U.S. patent application number 12/139269 was filed with the patent office on 2009-05-21 for nucleotide sequences and polypeptides encoded thereby useful for modifying plant characteristics and phenotypes.
This patent application is currently assigned to CERES, INC.. Invention is credited to Nickolai Alexandrov, Vyacheslav Brover, Ken Feldmann, Peter Mascia.
Application Number | 20090133156 12/139269 |
Document ID | / |
Family ID | 35431869 |
Filed Date | 2009-05-21 |
United States Patent
Application |
20090133156 |
Kind Code |
A1 |
Alexandrov; Nickolai ; et
al. |
May 21, 2009 |
NUCLEOTIDE SEQUENCES AND POLYPEPTIDES ENCODED THEREBY USEFUL FOR
MODIFYING PLANT CHARACTERISTICS AND PHENOTYPES
Abstract
Isolated polynucleotides and polypeptides encoded thereby are
described, together with the use of those products for making
transgenic plants.
Inventors: |
Alexandrov; Nickolai;
(Thousand Oaks, CA) ; Brover; Vyacheslav; (Simi
Valley, CA) ; Mascia; Peter; (Thousand Oaks, CA)
; Feldmann; Ken; (Newbury Park, CA) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
CERES, INC.
Thousand Oaks
CA
|
Family ID: |
35431869 |
Appl. No.: |
12/139269 |
Filed: |
June 13, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11172740 |
Jun 30, 2005 |
7396979 |
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12139269 |
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60583621 |
Jun 30, 2004 |
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60584829 |
Jun 30, 2004 |
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60584800 |
Jun 30, 2004 |
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Current U.S.
Class: |
800/278 ;
435/320.1; 435/419; 435/468; 435/6.15; 530/370; 536/23.6;
800/298 |
Current CPC
Class: |
C12N 15/8261 20130101;
C07K 14/415 20130101; C12N 15/8291 20130101; C12N 15/827 20130101;
Y02A 40/146 20180101 |
Class at
Publication: |
800/278 ;
800/298; 536/23.6; 435/320.1; 435/419; 530/370; 435/468; 435/6 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C12N 15/11 20060101 C12N015/11; C12N 15/29 20060101
C12N015/29; C12N 15/82 20060101 C12N015/82; C12N 5/10 20060101
C12N005/10; C07K 14/415 20060101 C07K014/415; C12Q 1/68 20060101
C12Q001/68 |
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
in the Sequence Listing or in the ortholog alignments of FIG. 1; 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 hybridizing 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 nucleotide sequence according to any polynucleotide
sequence in the Sequence Listing.
3. The isolated nucleic acid molecule according to claim 1, wherein
said amino acid sequence comprises any polypeptide sequence in the
Sequence Listing or in the ortholog alignments of FIG. 1.
4. 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.
5. The vector construct according to claim 4, wherein said first
nucleic acid is native to said second nucleic acid.
6. The vector construct according to claim 4, wherein said first
nucleic acid is heterologous to said second nucleic acid.
7. A host cell comprising an isolated nucleic acid molecule
according to claim 1 wherein said nucleic acid molecule is flanked
by exogenous sequence.
8. A host cell comprising a vector construct according to claim
4.
9. An isolated polypeptide comprising an amino acid sequence
exhibiting at least 85% sequence identity of an amino acid sequence
of the Sequence Listing or in the ortholog alignments of FIG.
1.
10. 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.
11. A method of transforming a host cell which comprises contacting
a host cell with a vector construct according to claim 4.
12. 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.
13. A host cell or organism which comprises a nucleic acid molecule
according to claim 1 which is exogenous or heterologous to said
plant or plant cell.
14. A host cell or organism according to claim 13, which is a
plant, plant cell, plant material or seed of a plant.
15. A plant which has been regenerated from a plant cell or seed
according to claim 14.
16. A plant, plant cell, plant material or seed of a plant which
comprises a nucleic acid molecule according to claim 1, wherein
said plant has improved characteristics as compared to a wild-type
plant cultivated under the same conditions.
17. The plant, plant cell, plant material or seed of a plant
according to claim 16, wherein the improved characteristic is the
one associated with and described in the miscellaneous feature
section of the sequence listing for the particular sequence.
18. A method for improving plant characteristics in a plant
comprising transforming a plant with a nucleic acid sequence
according to claim 1.
19. A transgenic plant having a gene construct comprising a nucleic
acid according to claim 1 encoding a component operably linked to a
plant promoter so that the component is ectopically overexpressed
in the transgenic plant, and the transgenic plant exhibits: i)
faster rate of growth, ii) greater fresh or dry weight at
maturation, iii) greater fruit or seed yield, iv) higher tolerance
to pH, v) higher tolerance to low phosphate concentration, or vi)
higher tolerance to low nitrogen concentration than a progenitor
plant which does not contain the polynucleotide construct, when the
transgenic plant and the progenitor plant are cultivated under
identical environmental conditions, wherein the component is any
one of the polypeptides set forth in the Sequence Listing or in the
ortholog alignment of FIG. 1.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This is a Divisional Application of U.S. Nonprovisional
application Ser. No. 11/272,740 filed on Jun. 30, 2005 which claims
priority under 35 U.S.C. .sctn. 119(e) on U.S. Provisional
Application No(s). 60/583,621; 60/584,829 and 60/584,800 filed on
Jun. 30, 2004, the entire contents of which are hereby incorporated
by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to isolated polynucleotides,
polypeptides encoded thereby, and the use of those products for
making transgenic plants or organisms, such as transgenic
plants.
BACKGROUND OF THE INVENTION
[0003] 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.
[0004] 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.
[0005] 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.
[0006] Plants specifically improved for agriculture, horticulture
and other industries can be obtained using molecular technologies.
As an example, great agronomic value can result from modulating the
size of a plant as a whole or of any of its organs. 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.
[0007] Similarly, modulation of the size and stature of an entire
plant, or a particular portion of a plant, allows production of
plants better suited for a particular industry. For example,
reductions in the height of specific ornamentals, crops and tree
species can be beneficial by allowing easier harvesting.
Alternatively, increasing height may be beneficial by providing
more biomass. Other examples of commercially desirable traits
include increasing the length of the floral stems of cut flowers,
increasing or altering leaf size and shape, enhancing the size of
seeds and/or fruits, enhancing yields by specifically stimulating
hormone (e.g. Brassinolide) synthesis and stimulating early
flowering or evoking late flowering by altering levels of
gibberellic acid or other hormones in specific cells. Changes in
organ size and biomass also result in changes in the mass of
constituent molecules such as secondary products.
[0008] To summarize, molecular genetic technologies provide the
ability to modulate and manipulate growth, development and
biochemistry of the entire plant as well as at the cell, tissue and
organ levels. Thus, plant morphology, development and biochemistry
are altered to maximize or minimize the desired plant trait.
SUMMARY OF THE INVENTION
[0009] The present invention, therefore, relates to isolated
polynucleotides, polypeptides encoded thereby, and the use of those
products for making transgenic organisms, such as plants, bacteria,
yeast, fungi and mammals, depending upon the desired
characteristics.
[0010] In the field of agriculture and forestry efforts are
constantly being made to produce plants with improved
characteristics, such as increased overall yield or increased yield
of biomass or chemical components, 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, people try to obtain plants with an
increased yield by breeding, but this is time-consuming and
labor-intensive. Furthermore, appropriate breeding programs must be
performed for each relevant plant species.
[0011] Recently, progress has 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 not usually being limited to one plant species, but
being transferable to other plant species as well. EP-A 0 511 979,
for example, discloses that the expression of a prokaryotic
asparagine synthetase in plant cells inter alia leads to an
increase in biomass production. Similarly, WO 96/21737 describes
the production of plants with increased yield from the expression
of deregulated or unregulated fructose-1,6-bisphosphatase due to an
increased rate of the photosynthesis. Nevertheless, there still is
a need for generally applicable processes that improve yield in
plants interesting for agriculture or forestry purposes.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1 shows alignments of the polypeptide sequences of the
invention with other sequences, showing conserved regions of
identical or similar residues.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0013] The following terms are utilized throughout this
application:
Chimeric: The term "chimeric" is used to describe genes, as defined
supra, or contructs 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. 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). Domains also
define areas of non-coding sequences such as promoters and miRNAs.
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. Misexpression: The term "misexpression" refers to an increase
or a decrease in the transcription of a coding region into a
complementary RNA sequence as compared to the parental wild-type.
This term also encompasses expression of a gene or coding region
for a different time period as compared to the wild-type and/or
from a non-natural location within the plant genome. 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. Regulatory Sequence The term "regulatory sequence,"
as used in the current invention, refers to any nucleotide sequence
that influences transcription or translation initiation and rate,
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. 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-11.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 in polynucleotide hybridization reactions can
be adjusted to favor hybridization of polynucleotides from
identical genes or related family members.
[0014] 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.
[0015] Stringency conditions can be selected 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
[0016] The genes and polynucleotides of the present invention are
of interest because when they are misexpressed (i.e. when expressed
at a non-natural location or in an increased amount) they produce
plants with important modified characteristics as discussed below.
These traits can be used to exploit or maximize plant products or
to minimize undesirable characteristics. 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, shape and size of
leaves can lead to increased production/harvest from leaf crops
such as lettuce, spinach, cabbage and tobacco. Likewise, a decrease
in plant height is beneficial in species that are particularly
susceptible to lodging or uprooting due to wind stress.
[0017] The polynucleotides and polypeptides of the invention were
isolated from Arabidopsis thaliana, corn, soybean, wheat, Brassica
and others as noted in the Sequence Listing. The polynucleotides
and polypeptides are useful to confer on transgenic plants the
properties identified for each sequence in the relevant portion
(miscellaneous feature section) of the Sequence Listing. The
miscellaneous feature section of the sequence listing contains, for
each sequence, a description of the domain or other characteristic
from which the sequence has the function known in the art for other
sequences. Some identified domains are indicated with "PFam Name",
signifying that the pfam name and description can be found in the
pfam database at http://pfam.wustl.edu. Other domains are indicated
by reference to a "GI Number" from the public sequence database
maintained by GenBank under the NCBI, including the non-redundant
(NR) database.
[0018] The sequences of the invention can be applied to substrates
for use in array applications such as, but not limited to, assays
of global gene expression, under varying conditions of development,
and growth conditions. The arrays are also used in diagnostic or
forensic methods
[0019] The polynucleotides, or fragments thereof, can also be used
as probes and primers. Probe length varies depending on the
application. For use as primers, probes are 12-40 nucleotides,
preferably 18-30 nucleotides long. For use in mapping, probes are
preferably 50 to 500 nucleotides, preferably 100-250 nucleotides
long. For Southern hybridizations, probes as long as several
kilobases are used.
[0020] The probes and/or primers are produced by synthetic
procedures such as the triester method of Matteucci et al. J. Am.
Chem. Soc. 103:3185 (1981) or according to Urdea et al. Proc. Natl.
Acad. 80:7461 (1981) or using commercially available automated
oligonucleotide synthesizers.
[0021] The polynucleotides of the invention can be utilized in a
number of methods known to those skilled in the art as probes
and/or primers to isolate and detect polynucleotides including,
without limitation: Southerns, Northerns, Branched DNA
hybridization assays, polymerase chain reaction microarray assays
and variations thereof. Specific methods given by way of examples,
and discussed below include:
[0022] hybridization
[0023] Methods of Mapping
[0024] Southern Blotting
[0025] Isolating cDNA from Related Organisms
[0026] Isolating and/or Identifying Homologous and Orthologous
Genes.
Also, the nucleic acid molecules of the invention can be used in
other methods, such as high density oligonucleotide hybridizing
assays, described, for example, in U.S. Pat. Nos. 6,004,753 and
5,945,306.
[0027] The polynucleotides or fragments thereof of the present
invention can be used as probes and/or primers for detection and/or
isolation of related polynucleotide sequences through
hybridization. Hybridization of one nucleic acid to another
constitutes a physical property that defines the polynucleotide of
the invention and the identified related sequences. Also, such
hybridization imposes structural limitations on the pair. A good
general discussion of the factors for determining hybridization
conditions is provided by Sambrook et al. ("Molecular Cloning, a
Laboratory Manual, 2nd ed., c. 1989 by Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; see esp., chapters 11
and 12). Additional considerations and details of the physical
chemistry of hybridization are provided by G. H. Keller and M. M.
Manak "DNA Probes", 2nd Ed. pp. 1-25, c. 1993 by Stockton Press,
New York, N.Y.
[0028] When using the polynucleotides to identify orthologous genes
in other species, the practitioner will preferably adjust the
amount of target DNA of each species so that, as nearly as is
practical, the same number of genome equivalents are present for
each species examined. This prevents faint signals from species
having large genomes, and thus small numbers of genome equivalents
per mass of DNA, from erroneously being interpreted as absence of
the corresponding gene in the genome.
[0029] The probes and/or primers of the instant invention can also
be used to detect or isolate nucleotides that are "identical" to
the probes or primers. Two nucleic acid sequences or polypeptides
are said to be "identical" if the sequence of nucleotides or amino
acid residues, respectively, in the two sequences is the same when
aligned for maximum correspondence as described below.
[0030] Isolated polynucleotides within the scope of the invention
also include allelic variants of the specific sequences presented
in the Sequence Listing. The probes and/or primers of the invention
are also used to detect and/or isolate polynucleotides exhibiting
at least 80% sequence identity with the sequences of the Sequence
Listing or fragments thereof. Related polynucleotide sequences can
also be identified according to the methods described in U.S.
Patent Publication 20040137466A1, dated Jul. 15, 2004 to Jofuku et
al.
[0031] With respect to nucleotide sequences, degeneracy of the
genetic code provides the possibility to substitute at least one
nucleotide of the nucleotide sequence of a gene with a different
nucleotide without changing the amino acid sequence of the
polypeptide. Hence, the DNA of the present invention also has any
base sequence that has been changed from a sequence in the Sequence
Listing by substitution in accordance with degeneracy of genetic
code. References describing codon usage include: Carels et al., J.
Mol. Evol. 46: 45 (1998) and Fennoy et al., Nucl. Acids Res.
21(23): 5294 (1993).
[0032] The polynucleotides of the invention are also used to create
various types of genetic and physical maps of the genome of corn,
Arabidopsis, soybean, rice, wheat, or other plants. Some are
absolutely associated with particular phenotypic traits, allowing
construction of gross genetic maps. Creation of such maps is based
on differences or variants, generally referred to as polymorphisms,
between different parents used in crosses. Common methods of
detecting polymorphisms that can be used are restriction fragment
length polymorphisms (RFLPs, single nucleotide polymorphisms (SNPs)
or simple sequence repeats (SSRs).
[0033] The use of RFLPs and of recombinant inbred lines for such
genetic mapping is described for Arabidopsis by Alonso-Blanco et
al. (Methods in Molecular Biology, vol. 82, "Arabidopsis
Protocols", pp. 137-146, J. M. Martinez-Zapater and J. Salinas,
eds., c. 1998 by Humana Press, Totowa, N.J.) and for corn by Burr
("Mapping Genes with Recombinant Inbreds", pp. 249-254. In
Freeling, M. and V. Walbot (Ed.), The Maize Handbook, c. 1994 by
Springer-Verlag New York, Inc.: New York, N.Y., USA; Berlin
Germany; Burr et al. Genetics (1998) 118: 519; Gardiner, J. et al.,
(1993) Genetics 134: 917). This procedure, however, is not limited
to plants and is used for other organisms (such as yeast) or for
individual cells.
[0034] The polynucleotides of the present invention are also used
for simple sequence repeat (SSR) mapping. Rice SSR mapping is
described by Morgante et al. (The Plant Journal (1993) 3: 165),
Panaud et al. (Genome (1995) 38: 1170); Senior et al. (Crop Science
(1996) 36: 1676), Taramino et al. (Genome (1996) 39: 277) and Ahn
et al. (Molecular and General Genetics (1993) 241: 483-90). SSR
mapping is achieved using various methods. In one instance,
polymorphisms are identified when sequence specific probes
contained within a polynucleotide flanking an SSR are made and used
in polymerase chain reaction (PCR) assays with template DNA from
two or more individuals of interest. Here, a change in the number
of tandem repeats between the SSR-flanking sequences produces
differently sized fragments (U.S. Pat. No. 5,766,847).
Alternatively, polymorphisms are identified by using the PCR
fragment produced from the SSR-flanking sequence specific primer
reaction as a probe against Southern blots representing different
individuals (U. H. Refseth et al., (1997) Electrophoresis 18:
1519).
[0035] The polynucleotides of the invention can further be used to
identify certain genes or genetic traits using, for example, known
AFLP technologies, such as in EP0534858 and U.S. Pat. No.
5,878,215.
[0036] The polynucleotides of the present invention are also used
for single nucleotide polymorphism (SNP) mapping.
[0037] Genetic and physical maps of crop species have many uses.
For example, these maps are used to devise positional cloning
strategies for isolating novel genes from the mapped crop species.
In addition, because the genomes of closely related species are
largely syntenic (i.e. they display the same ordering of genes
within the genome), these maps are used to isolate novel alleles
from relatives of crop species by positional cloning
strategies.
[0038] The various types of maps discussed above are used with the
polynucleotides of the invention to identify Quantitative Trait
Loci (QTLs). Many important crop traits, such as the solids content
of tomatoes, are quantitative traits and result from the combined
interactions of several genes. These genes reside at different loci
in the genome, often times on different chromosomes, and generally
exhibit multiple alleles at each locus. The polynucleotides of the
invention are used to identify QTLs and isolate specific alleles as
described by de Vicente and Tanksley (Genetics 134:585 (1993)).
Once a desired allele combination is identified, crop improvement
is accomplished either through biotechnological means or by
directed conventional breeding programs (for review see Tanksley
and McCouch, Science 277:1063 (1997)). In addition to isolating QTL
alleles in present crop species, the polynucleotides of the
invention are also used to isolate alleles from the corresponding
QTL of wild relatives.
[0039] In another embodiment, the polynucleotides are used to help
create physical maps of the genome of corn, Arabidopsis and related
species. Where polynucleotides are ordered on a genetic map, as
described above, they are used as probes to discover which clones
in large libraries of plant DNA fragments in YACs, BACs, etc.
contain the same polynucleotide or similar sequences, thereby
facilitating the assignment of the large DNA fragments to
chromosomal positions. Subsequently, the large BACs, YACs, etc. are
ordered unambiguously by more detailed studies of their sequence
composition (e.g. Marra et al. (1997) Genomic Research 7:1072-1084)
and by using their end or other sequences to find the identical
sequences in other cloned DNA fragments. The overlapping of DNA
sequences in this way allows building large contigs of plant
sequences to be built that, when sufficiently extended, provide a
complete physical map of a chromosome. Sometimes the
polynucleotides themselves provide the means of joining cloned
sequences into a contig. All scientific and patent publications
cited in this paragraph are hereby incorporated by reference.
[0040] U.S. Pat. Nos. 6,287,778 and 6,500,614, both hereby
incorporated by reference, describe scanning multiple alleles of a
plurality of loci using hybridization to arrays of
oligonucleotides. These techniques are useful for each of the types
of mapping discussed above.
[0041] Following the procedures described above and using a
plurality of the polynucleotides of the present invention, any
individual is genotyped. These individual genotypes are used for
the identification of particular cultivars, varieties, lines,
ecotypes and genetically modified plants or can serve as tools for
subsequent genetic studies involving multiple phenotypic
traits.
[0042] Identification and isolation of orthologous genes from
closely related species and alleles within a species is
particularly desirable because of their potential for crop
improvement. Many important crop traits, result from the combined
interactions of the products of several genes residing at different
loci in the genome. Generally, alleles at each of these loci make
quantitative differences to the trait. Once a more favorable allele
combination is identified, crop improvement is accomplished either
through biotechnological means or by directed conventional breeding
programs (Tanksley et al. Science 277:1063 (1997)).
[0043] FIG. 1 provides the results of ortholog analysis according
to the invention. This analysis provides a means for identifying
one or more sequences that are similar or orthologous or homologous
to one or more polynucleotides as noted herein, or one or more
target polypeptides encoded by the polynucleotides, or otherwise
noted herein and may include linking or associating a given plant
phenotype or gene function with a sequence. In the method, a
sequence database is provided (locally or across an internet or
intranet) and a query is made against the sequence database using
the relevant sequences herein and associated plant phenotypes or
gene functions.
[0044] In particular, the polypeptide sequences of the invention
(the "query sequences") were used to query against the Applicant's
own internal database of various plant sequences and against the
entire NCBI GenBank database. This search resulted in an alignment
for each query sequence with it's identified orthologous sequences,
and that group of sequences provided the basis for identifying a
respective consensus sequence. FIG. 1 sets forth the various
alignments, wherein each query sequence is identified as a
"Lead-Ceres Clone" followed by a numerical ID, the orthologs
identified from the Applicant's internal database are identified as
"Ceres Clone" followed by a numerical ID, and the orthologs
identified from GenBank are identified as "gi" followed by a
numerical ID. Each ortholog group, consisting of a query sequence
(Lead-Ceres Clone), the identified orthologs, and the respective
consensus sequence begins with a title that includes an
identification of the relevant Lead-Ceres Clone.
[0045] The alignments of FIG. 1 also include an identification of
the conserved domains or conserved regions, namely those domains or
regions that are conserved across the group of orthologous
sequences. One skilled in the art will recognize that each of the
sequences in a particular ortholog group will be useful for the
same purpose(s) as the Lead-Ceres Clone of that group, and that
other useful orthologs can be designed or identified by taking into
consideration the conserved regions or domains.
[0046] To aid in understanding the relationship of the various
sequence identifiers used in this application, Table 1 provides a
cross-reference for each polynucleotide sequence. In particular,
Table 1 matches each polynucleotide sequence in the Sequence
Listing ("SEQ ID NO:") with (1) a number referred to as the "Ceres
Clone ID" that is cited in the Sequence Listing as an internal
identifier for the Applicant; (2) a similar identifier also
utilized in the Sequence Listing and (3) the identifier utilized in
the Homolog Table of FIG. 1 that references the sequence as a
"Lead-Ceres Clone" and was used as the query sequence for
identifying a homologous group of sequences. The SEQ ID NOS. in the
Sequence Listing that are not in the Table 1 cross reference
represent the polypeptide sequences that are coded by the next
prior SEQ ID NO. or are part of the homolog group identified in
FIG. 1. For example, SEQ ID NO:1 is a polynucleotide sequence that
encodes the polypeptide of SEQ ID NO:2 while SEQ ID NOS: 3-4 are
the homologs of SEQ ID NO: 2 as shown in FIG. 1. Similarly, SEQ ID
NO:5 is a polynucleotide sequence that encodes the polypeptide of
SEQ ID NO:6 while SEQ ID NOS: 7-21 are the homologs of SEQ ID NO: 6
as shown in FIG. 1.
4. Use of the Genes to Make Transgenic Plants
[0047] 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 is made using standard recombinant DNA
techniques (Sambrook et al. 1989) and is introduced to the species
of interest by Agrobacterium-mediated transformation or by other
means of transformation as referenced below.
[0048] The vector backbone is any of those typical in the art such
as plasmids (such as Ti plasmids), viruses, artificial chromosomes,
BACs, YACs and PACs and vectors of the sort described by [0049] (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); [0050] (b) YAC: Burke et al., Science 236:806-812 (1987);
[0051] (c) PAC: Sternberg N. et al., Proc Natl Acad Sci USA.
January; 87(1): 103-7 (1990); [0052] (d) Bacteria-Yeast Shuttle
Vectors: Bradshaw et al., Nucl Acids Res 23: 4850-4856 (1995);
[0053] (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 N M (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
[0054] (g) Plasmid vectors: Sambrook et al., infra.
[0055] Typically, the construct comprises 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, glyphosate or phosphinotricin.
[0056] A plant promoter fragment is used that directs transcription
of the gene in all tissues of a regenerated plant and/or is a
constitutive promoter. Alternatively, the plant promoter directs
transcription of a sequence of the invention in a specific tissue
(tissue-specific promoter) or is otherwise under more precise
environmental control (inducible promoter).
[0057] If proper polypeptide production is desired, a
polyadenylation region at the 3'-end of the coding region is
typically included. The polyadenylation region is derived from the
natural gene, from a variety of other plant genes, or from T-DNA,
synthesized in the laboratory.
Transformation
[0058] 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).
[0059] The person skilled in the art knows processes for the
transformation of monocotyledonous and dicotyledonous plants. A
variety of techniques are available for introducing DNA into a
plant host cell. These techniques comprise transformation of plant
cells by DNA injection, DNA electroporation, use of bolistics
methods, protoplast fusion and via T-DNA using Agrobacterium
tumefaciens or Agrobacterium rhizogenes, as well as further
possibilities, or other bacterial hosts for Ti plasmid vectors. See
for example, Broothaerts et al., Gene Transfer to Plants by Diverse
Species of Bacteria, Nature, Vol. 433, pp. 629-633, 10 Feb.
2005.
[0060] DNA constructs of the invention are introduced into the cell
or the genome of the desired plant host by a variety of
conventional techniques. For example, the DNA construct is
introduced using techniques such as electroporation, microinjection
and polyethylene glycol precipitation of plant cell protoplasts or
protoplast fusion. Electroporation techniques are described in
Fromm et al. Proc. Natl. Acad. Sci. USA 82:5824 (1985).
Microinjection techniques are known in the art and well described
in the scientific and patent literature. The plasmids do not have
to fulfill specific requirements for use in DNA electroporation or
DNA injection into plant cells. Simple plasmids such as pUC
derivatives can be used.
[0061] The introduction of DNA constructs using polyethylene glycol
precipitation is described in Paszkowski et al. EMBO J. 3:2717
(1984). Introduction of foreign DNA using protoplast fusion is
described by Willmitzer (Willmitzer, L., 1993 Transgenic plants.
In: Biotechnology, A Multi-Volume Comprehensive Treatise (H. J.
Rehm, G. Reed, A. Puhler, P. Stadler, eds.), Vol. 2, 627-659, VCH
Weinheim-New York-Basel-Cambridge).
[0062] Alternatively, the DNA constructs of the invention are
introduced directly into plant tissue using ballistic methods, such
as DNA particle bombardment. Ballistic transformation techniques
are described in Klein et al. Nature 327:773 (1987). Introduction
of foreign DNA using ballistics is described by Willmitzer
(Willmitzer, L., 1993 Transgenic plants. In: Biotechnology, A
Multi-Volume Comprehensive Treatise (H. J. Rehm, G. Reed, A.
Puhler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim-New
York-Basel-Cambridge).
[0063] DNA constructs are also introduced with the help of
Agrobacteria. The use of Agrobacteria for plant cell transformation
is extensively examined and sufficiently disclosed in the
specification of EP-A 120 516, and 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
DePicker et al. (EMBO J. 4 (1985), 277-287). Using this technique,
the DNA constructs of the invention are combined with suitable
T-DNA flanking regions and introduced into a conventional
Agrobacterium tumefaciens host vector. The virulence functions of
the Agrobacterium tumefaciens host direct the insertion of the
construct and adjacent marker(s) into the plant cell DNA when the
cell is infected by the bacteria (McCormac et al., 1997, Mol.
Biotechnol. 8:199; Hamilton, 1997, Gene 200:107; Salomon et al.,
1984 EMBO J. 3:141; Herrera-Estrella et al., 1983 EMBO J. 2:987).
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, CM., 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, AP., Plant Mol.
Biol. 20:1203 (1992); Graves and Goldman, Plant Mol. Biol. 7:34
(1986) and Gould et al., Plant Physiology 95:426 (1991).
[0064] For plant cell T-DNA transfer of DNA, plant organs, e.g.
infloresences, plant explants, plant cells that have been cultured
in suspension or protoplasts are co-cultivated with Agrobacterium
tumefaciens or Agrobacterium rhizogenes or other suitable T-DNA
hosts. Whole plants are regenerated from the infected plant
material or seeds generated from infected plant material using a
suitable medium that contains antibiotics or biocides for the
selection of transformed cells or by spraying the biocide on plants
to select the transformed plants. Plants obtained in this way are
then examined for the presence of the DNA introduced. The
transformation of dicotyledonous plants via Ti-plasmid-vector
systems and Agrobacterium tumefaciens is well established.
[0065] Monocotyledonous plants are also transformed by means of
Agrobacterium based vectors (See 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). Maize
transformation in particular is described in the literature (see,
for example, 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. (1989, Bio/Technology 7, 581)
fertile plants are obtained from a mucus-free, soft (friable) maize
callus. Prioli and Sondahl (1989, Bio/Technology 7, 589) also
report regenerating fertile plants from maize protoplasts of the
maize Cateto inbred line, Cat 100-1.
[0066] Other cereal species have also been successfully
transformed, such as barley (Wan and Lemaux, see above; Ritala et
al., see above) and wheat (Nehra et al., 1994, Plant J. 5,
285-297).
[0067] Alternatives to Agrobacterium transformation for plants are
ballistics, protoplast fusion, electroporation of partially
permeabilized cells and use of glass fibers (>See 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)).
[0068] Introduced DNA is usually stable after integration into the
plant genome and is transmitted to the progeny of the transformed
cell or plant. Generally the transformed plant cell contains a
selectable marker that makes the transformed cells resistant to a
biocide or an antibiotic such as kanamycin, G 418, bleomycin,
hygromycin, phosphinotricin or others. Therefore, the individually
chosen marker should allow the selection of transformed cells from
cells lacking the introduced DNA.
[0069] The transformed cells grow within the plant in the usual way
(McCormick et al., 1986, Plant Cell Reports 5, 81-84) and the
resulting plants are cultured normally. Transformed plant cells
obtained by any of the above transformation techniques are 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.
[0070] 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 also occurs
from plant callus, explants, organs, or pairs 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)).
Useful and relevant procedures for transient expression are also
described in U.S. Application No. 60/537,070 filed on Jan. 16, 2004
and PCT Application No. PCT/US2005/001153 filed on Jan. 14,
2005.
[0071] After transformation, seeds are obtained from the plants and
used for testing stability and inheritance. Generally, two or more
generations are cultivated to ensure that the phenotypic feature is
stably maintained and transmitted.
[0072] 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.
[0073] The nucleotide sequences according to the invention
generally encode an appropriate protein 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, such as potato or cereals such as rice, maize,
wheat, barley, rye, triticale, oat, millet, etc., as well as
spinach, tobacco, sugar beet, soya, cotton etc.
[0074] In principle, the process according to the invention can be
applied to any plant.
[0075] 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. Examples are vegetable plants such as cucumber,
melon, pumpkin, eggplant, zucchini, tomato, spinach, cabbage
species, peas, beans, etc., as well as fruits such as pears,
apples, etc.
[0076] Thus, the invention has use over a broad range of plants,
preferably higher plants, pertaining to the classes of Angiospermae
and Gymnospermae. Plants of the subclasses of the Dicotylodenae and
the Monocotyledonae are particularly suitable. Dicotyledonous
plants belong to the orders of the Magniolales, Illiciales,
Laurales, Piperales Aristochiales, Nymphaeales, Ranunculales,
Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales,
Eucomiales, Leitneriales, Myricales, Fagales, Casuarinales,
Caryophyllales, Batales, Polygonales, Plumbaginales, Dilleniales,
Theales, Malvales, Urticales, Lecyihidales, Violales, Salicales,
Capparales, Ericales, Diapensales, Ebenales, Primulales, Rosales,
Fabales, Podostemales, Haloragales, Myrtales, Cornales, Proteales,
Santales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales,
Sapindales, Juglandales, Geraniales, Polygalales, Umbellales,
Gentianales, Polemoniales, Lamiales, Plantaginales,
Scrophulariales, Campanulales, Rubiales, Dipsacales, and Asterales.
Monocotyledonous plants belong to the orders of the Alismatales,
Hydrocharitales, Najadales, Triuridales, Commelinales,
Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales,
Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales,
Arales, Lilliales, and Orchidales. Plants belonging to the class of
the Gymnospermae are Pinales, Ginkgoales, Cycadales and
Gnetales.
[0077] The method of the invention is preferably used with plants
that are interesting for agriculture, horticulture, biomass for
bioconversion and/or forestry. Examples are tobacco, oilseed rape,
sugar beet, potato, tomato, cucumber, pepper, bean, pea, citrus
fruit, apple, pear, berries, plum, melon, eggplant, cotton,
soybean, sunflower, rose, poinsettia, petunia, guayule, cabbage,
spinach, alfalfa, artichoke, corn, wheat, rye, barley, grasses such
as switch grass or turf grass, millet, hemp, banana, poplar,
eucalyptus trees, conifers.
5. Phenotype Screens and Results
[0078] 5.1 Triparental Mating and Vacuum Infiltration
Transformation of Plants
[0079] The function/phenotype characteristics of the sequences of
the invention were determined by means of screens with transgenic
plants. Standard laboratory techniques are as described in Sambrook
et al. (1989) unless otherwise stated. Single colonies of
Agrobacterium C58C1Rif, E. coli helper strain HB101 and the E. coli
strain containing the transformation construct to be mobilized into
Agrobacterium are separately inoculated into appropriate growth
media and stationary cultures produced. Cultures are mixed gently,
plated on YEB (5 g Gibco beef extract, 1 g Bacto yeast extract, 1 g
Bacto peptone, 5 g sucrose, pH 7.4) solid growth media and
incubated overnight at 28.degree. C. The bacteria from the
triparental mating are collected in and serial dilutions made. An
aliquot of the each dilution is then plated and incubated for 2
days at 28.degree. C. on YEB plates supplemented with 100 .mu.g/ml
rifampicin and 100 .mu.g/ml carbenicillin for calculation of the
number of acceptor cells and on YEB plates supplemented with 100
.mu.g/ml rifampicin, 100 .mu.g/ml carbenicillin and 100 .mu.g/ml
spectinomycin for selection of transconjugant cells. The
cointegrate structure of purified transconjugants is verified via
Southern blot hybridization.
[0080] A transconjugant culture is prepared for vacuum infiltration
by inoculating 1 ml of a stationary culture arising from a single
colony into liquid YEB media and incubating at 28.degree. C. for
approximately 20 hours with shaking until the OD taken at 600 nm
was 0.8-1.0. The culture is then pelleted and the bacteria
resuspended in infiltration medium (0.5.times. MS salts, 5% w/v
sucrose, 10 .mu.g/l BAP, 200 .mu.l/l Silwet L-77, pH 5.8) to a
final OD.sub.600 of 1.0. This prepared transconjugant culture is
used within 20 minutes of preparation.
[0081] Wild-type plants for vacuum infiltration are grown in pots.
Briefly, seeds of Arabidopsis thaliana (ecotype Wassilewskija) are
sown in pots and left at 4.degree. C. for two to four days to
vernalize. They are then transferred to 22-25.degree. C. and grown
under long-day (16 hr light: 8 hr dark) conditions, sub-irrigated
with water. After bolting, the primary inflorescence is removed
and, after four to eight days, the pots containing the plants are
inverted in the vacuum chamber to submerge all of the plants in the
prepared transconjugant culture. Vacuum is drawn for two minutes
before pots are removed, covered with plastic wrap and incubated in
a cool room under darkness or very low light for one to two days.
The plastic wrap is then removed; the plants returned to their
previous growing conditions and subsequently produced (T1) seed
collected.
[0082] 5.2 Selection of T-DNA Insertion Lines
[0083] Seeds from the initial vacuum infiltrated plants are sown on
flats of Metromix 350 soil. Flats are vernalized for four to five
days at 4.degree. C. before being transferred to 22-25.degree. C.
and grown under long-day (16 hr light: 8 hr dark) conditions,
sub-irrigated with water. Approximately seven to ten days after
germination, the (T1) seedlings are sprayed with 0.02% Finale
herbicide (AgrEvo). After another five to seven days, herbicide
treatment is repeated. Herbicide resistant T1 plants are allowed to
self-pollinate and T2 seed are collected from each individual. In
the few cases where the T1 plant produced few seed, the T2 seed is
planted in bulk, the T2 plants allowed to self-pollinate and T3
seed collected.
[0084] 5.3 Phenotype Screening
[0085] Seeds from each T2 (or T3) line are planted in a 4-inch pot
containing either Sunshine mix or Metromix 350 soil. Pots are
vernalized for four to five days at 4.degree. C. before being
transferred to 22-25.degree. C. and grown under long-day (16 hr
light: 8 hr dark) conditions, sub-irrigated with water. A first
phenotype screen is conducted by visually inspecting the seedlings
five to seven days after germination and aberrant phenotypes noted.
Plants are then sprayed with Finale herbicide within four days
(i.e. about seven to nine days after germination). The second
visual screen is conducted on surviving T2 (or T3) plants about
sixteen to seventeen days after germination and the final screen
was conducted after the plants have bolted and formed siliques.
Here, the third and fourth green siliques are collected and
aberrant phenotypes noted.
[0086] Alternatively, seed are surface sterilized and transferred
to agar solidified medium containing Murashige and Skoog salts
(1.times.), 1% sucrose (wt/v) pH 5.7 before autoclaving. Seed re
cold treated for 48 hours and transferred to long days [16 hours
light and 8 hours dark], 25.degree. C. Plants are screened at 5 and
10 days.
[0087] 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.
[0088] Each of the references from the patent and periodical
literature cited herein is hereby expressly incorporated in its
entirety by such citation.
TABLE-US-00001 TABLE 1 SEQUECE IDENTIFIER CROSS-REFERENCE CERES
CLONE SEQ ID NO ID CERES ID IN SEQUENCE LISTING HOMOLOG_TABLE_ID
SEQ ID NO: 1 40252 Identifier: Ceres CLONE ID no. 40252
Lead_CeresClone40252 SEQ ID NO: 5 32791 Identifier: Ceres CLONE ID
no. 32791 Lead_CeresClone32791 SEQ ID NO: 22 39319 Identifier:
Ceres CLONE ID no. 39319 Lead_CeresClone39319 SEQ ID NO: 37 41337
Identifier: Ceres CLONE ID no. 41337 Lead_CeresClone41337 SEQ ID
NO: 46 314 Identifier: Ceres CLONE ID no. 314 Lead_CeresClone314
SEQ ID NO: 54 332 Identifier: Ceres CLONE ID no. 332
Lead_CeresClone332 SEQ ID NO: 66 907 Identifier: Ceres CLONE ID no.
907 Lead_CeresClone907 SEQ ID NO: 71 1241 Identifier: Ceres CLONE
ID no. 1241 Lead_CeresClone1241 SEQ ID NO: 79 1610 Identifier:
Ceres CLONE ID no. 1610 Lead_CeresClone1610 SEQ ID NO: 95 2403
Identifier: Ceres CLONE ID no. 2403 Lead_CeresClone2403 SEQ ID NO:
107 2835 Identifier: Ceres CLONE ID no. 2835 Lead_CeresClone2835
SEQ ID NO: 114 3000 Identifier: Ceres CLONE ID no. 3000
Lead_CeresClone3000 SEQ ID NO: 123 3036 Identifier: Ceres CLONE ID
no. 3036 Lead_CeresClone3036 SEQ ID NO: 128 3363 Identifier: Ceres
CLONE ID no. 3363 Lead_CeresClone3363 SEQ ID NO: 147 3510
Identifier: Ceres CLONE ID no. 3510 Lead_CeresClone3510 SEQ ID NO:
161 3699 Identifier: Ceres CLONE ID no. 3699 Lead_CeresClone3699
SEQ ID NO: 176 3858 Identifier: Ceres CLONE ID no. 3858
Lead_CeresClone3858 SEQ ID NO: 191 5597 Identifier: Ceres CLONE ID
no. 5597 Lead_CeresClone5597 SEQ ID NO: 199 5605 Identifier: Ceres
CLONE ID no. 5605 Lead_CeresClone5605 SEQ ID NO: 210 6685
Identifier: Ceres CLONE ID no. 6685 Lead_CeresClone6685 SEQ ID NO:
215 8265 Identifier: Ceres CLONE ID no. 8265 Lead_CeresClone8265
SEQ ID NO: 221 8490 Identifier: Ceres CLONE ID no. 8490
Lead_CeresClone8490 SEQ ID NO: 230 9683 Identifier: Ceres CLONE ID
no. 9683 Lead_CeresClone9683 SEQ ID NO: 234 9897 Identifier: Ceres
CLONE ID no. 9897 Lead_CeresClone9897 SEQ ID NO: 242 12272
Identifier: Ceres CLONE ID no. 12272 Lead_CeresClone12272 SEQ ID
NO: 251 12935 Identifier: Ceres CLONE ID no. 12935
Lead_CeresClone12935 SEQ ID NO: 262 13092 Identifier: Ceres CLONE
ID no. 13092 Lead_CeresClone13092 SEQ ID NO: 270 13263 Identifier:
Ceres CLONE ID no. 13263 Lead_CeresClone13263 SEQ ID NO: 276 14583
Identifier: Ceres CLONE ID no. 14583 Lead_CeresClone14583 SEQ ID
NO: 283 14909 Identifier: Ceres CLONE ID no. 14909
Lead_CeresClone14909 SEQ ID NO: 295 16412 Identifier: Ceres CLONE
ID no. 16412 Lead_CeresClone16412 SEQ ID NO: 304 16461 Identifier:
Ceres CLONE ID no. 16461 Lead_CeresClone16461 SEQ ID NO: 309 17409
Identifier: Ceres CLONE ID no. 17409 Lead_CeresClone17409 SEQ ID
NO: 316 17482 Identifier: Ceres CLONE ID no. 17482
Lead_CeresClone17482 SEQ ID NO: 321 17632 Identifier: Ceres CLONE
ID no. 17632 Lead_CeresClone17632 SEQ ID NO: 328 18612 Identifier:
Ceres CLONE ID no. 18612 Lead_CeresClone18612 SEQ ID NO: 335 18820
Identifier: Ceres CLONE ID no. 18820 Lead_CeresClone18820 SEQ ID
NO: 341 19188 Identifier: Ceres CLONE ID no. 19188
Lead_CeresClone19188 SEQ ID NO: 351 20257 Identifier: Ceres CLONE
ID no. 20257 Lead_CeresClone20257 SEQ ID NO: 362 21068 Identifier:
Ceres CLONE ID no. 21068 Lead_CeresClone21068 SEQ ID NO: 367 22461
Identifier: Ceres CLONE ID no. 22461 Lead_CeresClone22461 SEQ ID
NO: 372 23203 Identifier: Ceres CLONE ID no. 23203
Lead_CeresClone23203 SEQ ID NO: 382 26907 Identifier: Ceres CLONE
ID no. 26907 Lead_CeresClone26907 SEQ ID NO: 393 27460 Identifier:
Ceres CLONE ID no. 27460 Lead_CeresClone27460 SEQ ID NO: 396 32348
Identifier: Ceres CLONE ID no. 32348 Lead_CeresClone32348 SEQ ID
NO: 407 32548 Identifier: Ceres CLONE ID no. 32548
Lead_CeresClone32548 SEQ ID NO: 417 32753 Identifier: Ceres CLONE
ID no. 32753 Lead_CeresClone32753 SEQ ID NO: 422 34167 Identifier:
Ceres CLONE ID no. 34167 Lead_CeresClone34167 SEQ ID NO: 430 34385
Identifier: Ceres CLONE ID no. 34385 Lead_CeresClone34385 SEQ ID
NO: 449 36518 Identifier: Ceres CLONE ID no. 36518
Lead_CeresClone36518 SEQ ID NO: 456 36891 Identifier: Ceres CLONE
ID no. 36891 Lead_CeresClone36891 SEQ ID NO: 471 36904 Identifier:
Ceres CLONE ID no. 36904 Lead_CeresClone36904 SEQ ID NO: 478 37288
Identifier: Ceres CLONE ID no. 37288 Lead_CeresClone37288 SEQ ID
NO: 484 37298 Identifier: Ceres CLONE ID no. 37298
Lead_CeresClone37298 SEQ ID NO: 495 37663 Identifier: Ceres CLONE
ID no. 37663 Lead_CeresClone37663 SEQ ID NO: 498 38101 Identifier:
Ceres CLONE ID no. 38101 Lead_CeresClone38101 SEQ ID NO: 511 38419
Identifier: Ceres CLONE ID no. 38419 Lead_CeresClone38419 SEQ ID
NO: 518 38470 Identifier: Ceres CLONE ID no. 38470
Lead_CeresClone38470 SEQ ID NO: 526 38690 Identifier: Ceres CLONE
ID no. 38690 Lead_CeresClone38690 SEQ ID NO: 530 39286 Identifier:
Ceres CLONE ID no. 39286 Lead_CeresClone39286 SEQ ID NO: 536 40508
Identifier: Ceres CLONE ID no. 40508 Lead_CeresClone40508 SEQ ID
NO: 543 40729 Identifier: Ceres CLONE ID no. 40729
Lead_CeresClone40729 SEQ ID NO: 551 41306 Identifier: Ceres CLONE
ID no. 41306 Lead_CeresClone41306 SEQ ID NO: 554 41439 Identifier:
Ceres CLONE ID no. 41439 Lead_CeresClone41439 SEQ ID NO: 569 42141
Identifier: Ceres CLONE ID no. 42141 Lead_CeresClone42141 SEQ ID
NO: 578 92459 Identifier: Ceres CLONE ID no. 92459
Lead_CeresClone92459 SEQ ID NO: 585 92670 Identifier: Ceres CLONE
ID no. 92670 Lead_CeresClone92670 SEQ ID NO: 598 94231 Identifier:
Ceres CLONE ID no. 94231 Lead_CeresClone94231 SEQ ID NO: 611 95135
Identifier: Ceres CLONE ID no. 95135 Lead_CeresClone95135 SEQ ID
NO: 623 97434 Identifier: Ceres CLONE ID no. 97434
Lead_CeresClone97434 SEQ ID NO: 640 97480 Identifier: Ceres CLONE
ID no. 97480 Lead_CeresClone97480 SEQ ID NO: 654 97958 Identifier:
Ceres CLONE ID no. 97958 Lead_CeresClone97958 SEQ ID NO: 672 98855
Identifier: Ceres CLONE ID no. 98855 Lead_CeresClone98855 SEQ ID
NO: 681 99657 Identifier: Ceres CLONE ID no. 99657
Lead_CeresClone99657 SEQ ID NO: 699 100465 Identifier: Ceres CLONE
ID no. 100465 Lead_CeresClone100465 SEQ ID NO: 703 107731
Identifier: Ceres CLONE ID no. 107731 Lead_CeresClone107731 SEQ ID
NO: 717 110454 Identifier: Ceres CLONE ID no. 110454
Lead_CeresClone110454 SEQ ID NO: 733 116843 Identifier: Ceres CLONE
ID no. 116843 Lead_CeresClone116843 SEQ ID NO: 738 119256
Identifier: Ceres CLONE ID no. 119256 Lead_CeresClone119256 SEQ ID
NO: 746 123905 Identifier: Ceres CLONE ID no. 123905
Lead_CeresClone123905 SEQ ID NO: 753 141805 Identifier: Ceres CLONE
ID no. 141805 Lead_CeresClone141805 SEQ ID NO: 759 141890
Identifier: Ceres CLONE ID no. 141890 Lead_CeresClone141890 SEQ ID
NO: 762 147358 Identifier: Ceres CLONE ID no. 147358
Lead_CeresClone147358 SEQ ID NO: 775 148943 Identifier: Ceres CLONE
ID no. 148943 Lead_CeresClone148943 SEQ ID NO: 785 157547
Identifier: Ceres CLONE ID no. 157547 Lead_CeresClone157547 SEQ ID
NO: 791 158333 Identifier: Ceres CLONE ID no. 158333
Lead_CeresClone158333 SEQ ID NO: 797 227651 Identifier: Ceres CLONE
ID no. 227651 Lead_CeresClone227651 SEQ ID NO: 815 235672
Identifier: Ceres CLONE ID no. 235672 Lead_CeresClone235672 SEQ ID
NO: 819 241131 Identifier: Ceres CLONE ID no. 241131
Lead_CeresClone241131 SEQ ID NO: 825 262460 Identifier: Ceres CLONE
ID no. 262460 Lead_CeresClone262460 SEQ ID NO: 832 270555
Identifier: Ceres CLONE ID no. 270555 Lead_CeresClone270555 SEQ ID
NO: 835 481710 Identifier: Ceres CLONE ID no. 481710
Lead_CeresClone481710 SEQ ID NO: 846 482122 Identifier: Ceres CLONE
ID no. 482122 Lead_CeresClone482122 SEQ ID NO: 860 536457
Identifier: Ceres CLONE ID no. 536457 Lead_CeresClone536457 SEQ ID
NO: 871 536796 Identifier: Ceres CLONE ID no. 536796
Lead_CeresClone536796 SEQ ID NO: 877 572121 Identifier: Ceres CLONE
ID no. 572121 Lead_CeresClone572121 SEQ ID NO: 880 641355
Identifier: Ceres CLONE ID no. 641355 Lead_CeresClone641355 SEQ ID
NO: 894 660003 Identifier: Ceres CLONE ID no. 660003
Lead_CeresClone660003 SEQ ID NO: 898 664365 Identifier: Ceres CLONE
ID no. 664365 Lead_CeresClone664365 SEQ ID NO: 908 708342
Identifier: Ceres CLONE ID no. 708342 Lead_CeresClone708342 SEQ ID
NO: 914 969750 Identifier: Ceres CLONE ID no. 969750
Lead_CeresClone969750 SEQ ID NO: 919 1001432 Identifier: Ceres
CLONE ID no. 1001432 Lead_CeresClone1001432 SEQ ID NO: 930 1002819
Identifier: Ceres CLONE ID no. 1002819 Lead_CeresClone1002819 SEQ
ID NO: 935 1007549 Identifier: Ceres CLONE ID no. 1007549
Lead_CeresClone1007549 SEQ ID NO: 948 1043081 Identifier: Ceres
CLONE ID no. 1043081 Lead_CeresClone1043081 SEQ ID NO: 965 99298
Identifier: Ceres CLONE ID no. 99298 Lead_CeresClone99298 SEQ ID
NO: 974 100245 Identifier: Ceres CLONE ID no. 100245
Lead_CeresClone100245 SEQ ID NO: 989 101798 Identifier: Ceres CLONE
ID no. 101798 Lead_CeresClone101798 SEQ ID NO: 1005 38370
Identifier: Ceres CLONE ID no. 38370 Lead_CeresClone38370 SEQ ID
NO: 1012 1496 Identifier: Ceres CLONE ID no. 1496
Lead_CeresClone1496 SEQ ID NO: 1031 2561 Identifier: Ceres CLONE ID
no. 2561 Lead_CeresClone2561 SEQ ID NO: 1042 3618 Identifier: Ceres
CLONE ID no. 3618 Lead_CeresClone3618 SEQ ID NO: 1048 7191
Identifier: Ceres CLONE ID no. 7191 Lead_CeresClone7191 SEQ ID NO:
1057 8254 Identifier: Ceres CLONE ID no. 8254 Lead_CeresClone8254
SEQ ID NO: 1062 8877 Identifier: Ceres CLONE ID no. 8877
Lead_CeresClone8877 SEQ ID NO: 1075 8916 Identifier: Ceres CLONE ID
no. 8916 Lead_CeresClone8916 SEQ ID NO: 1078 10879 Identifier:
Ceres CLONE ID no. 10879 Lead_CeresClone10879 SEQ ID NO: 1086 19116
Identifier: Ceres CLONE ID no. 19116 Lead_CeresClone19116 SEQ ID
NO: 1092 19319 Identifier: Ceres CLONE ID no. 19319
Lead_CeresClone19319 SEQ ID NO: 1100 19486 Identifier: Ceres CLONE
ID no. 19486 Lead_CeresClone19486 SEQ ID NO: 1106 19510 Identifier:
Ceres CLONE ID no. 19510 Lead_CeresClone19510 SEQ ID NO: 1125 23322
Identifier: Ceres CLONE ID no. 23322 Lead_CeresClone23322 SEQ ID
NO: 1136 25538 Identifier: Ceres CLONE ID no. 25538
Lead_CeresClone25538
SEQ ID NO: 1145 25607 Identifier: Ceres CLONE ID no. 25607
Lead_CeresClone25607 SEQ ID NO: 1155 25758 Identifier: Ceres CLONE
ID no. 25758 Lead_CeresClone25758 SEQ ID NO: 1162 25886 Identifier:
Ceres CLONE ID no. 25886 Lead_CeresClone25886 SEQ ID NO: 1181 27464
Identifier: Ceres CLONE ID no. 27464 Lead_CeresClone27464 SEQ ID
NO: 1190 28602 Identifier: Ceres CLONE ID no. 28602
Lead_CeresClone28602 SEQ ID NO: 1204 35493 Identifier: Ceres CLONE
ID no. 35493 Lead_CeresClone35493 SEQ ID NO: 1217 37229 Identifier:
Ceres CLONE ID no. 37229 Lead_CeresClone37229 SEQ ID NO: 1227 37493
Identifier: Ceres CLONE ID no. 37493 Lead_CeresClone37493 SEQ ID
NO: 1235 38105 Identifier: Ceres CLONE ID no. 38105
Lead_CeresClone38105 SEQ ID NO: 1242 38214 Identifier: Ceres CLONE
ID no. 38214 Lead_CeresClone38214 SEQ ID NO: 1252 41320 Identifier:
Ceres CLONE ID no. 41320 Lead_CeresClone41320 SEQ ID NO: 1262 42533
Identifier: Ceres CLONE ID no. 42533 Lead_CeresClone42533 SEQ ID
NO: 1271 42925 Identifier: Ceres CLONE ID no. 42925
Lead_CeresClone42925 SEQ ID NO: 1282 95453 Identifier: Ceres CLONE
ID no. 95453 Lead_CeresClone95453 SEQ ID NO: 1295 96020 Identifier:
Ceres CLONE ID no. 96020 Lead_CeresClone96020 SEQ ID NO: 1302 97415
Identifier: Ceres CLONE ID no. 97415 Lead_CeresClone97415 SEQ ID
NO: 1313 101255 Identifier: Ceres CLONE ID no. 101255
Lead_CeresClone101255 SEQ ID NO: 1321 103581 Identifier: Ceres
CLONE ID no. 103581 Lead_CeresClone103581 SEQ ID NO: 1336 109514
Identifier: Ceres CLONE ID no. 109514 Lead_CeresClone109514 SEQ ID
NO: 1346 115946 Identifier: Ceres CLONE ID no. 115946
Lead_CeresClone115946 SEQ ID NO: 1351 115975 Identifier: Ceres
CLONE ID no. 115975 Lead_CeresClone115975 SEQ ID NO: 1359 117369
Identifier: Ceres CLONE ID no. 117369 Lead_CeresClone117369 SEQ ID
NO: 1370 118337 Identifier: Ceres CLONE ID no. 118337
Lead_CeresClone118337 SEQ ID NO: 1383 150912 Identifier: Ceres
CLONE ID no. 150912 Lead_CeresClone150912 SEQ ID NO: 1389 152141
Identifier: Ceres CLONE ID no. 152141 Lead_CeresClone152141 SEQ ID
NO: 1403 157730 Identifier: Ceres CLONE ID no. 157730
Lead_CeresClone157730 SEQ ID NO: 1411 225597 Identifier: Ceres
CLONE ID no. 225597 Lead_CeresClone225597 SEQ ID NO: 1415 264705
Identifier: Ceres CLONE ID no. 264705 Lead_CeresClone264705 SEQ ID
NO: 1436 627596 Identifier: Ceres CLONE ID no. 627596
Lead_CeresClone627596 SEQ ID NO: 1450 729085 Identifier: Ceres
CLONE ID no. 729085 Lead_CeresClone729085 SEQ ID NO: 1463 1011386
Identifier: Ceres CLONE ID no. 1011386 Lead_CeresClone1011386 SEQ
ID NO: 1467 6082 Identifier: Ceres CLONE ID no. 6082
Lead_CeresClone6082 SEQ ID NO: 1474 13812 Identifier: Ceres CLONE
ID no. 13812 Lead_CeresClone13812 SEQ ID NO: 1479 32811 Identifier:
Ceres CLONE ID no. 32811 Lead_CeresClone32811 SEQ ID NO: 1485
224062 Identifier: Ceres CLONE ID no. 224062 Lead_CeresClone224062
SEQ ID NO: 1494 254065 Identifier: Ceres CLONE ID no. 254065
Lead_CeresClone254065 SEQ ID NO: 1502 22339 Identifier: Ceres CLONE
ID no. 22339 Lead_CeresClone22339 SEQ ID NO: 1516 99784 Identifier:
Ceres CLONE ID no. 99784 Lead_CeresClone99784 SEQ ID NO: 1532
100319 Identifier: Ceres CLONE ID no. 100319 Lead_CeresClone100319
SEQ ID NO: 1539 124720 Identifier: Ceres CLONE ID no. 124720
Lead_CeresClone124720 SEQ ID NO: 1548 288251 Identifier: Ceres
CLONE ID no. 288251 Lead_CeresClone288251 SEQ ID NO: 1555 8014
Identifier: Ceres CLONE ID no. 8014 Lead_CeresClone8014 SEQ ID NO:
1562 16204 Identifier: Ceres CLONE ID no. 16204
Lead_CeresClone16204 SEQ ID NO: 1573 101250 Identifier: Ceres CLONE
ID no. 101250 Lead_CeresClone101250 SEQ ID NO: 1579 283597
Identifier: Ceres CLONE ID no. 283597 Lead_CeresClone283597 SEQ ID
NO: 1586 292789 Identifier: Ceres CLONE ID no. 292789
Lead_CeresClone292789 SEQ ID NO: 1606 4289 Identifier: Ceres CLONE
ID no. 4289 Lead_CeresClone4289 SEQ ID NO: 1610 7925 Identifier:
Ceres CLONE ID no. 7925 Lead_CeresClone7925 SEQ ID NO: 1613 10857
Identifier: Ceres CLONE ID no. 10857 Lead_CeresClone10857 SEQ ID
NO: 1619 19481 Identifier: Ceres CLONE ID no. 19481
Lead_CeresClone19481 SEQ ID NO: 1626 28979 Identifier: Ceres CLONE
ID no. 28979 Lead_CeresClone28979 SEQ ID NO: 1638 113719
Identifier: Ceres CLONE ID no. 113719 Lead_CeresClone113719 SEQ ID
NO: 1651 147593 Identifier: Ceres CLONE ID no. 147593
Lead_CeresClone147593 SEQ ID NO: 1661 150798 Identifier: Ceres
CLONE ID no. 150798 Lead_CeresClone150798 SEQ ID NO: 1666 152076
Identifier: Ceres CLONE ID no. 152076 Lead_CeresClone152076 SEQ ID
NO: 1673 154031 Identifier: Ceres CLONE ID no. 154031
Lead_CeresClone154031 SEQ ID NO: 1677 246416 Identifier: Ceres
CLONE ID no. 246416 Lead_CeresClone246416 SEQ ID NO: 1696 949
Identifier: Ceres CLONE ID no. 949 Lead_CeresClone949 SEQ ID NO:
1710 2036 Identifier: Ceres CLONE ID no. 2036 Lead_CeresClone2036
SEQ ID NO: 1719 18857 Identifier: Ceres CLONE ID no. 18857
Lead_CeresClone18857 SEQ ID NO: 1728 23518 Identifier: Ceres CLONE
ID no. 23518 Lead_CeresClone23518 SEQ ID NO: 1745 156655
Identifier: Ceres CLONE ID no. 156655 Lead_CeresClone156655 SEQ ID
NO: 1750 2273 Identifier: Ceres CLONE ID no. 2273
Lead_CeresClone2273 SEQ ID NO: 1756 5198 Identifier: Ceres CLONE ID
no. 5198 Lead_CeresClone5198 SEQ ID NO: 1765 13767 Identifier:
Ceres CLONE ID no. 13767 Lead_CeresClone13767 SEQ ID NO: 1773 29150
Identifier: Ceres CLONE ID no. 29150 Lead_CeresClone29150 SEQ ID
NO: 1777 34480 Identifier: Ceres CLONE ID no. 34480
Lead_CeresClone34480 SEQ ID NO: 1785 38625 Identifier: Ceres CLONE
ID no. 38625 Lead_CeresClone38625 SEQ ID NO: 1791 39351 Identifier:
Ceres CLONE ID no. 39351 Lead_CeresClone39351 SEQ ID NO: 1800
153053 Identifier: Ceres CLONE ID no. 153053 Lead_CeresClone153053
SEQ ID NO: 1805 159318 Identifier: Ceres CLONE ID no. 159318
Lead_CeresClone159318 SEQ ID NO: 1811 241379 Identifier: Ceres
CLONE ID no. 241379 Lead_CeresClone241379 SEQ ID NO: 1822 5220
Identifier: Ceres CLONE ID no. 5220 Lead_CeresClone5220 SEQ ID NO:
1826 11214 Identifier: Ceres CLONE ID no. 11214
Lead_CeresClone11214 SEQ ID NO: 1841 563522 Identifier: Ceres CLONE
ID no. 563522 Lead_CeresClone563522 SEQ ID NO: 1860 21563
Identifier: Ceres CLONE ID no. 21563 Lead_CeresClone21563 SEQ ID
NO: 1868 6397 Identifier: Ceres CLONE ID no. 6397
Lead_CeresClone6397 SEQ ID NO: 1872 14555 Identifier: Ceres CLONE
ID no. 14555 Lead_CeresClone14555 SEQ ID NO: 1882 4067 Identifier:
Ceres CLONE ID no. 4067 Lead_CeresClone4067 SEQ ID NO: 1895 4734
Identifier: Ceres CLONE ID no. 4734 Lead_CeresClone4734 SEQ ID NO:
1903 28643 Identifier: Ceres CLONE ID no. 28643
Lead_CeresClone28643 SEQ ID NO: 1917 733804 Identifier: Ceres CLONE
ID no. 733804 Lead_CeresClone733804 SEQ ID NO: 1929 9221
Identifier: Ceres CLONE ID no. 9221 Lead_CeresClone9221 SEQ ID NO:
1943 11929 Identifier: Ceres CLONE ID no. 11929
Lead_CeresClone11929 SEQ ID NO: 1954 12071 Identifier: Ceres CLONE
ID no. 12071 Lead_CeresClone12071 SEQ ID NO: 1961 13625 Identifier:
Ceres CLONE ID no. 13625 Lead_CeresClone13625 SEQ ID NO: 1971 16865
Identifier: Ceres CLONE ID no. 16865 Lead_CeresClone16865 SEQ ID
NO: 1988 18246 Identifier: Ceres CLONE ID no. 18246
Lead_CeresClone18246 SEQ ID NO: 1994 31044 Identifier: Ceres CLONE
ID no. 31044 Lead_CeresClone31044 SEQ ID NO: 2001 38635 Identifier:
Ceres CLONE ID no. 38635 Lead_CeresClone38635 SEQ ID NO: 2008 39155
Identifier: Ceres CLONE ID no. 39155 Lead_CeresClone39155 SEQ ID
NO: 2011 107988 Identifier: Ceres CLONE ID no. 107988
Lead_CeresClone107988 SEQ ID NO: 2017 109912 Identifier: Ceres
CLONE ID no. 109912 Lead_CeresClone109912 SEQ ID NO: 2020 154718
Identifier: Ceres CLONE ID no. 154718 Lead_CeresClone154718 SEQ ID
NO: 2024 226122 Identifier: Ceres CLONE ID no. 226122
Lead_CeresClone226122 SEQ ID NO: 2039 691319 Identifier: Ceres
CLONE ID no. 691319 Lead_CeresClone691319 SEQ ID NO: 2045 641
Identifier: Ceres CLONE ID no. 641 Lead_CeresClone641 SEQ ID NO:
2050 3819 Identifier: Ceres CLONE ID no. 3819 Lead_CeresClone3819
SEQ ID NO: 2069 3853 Identifier: Ceres CLONE ID no. 3853
Lead_CeresClone3853 SEQ ID NO: 2074 8133 Identifier: Ceres CLONE ID
no. 8133 Lead_CeresClone8133 SEQ ID NO: 2079 15343 Identifier:
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Identifier: Ceres CLONE ID no. 22007 Lead_CeresClone22007 SEQ ID
NO: 2088 23771 Identifier: Ceres CLONE ID no. 23771
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Identifier: Ceres CLONE ID no. 34210 Lead_CeresClone34210 SEQ ID
NO: 2130 38757 Identifier: Ceres CLONE ID no. 38757
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Identifier: Ceres CLONE ID no. 39127 Lead_CeresClone39127 SEQ ID
NO: 2154 95855 Identifier: Ceres CLONE ID no. 95855
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ID no. 99763 Lead_CeresClone99763 SEQ ID NO: 2170 267657
Identifier: Ceres CLONE ID no. 267657 Lead_CeresClone267657 SEQ ID
NO: 2173 545208 Identifier: Ceres CLONE ID no. 545208
Lead_CeresClone545208 SEQ ID NO: 2178 546490 Identifier: Ceres
CLONE ID no. 546490 Lead_CeresClone546490 SEQ ID NO: 2181 566317
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Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090133156A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090133156A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References