_P Treatment 1 uM BR vs. No Treatment Shoot
Meristem 108481 At_Shoot_Apices_cDNA_P Plant Line Ws-2 108481
At_Shoot_Apices_cDNA_P Treatment 1 uM BRZ vs. No Treatment Leaves
108488 At_50mM_NH4NO3_L-to-H_Rosette_cDNA_P Timepoint (hr) 2 Heat
108523 Zm_42deg_Heat_P Temperature Heat (42 deg C.) 108523
Zm_42deg_Heat_P Timepoint (hr) 6 108523 Zm_42deg_Heat_P Tissue
Aerial Imbibition & Germination 108528 Zm_Imbibed_Seeds_P Age 5
vs. 2 108528 Zm_Imbibed_Seeds_P Tissue Aerial vs. Embryo 108528
Zm_Imbibed_Seeds_P Treatment Imbibition Imbibition &
Germination 108530 Zm_Imbibed_Seeds_P Age 6 vs. 2 108530
Zm_Imbibed_Seeds_P Tissue Aerial vs. Embryo 108530
Zm_Imbibed_Seeds_P Treatment Imbibition Imbibition &
Germination, 108543 Zm_Imbibed_Embryo_Endosperm_P Age 2
Reproductive 108543 Zm_Imbibed_Embryo_Endosperm_P Tissue Embryo vs.
Whole Plant 108543 Zm_Imbibed_Embryo_Endosperm_P Treatment Imbibed
Imbibition & Germination 108546 Zm_Imbibed_Seeds_P Age 3 vs.2
108546 Zm_Imbibed_Seeds_P Tissue Roots vs. Embryo 103546
Zm_Imbibed_Seeds_P Treatment Imbibition Jasmonale 108569
At_0.001%_MeJA_cDNA_P Timepoint (hr) 6 108569 At_0.001%_MeJA_cDNA_P
Tissue Aerial 108569 At_0.001%_MeJA_cDNA_P Treatment 0.001% MeJA
vs. No Treatment Heat 108577 At_42deg_Heat_cDNA_P Temperature 42
vs. 22 108577 At_42deg_Heat_cDNA_P Timepoint (hr) 6 108577
At_42deg_Heat_cDNA_P Tissue Aerial Cold 108579 At_4deg_Cold_cDNA_P
Temperature 4 vs. 22 108579 At_4deg_Cold_cDNA_P Timepoint (hr) 6
108579 At_4deg_Cold_cDNA_P Tissue Aerial Root and Root Hairs 108594
At_Ler-rhl_Root_cDNA_P Plant Line Ler_rhl 108594
At_Ler-rhl_Root_cDNA_P Tissue Root ABA, Drought, Germination 108614
At_100uM_ABA_Mutants_cDNA_P Plant Line CS24 108614
At_100uM_ABA_Mutants_cDNA_P Timepoint (hr) 6 108614
At_100uM_ABA_Mutants_cDNA_P Tissue Aerial 108614
At_100uM_ABA_Mutants_cDNA_P Treatment 100 uM ABA vs. No Treatment
ABA, Drought, Germination 108622 At_100uM_ABA_Mutants_cDNA_P Plant
Line CS22 108622 At_100uM_ABA_Mutants_cDNA_P Timepoint (hr) 6
108622 At_100uM_ABA_Mutants_cDNA_P Tissue Aerial 108622
At_100uM_ABA_Mutants_cDNA_P Treatment 100 uM ABA vs. No Treatment
Viability 108629 At_Herbicide_v3_1_cDNA_P Timepoint (hr) 1 108629
At_Herbicide_v3_1_cDNA_P Treatment Glean vs. No Treatment Viability
108630 At_Herbicide_v3_1_cDNA_P Timepoint (hr) 1 108630
At_Herbicide_v3_1_cDNA_P Treatment Trimec vs. No Treatment
Salicylic Add 108668 At_2mM_SA_cDNA_P Plant Line WS 108668
At_2mM_SA_cDNA_P Timepoint (hr) 6 108665 At_2mM_SA_cDNA_P Treatment
2 mM SA vs. No Treatment Reproductive and Seed & 108687
Zm_Embryos-Flowers_P Tissue Embryo Fruit Development 108688
Zm_Embryos-Flowers_P Tissue Immature Flowers ABA, Drought,
Germination 20000069 At_100uM_ABA_Mutants_cDNA_P Plant Line CS23
20000069 At_100uM_ABA_Mutants_cDNA_P Timepoint (hr) 6 20000069
At_100uM_ABA_Mutants_cDNA_P Tissue Aerial 20000069
At_100uM_ABA_Mutants_cDNA_P Treatment 100 uM ABA vs. No Treatment
ABA, Drought, Germination 20000070 At_100uM_ABA_Mutants_cDNA_P
Plant Line CS24 20000070 At_100uM_ABA_Mutants_cDNA_P Timepoint (hr)
6 20000070 At_100uM_ABA_Mutants_cDNA_P Tissue Aerial 20000070
At_100uM_ABA_Mutants_cDNA_P Treatment 100 uM ABA vs. No Treatment
ASA, Drought, Germination 20000071 At_100uM_ABA_Mutants_cDNA_P
Plant Line CS8104 20000071 At_100uM_ABA_Mutants_cDNA_P Timepoint
(hr) 3 20000071 At_100uM_ABA_Mutants_cDNA_P Tissue Aerial 20000071
At_100uM_ABA_Mutants_cDNA_P Treatment 100 uM ABA vs. No Treatment
ABA, Drought, Germination 20000072 At_100uM_ABA_Mutants_cDNA_P
Plant Line CS8105 20000072 At_100uM_ABA_Mutants_cDNA_P Timepoint
(hr) 6 20000072 At_100uM_ABA_Mutants_cDNA_P Tissue Aerial 20000072
At_100uM_ABA_Mutants_cDNA_P Treatment 100 uM ABA vs. No Treatment
ABA, Drought, Germination 20000086 At_100uM_ABA_Mutants_cDNA_P
Plant Line CS22 20000086 At_100uM_ABA_Mutants_cDNA_P Timepoint (hr)
6 20000086 At_100uM_ABA_Mutants_cDNA_P Tissue aerial 20000086
At_100uM_ABA_Mutants_cDNA_P Treatment 100 uM ABA vs. No Treatment
ABA, Draught, Germination 20000087 At_100uM_ABA_Mutants_cDNA_P
Plant Line WS 20000087 At_100uM_ABA_Mutants_cDNA_P Timepoint (hr) 6
20000087 At_100uM_ABA_Mutants_cDNA_P Tissue aerial 20000087
At_100uM_ABA_Mutants_cDNA_P Treatment 100 uM ABA vs. No Treatment
ABA, Drought, Germination 20000088 At_100uM_ABA_Mutants_cDNA_P
Plant Line Landsberg 20000088 At_100uM_ABA_Mutants_cDNA_P Timepoint
(hr) 6 20000088 At_100uM_ABA_Mutants_cDNA_P Tissue aeriel 20000088
At_100uM_ABA_Mutants_cDNA_P Treatment 100 uM ABA vs. No Treatment
Salicylic Acid 20000090 At_2mM_SA_CS3726-Columbia_cDNA_P Plant Line
Columbia 20000090 At_2mM_SA_CS3726-Columbia_cDNA_P Time point (hr)
6 20000090 At_2mM_SA_CS3726-Columbia_cDNA_P Tissue Aerial 20000090
At_2mM_SA_CS3726-Columbia_cDNA_P Treatment 2 mM SA vs. No Treatment
Heat 20000111 At_42deg_Heat_cDNA_P Temperature 42 vs. 23 20000111
At_42deg_Heat_cDNA_P Timepoint (hr) 5 20000111 At_42deg_Heat_cDNA_P
Tissue Aerial Heat 20000113 At_42deg_Heat_cDNA_P Temperature 42 vs.
23 20000113 At_42deg_Heat_cDNA_P Timepoint (hr) 8 20000113
At_42deg_Heat_cDNA_P Tissue Aerial ABA, Drought, Germination
20000117 At_100uM_ABA_Mutants_cDNA_P Plant Line columbia 20000117
At_100uM_ABA_Mutants_cDNA_P Timepoint (hr) 6 20000117
At_100uM_ABA_Mutants_cDNA_P Tissue aerial 20000117
At_100uM_ABA_Mutants_cDNA_P Treatment 100 uM ABA vs. No Treatment
Heat 20000171 At_42deg_Heat_P Probe Method mRNA vs. mRNA 20000171
At_42deg_Heat_P Temperature 42 vs. 22 20000171 At_42deg_Heat_P
Timepoint (hr) 1 20000171 At_42deg_Heat_P Tissue Aerial Heat
20000173 At_42deg_Heat_P Probe Method mRNA vs. mRNA 20000173
At_42deg_Heat_P Temperature 42 vs. 22 20000173 At_42deg_Heat_P
Timepoint (hr) 6 20000173 At_42deg_Heat_P Tissue Aerial Early
Seedling Phase 20000179 At_Germinating_Seeds_P Age 6 vs. 0 20000179
At_Germinating_Seeds_P Tissue Germinating Seeds Early Seedling
Phase 20000180 At_Germinating_Seeds_P Age 24 vs. 0 20000180
At_Germinating_Seeds_P Tissue Germinating Seeds Salicylic Acid
20000182 At_2mM_SA_P Timepoint (hr) 6 20000182 At_2mM_SA_P Tissue
Aerial 20000182 At_2mM_SA_P Treatment 2 mM SA vs. No Treatment
Leaves, Shoot Meristem 20000184 At_Shoots_P Age 7 20000184
At_Shoots_P Tissue Shoots vs. Whole Plant Root 20000185 At_Roots_P
Age 7 20000185 At_Roots_P Tissue Roots vs. Whole Plant Cold
20000213 At_4deg_Cold_P Temperature 4 vs. 22 20000213
At_4deg_Cold_P Timepoint (hr) 2 Seed and Fruit Development 20000234
At_Siliques_P Tissue <5 mm Siliques vs. Whole Plant Seed and
Fruit Development 20000235 At_Siliques_YF_6-05-02_P Tissue 5-10 mm
Siliques vs. Whole Plant Seed and Fruit Development 20000236
At_Siliques_P Tissue >10 mm Siliques vs. Whole Plant
Reproductive and Seed & 20000264 At_Open_Flower_P Tissue Open
Flower vs. Whole Plant Fruit Development Reproductive and Seed
& 20000265 At_Open_Flower_P Tissue Closed Bud vs. Whole Rant
Fruit Development Reproductive and Seed & 20000286
At_Open_Flower_P Tissue Half Open vs. Whole Plant Fruit Development
Drought 20000437 At_Drought_P Timepoint (hr) 24 20000437
At_Drought_P Tissue Whole Plant 20000437 At_Drought_P Treatment
Drought vs. No Draught Leaves, Shoot Meristem 20000438 At_Shoots_P
Age 14 20000438 At_Shoots_P Tissue Shoots vs. Whole Plant Roots
20000439 At_Roots_P Age 14 20000439 At_Roots_P Tissue Roots vs.
Whole Plant Brassinolide 20000441 At_1uM_BR-BRZ_P Tissue Shoot
Apices 20000441 At_1uM_BR-BRZ_P Treatment 1 uM BR vs. No Treatment
20000443 At_1uM_BR-BRZ_P Tissue Shoot Apices 20000443
At_1uM_BR-BRZ_P Treatment 1 uM BRZ vs. No Treatment Salicylic Acid
20000478 Zm_5mM_SA_P Age 8 20000478 Zm_5mM_SA_P Plant Line Hybrid
20000478 Zm_5mM_SA_P Timepoint (hr) 72 20000478 Zm_5mM_SA_P Tissue
Aerial 20000478 Zm_5mM_SA_P Treatment 5 mM SA vs. No Treatment
Reproductive and Seed & 20000493 Zm_Hybrid_Seed_Dev_P DAP 20
vs. 12 Fruit Development 20000493 Zm_Hybrid_Seed_Dev_P Plant Line
Hybrid 20000493 Zm_Hybrid_Seed_Dev_P Tissue Endosperm vs. Unfert
Floret Guard Cells 20000495 At_Guard_Cells_P Harvest Date Aug. 2,
2002 20000495 At_Guard_Cells_P Organism A. thaliana 20000495
At_Guard_Cells_P Tissue Guard Cells vs. Leaves PEG 20000527
At_10%_PEG_P Age 20 20000527 At_10%_PEG_P Tissue Aerial 20000527
At_10%_PEG_P Treatment 10% PEG vs. No Treatment ABA, Drought,
Germination 20000573 At_100uM_ABA_Mutants_P Organism A. thaliana
20000573 At_100uM_ABA_Mutants_P Plant Line CS22 vs. Ler wt 20000573
At_100uM_ABA_Mutants_P Timepoint (hr) N/A 20000573
At_100uM_ABA_Mutants_P Tissue Whole Plant 20000573
At_100uM_ABA_Mutants_P Treatment None Viability 20000629
Zm_Herbicide-Treatments_P Timepoint (hr) 12 20000629
Zm_Herbicide-Treatments_P Tissue Aerial 20000629
Zm_Herbicide-Treatments_P Treatment Trimec vs. No Treatment Drought
20000638 At_Drought_cDNA_P Timepoint (hr) 144 20000838
At_Drought_cDNA_P Tissue sdf Reproductive 20000794 At_Petals_P Age
23-25 days 20000794 At_Petals_P Tissue Petals vs. Whole plant Shade
20001247 At_Far-red-induction_P Age 7 20001247
At_Far-red-induction_P Light Far Red vs. White 20001247
At_Far-red-induction_P Plant Line Columbia 20001247
At_Far-red-induction_P Timepoint (hr) 1 Shade 20001248
At_Far-red-induction_P Age 7 20001248 At_Far-red-induction_P Light
Far Red vs. White 20001248 At_Far-red-induction_P Plant Line
Columbia 20001248 At_Far-red-induction_P Timepoint (hr) 4 Shade
20001450 At_Far-red-induction_P Age 7 20001450
At_Far-red-inducfion_P Light Far Red vs. White 20001450
At_Far-red-induction_P Plant Line Columbia 20001450
At_Far-red-induction_P Timepoint (hr) 8 Shade 20001451
At_Far-red-induction_P Age 7 20001451 At_Far-red-induction_P Light
Far Red vs. White 20001451 At_Far-red-induction_P Plant Line
Columbia 20001451 At_Far-red-induction_P Timepoint (hr) 24 Nitrogen
20001459 At_50mM_NH4NO3_L-to-H_P Timepoint (hr) 4 20001459
At_50mM_NH4NO3_L-to-H_P Tissue Siliques
20001459 At_50mM_NH4NO3_L-to-H_P Treatment 50 mM NH4NO3 vs. 100 mM
Manitol Viability 20000530 Zm_2-4D_YF_8-26-02_P Organism Zea Mays
20000530 Zm_2-4D_YF_8-26-02_P Timepoint (hr) 48 20000530
Zm_2-4D_YF_8-26-02_P Tissue Aerial 20000530 Zm_2-4D_YF_8-26-02_P
Treatment 2,4-D vs. No Treatment Guard Cells 20000570
At_Guard_Cells_JD_9-9-02_cDNA_P Harvest Date Jul. 19, 2002 20000570
At_Guard_Cells_JD_9-9-02_cDNA_P Organism Canola 20000570
At_Guard_Cells_JD_9-9-02_cDNA_P Tissue Guard Cells vs. Leaves
TABLE-US-00010 TABLE 10 MA_DIFF TABLE RESULTS FOR DIFFERENTIAL
EXPRESSION ANALYSIS Value (average Differential Clone cDNA
Biomaterial Expt_Rep_ID Short_Name.sub.-- log ratio) Differential
(+/-) 96 12323601 1580810 20000527 At_10%_PEG_P 1.554916476 1 + 96
12323601 1580810 20000573 At_100uM_ABA_Mutants_P 1.433853789 1 + 96
12323601 1580810 20000441 At_1uM_BR-BRZ_P -3.010706617 -1 - 96
12323601 1580810 20000443 At_1uM_BR-BRZ_P -2.748840687 -1 - 96
12323601 1580810 20000171 At_42deg_Heat_P 1.514139809 1 + 96
12323601 1580810 20000173 At_42deg_Heat_P -1.71925392 -1 - 96
12323601 23030 108577 At_42deg_Heat_cDNA_P -3.338050794 -1 - 96
12323801 1580810 20000213 At_4deg_Cold_P 2.813628804 1 + 96
12323601 23030 108579 At_4deg_Cold_cDNA_P 3.999311124 1 + 96
12323601 1580810 20001459 At_50mM_NH4NO3_L-to-H_P -1.715098188 -1 -
96 12323601 1580810 20000437 At_Drought_P 4.220227281 1 + 96
12323601 1580810 20001247 At_Far-red-induction_P -4.634953394 -1 -
96 12323601 1580810 20001248 At_Far-red-induction_P 5.592598825 1 +
96 12323601 1580810 20001450 At_Far-red-induction_P 1.649915315 1 +
96 12323601 1580810 20000180 At_Germinating_Seeds_P -2.680555133 -1
- 96 12323601 1580810 20000495 At_Guard_Cells_P -3.247865708 -1 -
96 12323601 1580810 20000264 At_Open_Flower_P -2.752089532 -1 - 96
12323601 1580810 20000185 At_Roots_P -4.966099796 -1 - 96 12323601
1580810 20000439 At_Roots_P -4.736820319 -1 - 96 12323601 1580810
20000438 At_Shoots_P -4.72150623 -1 - 96 12323601 1580810 20000234
At_Siliques_P -2.874162085 -1 - 96 12323601 1580810 20000235
At_Siliques_P -2.246390758 -1 - 96 12323601 1580810 20000236
At_Siliques_P -2.46053553 -1 - 8161 12321174 19239 108569
At_0.001%_MeJA_cDNA_P -1.173013726 -1 - 8161 12321174 1580012
20000527 At_10%_PEG_P 1.259078847 1 + 8161 12321174 19239 20000069
At_100uM_ABA_Mutants_cDNA_P 4.740481926 1 + 8161 12321174 19239
20000070 At_100uM_ABA_Mutants_cDNA_P 3.670069907 1 + 8161 12321174
19239 20000071 At_100uM_ABA_Mutants_cDNA_P 3.608787472 1 + 8161
12321174 19239 20000086 At_100uM_ABA_Mutants_cDNA_P 3.027039775 1 +
8161 12321174 19239 20000087 At_100uM_ABA_Mutants_cDNA_P
2.423296688 1 + 8161 12321174 19239 20000088
At_100uM_ABA_Mutants_cDNA_P 2.856206036 1 + 8161 12321174 19239
20000117 At_100uM_ABA_Mutants_cDNA_P 3.485993547 1 + 8161 12321174
19239 108614 At_100uM_ABA_Mutants_cDNA_P -1.365108521 -1 - 8161
12321174 19239 108622 At_100uM_ABA_Mutants_cDNA_P -1.321545662 -1 -
8161 12321174 1580012 20000441 At_1uM_BR-BRZ_P -2.735405149 -1 -
8161 12321174 1580012 20000443 At_1uM_BR-BRZ_P -2.242959206 -1 -
8161 12321174 19239 20000090 At_2mM_SA_CS3726-Columbia_cDNA_P
2.729191739 1 + 8161 12321174 19239 108668 At_2mM_SA_cDNA_P
-1.508606549 -1 - 8161 12321174 1580012 20000182 At_2mM_SA_P
-1.704743738 -1 - 8161 12321174 19239 20000111 At_42deg_Heat_cDNA_P
-2.464590235 -1 - 8161 12321174 19239 20000113 At_42deg_Heat_cDNA_P
-1.876879573 -1 - 8161 12321174 1580012 20000173 At_42deg_Heat_P
-2.821092623 -1 - 8161 12321174 1580012 20000213 At_4deg_Cold_P
4.599973491 1 + 8161 12321174 19239 108579 At_4deg_Cold_cDNA_P
3.707962628 1 + 8161 12321174 19239 108488
At_50mM_NH4NO3_L-to-H_Rosette_cDNA_P -1.429425437 -1 - 8161
12321174 1580012 20001459 At_50mM_NH4NO3_L-to-H_P -2.78961071 -1 -
8161 12321174 19239 108473 At_Drought_Flowers_cDNA_P 1.708925799 1
+ 8161 12321174 1580012 20000437 At_Drought_P 4.822027774 1 + 8161
12321174 1580012 20001247 At_Far-red-induction_P -4.212204824 -1 -
8161 12321174 1580012 20001248 At_Far-red-induction_P 6.169999757 1
+ 8161 12321174 1580012 20001450 At_Far-red-induction_P 1.763281094
1 + 8161 12321174 1580012 20001451 At_Far-red-induction_P
1.395085228 1 + 8161 12321174 1580012 20000179
At_Germinating_Seeds_P -1.441537409 -1 - 8161 12321174 1580012
20000180 At_Germinating_Seeds_P -3.147732829 -1 - 8161 12321174
19239 108461 At_Germinating_Seeds_cDNA_P -1.646872266 -1 - 8161
12321174 19239 108462 At_Germinating_Seeds_cDNA_P -1.665185357 -1 -
8161 12321174 19239 108463 At_Germinating_Seeds_cDNA_P -1.426993122
-1 - 8161 12321174 19239 108464 At_Germinating_Seeds_cDNA_P
-1.828990435 -1 - 8161 12321174 1580012 20000495 At_Guard_Cells_P
-2.920579386 -1 - 8161 12321174 19239 20000570
At_Guard_Cells_cDNA_P -1.49484136 -1 - 8161 12321174 19239 107881
At_Herbicide_v2_cDNA_P -1.761216284 -1 - 8161 12321174 19239 107891
At_Herbicide_v2_cDNA_P -2.164326634 -1 - 8161 12321174 19239 108465
At_Herbicide_v3_1_cDNA_P 4.557494714 1 + 8161 12321174 19239 108629
At_Herbicide_v3_1_cDNA_P 1.998365625 1 + 8161 12321174 19239 108594
At_Ler-rhl_Root_cDNA_P 1.196805915 1 + 8161 12321174 1580012
20000264 At_Open_Flower_P -3.159834613 -1 - 8161 12321174 1580012
20000265 At_Open_Flower_P -2.481345749 -1 - 8161 12321174 1580012
20000286 At_Open_Flower_P -2.087635814 -1 - 8161 12321174 1580012
20000794 At_Petals_P -1.300481698 -1 - 8161 12321174 19239 108434
At_Root_Tips_cDNA_P -2.458487785 -1 - 8161 12321174 1580012
20000185 At_Roots_P -5.279075251 -1 - 8161 12321174 1580012
20000439 At_Roots_P -5.030661468 -1 - 8161 12321174 19239 108480
At_Shoot_Apices_cDNA_P -2.309034231 -1 - 8161 12321174 19239 108481
At_Shoot_Apices_cDNA_P -1.817142133 -1 - 8161 12321174 1580012
20000184 At_Shoots_P -5.762449008 -1 - 8161 12321174 1580012
20000438 At_Shoots_P -5.999883819 -1 - 8161 12321174 1580012
20000234 At_Siliques_P -3.209042334 -1 - 8161 12321174 1580012
20000235 At_Siliques_P -3.022090865 -1 - 8161 12321174 1580012
20000236 At_Siliques_P -2.340846461 -1 - 8161 12321174 19239 108435
At_stm_Mutants_cDNA_P -2.665998172 -1 - 8161 12321174 19239 108437
At_Tissue_Specific_Expression_cDNA_P -1.916511208 -1 - 8161
12321174 19239 108438 At_Tissue_Specific_Expression_cDNA_P
-1.518965097 -1 - 8490 13491409 1580810 20000527 At_10%_PEG_P
1.554916476 1 + 8490 13491409 19237 20000070
At_100uM_ABA_Mutants_cDNA_P 5.066668574 1 + 8490 13491409 19237
20000072 At_100uM_ABA_Mutants_cDNA_P 4.640392336 1 + 8490 13491409
19237 20000086 At_100uM_ABA_Mutants_cDNA_P 3.798180577 1 + 8490
13491409 19237 20000087 At_100uM_ABA_Mutants_cDNA_P 3.425132193 1 +
8490 13491409 19237 20000088 At_100uM_ABA_Mutants_cDNA_P
3.271355571 1 + 8490 13491409 1580810 20000573
At_100uM_ABA_Mutants_P 1.433853789 1 + 8490 13491409 1580810
20000441 At_1uM_BR-BRZ_P -3.010706617 -1 - 8490 13491409 1580810
20000443 At_1uM_BR-BRZ_P -2.748840687 -1 - 8490 13491409 19237
20000090 At_2mM_SA_CS3726-Columbia_cDNA_P 3.600372905 1 + 8490
13491409 19237 20000111 At_42deg_Heat_cDNA_P -2.04796601 -1 - 8490
13491409 1580810 20000171 At_42deg_Heat_P 1.514139809 1 + 8490
13491409 1580810 20000173 At_42deg_Heat_P -1.71925392 -1 - 8490
13491409 1580810 20000213 At_4deg_Cold_P 2.813628804 1 + 8490
13491409 1580810 20001459 At_50mM_NH4NO3_L-to-H_P -1.715098188 -1 -
8490 13491409 19237 108473 At_Drought_Flowers_cDNA_P 1.214971498 1
+ 8490 13491409 1580810 20000437 At_Drought_P 4.220227281 1 + 8490
13491409 19237 20000636 At_Drought_cDNA_P 1.707634838 1 + 8490
13491409 1580810 20001247 At_Far-red-induction_P -4.634953394 -1 -
8490 13491409 1580810 20001248 At_Far-red-induction_P 5.592598825 1
+ 8490 13491409 1580810 20001450 At_Far-red-induction_P 1.649915315
1 + 8490 13491409 1580810 20000180 At_Germinating_Seeds_P
-2.680555133 -1 - 8490 13491409 19237 108461
At_Germinating_Seeds_cDNA_P -2.458568535 -1 - 8490 13491409 19237
108462 At_Germinating_Seeds_cDNA_P -2.330805635 -1 - 8490 13491409
19237 108463 At_Germinating_Seeds_cDNA_P -2.324720192 -1 - 8490
13491409 19237 108464 At_Germinating_Seeds_cDNA_P -2.37426655 -1 -
8490 13491409 1580810 20000495 At_Guard_Cells_P -3.247865708 -1 -
8490 13491409 19237 107881 At_Herbicide_v2_cDNA_P -3.271686652 -1 -
8490 13491409 19237 107891 At_Herbicide_v2_cDNA_P -2.602710336 -1 -
8490 13491409 19237 108465 At_Herbicide_v3_1_cDNA_P 4.904232807 1 +
8490 13491409 19237 108629 At_Herbicide_v3_1_cDNA_P 1.945047545 1 +
8490 13491409 19237 108630 At_Herbicide_v3_1_cDNA_P 1.421005702 1 +
8490 13491409 1580810 20000264 At_Open_Flower_P -2.752089532 -1 -
8490 13491409 19237 108434 At_Root_Tips_cDNA_P -2.359223661 -1 -
8490 13491409 1580810 20000185 At_Roots_P -4.966099796 -1 - 8490
13491409 1580810 20000439 At_Roots_P -4.736820319 -1 - 8490
13491409 19237 108480 At_Shoot_Apices_cDNA_P -2.408359482 -1 - 8490
13491409 19237 108481 At_Shoot_Apices_cDNA_P -3.133713759 -1 - 8490
13491409 1580810 20000438 At_Shoots_P -4.72150623 -1 - 8490
13491409 1580810 20000234 At_Siliques_P -2.874162085 -1 - 8490
13491409 1580810 20000235 At_Siliques_P -2.246390758 -1 - 8490
13491409 1580810 20000236 At_Siliques_P -2.46053553 -1 - 8490
13491409 19237 108435 At_stm_Mutants_cDNA_P -2.551559281 -1 - 8490
13491409 19237 108429 At_Tissue_Specific_Expression_cDNA_P
-1.022462895 -1 - 8490 13491409 19237 108437
At_Tissue_Specific_Expression_cDNA_P -1.501818945 -1 - 8490
13491409 19237 108438 At_Tissue_Specific_Expression_cDNA_P
-1.739999423 -1 - 8490 13491409 19237 108439
At_Tissue_Specific_Expression_cDNA_P -2.657664047 -1 - 305463
12355477 1609791 20000478 Zm_5mM_SA_P 1.626535585 1 + 305483
12355477 1609791 20000629 Zm_Herbicide-Treatments_P 1.058894478 1 +
305463 12355477 1609791 20000493 Zm_Hybrid_Seed_Dev_P 1.987652422 1
+ 305463 12355477 1609791 108543 Zm_Imbibed_Embryo_Endosperm_P
-1.733891996 -1 - 305463 12355477 1609791 108528 Zm_Imbibed_Seeds_P
1.608201864 1 + 305463 12355477 1609791 108530 Zm_Imbibed_Seeds_P
1.26990335 1 + 305463 12355477 1609791 108546 Zm_Imbibed_Seeds_P
1.460500636 1 + 486033 12436299 1608109 20000530 Zm_2-4D_P
1.265765889 1 + 486033 12436299 1608109 108523 Zm_42deg_Heat_P
-1.154793559 -1 - 486033 12436299 1608109 108687
Zm_Embryos-Flowers_P 1.691512498 1 + 486033 12436299 1608109 108688
Zm_Embryos-Flowers_P -2.071866621 -1 -
Sequence CWU 1
1
5011453DNAZea mays subsp. maysmisc_feature(1)..(1453)ceres Seq. ID
no. 12355477 1aatccctcgc ctgcaactgg ctctctgtcc ccttctgctc
cccccacggt tccccagagc 60ccgagccaaa tctaggggct tccttcatcc gagcgtggtt
tcaattctag gggtagtcac 120ctcacctgaa ttccgcccaa ataaattcgt
cgctgccttg tgatccttgg ggtttccttg 180gttcttgagt tgcgatcttc
tgctggttcg tgtcccccaa tccgtaatca atccggcgtc 240taggaaacca
attgctgctc agttctctta tttgctcctc gccttccttc ctccagcctg
300gttaaaatat cgaaagggga ttttttttta aaaatctgct catcgaggaa
gcagggaaga 360caagaattgt tgcatcggat aaaggtcggg tgaaaataca
agcaaatcct gggaactcgc 420gtccctttgc taggtggttc tttcctgata
caaagaacac aatgggcgat gtgtccttga 480acggacccat taaggctgct
gagccaggtg ccggtggcat tgccaagggc aatcaagttc 540tggacacgat
gtccgccggg tggacagacg agagacacag gctgtatata agctctatgg
600aggcctcttt cgtcgatcaa ctgtacaacc acgggagccg tccgcgcaac
gcaaacggca 660ccgccttcaa ggctctccgc agggagtacg tcgagtatga
gaagaccgat gctcctgtgc 720gaaggggggc taagtgctgc ggcgttcctg
caaatccttg gatgcagcat ttcaggccac 780gtagtgatgg cggtaataac
gcgcgaggcg atgggctcgg ggattctgtg ggcgatcttg 840aatctggcac
tgaggcaaac cggaagagcc tctcagcgtc tcatggaagg gaacgggacg
900cttgtgaggg agaaccccag cttctccatg aaagtagaga ggtctctgat
caaaattttg 960ctgacgacga ggctgaagct gaaacagaat caatgaaagc
atacaagaaa aggagattaa 1020gcaggacaat gatcaactaa atttgcaggg
tcaattagct tagcctgttg caggaattga 1080gatgactgtc ctaaaaggag
gcagtaagat gatgggacat gtcttacgaa attttcagct 1140gttgcctctt
ggtagccaag gcactttgaa tccgaaggaa ggtgttgaag ggtagttgtt
1200agtgatcttg tgatgatata acgagctctg gagcagttag catcggcatt
ttagtggatt 1260atgttcttgt tatgtgtatc tgtctatttt tcagtcctca
tcggtagtgc tgcatagtac 1320ctcgctctct cgtcagaagg atattaggct
aggtcactgt tattaaattt ttcaataaca 1380gtgaagtgta catgtgtttg
ccaaatggtg agaatcatta ttgatttcca attcacaaac 1440tattctttat gcc
14532576DNAZea mays subsp. mays 2atgggcgatg tgtccttgaa cggacccatt
aaggctgctg agccaggtgc cggtggcatt 60gccaagggca atcaagttct ggacacgatg
tccgccgggt ggacagacga gagacacagg 120ctgtatataa gctctatgga
ggcctctttc gtcgatcaac tgtacaacca cgggagccgt 180ccgcgcaacg
caaacggcac cgccttcaag gctctccgca gggagtacgt cgagtatgag
240aagaccgatg ctcctgtgcg aaggggggct aagtgctgcg gcgttcctgc
aaatccttgg 300atgcagcatt tcaggccacg tagtgatggc ggtaataacg
cgcgaggcga tgggctcggg 360gattctgtgg gcgatcttga atctggcact
gaggcaaacc ggaagagcct ctcagcgtct 420catggaaggg aacgggacgc
ttgtgaggga gaaccccagc ttctccatga aagtagagag 480gtctctgatc
aaaattttgc tgacgacgag gctgaagctg aaacagaatc aatgaaagca
540tacaagaaaa ggagattaag caggacaatg atcaac 5763192PRTZea mays
subsp. mayspeptide(1)..(192)ceres Seq. ID no. 12355478 3Met Gly Asp
Val Ser Leu Asn Gly Pro Ile Lys Ala Ala Glu Pro Gly1 5 10 15Ala Gly
Gly Ile Ala Lys Gly Asn Gln Val Leu Asp Thr Met Ser Ala 20 25 30Gly
Trp Thr Asp Glu Arg His Arg Leu Tyr Ile Ser Ser Met Glu Ala 35 40
45Ser Phe Val Asp Gln Leu Tyr Asn His Gly Ser Arg Pro Arg Asn Ala
50 55 60Asn Gly Thr Ala Phe Lys Ala Leu Arg Arg Glu Tyr Val Glu Tyr
Glu65 70 75 80Lys Thr Asp Ala Pro Val Arg Arg Gly Ala Lys Cys Cys
Gly Val Pro 85 90 95Ala Asn Pro Trp Met Gln His Phe Arg Pro Arg Ser
Asp Gly Gly Asn 100 105 110Asn Ala Arg Gly Asp Gly Leu Gly Asp Ser
Val Gly Asp Leu Glu Ser 115 120 125Gly Thr Glu Ala Asn Arg Lys Ser
Leu Ser Ala Ser His Gly Arg Glu 130 135 140Arg Asp Ala Cys Glu Gly
Glu Pro Gln Leu Leu His Glu Ser Arg Glu145 150 155 160Val Ser Asp
Gln Asn Phe Ala Asp Asp Glu Ala Glu Ala Glu Thr Glu 165 170 175Ser
Met Lys Ala Tyr Lys Lys Arg Arg Leu Ser Arg Thr Met Ile Asn 180 185
1904489DNAZea mays subsp. mays 4atgtccgccg ggtggacaga cgagagacac
aggctgtata taagctctat ggaggcctct 60ttcgtcgatc aactgtacaa ccacgggagc
cgtccgcgca acgcaaacgg caccgccttc 120aaggctctcc gcagggagta
cgtcgagtat gagaagaccg atgctcctgt gcgaaggggg 180gctaagtgct
gcggcgttcc tgcaaatcct tggatgcagc atttcaggcc acgtagtgat
240ggcggtaata acgcgcgagg cgatgggctc ggggattctg tgggcgatct
tgaatctggc 300actgaggcaa accggaagag cctctcagcg tctcatggaa
gggaacggga cgcttgtgag 360ggagaacccc agcttctcca tgaaagtaga
gaggtctctg atcaaaattt tgctgacgac 420gaggctgaag ctgaaacaga
atcaatgaaa gcatacaaga aaaggagatt aagcaggaca 480atgatcaac
4895163PRTZea mays subsp. mayspeptide(1)..(163)ceres Seq. ID no.
12355479 5Met Ser Ala Gly Trp Thr Asp Glu Arg His Arg Leu Tyr Ile
Ser Ser1 5 10 15Met Glu Ala Ser Phe Val Asp Gln Leu Tyr Asn His Gly
Ser Arg Pro 20 25 30Arg Asn Ala Asn Gly Thr Ala Phe Lys Ala Leu Arg
Arg Glu Tyr Val 35 40 45Glu Tyr Glu Lys Thr Asp Ala Pro Val Arg Arg
Gly Ala Lys Cys Cys 50 55 60Gly Val Pro Ala Asn Pro Trp Met Gln His
Phe Arg Pro Arg Ser Asp65 70 75 80Gly Gly Asn Asn Ala Arg Gly Asp
Gly Leu Gly Asp Ser Val Gly Asp 85 90 95Leu Glu Ser Gly Thr Glu Ala
Asn Arg Lys Ser Leu Ser Ala Ser His 100 105 110Gly Arg Glu Arg Asp
Ala Cys Glu Gly Glu Pro Gln Leu Leu His Glu 115 120 125Ser Arg Glu
Val Ser Asp Gln Asn Phe Ala Asp Asp Glu Ala Glu Ala 130 135 140Glu
Thr Glu Ser Met Lys Ala Tyr Lys Lys Arg Arg Leu Ser Arg Thr145 150
155 160Met Ile Asn6441DNAZea mays subsp. mays 6atggaggcct
ctttcgtcga tcaactgtac aaccacggga gccgtccgcg caacgcaaac 60ggcaccgcct
tcaaggctct ccgcagggag tacgtcgagt atgagaagac cgatgctcct
120gtgcgaaggg gggctaagtg ctgcggcgtt cctgcaaatc cttggatgca
gcatttcagg 180ccacgtagtg atggcggtaa taacgcgcga ggcgatgggc
tcggggattc tgtgggcgat 240cttgaatctg gcactgaggc aaaccggaag
agcctctcag cgtctcatgg aagggaacgg 300gacgcttgtg agggagaacc
ccagcttctc catgaaagta gagaggtctc tgatcaaaat 360tttgctgacg
acgaggctga agctgaaaca gaatcaatga aagcatacaa gaaaaggaga
420ttaagcagga caatgatcaa c 4417147PRTZea mays subsp.
mayspeptide(1)..(147)ceres Seq. ID no. 12355480 7Met Glu Ala Ser
Phe Val Asp Gln Leu Tyr Asn His Gly Ser Arg Pro1 5 10 15Arg Asn Ala
Asn Gly Thr Ala Phe Lys Ala Leu Arg Arg Glu Tyr Val 20 25 30Glu Tyr
Glu Lys Thr Asp Ala Pro Val Arg Arg Gly Ala Lys Cys Cys 35 40 45Gly
Val Pro Ala Asn Pro Trp Met Gln His Phe Arg Pro Arg Ser Asp 50 55
60Gly Gly Asn Asn Ala Arg Gly Asp Gly Leu Gly Asp Ser Val Gly Asp65
70 75 80Leu Glu Ser Gly Thr Glu Ala Asn Arg Lys Ser Leu Ser Ala Ser
His 85 90 95Gly Arg Glu Arg Asp Ala Cys Glu Gly Glu Pro Gln Leu Leu
His Glu 100 105 110Ser Arg Glu Val Ser Asp Gln Asn Phe Ala Asp Asp
Glu Ala Glu Ala 115 120 125Glu Thr Glu Ser Met Lys Ala Tyr Lys Lys
Arg Arg Leu Ser Arg Thr 130 135 140Met Ile Asn14581494DNAZea mays
subsp. maysmisc_feature(1)..(1494)ceres Seq. ID no. 12410516
8gtgtttcatt tttaatgacc attctctcat ctgctgctgg ctgcggctat atacccccct
60ctctctgtct ctctatctcc ttctgttctt agacgtttct ccatagcctg agccaaatct
120agggggcttg cttcatctgc tgtccgatcg tggtttggtt tctcggggct
ggcgcggtca 180agagcgcacc tgaattccac cgaaatccgc cacggtagtt
cttgcctagg tgtgtcgttg 240gtcgttgcct tgtgaccctt gcggattttc
ttgtttcttt ttgagttgcg atctttgcag 300gttagtctcc cccccaatcc
gtaatcatcc ggcgtctagg aaactgcagt ccagttttct 360tatttgttcg
tctcgtgcct tctccccatc ctggttagaa agaatatcgg aagggggatt
420tttttttttg cctgttcgta gaggaagcag tgaagacata attgttgcat
ctgataaagc 480tcgggcgaaa tacacgcaaa tccttggaat tttgcatccc
tttgctggct cttttctgat 540tcagagaacc caatggggga tgtgtccttg
aatcgacccg ttaaggccga gccaactgcc 600ggtggcattg ccaagggaaa
ccgagttctg gacacgatgt ccgccgggtg gacggacgag 660agacacatgc
tgtatataag ctccatggag gcttcttttg tcgatcagct atacaaccat
720ggaaaccatc cgcacgacgc aaatggcgct ggcttcaagg ttctccgcag
gggggtgtgg 780gagtacatcg agtatgagaa gaccagtgcc cctgtgcgaa
gtggggctaa atgctgcgtc 840cctgcaaatc cttggatccg gcatttcagg
ccacgtgact gcggtagtaa cgcacagagt 900gacgcggtcg aggcctcagt
gggcgaccat gagtcgggta ctcaggcaag ccgcaagagc 960ccttcagtgt
ctcatggaag ggaacgggga gcttgtaagg gagaacccca gattctacat
1020gaaagtacag aggtctctga tcaaaatttt gctgacgatg aggctgaagc
tgaaacagaa 1080tcaatgaaag catgcaagaa aaggagacta agcagggctt
tgcactccgg tgctgaatga 1140tcaagtaaat tcgcaggaac aattagctta
gcctgttgca agaatcgata tgatttatcc 1200taaaagaagg tgttaagatg
atgggacatg gctttcaaaa ctttcagctg ttgcctgctg 1260gtagccaaga
cacactgaat ccgaaggaag gcgttgaagg gtagctgtta gtgattttgt
1320gatataaaga gtactggggc agttagcatc ggcattttta gcggatttaa
gttcttgtta 1380tgtatatctg tcttctgtct tcatcagtag tgctgcttag
tacctcactc tctcgtcagc 1440aggatatttc tatatattgt ctgtacttgg
tagatatatg tattggttga tccg 14949585DNAZea mays subsp. mays
9atgggggatg tgtccttgaa tcgacccgtt aaggccgagc caactgccgg tggcattgcc
60aagggaaacc gagttctgga cacgatgtcc gccgggtgga cggacgagag acacatgctg
120tatataagct ccatggaggc ttcttttgtc gatcagctat acaaccatgg
aaaccatccg 180cacgacgcaa atggcgctgg cttcaaggtt ctccgcaggg
gggtgtggga gtacatcgag 240tatgagaaga ccagtgcccc tgtgcgaagt
ggggctaaat gctgcgtccc tgcaaatcct 300tggatccggc atttcaggcc
acgtgactgc ggtagtaacg cacagagtga cgcggtcgag 360gcctcagtgg
gcgaccatga gtcgggtact caggcaagcc gcaagagccc ttcagtgtct
420catggaaggg aacggggagc ttgtaaggga gaaccccaga ttctacatga
aagtacagag 480gtctctgatc aaaattttgc tgacgatgag gctgaagctg
aaacagaatc aatgaaagca 540tgcaagaaaa ggagactaag cagggctttg
cactccggtg ctgaa 58510195PRTZea mays subsp.
mayspeptide(1)..(195)ceres Seq. ID no. 12410517 10Met Gly Asp Val
Ser Leu Asn Arg Pro Val Lys Ala Glu Pro Thr Ala1 5 10 15Gly Gly Ile
Ala Lys Gly Asn Arg Val Leu Asp Thr Met Ser Ala Gly 20 25 30Trp Thr
Asp Glu Arg His Met Leu Tyr Ile Ser Ser Met Glu Ala Ser 35 40 45Phe
Val Asp Gln Leu Tyr Asn His Gly Asn His Pro His Asp Ala Asn 50 55
60Gly Ala Gly Phe Lys Val Leu Arg Arg Gly Val Trp Glu Tyr Ile Glu65
70 75 80Tyr Glu Lys Thr Ser Ala Pro Val Arg Ser Gly Ala Lys Cys Cys
Val 85 90 95Pro Ala Asn Pro Trp Ile Arg His Phe Arg Pro Arg Asp Cys
Gly Ser 100 105 110Asn Ala Gln Ser Asp Ala Val Glu Ala Ser Val Gly
Asp His Glu Ser 115 120 125Gly Thr Gln Ala Ser Arg Lys Ser Pro Ser
Val Ser His Gly Arg Glu 130 135 140Arg Gly Ala Cys Lys Gly Glu Pro
Gln Ile Leu His Glu Ser Thr Glu145 150 155 160Val Ser Asp Gln Asn
Phe Ala Asp Asp Glu Ala Glu Ala Glu Thr Glu 165 170 175Ser Met Lys
Ala Cys Lys Lys Arg Arg Leu Ser Arg Ala Leu His Ser 180 185 190Gly
Ala Glu 19511501DNAZea mays subsp. mays 11atgtccgccg ggtggacgga
cgagagacac atgctgtata taagctccat ggaggcttct 60tttgtcgatc agctatacaa
ccatggaaac catccgcacg acgcaaatgg cgctggcttc 120aaggttctcc
gcaggggggt gtgggagtac atcgagtatg agaagaccag tgcccctgtg
180cgaagtgggg ctaaatgctg cgtccctgca aatccttgga tccggcattt
caggccacgt 240gactgcggta gtaacgcaca gagtgacgcg gtcgaggcct
cagtgggcga ccatgagtcg 300ggtactcagg caagccgcaa gagcccttca
gtgtctcatg gaagggaacg gggagcttgt 360aagggagaac cccagattct
acatgaaagt acagaggtct ctgatcaaaa ttttgctgac 420gatgaggctg
aagctgaaac agaatcaatg aaagcatgca agaaaaggag actaagcagg
480gctttgcact ccggtgctga a 50112167PRTZea mays subsp.
mayspeptide(1)..(167)ceres Seq. ID no. 12410518 12Met Ser Ala Gly
Trp Thr Asp Glu Arg His Met Leu Tyr Ile Ser Ser1 5 10 15Met Glu Ala
Ser Phe Val Asp Gln Leu Tyr Asn His Gly Asn His Pro 20 25 30His Asp
Ala Asn Gly Ala Gly Phe Lys Val Leu Arg Arg Gly Val Trp 35 40 45Glu
Tyr Ile Glu Tyr Glu Lys Thr Ser Ala Pro Val Arg Ser Gly Ala 50 55
60Lys Cys Cys Val Pro Ala Asn Pro Trp Ile Arg His Phe Arg Pro Arg65
70 75 80Asp Cys Gly Ser Asn Ala Gln Ser Asp Ala Val Glu Ala Ser Val
Gly 85 90 95Asp His Glu Ser Gly Thr Gln Ala Ser Arg Lys Ser Pro Ser
Val Ser 100 105 110His Gly Arg Glu Arg Gly Ala Cys Lys Gly Glu Pro
Gln Ile Leu His 115 120 125Glu Ser Thr Glu Val Ser Asp Gln Asn Phe
Ala Asp Asp Glu Ala Glu 130 135 140Ala Glu Thr Glu Ser Met Lys Ala
Cys Lys Lys Arg Arg Leu Ser Arg145 150 155 160Ala Leu His Ser Gly
Ala Glu 16513471DNAZea mays subsp. mays 13atgctgtata taagctccat
ggaggcttct tttgtcgatc agctatacaa ccatggaaac 60catccgcacg acgcaaatgg
cgctggcttc aaggttctcc gcaggggggt gtgggagtac 120atcgagtatg
agaagaccag tgcccctgtg cgaagtgggg ctaaatgctg cgtccctgca
180aatccttgga tccggcattt caggccacgt gactgcggta gtaacgcaca
gagtgacgcg 240gtcgaggcct cagtgggcga ccatgagtcg ggtactcagg
caagccgcaa gagcccttca 300gtgtctcatg gaagggaacg gggagcttgt
aagggagaac cccagattct acatgaaagt 360acagaggtct ctgatcaaaa
ttttgctgac gatgaggctg aagctgaaac agaatcaatg 420aaagcatgca
agaaaaggag actaagcagg gctttgcact ccggtgctga a 47114157PRTZea mays
subsp. mayspeptide(1)..(157)ceres Seq. ID no. 12410519 14Met Leu
Tyr Ile Ser Ser Met Glu Ala Ser Phe Val Asp Gln Leu Tyr1 5 10 15Asn
His Gly Asn His Pro His Asp Ala Asn Gly Ala Gly Phe Lys Val 20 25
30Leu Arg Arg Gly Val Trp Glu Tyr Ile Glu Tyr Glu Lys Thr Ser Ala
35 40 45Pro Val Arg Ser Gly Ala Lys Cys Cys Val Pro Ala Asn Pro Trp
Ile 50 55 60Arg His Phe Arg Pro Arg Asp Cys Gly Ser Asn Ala Gln Ser
Asp Ala65 70 75 80Val Glu Ala Ser Val Gly Asp His Glu Ser Gly Thr
Gln Ala Ser Arg 85 90 95Lys Ser Pro Ser Val Ser His Gly Arg Glu Arg
Gly Ala Cys Lys Gly 100 105 110Glu Pro Gln Ile Leu His Glu Ser Thr
Glu Val Ser Asp Gln Asn Phe 115 120 125Ala Asp Asp Glu Ala Glu Ala
Glu Thr Glu Ser Met Lys Ala Cys Lys 130 135 140Lys Arg Arg Leu Ser
Arg Ala Leu His Ser Gly Ala Glu145 150 15515409DNABrassica
napusmisc_feature(1)..(409)ceres Seq. ID no. 4788142 15ttttttcttt
ttcaccttct cctcctcctt ctctcctttc ttctgatatt ttcctctctc 60tagtcttaac
aagatagata ggtagcaaat ggttggtgac tacagagaga actatagccc
120aagctccgac gattcttctt ctgtagggga agagacgact tcttcaatgt
attctgcgag 180gaatgaagat acgcctacag aatggaccga tgagaagcat
agtttgtatc ttaaatcaat 240ggaagcttcc ttcgttgatc agctgtacaa
ctccctcggt gcgctcggct ccaaaaacaa 300caaggatact gtcggaccat
cgagaaggtt cggtgatggt ggaaaacctt ctgaagaaca 360ggtatgaata
ggacactttc ccctgtcttt ttccatgtgc gatgttgtg 40916276DNABrassica
napus 16atggttggtg actacagaga gaactatagc ccaagctccg acgattcttc
ttctgtaggg 60gaagagacga cttcttcaat gtattctgcg aggaatgaag atacgcctac
agaatggacc 120gatgagaagc atagtttgta tcttaaatca atggaagctt
ccttcgttga tcagctgtac 180aactccctcg gtgcgctcgg ctccaaaaac
aacaaggata ctgtcggacc atcgagaagg 240ttcggtgatg gtggaaaacc
ttctgaagaa caggta 2761792PRTBrassica napuspeptide(1)..(92)ceres
Seq. ID no. 4788143 17Met Val Gly Asp Tyr Arg Glu Asn Tyr Ser Pro
Ser Ser Asp Asp Ser1 5 10 15Ser Ser Val Gly Glu Glu Thr Thr Ser Ser
Met Tyr Ser Ala Arg Asn 20 25 30Glu Asp Thr Pro Thr Glu Trp Thr Asp
Glu Lys His Ser Leu Tyr Leu 35 40 45Lys Ser Met Glu Ala Ser Phe Val
Asp Gln Leu Tyr Asn Ser Leu Gly 50 55 60Ala Leu Gly Ser Lys Asn Asn
Lys Asp Thr Val Gly Pro Ser Arg Arg65 70 75 80Phe Gly Asp Gly Gly
Lys Pro Ser Glu Glu Gln Val 85 9018198DNABrassica napus
18atgtattctg cgaggaatga agatacgcct acagaatgga ccgatgagaa gcatagtttg
60tatcttaaat caatggaagc ttccttcgtt gatcagctgt acaactccct cggtgcgctc
120ggctccaaaa acaacaagga tactgtcgga ccatcgagaa ggttcggtga
tggtggaaaa 180ccttctgaag aacaggta 1981966PRTBrassica
napuspeptide(1)..(66)ceres Seq. ID no. 4788144 19Met Tyr Ser Ala
Arg Asn Glu Asp Thr Pro Thr Glu Trp Thr Asp Glu1 5 10 15Lys His Ser
Leu Tyr Leu Lys Ser Met Glu Ala Ser Phe Val Asp Gln 20 25 30Leu
Tyr Asn Ser Leu Gly Ala Leu Gly Ser Lys Asn Asn Lys Asp Thr 35 40
45Val Gly Pro Ser Arg Arg Phe Gly Asp Gly Gly Lys Pro Ser Glu Glu
50 55 60Gln Val6520186DNABrassica napus 20atgaagatac gcctacagaa
tggaccgatg agaagcatag tttgtatctt aaatcaatgg 60aagcttcctt cgttgatcag
ctgtacaact ccctcggtgc gctcggctcc aaaaacaaca 120aggatactgt
cggaccatcg agaaggttcg gtgatggtgg aaaaccttct gaagaacagg 180tatgaa
1862162PRTBrassica napuspeptide(1)..(62)ceres Seq. ID no. 4788145
21Met Lys Ile Arg Leu Gln Asn Gly Pro Met Arg Ser Ile Val Cys Ile1
5 10 15Leu Asn Gln Trp Lys Leu Pro Ser Leu Ile Ser Cys Thr Thr Pro
Ser 20 25 30Val Arg Ser Ala Pro Lys Thr Thr Arg Ile Leu Ser Asp His
Arg Glu 35 40 45Gly Ser Val Met Val Glu Asn Leu Leu Lys Asn Arg Tyr
Glu 50 55 6022486DNABrassica napusmisc_feature(1)..(486)ceres Seq.
ID no. 4796909 22tttccgtctt tctttttcac cttctcctcc tccttctctc
ctttcttctg atattttcct 60ctctctagtc ttaacaagat agataggtag caaatggttg
gtgactacag agagaactat 120agcccaagct ccgacgattc ttcttctgta
ggggaagaga cgacttcttc aatgtattct 180gcgaggaatg aagatacgcc
tacagaatgg accgatgaga agcatagttt gtatcttaaa 240tcaatggaag
cttccttcgt tgatcagctg tacaactccc tcggtgcgct cggctccaaa
300aacaacaagg atactgtcgg accatcgaga aggttcggtg atggtggaaa
accttctgaa 360gaacagaaga tgaatgtgag gcagcctgag tatcgtctca
atggaagaca cggtcgtcgc 420tctcacgagt ttcttaggag tccatggatc
aagcactata agccttcacc aaagtcccta 480acagat 48623393DNABrassica
napus 23atggttggtg actacagaga gaactatagc ccaagctccg acgattcttc
ttctgtaggg 60gaagagacga cttcttcaat gtattctgcg aggaatgaag atacgcctac
agaatggacc 120gatgagaagc atagtttgta tcttaaatca atggaagctt
ccttcgttga tcagctgtac 180aactccctcg gtgcgctcgg ctccaaaaac
aacaaggata ctgtcggacc atcgagaagg 240ttcggtgatg gtggaaaacc
ttctgaagaa cagaagatga atgtgaggca gcctgagtat 300cgtctcaatg
gaagacacgg tcgtcgctct cacgagtttc ttaggagtcc atggatcaag
360cactataagc cttcaccaaa gtccctaaca gat 39324131PRTBrassica
napuspeptide(1)..(131)ceres Seq. ID no. 4796910 24Met Val Gly Asp
Tyr Arg Glu Asn Tyr Ser Pro Ser Ser Asp Asp Ser1 5 10 15Ser Ser Val
Gly Glu Glu Thr Thr Ser Ser Met Tyr Ser Ala Arg Asn 20 25 30Glu Asp
Thr Pro Thr Glu Trp Thr Asp Glu Lys His Ser Leu Tyr Leu 35 40 45Lys
Ser Met Glu Ala Ser Phe Val Asp Gln Leu Tyr Asn Ser Leu Gly 50 55
60Ala Leu Gly Ser Lys Asn Asn Lys Asp Thr Val Gly Pro Ser Arg Arg65
70 75 80Phe Gly Asp Gly Gly Lys Pro Ser Glu Glu Gln Lys Met Asn Val
Arg 85 90 95Gln Pro Glu Tyr Arg Leu Asn Gly Arg His Gly Arg Arg Ser
His Glu 100 105 110Phe Leu Arg Ser Pro Trp Ile Lys His Tyr Lys Pro
Ser Pro Lys Ser 115 120 125Leu Thr Asp 13025315DNABrassica napus
25atgtattctg cgaggaatga agatacgcct acagaatgga ccgatgagaa gcatagtttg
60tatcttaaat caatggaagc ttccttcgtt gatcagctgt acaactccct cggtgcgctc
120ggctccaaaa acaacaagga tactgtcgga ccatcgagaa ggttcggtga
tggtggaaaa 180ccttctgaag aacagaagat gaatgtgagg cagcctgagt
atcgtctcaa tggaagacac 240ggtcgtcgct ctcacgagtt tcttaggagt
ccatggatca agcactataa gccttcacca 300aagtccctaa cagat
31526105PRTBrassica napuspeptide(1)..(105)ceres Seq. ID no. 4796911
26Met Tyr Ser Ala Arg Asn Glu Asp Thr Pro Thr Glu Trp Thr Asp Glu1
5 10 15Lys His Ser Leu Tyr Leu Lys Ser Met Glu Ala Ser Phe Val Asp
Gln 20 25 30Leu Tyr Asn Ser Leu Gly Ala Leu Gly Ser Lys Asn Asn Lys
Asp Thr 35 40 45Val Gly Pro Ser Arg Arg Phe Gly Asp Gly Gly Lys Pro
Ser Glu Glu 50 55 60Gln Lys Met Asn Val Arg Gln Pro Glu Tyr Arg Leu
Asn Gly Arg His65 70 75 80Gly Arg Arg Ser His Glu Phe Leu Arg Ser
Pro Trp Ile Lys His Tyr 85 90 95Lys Pro Ser Pro Lys Ser Leu Thr Asp
100 10527243DNABrassica napus 27atggaagctt ccttcgttga tcagctgtac
aactccctcg gtgcgctcgg ctccaaaaac 60aacaaggata ctgtcggacc atcgagaagg
ttcggtgatg gtggaaaacc ttctgaagaa 120cagaagatga atgtgaggca
gcctgagtat cgtctcaatg gaagacacgg tcgtcgctct 180cacgagtttc
ttaggagtcc atggatcaag cactataagc cttcaccaaa gtccctaaca 240gat
2432881PRTBrassica napuspeptide(1)..(81)ceres Seq. ID no. 4796912
28Met Glu Ala Ser Phe Val Asp Gln Leu Tyr Asn Ser Leu Gly Ala Leu1
5 10 15Gly Ser Lys Asn Asn Lys Asp Thr Val Gly Pro Ser Arg Arg Phe
Gly 20 25 30Asp Gly Gly Lys Pro Ser Glu Glu Gln Lys Met Asn Val Arg
Gln Pro 35 40 45Glu Tyr Arg Leu Asn Gly Arg His Gly Arg Arg Ser His
Glu Phe Leu 50 55 60Arg Ser Pro Trp Ile Lys His Tyr Lys Pro Ser Pro
Lys Ser Leu Thr65 70 75 80Asp291014DNAArabidopsis
thalianamisc_feature(1)..(1014)ceres Seq. ID no. 12321174
29ctctctctct taaagctctc ttctttggct ctttcgaaga agaaccattt ttatttccta
60agagagacga cggagttctt ttctaaagca ccggagagga ggagaagcaa cgatggagaa
120tgattgcacg gtgaatattg tctctctgga gaaggatcgc gatgtttcgg
aggcgtcggc 180tgaatctcag agcgagtcga ctctttcgaa ctcgctcgat
tccggtgtta cggctgagac 240ctctcgttct gatgctgatt ccaaactgga
tgaatgtact gcttggacga atgagaaaca 300caactcatat cttgattatt
tagagagctc gtttgttagg caattatact ccttgcttgg 360aggtgggact
cagagacttt ctagaactcg tgatgtgcag tctaactctc ataaatcagc
420tgatcagttt accgtcctac aaaatggttg ctggcagaag gttaactttg
gaaagaaaca 480atcttgtttg gagacttcat ctgagtttcg ttttcacaga
aattcattga gaaataagcc 540tgaaaattcc aacggaaatt acaccatggg
aactactgtc caaggagatg tgttatgtca 600tgacgaaacc aaacactcag
aggcgtcagg gcagaatttc agagaagaag aagaagaaga 660agagaaggga
gaggtgagca aaaaacgaga aagagaagca aataacgatg atagttcatt
720gaaggaggat caggttgtgc cggtaaggat ggtgaagccc agaacgtgaa
agcattagga 780agtgtagatg aaatactatg aatagagata aagaaataga
agaaggtgtg gttacgaatg 840tggagagggt tttgtttgtt gtatagcgtg
aggctaaaga gagccttcct tataaaggga 900tccaatggga tatggaaata
ggattggtgt ttgttttcgt taaattttgt ctaatgttaa 960ctaggggaaa
agttatctga tagtattagc atcttatggc aattttattc tttt
101430654DNAArabidopsis thaliana 30atggagaatg attgcacggt gaatattgtc
tctctggaga aggatcgcga tgtttcggag 60gcgtcggctg aatctcagag cgagtcgact
ctttcgaact cgctcgattc cggtgttacg 120gctgagacct ctcgttctga
tgctgattcc aaactggatg aatgtactgc ttggacgaat 180gagaaacaca
actcatatct tgattattta gagagctcgt ttgttaggca attatactcc
240ttgcttggag gtgggactca gagactttct agaactcgtg atgtgcagtc
taactctcat 300aaatcagctg atcagtttac cgtcctacaa aatggttgct
ggcagaaggt taactttgga 360aagaaacaat cttgtttgga gacttcatct
gagtttcgtt ttcacagaaa ttcattgaga 420aataagcctg aaaattccaa
cggaaattac accatgggaa ctactgtcca aggagatgtg 480ttatgtcatg
acgaaaccaa acactcagag gcgtcagggc agaatttcag agaagaagaa
540gaagaagaag agaagggaga ggtgagcaaa aaacgagaaa gagaagcaaa
taacgatgat 600agttcattga aggaggatca ggttgtgccg gtaaggatgg
tgaagcccag aacg 65431218PRTArabidopsis
thalianapeptide(1)..(218)ceres Seq. ID no. 12321175 31Met Glu Asn
Asp Cys Thr Val Asn Ile Val Ser Leu Glu Lys Asp Arg1 5 10 15Asp Val
Ser Glu Ala Ser Ala Glu Ser Gln Ser Glu Ser Thr Leu Ser 20 25 30Asn
Ser Leu Asp Ser Gly Val Thr Ala Glu Thr Ser Arg Ser Asp Ala 35 40
45Asp Ser Lys Leu Asp Glu Cys Thr Ala Trp Thr Asn Glu Lys His Asn
50 55 60Ser Tyr Leu Asp Tyr Leu Glu Ser Ser Phe Val Arg Gln Leu Tyr
Ser65 70 75 80Leu Leu Gly Gly Gly Thr Gln Arg Leu Ser Arg Thr Arg
Asp Val Gln 85 90 95Ser Asn Ser His Lys Ser Ala Asp Gln Phe Thr Val
Leu Gln Asn Gly 100 105 110Cys Trp Gln Lys Val Asn Phe Gly Lys Lys
Gln Ser Cys Leu Glu Thr 115 120 125Ser Ser Glu Phe Arg Phe His Arg
Asn Ser Leu Arg Asn Lys Pro Glu 130 135 140Asn Ser Asn Gly Asn Tyr
Thr Met Gly Thr Thr Val Gln Gly Asp Val145 150 155 160Leu Cys His
Asp Glu Thr Lys His Ser Glu Ala Ser Gly Gln Asn Phe 165 170 175Arg
Glu Glu Glu Glu Glu Glu Glu Lys Gly Glu Val Ser Lys Lys Arg 180 185
190Glu Arg Glu Ala Asn Asn Asp Asp Ser Ser Leu Lys Glu Asp Gln Val
195 200 205Val Pro Val Arg Met Val Lys Pro Arg Thr 210
215321027DNAArabidopsis thalianamisc_feature(1)..(1027)ceres Seq.
ID no. 12323601 32agatattttg tttctctctt tctctctgat atttttcatt
ttcttcttct tctctctctc 60tctccacaaa gataagccaa caatggttgg tgattacaga
ggacgcttta gtagccgtcg 120tttctccgac gactctgacg attcttccga
cgatgcttct tccgtggagg gagagaccac 180ttcttccatg tactctgcgg
ggaaagagta tatggaaaca gaatggacta atgagaagca 240tagtttatat
cttaaatcta tggaagcttc attcgtagat cagttatata actcgctcgg
300agctctcggg aagaacgaga atgtatccga atcaacgagg ttcggtagcg
gtagaaaacc 360gtctcaagaa cagttcaagg ttcttcatga tggtttctgg
cagaagatta atgtgaaaca 420acctgaacat cggattaacg gaaggcacgg
tggtaattct catgagtttc ttaggagtcc 480atggattaag cattataaac
ctttagtaaa gacacaaatc ccggtaacgg atgagcccga 540aaatcaagtt
gttagcagct ctaatgggaa gaagggaata tgcagctctg gctcagcctc
600tagtctcaag cagctaagct ctcattcgcg tgaccacgac caaatcagcg
ttggagaagc 660agaggtatcg gatcagaact ttgttaacga aggaataaaa
ggcgaaaacg gaagctcgaa 720gaagatgaag acggtgatga tgagtgaatc
gtcgagtacc gatcaggttg ttccactcaa 780taagctcttg caacatgacg
taaatttgaa gtctgtttct tgagaggtca gatggtgaag 840ctttatatga
ggagagaatt ttgtaatgta tatatatttg cataacttat aagtcaaatt
900tactatcctt agttacaagt ttcttcatca tatatcccta actataaata
tatttatatg 960ctcatgtgag tggattcatt tgtactgtaa aacccttaga
aagacgtcaa attagtattt 1020gatggtc 102733819DNAArabidopsis thaliana
33gatattttgt ttctctcttt ctctctgata tttttcattt tcttcttctt ctctctctct
60ctccacaaag ataagccaac aatggttggt gattacagag gacgctttag tagccgtcgt
120ttctccgacg actctgacga ttcttccgac gatgcttctt ccgtggaggg
agagaccact 180tcttccatgt actctgcggg gaaagagtat atggaaacag
aatggactaa tgagaagcat 240agtttatatc ttaaatctat ggaagcttca
ttcgtagatc agttatataa ctcgctcgga 300gctctcggga agaacgagaa
tgtatccgaa tcaacgaggt tcggtagcgg tagaaaaccg 360tctcaagaac
agttcaaggt tcttcatgat ggtttctggc agaagattaa tgtgaaacaa
420cctgaacatc ggattaacgg aaggcacggt ggtaattctc atgagtttct
taggagtcca 480tggattaagc attataaacc tttagtaaag acacaaatcc
cggtaacgga tgagcccgaa 540aatcaagttg ttagcagctc taatgggaag
aagggaatat gcagctctgg ctcagcctct 600agtctcaagc agctaagctc
tcattcgcgt gaccacgacc aaatcagcgt tggagaagca 660gaggtatcgg
atcagaactt tgttaacgaa ggaataaaag gcgaaaacgg aagctcgaag
720aagatgaaga cggtgatgat gagtgaatcg tcgagtaccg atcaggttgt
tccactcaat 780aagctcttgc aacatgacgt aaatttgaag tctgtttct
81934273PRTArabidopsis thalianapeptide(1)..(273)ceres Seq. ID no.
12323602 34Asp Ile Leu Phe Leu Ser Phe Ser Leu Ile Phe Phe Ile Phe
Phe Phe1 5 10 15Phe Ser Leu Ser Leu His Lys Asp Lys Pro Thr Met Val
Gly Asp Tyr 20 25 30Arg Gly Arg Phe Ser Ser Arg Arg Phe Ser Asp Asp
Ser Asp Asp Ser 35 40 45Ser Asp Asp Ala Ser Ser Val Glu Gly Glu Thr
Thr Ser Ser Met Tyr 50 55 60Ser Ala Gly Lys Glu Tyr Met Glu Thr Glu
Trp Thr Asn Glu Lys His65 70 75 80Ser Leu Tyr Leu Lys Ser Met Glu
Ala Ser Phe Val Asp Gln Leu Tyr 85 90 95Asn Ser Leu Gly Ala Leu Gly
Lys Asn Glu Asn Val Ser Glu Ser Thr 100 105 110Arg Phe Gly Ser Gly
Arg Lys Pro Ser Gln Glu Gln Phe Lys Val Leu 115 120 125His Asp Gly
Phe Trp Gln Lys Ile Asn Val Lys Gln Pro Glu His Arg 130 135 140Ile
Asn Gly Arg His Gly Gly Asn Ser His Glu Phe Leu Arg Ser Pro145 150
155 160Trp Ile Lys His Tyr Lys Pro Leu Val Lys Thr Gln Ile Pro Val
Thr 165 170 175Asp Glu Pro Glu Asn Gln Val Val Ser Ser Ser Asn Gly
Lys Lys Gly 180 185 190Ile Cys Ser Ser Gly Ser Ala Ser Ser Leu Lys
Gln Leu Ser Ser His 195 200 205Ser Arg Asp His Asp Gln Ile Ser Val
Gly Glu Ala Glu Val Ser Asp 210 215 220Gln Asn Phe Val Asn Glu Gly
Ile Lys Gly Glu Asn Gly Ser Ser Lys225 230 235 240Lys Met Lys Thr
Val Met Met Ser Glu Ser Ser Ser Thr Asp Gln Val 245 250 255Val Pro
Leu Asn Lys Leu Leu Gln His Asp Val Asn Leu Lys Ser Val 260 265
270Ser35738DNAArabidopsis thaliana 35atggttggtg attacagagg
acgctttagt agccgtcgtt tctccgacga ctctgacgat 60tcttccgacg atgcttcttc
cgtggaggga gagaccactt cttccatgta ctctgcgggg 120aaagagtata
tggaaacaga atggactaat gagaagcata gtttatatct taaatctatg
180gaagcttcat tcgtagatca gttatataac tcgctcggag ctctcgggaa
gaacgagaat 240gtatccgaat caacgaggtt cggtagcggt agaaaaccgt
ctcaagaaca gttcaaggtt 300cttcatgatg gtttctggca gaagattaat
gtgaaacaac ctgaacatcg gattaacgga 360aggcacggtg gtaattctca
tgagtttctt aggagtccat ggattaagca ttataaacct 420ttagtaaaga
cacaaatccc ggtaacggat gagcccgaaa atcaagttgt tagcagctct
480aatgggaaga agggaatatg cagctctggc tcagcctcta gtctcaagca
gctaagctct 540cattcgcgtg accacgacca aatcagcgtt ggagaagcag
aggtatcgga tcagaacttt 600gttaacgaag gaataaaagg cgaaaacgga
agctcgaaga agatgaagac ggtgatgatg 660agtgaatcgt cgagtaccga
tcaggttgtt ccactcaata agctcttgca acatgacgta 720aatttgaagt ctgtttct
73836246PRTArabidopsis thalianapeptide(1)..(246)ceres Seq. ID no.
12323603 36Met Val Gly Asp Tyr Arg Gly Arg Phe Ser Ser Arg Arg Phe
Ser Asp1 5 10 15Asp Ser Asp Asp Ser Ser Asp Asp Ala Ser Ser Val Glu
Gly Glu Thr 20 25 30Thr Ser Ser Met Tyr Ser Ala Gly Lys Glu Tyr Met
Glu Thr Glu Trp 35 40 45Thr Asn Glu Lys His Ser Leu Tyr Leu Lys Ser
Met Glu Ala Ser Phe 50 55 60Val Asp Gln Leu Tyr Asn Ser Leu Gly Ala
Leu Gly Lys Asn Glu Asn65 70 75 80Val Ser Glu Ser Thr Arg Phe Gly
Ser Gly Arg Lys Pro Ser Gln Glu 85 90 95Gln Phe Lys Val Leu His Asp
Gly Phe Trp Gln Lys Ile Asn Val Lys 100 105 110Gln Pro Glu His Arg
Ile Asn Gly Arg His Gly Gly Asn Ser His Glu 115 120 125Phe Leu Arg
Ser Pro Trp Ile Lys His Tyr Lys Pro Leu Val Lys Thr 130 135 140Gln
Ile Pro Val Thr Asp Glu Pro Glu Asn Gln Val Val Ser Ser Ser145 150
155 160Asn Gly Lys Lys Gly Ile Cys Ser Ser Gly Ser Ala Ser Ser Leu
Lys 165 170 175Gln Leu Ser Ser His Ser Arg Asp His Asp Gln Ile Ser
Val Gly Glu 180 185 190Ala Glu Val Ser Asp Gln Asn Phe Val Asn Glu
Gly Ile Lys Gly Glu 195 200 205Asn Gly Ser Ser Lys Lys Met Lys Thr
Val Met Met Ser Glu Ser Ser 210 215 220Ser Thr Asp Gln Val Val Pro
Leu Asn Lys Leu Leu Gln His Asp Val225 230 235 240Asn Leu Lys Ser
Val Ser 24537633DNAArabidopsis thaliana 37atgtactctg cggggaaaga
gtatatggaa acagaatgga ctaatgagaa gcatagttta 60tatcttaaat ctatggaagc
ttcattcgta gatcagttat ataactcgct cggagctctc 120gggaagaacg
agaatgtatc cgaatcaacg aggttcggta gcggtagaaa accgtctcaa
180gaacagttca aggttcttca tgatggtttc tggcagaaga ttaatgtgaa
acaacctgaa 240catcggatta acggaaggca cggtggtaat tctcatgagt
ttcttaggag tccatggatt 300aagcattata aacctttagt aaagacacaa
atcccggtaa cggatgagcc cgaaaatcaa 360gttgttagca gctctaatgg
gaagaaggga atatgcagct ctggctcagc ctctagtctc 420aagcagctaa
gctctcattc gcgtgaccac gaccaaatca gcgttggaga agcagaggta
480tcggatcaga actttgttaa cgaaggaata aaaggcgaaa acggaagctc
gaagaagatg 540aagacggtga tgatgagtga atcgtcgagt accgatcagg
ttgttccact caataagctc 600ttgcaacatg acgtaaattt gaagtctgtt tct
63338211PRTArabidopsis thalianapeptide(1)..(211)ceres Seq. ID no.
12323604 38Met Tyr Ser Ala Gly Lys Glu Tyr Met Glu Thr Glu Trp Thr
Asn Glu1 5 10 15Lys His Ser Leu Tyr Leu Lys Ser Met Glu Ala Ser Phe
Val Asp Gln 20 25 30Leu Tyr Asn Ser Leu Gly Ala Leu Gly Lys Asn Glu
Asn Val Ser Glu
35 40 45Ser Thr Arg Phe Gly Ser Gly Arg Lys Pro Ser Gln Glu Gln Phe
Lys 50 55 60Val Leu His Asp Gly Phe Trp Gln Lys Ile Asn Val Lys Gln
Pro Glu65 70 75 80His Arg Ile Asn Gly Arg His Gly Gly Asn Ser His
Glu Phe Leu Arg 85 90 95Ser Pro Trp Ile Lys His Tyr Lys Pro Leu Val
Lys Thr Gln Ile Pro 100 105 110Val Thr Asp Glu Pro Glu Asn Gln Val
Val Ser Ser Ser Asn Gly Lys 115 120 125Lys Gly Ile Cys Ser Ser Gly
Ser Ala Ser Ser Leu Lys Gln Leu Ser 130 135 140Ser His Ser Arg Asp
His Asp Gln Ile Ser Val Gly Glu Ala Glu Val145 150 155 160Ser Asp
Gln Asn Phe Val Asn Glu Gly Ile Lys Gly Glu Asn Gly Ser 165 170
175Ser Lys Lys Met Lys Thr Val Met Met Ser Glu Ser Ser Ser Thr Asp
180 185 190Gln Val Val Pro Leu Asn Lys Leu Leu Gln His Asp Val Asn
Leu Lys 195 200 205Ser Val Ser 21039960DNAArabidopsis
thalianamisc_feature(1)..(960)ceres Seq. ID no. 13491409
39atttttgttt ctctctttct ctctgatatt tttcattttc ttcttcttct ctctctctct
60ccacaaagat aagccaacaa tggttggtga ttacagagga cgctttagta gccgtcgttt
120ctccgatgac tctgacgatt cttccgacga tgcttcttcc gtggagggag
agaccacttc 180ttccatgtac tctgcgggga aagagtatat ggaaacagaa
tggactaatg agaagcatag 240tttatatctt aaatctatgg aagcttcatt
cgtagatcag ttatataact cgctcggagc 300tctcgggaag aacgagaatg
tatccgaatc aacgaggttc ggtagcggta gaaaaccgtc 360tcaagaacag
ttcaaggttc ttcatgatgg tttctggcag aagattaatg tgaaacaacc
420tgaacatcgg attaacggaa ggcacggtgg taattctcat gagtttctta
ggagtccatg 480gattaagcat tataaacctt tagtaaagac acaaatcccg
gtaacggatg agcccgaaaa 540tcaagttgtt agcagctcta atgggaagaa
gggaatatgc agctctggct cagcctctag 600tctcaagcag ctaagctctc
attcgcgtga ccacgaccaa atcagcgttg gagaagcaga 660ggtatcggat
cagaactttg ttaacgaagg aataaaaggc gaaaacggaa gctcgaagaa
720gatgaagacg gtgatgatga gtgaatcgtc gagtaccgat caggttgttc
cactcaataa 780actcttgcaa catgacgtaa atttgaagtc tgtttcttga
gaggtcagat ggtgaagctt 840tatatgagga gagaattttg taatgtatat
atatttgcat aacttataag tcaaatttac 900tatccttagt tacaagtttc
ttcatcatat atccctaact ataaatatat ttatatgccc 96040816DNAArabidopsis
thaliana 40tttttgtttc tctctttctc tctgatattt ttcattttct tcttcttctc
tctctctctc 60cacaaagata agccaacaat ggttggtgat tacagaggac gctttagtag
ccgtcgtttc 120tccgatgact ctgacgattc ttccgacgat gcttcttccg
tggagggaga gaccacttct 180tccatgtact ctgcggggaa agagtatatg
gaaacagaat ggactaatga gaagcatagt 240ttatatctta aatctatgga
agcttcattc gtagatcagt tatataactc gctcggagct 300ctcgggaaga
acgagaatgt atccgaatca acgaggttcg gtagcggtag aaaaccgtct
360caagaacagt tcaaggttct tcatgatggt ttctggcaga agattaatgt
gaaacaacct 420gaacatcgga ttaacggaag gcacggtggt aattctcatg
agtttcttag gagtccatgg 480attaagcatt ataaaccttt agtaaagaca
caaatcccgg taacggatga gcccgaaaat 540caagttgtta gcagctctaa
tgggaagaag ggaatatgca gctctggctc agcctctagt 600ctcaagcagc
taagctctca ttcgcgtgac cacgaccaaa tcagcgttgg agaagcagag
660gtatcggatc agaactttgt taacgaagga ataaaaggcg aaaacggaag
ctcgaagaag 720atgaagacgg tgatgatgag tgaatcgtcg agtaccgatc
aggttgttcc actcaataaa 780ctcttgcaac atgacgtaaa tttgaagtct gtttct
81641272PRTArabidopsis thalianapeptide(1)..(272)ceres Seq. ID no.
13491410 41Phe Leu Phe Leu Ser Phe Ser Leu Ile Phe Phe Ile Phe Phe
Phe Phe1 5 10 15Ser Leu Ser Leu His Lys Asp Lys Pro Thr Met Val Gly
Asp Tyr Arg 20 25 30Gly Arg Phe Ser Ser Arg Arg Phe Ser Asp Asp Ser
Asp Asp Ser Ser 35 40 45Asp Asp Ala Ser Ser Val Glu Gly Glu Thr Thr
Ser Ser Met Tyr Ser 50 55 60Ala Gly Lys Glu Tyr Met Glu Thr Glu Trp
Thr Asn Glu Lys His Ser65 70 75 80Leu Tyr Leu Lys Ser Met Glu Ala
Ser Phe Val Asp Gln Leu Tyr Asn 85 90 95Ser Leu Gly Ala Leu Gly Lys
Asn Glu Asn Val Ser Glu Ser Thr Arg 100 105 110Phe Gly Ser Gly Arg
Lys Pro Ser Gln Glu Gln Phe Lys Val Leu His 115 120 125Asp Gly Phe
Trp Gln Lys Ile Asn Val Lys Gln Pro Glu His Arg Ile 130 135 140Asn
Gly Arg His Gly Gly Asn Ser His Glu Phe Leu Arg Ser Pro Trp145 150
155 160Ile Lys His Tyr Lys Pro Leu Val Lys Thr Gln Ile Pro Val Thr
Asp 165 170 175Glu Pro Glu Asn Gln Val Val Ser Ser Ser Asn Gly Lys
Lys Gly Ile 180 185 190Cys Ser Ser Gly Ser Ala Ser Ser Leu Lys Gln
Leu Ser Ser His Ser 195 200 205Arg Asp His Asp Gln Ile Ser Val Gly
Glu Ala Glu Val Ser Asp Gln 210 215 220Asn Phe Val Asn Glu Gly Ile
Lys Gly Glu Asn Gly Ser Ser Lys Lys225 230 235 240Met Lys Thr Val
Met Met Ser Glu Ser Ser Ser Thr Asp Gln Val Val 245 250 255Pro Leu
Asn Lys Leu Leu Gln His Asp Val Asn Leu Lys Ser Val Ser 260 265
27042738DNAArabidopsis thaliana 42atggttggtg attacagagg acgctttagt
agccgtcgtt tctccgatga ctctgacgat 60tcttccgacg atgcttcttc cgtggaggga
gagaccactt cttccatgta ctctgcgggg 120aaagagtata tggaaacaga
atggactaat gagaagcata gtttatatct taaatctatg 180gaagcttcat
tcgtagatca gttatataac tcgctcggag ctctcgggaa gaacgagaat
240gtatccgaat caacgaggtt cggtagcggt agaaaaccgt ctcaagaaca
gttcaaggtt 300cttcatgatg gtttctggca gaagattaat gtgaaacaac
ctgaacatcg gattaacgga 360aggcacggtg gtaattctca tgagtttctt
aggagtccat ggattaagca ttataaacct 420ttagtaaaga cacaaatccc
ggtaacggat gagcccgaaa atcaagttgt tagcagctct 480aatgggaaga
agggaatatg cagctctggc tcagcctcta gtctcaagca gctaagctct
540cattcgcgtg accacgacca aatcagcgtt ggagaagcag aggtatcgga
tcagaacttt 600gttaacgaag gaataaaagg cgaaaacgga agctcgaaga
agatgaagac ggtgatgatg 660agtgaatcgt cgagtaccga tcaggttgtt
ccactcaata aactcttgca acatgacgta 720aatttgaagt ctgtttct
73843246PRTArabidopsis thalianapeptide(1)..(246)ceres Seq. ID no.
13491411 43Met Val Gly Asp Tyr Arg Gly Arg Phe Ser Ser Arg Arg Phe
Ser Asp1 5 10 15Asp Ser Asp Asp Ser Ser Asp Asp Ala Ser Ser Val Glu
Gly Glu Thr 20 25 30Thr Ser Ser Met Tyr Ser Ala Gly Lys Glu Tyr Met
Glu Thr Glu Trp 35 40 45Thr Asn Glu Lys His Ser Leu Tyr Leu Lys Ser
Met Glu Ala Ser Phe 50 55 60Val Asp Gln Leu Tyr Asn Ser Leu Gly Ala
Leu Gly Lys Asn Glu Asn65 70 75 80Val Ser Glu Ser Thr Arg Phe Gly
Ser Gly Arg Lys Pro Ser Gln Glu 85 90 95Gln Phe Lys Val Leu His Asp
Gly Phe Trp Gln Lys Ile Asn Val Lys 100 105 110Gln Pro Glu His Arg
Ile Asn Gly Arg His Gly Gly Asn Ser His Glu 115 120 125Phe Leu Arg
Ser Pro Trp Ile Lys His Tyr Lys Pro Leu Val Lys Thr 130 135 140Gln
Ile Pro Val Thr Asp Glu Pro Glu Asn Gln Val Val Ser Ser Ser145 150
155 160Asn Gly Lys Lys Gly Ile Cys Ser Ser Gly Ser Ala Ser Ser Leu
Lys 165 170 175Gln Leu Ser Ser His Ser Arg Asp His Asp Gln Ile Ser
Val Gly Glu 180 185 190Ala Glu Val Ser Asp Gln Asn Phe Val Asn Glu
Gly Ile Lys Gly Glu 195 200 205Asn Gly Ser Ser Lys Lys Met Lys Thr
Val Met Met Ser Glu Ser Ser 210 215 220Ser Thr Asp Gln Val Val Pro
Leu Asn Lys Leu Leu Gln His Asp Val225 230 235 240Asn Leu Lys Ser
Val Ser 24544633DNAArabidopsis thaliana 44atgtactctg cggggaaaga
gtatatggaa acagaatgga ctaatgagaa gcatagttta 60tatcttaaat ctatggaagc
ttcattcgta gatcagttat ataactcgct cggagctctc 120gggaagaacg
agaatgtatc cgaatcaacg aggttcggta gcggtagaaa accgtctcaa
180gaacagttca aggttcttca tgatggtttc tggcagaaga ttaatgtgaa
acaacctgaa 240catcggatta acggaaggca cggtggtaat tctcatgagt
ttcttaggag tccatggatt 300aagcattata aacctttagt aaagacacaa
atcccggtaa cggatgagcc cgaaaatcaa 360gttgttagca gctctaatgg
gaagaaggga atatgcagct ctggctcagc ctctagtctc 420aagcagctaa
gctctcattc gcgtgaccac gaccaaatca gcgttggaga agcagaggta
480tcggatcaga actttgttaa cgaaggaata aaaggcgaaa acggaagctc
gaagaagatg 540aagacggtga tgatgagtga atcgtcgagt accgatcagg
ttgttccact caataaactc 600ttgcaacatg acgtaaattt gaagtctgtt tct
63345211PRTArabidopsis thalianapeptide(1)..(211)ceres Seq. ID no.
13491412 45Met Tyr Ser Ala Gly Lys Glu Tyr Met Glu Thr Glu Trp Thr
Asn Glu1 5 10 15Lys His Ser Leu Tyr Leu Lys Ser Met Glu Ala Ser Phe
Val Asp Gln 20 25 30Leu Tyr Asn Ser Leu Gly Ala Leu Gly Lys Asn Glu
Asn Val Ser Glu 35 40 45Ser Thr Arg Phe Gly Ser Gly Arg Lys Pro Ser
Gln Glu Gln Phe Lys 50 55 60Val Leu His Asp Gly Phe Trp Gln Lys Ile
Asn Val Lys Gln Pro Glu65 70 75 80His Arg Ile Asn Gly Arg His Gly
Gly Asn Ser His Glu Phe Leu Arg 85 90 95Ser Pro Trp Ile Lys His Tyr
Lys Pro Leu Val Lys Thr Gln Ile Pro 100 105 110Val Thr Asp Glu Pro
Glu Asn Gln Val Val Ser Ser Ser Asn Gly Lys 115 120 125Lys Gly Ile
Cys Ser Ser Gly Ser Ala Ser Ser Leu Lys Gln Leu Ser 130 135 140Ser
His Ser Arg Asp His Asp Gln Ile Ser Val Gly Glu Ala Glu Val145 150
155 160Ser Asp Gln Asn Phe Val Asn Glu Gly Ile Lys Gly Glu Asn Gly
Ser 165 170 175Ser Lys Lys Met Lys Thr Val Met Met Ser Glu Ser Ser
Ser Thr Asp 180 185 190Gln Val Val Pro Leu Asn Lys Leu Leu Gln His
Asp Val Asn Leu Lys 195 200 205Ser Val Ser 210461031DNAArtificial
Sequenceclone nucleotide 486033 46agttcgcttt ggcctccgct tgccccctcc
ctctcgcgtc tctatacatc gccgctgttg 60tgttgcagtt cagtttgcat cctgagctct
ctcctggacc agccgagatt tctctctctg 120cgcatctcta attcatcttc
gtcgagagga gctgttcctc ttctttgccg cctcgaatct 180gggactggtc
ggttttctgg atccctgctg cctgtcgggt tctcgagagg tgtaaaatcc
240aatggagggt gtgtcatcgt tgaaccagcc gttgatcaac gacgaccggc
agcccgtgcc 300cagcagtatc gccaagggtg atcaaatcca aggcctgttg
tcgggtgaat ggacaaatga 360gcggcacagc tcgtacataa gctccatgga
ggcatctttc gtggagcaac tccgtagtgg 420ttccaaggcc atccaggagg
gcttgtgcca gagcatgagg attccgaggg atgatgctcg 480cagccatgac
gtccctgaga gtccgtgggt ggtggtgagg cgtttcaggc cacgcggtgt
540ccaccatggc gatggaatgg aagtggaacc tttggtcgat ggttatggat
caggtactga 600cacggcccng agagaaggtc cggacccacg caagatagcg
aaggcttctg ctattattga 660agtcacggac cagaattttc ctgaggaggg
gattcaatcc agtaacggtg catgcaagag 720acagaaatct actcctggca
atgcatcaaa tggccagggt acttaacaag atagtggaag 780ccaagccatg
ccctctctga agccttcagg aggccatggg ggaaacgaga cttgtctgca
840gtactacgtg atgacaggtc gtgctgcagc tgcaagtagt ttggcttacc
aaaatatgat 900atcgtcgtcc tttctgcggt gtggagagta gaatatgcat
atccacatct gcagagagca 960ccggttctct tcttcttgtt gctgttacta
ttttgtgcca tggagcaaat ttatttggta 1020aatttgagct g
103147174PRTArtificial Sequenceclone peptide 486033 47Met Glu Gly
Val Ser Ser Leu Asn Gln Pro Leu Ile Asn Asp Asp Arg1 5 10 15Gln Pro
Val Pro Ser Ser Ile Ala Lys Gly Asp Gln Ile Gln Gly Leu 20 25 30Leu
Ser Gly Glu Trp Thr Asn Glu Arg His Ser Ser Tyr Ile Ser Ser 35 40
45Met Glu Ala Ser Phe Val Glu Gln Leu Arg Ser Gly Ser Lys Ala Ile
50 55 60Gln Glu Gly Leu Cys Gln Ser Met Arg Ile Pro Arg Asp Asp Ala
Arg65 70 75 80Ser His Asp Val Pro Glu Ser Pro Trp Val Val Val Arg
Arg Phe Arg 85 90 95Pro Arg Gly Val His His Gly Asp Gly Met Glu Val
Glu Pro Leu Val 100 105 110Asp Gly Tyr Gly Ser Gly Thr Asp Thr Ala
Xaa Arg Glu Gly Pro Asp 115 120 125Pro Arg Lys Ile Ala Lys Ala Ser
Ala Ile Ile Glu Val Thr Asp Gln 130 135 140Asn Phe Pro Glu Glu Gly
Ile Gln Ser Ser Asn Gly Ala Cys Lys Arg145 150 155 160Gln Lys Ser
Thr Pro Gly Asn Ala Ser Asn Gly Gln Gly Thr 165
17048210PRTArtificial SequenceConsensus sequence derived from
various organisms 48Val Xaa Xaa Glu Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa
Gly Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Thr Xaa Glu
Xaa His Xaa Xaa Tyr 20 25 30Xaa Xaa Ser Met Glu Ala Ser Phe Val Xaa
Gln Leu Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Lys Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Trp Xaa Xaa 100 105 110Xaa Xaa Xaa
Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp 115 120 125Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135
140Xaa Xaa Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa145 150 155 160Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 165 170 175Xaa Xaa Xaa Xaa Glu Val Xaa Asp Gln Asn
Phe Xaa Xaa Xaa Xaa Xaa 180 185 190Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys
Xaa Xaa Lys Xaa Xaa Xaa Xaa Ser 195 200 205Xaa Xaa
21049241PRTArtificial SequenceConsensus sequence derived from
various organisms 49Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Trp 20 25 30Thr Xaa Glu Xaa His Xaa Xaa Tyr Xaa Xaa
Xaa Xaa Glu Xaa Ser Phe 35 40 45Val Xaa Gln Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135
140Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa145 150 155 160Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 165 170 175Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 180 185 190Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215 220Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Glu Xaa Xaa Xaa Gln Asn Phe Xaa Xaa225 230 235
240Xaa5019DNAArtificial SequenceOligo primer used in the generation
of labeled probes for hybridization from first-strand cDNA
50tttttttttt ttttttttv 19
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