U.S. patent application number 14/678629 was filed with the patent office on 2015-10-22 for expression of microbial proteins in plants for production of plants with improved properties.
This patent application is currently assigned to MONSANTO TECHNOLOGY LLC. The applicant listed for this patent is Monsanto Technology LLC. Invention is credited to Yongwei Cao, Xianfeng Chen, Barry S. Goldman, Gregory J. Hinkle, Steven C. Slater.
Application Number | 20150299720 14/678629 |
Document ID | / |
Family ID | 29739483 |
Filed Date | 2015-10-22 |
United States Patent
Application |
20150299720 |
Kind Code |
A1 |
Cao; Yongwei ; et
al. |
October 22, 2015 |
EXPRESSION OF MICROBIAL PROTEINS IN PLANTS FOR PRODUCTION OF PLANTS
WITH IMPROVED PROPERTIES
Abstract
Recombinant constructs and methods useful for improvement of
plants are provided. In particular, recombinant constructs
comprising promoters functional in plant cells positioned for
expression of polynucleotides encoding polypeptides from microbial
sources are provided. The disclosed constructs and methods find use
in production of transgenic plants to provide plants, particularly
crop plants, having improved properties.
Inventors: |
Cao; Yongwei; (Chesterfield,
MO) ; Hinkle; Gregory J.; (Plymouth, MA) ;
Slater; Steven C.; (Middleton, WI) ; Chen;
Xianfeng; (Wildwood, MO) ; Goldman; Barry S.;
(St. Louis, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Monsanto Technology LLC |
St. Louis |
MO |
US |
|
|
Assignee: |
MONSANTO TECHNOLOGY LLC
St. Louis
MO
|
Family ID: |
29739483 |
Appl. No.: |
14/678629 |
Filed: |
April 3, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11980183 |
Oct 29, 2007 |
9024113 |
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14678629 |
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10369493 |
Feb 20, 2003 |
7314974 |
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11980183 |
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60360039 |
Feb 21, 2002 |
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Current U.S.
Class: |
800/279 ;
435/320.1; 800/278; 800/281; 800/289; 800/290; 800/298; 800/306;
800/312; 800/314; 800/317.3; 800/320; 800/320.1; 800/320.2;
800/320.3; 800/322 |
Current CPC
Class: |
Y02A 40/146 20180101;
C12N 15/8273 20130101; C12N 15/8279 20130101; C12N 15/8243
20130101; C12N 15/8247 20130101; C12N 15/8241 20130101; C12N
15/8261 20130101; C12N 15/8251 20130101; C12N 15/8274 20130101;
C07K 14/195 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Claims
1. A recombinant DNA construct comprising a promoter functional in
a plant cell, wherein said promoter is positioned to provide for
expression of a polynucleotide encoding a polypeptide, and wherein
said polynucleotide is selected from the group consisting of: (a) a
polynucleotide comprising a nucleic acid sequence having at least
80% sequence identity to a reference nucleic acid sequence selected
from the group consisting of SEQ ID NO:1 through SEQ ID NO:23687;
(b) a polynucleotide encoding a polypeptide having an amino acid
sequence having at least 80% sequence identity to a reference amino
acid sequence selected from the group consisting of SEQ ID NO:23688
through SEQ ID NO:47374; and (c) a polynucleotide encoding a
polypeptide that is a functional homolog of a polypeptide selected
from the group consisting of SEQ ID NO:23688 through SEQ ID
NO:47374.
2. A transgenic plant comprising a recombinant DNA construct,
wherein said construct comprises a promoter functional in a plant
cell, wherein said promoter is positioned to provide for expression
of a polynucleotide encoding a polypeptide, and wherein said
polynucleotide is selected from the group consisting of: (a) a
polynucleotide comprising a nucleic acid sequence selected from the
group consisting of SEQ ID NO:1 through SEQ ID NO:23687; (b) a
polynucleotide encoding a polypeptide having an amino acid sequence
selected from the group consisting of SEQ ID NO:23688 through SEQ
ID NO:47374: and (c) a polynucleotide encoding a polypeptide that
is a functional homolog of a polypeptide selected from the group
consisting of SEQ ID NO:23688 through SEQ ID NO:47374.
3. The transgenic plant according to claim 2, wherein said plant is
a crop plant.
4. The transgenic plant according to claim 3, wherein said plant is
maize.
5. The transgenic plant according to claim 3, wherein said plant is
soybean.
6.-16. (canceled)
17. The transgenic plant according to claim 2, wherein said plant
is soybean, cotton, canola, wheat, sunflower, sorghum, alfalfa,
barley, millet, rice, tobacco, a fruit, a vegetable, or turf grass
plant.
18. The transgenic plant according to claim 2, wherein said nucleic
acid sequence has at least 90% sequence identity to a reference
nucleic acid sequence selected from the group consisting of SEQ ID
NO:1 through SEQ ID NO:23687.
19. The transgenic plant according to claim 18, wherein said
nucleic acid sequence has least 95% sequence identity to a
reference nucleic acid sequence selected from the group consisting
of SEQ ID NO:1 through SEQ ID NO:23687.
20. The transgenic plant according to claim 19, wherein said
nucleic acid sequence has at least 98% sequence identity to a
reference nucleic acid sequence selected from the group consisting
of SEQ ID NO:1 through SEQ ID NO:23687.
21. The transgenic plant according to claim 20, wherein said
nucleic acid sequence has at least 99% sequence identity to a
reference nucleic acid sequence selected from the group consisting
of SEQ ID NO:1 through SEQ ID NO:23687.
22. The transgenic plant according to claim 21, wherein said
nucleic acid sequence is selected from the group consisting of SEQ
ID NO:1 through SEQ ID NO:23687.
23. The transgenic plant according to claim 2, wherein said amino
acid sequence has at least 90% sequence identity to a reference
amino acid sequence selected from the group consisting of SEQ ID
NO:23688 through SEQ ID NO:47374.
24. The transgenic plant according to claim 23, wherein said amino
acid sequence has at least 95% sequence identity to a reference
amino acid sequence selected from the group consisting of SEQ ID
NO:23688 through SEQ ID NO:47374.
25. The transgenic plant according to claim 24, wherein said amino
acid sequence is selected from the group consisting of SEQ ID
NO:23688 through SEQ ID NO:47374.
26. The transgenic plant according to claim 2, wherein said
functional homolog comprises at least 50 consecutive amino acids
having at least 80% identity to a 50 amino acid polypeptide
fragment of a reference sequence selected from the group consisting
of SEQ ID NO:23688 through SEQ ID NO:47374.
27. The transgenic plant according to claim 2, wherein said
polynucleotide further encodes a transit peptide for targeting to a
plastid.
28. A method of producing a transgenic plant having an improved
property, wherein said method comprises transforming a plant with a
recombinant construct comprising a promoter functional in a plant
cell, wherein said promoter is positioned to provide for expression
of a polynucleotide associated with said improved property, and
growing said transformed plant, wherein said polynucleotide is
selected from the group consisting of: (a) a polynucleotide
comprising a nucleic acid sequence having at least 80% sequence
identity to a reference nucleic acid sequence selected from the
group consisting of SEQ ID NO:1 through SEQ ID NO:23687; (b) a
polynucleotide encoding a polypeptide having an amino acid sequence
having at least 80% sequence identity to a reference amino acid
sequence selected from the group consisting of SEQ ID NO:23688
through SEQ ID NO:47374; and (c) a polynucleotide encoding a
polypeptide that is a functional homolog of a polypeptide selected
from the group consisting of SEQ ID NO:23688 through SEQ ID
NO:47374.
29. The method according to claim 28, wherein said transgenic plant
is soy, cotton, canola, wheat, sunflower, sorghum, alfalfa, barley,
millet, rice, tobacco, a fruit, a vegetable, or turf grass.
30. The method according to claim 28, wherein said improved quality
is selected from the group consisting of cold tolerance, disease
control, drought tolerance, plant growth, heat tolerance, herbicide
tolerance, osmotic tolerance, pathogen tolerance, pest tolerance,
increased seed oil yield, increased seed protein yield, and
increased yield.
31. The method according to claim 28, wherein said functional
homolog comprises at least.50 consecutive amino acids having at
least 80% identity to a 50 amino acid polypeptide fragment of a
reference sequence selected from the group consisting of SEQ ID
NO:23688 through SEQ ID NO:47374.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of and claims priority to
Ser. No. 10/369,493, filed Feb. 20, 2003 (pending with allowed
claims) which claims priority under 35 U.S.C. .sctn.119(e) of U.S.
Provisional Application No. 60/360,039 filed Feb. 21, 2002, the
disclosures of which applications are incorporated herein by
reference in their entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] Two copies of the sequence listing (Copy 1 and Copy 2) on
CD-ROMs, each containing the file named pa.sub.--00433.rpt, which
is 136,695,099 bytes (measured in MS-WINDOWS) and was created on
Feb. 7, 2003, are herein incorporated by reference. The compliant
computer readable copy of the sequence listing already on file in
Ser. No. 10/369,493 is identical to the sequence listing in this
application and should be used in this application.
Incorporation of Tables
[0003] Tables 1 through 21 referenced below in the detailed
description are found in parent application Ser. No. 10/369,483 and
are incorporated herein by reference.
FIELD OF THE INVENTION
[0004] Disclosed herein are inventions in the field of plant
biochemistry and genetics. More specifically methods of producing
transgenic plants having improved properties as the result of
expression of microbial polypeptides are provided.
SUMMARY OF THE INVENTION
[0005] This invention provides recombinant DNA constructs which
provide for expression in plant cells of polypeptides encoded by
microbial genes. Expression of such polypeptides in transgenic
plants leads to plants having improved phenotypic properties and/or
improved response to stressful environmental conditions. Of
particular interest are recombinant DNA constructs, wherein said
constructs comprise a promoter functional in a plant cell, wherein
said promoter is positioned to provide for expression of a
polynucleotide encoding a polypeptide from a microbial source,
wherein said polynucleotide is selected from the group consisting
of: [0006] (a) a polynucleotide comprising a nucleic acid sequence
selected from the group consisting of SEQ ID NO:1 through SEQ ID
NO:23687; [0007] (b) a polynucleotide encoding a polypeptide having
an amino acid sequence selected from the group consisting of SEQ ID
NO:23688 through SEQ ID NO:47374; [0008] (c) a polynucleotide
having at least 70% sequence identity to a polynucleotide of (a) or
(b); [0009] (d) a polynucleotide encoding a polypeptide having at
least 80% sequence identity to a polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO:23688
through SEQ ID NO:47374, wherein said encoded polypeptide is a
functional homolog of said polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO:23688
through SEQ ID NO:47374.
[0010] Such constructs are useful for production of transgenic
plants having at least one improved biological property as the
result of expression of a polypeptide using a construct of this
invention. Improved properties of interest include yield, disease
resistance, growth rate, stress tolerance and others as set forth
in more detail herein.
[0011] The present invention also provides a method of improving a
biological property of a plant by inserting into cells of said
plant a recombinant DNA construct of the present invention.
[0012] This invention also provides transformed plants, preferably
transformed crop plants, comprising a recombinant DNA construct of
the present invention, and having an improved biological property
as the result of the expression of a microbial polypeptide from
said recombinant DNA construct.
DETAILED DESCRIPTION OF THE INVENTION
[0013] The present invention provides recombinant DNA constructs,
wherein said constructs comprise a promoter functional in a plant
cell, positioned to provide for expression of a polynucleotide
encoding a polypeptide from a microbial source. Microbial
polypeptides of interest for expression from such constructs are
provided herein and are selected for their ability to impart
improved properties to transformed plants as the result of
modification of any one or more of a variety of plant
phenotypes.
[0014] The constructs of the present invention find use in
generation of transgenic plants to provide for expression of
polypeptides encoded by polynucleotides, including the native
microbial polynucleotides described herein. As a result of such
biotechnological applications, plants, particularly crop plants,
having improved properties are obtained. Crop plants of interest in
the present invention include, but are not limited to soy, cotton,
canola, maize, wheat, sunflower, sorghum, alfalfa, barley, millet,
rice, tobacco, fruit and vegetable crops, and turf grass. Of
particular interest are expression of the disclosed polypeptides to
provide plants having improved yield resulting from improved
utilization of key biochemical compounds, such as nitrogen,
phosphorous or carbohydrate, or resulting from improved responses
to environmental stresses, such as cold, heat, drought or salt, or
improved response to attack by pests or pathogens. Constructs of
the present invention may also be used to provide plants having
improved growth and development, and ultimately increased yield, as
the result of modified expression of growth regulators or
modification of cell cycle or photosynthesis pathways. Other traits
of interest that may be modified in plants using constructs of the
present invention include seed oil and protein quantity and
quality, herbicide tolerance and rate of homologous
recombination.
[0015] The polynucleotides or polypeptides from a microbial source
as used in this invention may be isolated from the source organism
or may be obtained in some other manner, for example by de novo
synthesis of polynucleotides. Thus, as used herein, the term
"microbial source" indicates that the molecule was identified as
naturally existing in a microbe, but does not necessarily indicate
the molecule itself was specifically isolated from the source
organism.
[0016] As used herein a "transgenic" organism is one whose genome
has been altered by the incorporation of foreign genetic material
or additional copies of native genetic material, e.g. by
transformation or recombination.
[0017] It is understood that the molecules of the invention may be
labeled with reagents that facilitate detection of the molecule. As
used herein, a label can be any reagent that facilitates detection,
including fluorescent labels (Prober, et al., Science 238:336-340
(1987); Albarella et al., EP 144914), chemical labels (Sheldon et
al., U.S. Pat. No. 4,582,789; Albarella et al., U.S. Pat. No.
4,563,417), or modified bases (Miyoshi et al., EP 119448),
including nucleotides with radioactive elements, e.g. .sup.32P,
.sup.33P, .sup.35S or .sup.125I such as .sup.32P
deoxycytidine-5'-triphosphate (.sup.32PdCTP).
[0018] Polynucleotides are capable of specifically hybridizing to
other polynucleotides under certain circumstances. As used herein,
two polynucleotides are said to be capable of specifically
hybridizing to one another if the two molecules are capable of
forming an anti-parallel, double-stranded nucleic acid structure. A
nucleic acid molecule is said to be the "complement" of another
nucleic acid molecule if the molecules exhibit complete
complementarity. As used herein, molecules are said to exhibit
"complete complementarity" when every nucleotide in each of the
molecules is complementary to the corresponding nucleotide of the
other. Two molecules are said to be "minimally complementary" if
they can hybridize to one another with sufficient stability to
permit them to remain annealed to one another under at least
conventional "low-stringency" conditions. Similarly, the molecules
are said to be "complementary" if they can hybridize to one another
with sufficient stability to permit them to remain annealed to one
another under conventional "high-stringency" conditions.
Conventional stringency conditions are described by Sambrook et
al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring
Harbor Press, Cold Spring Harbor, New York (1989), and by Haynes et
al., Nucleic Acid Hybridization, A Practical Approach, IRL Press,
Washington, D.C. (1985).
[0019] Departures from complete complementarity are therefore
permissible, as long as such departures do not completely preclude
the capacity of the molecules to form a double-stranded structure.
Thus, in order for a nucleic acid molecule to serve as a primer or
probe it need only be sufficiently complementary in sequence to be
able to form a stable double-stranded structure under the
particular solvent and salt concentrations employed. Appropriate
stringency conditions which promote DNA hybridization are, for
example, 6.0 X sodium chloride/sodium citrate (SSC) at about
45.degree. C., followed by a wash of 2.0 X SSC at 50.degree. C.
Such conditions are known to those skilled in the art and can be
found, for example in Current Protocols in Molecular Biology, John
Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Salt concentration and
temperature in the wash step can be adjusted to alter hybridization
stringency. For example, conditions may vary from low stringency of
about 2.0 x SSC at 40.degree. C. to moderately stringent conditions
of about 2.0 x SSC at 50.degree. C. to high stringency conditions
of about 0.2 x SSC at 50.degree. C. As used herein "sequence
identity" refers to the extent to which two optimally aligned
polynucleotide or peptide sequences are invariant throughout a
window of alignment of components, e.g. nucleotides or amino acids.
An "identity fraction" for aligned segments of a test sequence and
a reference sequence is the number of identical components which
are shared by the two aligned sequences divided by the total number
of components in the reference sequence segment, i.e. the entire
reference sequence or a smaller defined part of the reference
sequence. "Percent identity" is the identity fraction times 100.
Comparison of sequences to determine percent identity can be
accomplished by a number of well-known methods, including for
example by using mathematical algorithms, such as those in the
BLAST suite of sequence analysis programs.
[0020] Polynucleotides--This invention utilizes polynucleotides
that encode polypeptides identified from a microbial source. The
encoded polypeptides may be the complete protein encoded by an
identified microbial gene, or may be fragments of the encoded
protein. Preferably, polynucleotides utilized herein encode
polypeptides constituting a substantial portion of the complete
protein, and more preferentially, constituting a sufficient portion
of the complete protein to provide the relevant biological
activity.
[0021] Of particular interest are polynucleotides that encode
polypeptides involved in one or more important biological functions
that are common between microbes and plants. Such polynucleotides
may be expressed in transgenic plants to produce plants having
improved phenotypic properties and/or improved response to
stressful environmental conditions. See, for example, Tables 3-21
for a list of improved plant properties and responses and the SEQ
ID NOs representing exemplary polynucleotides that may be expressed
in transgenic plants to impart such improvements.
[0022] Polynucleotides of the present invention are generally used
to impart such biological properties by providing for enhanced
protein activity in a transgenic organism, preferably a transgenic
plant. Enhanced protein activity is evaluated by reference to a
wild type cell or organism and can be determined by direct or
indirect measurement. Direct measurement of protein activity might
include an analytical assay for the protein, per se, or enzymatic
product of protein activity. Indirect assay might include
measurement of a property affected by the protein.
[0023] The polynucleotides that find use in this invention
represent genes from a variety of bacterial and fungal sources as
shown in Table 1. Nucleic acid sequences of polynucleotides for use
in the constructs of the present invention are exemplified herein
by the native microbial polynucleotide sequences provided as SEQ ID
NO: 1 through SEQ ID NO: 23687.
[0024] Also of interest for use in the constructs of the present
invention are variants of the polynucleotides provided herein. Such
variants may be naturally occurring, including homologous.
polynucleotides from the same or a different species, or may be
non-natural variants, for example polynucleotides synthesized using
chemical synthesis methods, or generated using recombinant DNA
techniques. Degeneracy of the genetic code provides the possibility
to substitute at least one base of the protein encoding sequence of
a gene with a different base without causing the amino acid
sequence of the polypeptide produced from the gene to be changed.
Hence, the DNA utilized in the present invention may also have any
base sequence that has been changed from SEQ ID NO: 1 through SEQ
ID NO: 23687 by substitution in accordance with degeneracy of the
genetic code.
[0025] Also of interest regarding variant sequences for expression
of microbial polypeptides in plants is the use of polynucleotides
optimized for efficient expression of the encoded polypeptide in
plants. For example, the encoding polynucleotides can be
synthesized or modified using plant preferred codons for improved
expression. It is recognized that all or any part of the encoding
sequence may be optimized by synthesis or modification, and that
partially optimized polynucleotides are also of interest for
expression of microbial polypeptides for modification of plant
properties. Codon usage tables may be used to identify plant
preferred codons, for example by identifying the codons of highest
frequency in the proteins expressed in the largest amount in the
particular plant species of interest, and to avoid the use of
codons that are rarely found in plants. References describing codon
usage include: U.S. Pat. No. 5,500,365, Nakamura et al. (2000)
Nucl. Acids Res.28 :292, Perlak et al. (1991) Proc. Natl. Acad.
Sci. USA 88:3324-3328, Carels et al. (1998) J. Mol. Evol. 46: 45
and Fennoy et al. (1993) Nucl. Acids Res. 21(23):5294. Codon usage
tables for a number of plant species are also available from the
Department of Plant Gene Research at the Kazusa DNA Research
Institute, Japan, for example at www.kazusa.or.jp/codon/.
Additional sequence modifications are known to enhance gene
expression in plant hosts. These include elimination of sequences
encoding spurious polyadenylation signals, exon-intron splice site
signals, transposon-like repeats, hairpin secondary mRNA structures
and other such well-characterized sequences which may be
deleterious to gene expression. The G-C content of the sequence may
also be adjusted to levels generally used by the target plant host.
For example, some microbial genes are very rich (>60%) in
adenine (A) and thymine (T) while plant genes are on the order of
45-55% A+T. It is also preferred that regions comprising many
consecutive A+T bases or G+C bases are disrupted since these
regions are predicted to have a higher likelihood to form hairpin
structure due to self-complementarity. Therefore, insertion of
heterogeneous base pairs would reduce the likelihood of
self-complementary secondary structure formation which are known to
inhibit transcription and/or translation in some organisms. In most
cases, the adverse effects may be minimized by using sequences
which do not contain more than five consecutive A+T or G+C.
[0026] As discussed above, modification of the DNA sequences of
wild-type genes and construction oft a completely synthetic gene
for a given amino acid sequence are both desirable methods for
increasing the expression level of non-plant genes in plant cells.
In general, regions with multiple consecutive A+T or G+C
nucleotides should be avoided. Codons should be selected avoiding
the TA and CG doublets where possible. Codon usage can be
normalized against a plant preferred codon usage table and the G+C
content preferably adjusted to about 50%. The resulting sequence
should also be examined to ensure that there are minimal putative
plant polyadenylation signals and ATTTA sequences.
[0027] Polynucleotides for use in constructs of the present
invention that are variants of the polynucleotides described herein
will generally demonstrate significant identity with the
polynucleotides provided herein. Of particular interest are
polynucleotide homologs having at least about 60% sequence
identity, at least about 70% sequence identity, at least about 80%
sequence identity, at least about 85% sequence identity, and more
preferably at least about 90%, 95% or even greater, such as 98% or
99% sequence identity with polynucleotide sequences described
herein.
[0028] Protein and Polypeptide Molecules--This invention
encompasses recombinant DNA expression constructs that provide for
production of polypeptides from microbial sources in plants and/or
plant cells. Amino acid sequences of the polypeptides of interest
for expression using constructs of the present invention are
provided herein as SEQ ID NO:23688 through SEQ ID NO:47374.
[0029] As used herein, the term "polypeptide" means an unbranched
chain of amino acid residues that are covalently linked by an amide
linkage between the carboxyl group of one amino acid and the amino
group of another. The term polypeptide can encompass whole proteins
(i.e. a functional protein encoded by a particular gene), as well
as fragments of proteins. Of particular interest are polypeptides
which represent whole proteins or a sufficient portion of the
entire protein to impart the relevant biological activity of the
protein. The term "protein" also includes molecules consisting of
one or more polypeptide chains. Thus, a polypeptide for use in
constructs of the present invention may also constitute an entire
gene product, but only a portion of a functional oligomeric protein
having multiple polypeptide chains.
[0030] Of particular interest for expression from constructs of the
present invention are polypeptides involved in one or more
important biological properties in plants. Such polypeptides may be
produced in transgenic plants to provide plants having improved
phenotypic properties and/or improved response to stressful
environmental conditions. See, Tables 3-21 for improved plant
properties and responses and the SEQ ID NOs for the polypeptides
whose expression in transgenic plants is of interest to impart such
improvements. A summary of such improved properties and
polypeptides of interest is provided below.
[0031] Yield/Nitrogen: Yield improvement by improved nitrogen flow,
sensing, uptake, storage and/or transport. Polypeptides useful for
imparting such properties include those involved in aspartate and
glutamate biosynthesis, polypeptides involved in aspartate and
glutamate transport, polypeptides associated with the TOR (Target
of Rapamycin) pathway, nitrate transporters, ammonium transporters,
chlorate transporters and polypeptides involved in tetrapyrrole
biosynthesis.
[0032] Yield/Carbohydrate: Yield improvement by effects on
carbohydrate metabolism, for example by increased sucrose
production and/or transport. Polypeptides useful for improved yield
by effects on carbohydrate metabolism include polypeptides involved
in sucrose or starch metabolism, carbon assimilation or
carbohydrate transport, including, for example sucrose transporters
or glucose/hexose transporters, enzymes involved in
glycolysis/gluconeogenesis, the pentose phosphate cycle, or
raffinose biosynthesis, and polypeptides involved in glucose
signaling, such as SNFl complex proteins.
[0033] Yield/Photosynthesis: Yield improvement resulting from
increased photosynthesis. Polypeptides useful for increasing the
rate of photosynthesis include phytochrome, photosystem I and II
proteins, electron carriers, ATP synthase, NADH dehydrogenase and
cytochrome oxidase.
[0034] Yield/Phosphorus: Yield improvement resulting from increased
phosphorus uptake, transport or utilization. Polypeptides useful
for improving yield in this manner include phosphatases and
phosphate transporters.
[0035] Yield/Stress tolerance: Yield improvement resulting from
improved plant growth and development by helping plants to tolerate
stressful growth conditions. Polypeptides useful for improved
stress tolerance under a variety of stress conditions include
polypeptides involved in gene regulation, such as
serine/threonine-protein kinases, MAP kinases, MAP kinase kinases,
and MAP kinase kinase kinases; polypeptides that act as receptors
for signal transduction and regulation, such as receptor protein
kinases; intracellular signaling proteins, such as protein
phosphatases, GTP binding proteins, and phospholipid signaling
proteins; polypeptides involved in arginine biosynthesis;
polypeptides involved in ATP metabolism, including for example
ATPase, adenylate transporters, and polypeptides involved in ATP
synthesis and transport; polypeptides involved in glycine betaine,
jasmonic acid, flavonoid or steroid biosynthesis; and homoglobin.
Enhanced activity of such polypeptides in transgenic plants will
provide changes in the ability of a plant to respond to a variety
of environmental stresses, such as chemical stress, drought stress
and pest stress.
[0036] Cold tolerance: Polypeptides of interest for improving plant
tolerance to cold or freezing temperatures include polypeptides
involved in biosynthesis of trehalose or raffinose, polypeptides
encoded by cold induced genes, fatty acyl desaturases and other
polypeptides involved in glycerolipid or membrane lipid
biosynthesis, which find use in modification of membrane fatty acid
composition, alternative oxidase, calcium-dependent protein
kinases, LEA proteins and uncoupling protein.
[0037] Heat tolerance: Polypeptides of interest for improving plant
tolerance to heat include polypeptides involved in biosynthesis of
trehalose, polypeptides involyed in glycerolipid biosynthesis or
membrane lipid metabolism (for altering membrane fatty acid
composition), heat shock proteins and mitochondrial NDK.
[0038] Osmotic tolerance: Polypeptides of interest for improving
plant tolerance to extreme osmotic conditions include polypeptides
involved in proline biosynthesis.
[0039] Drought tolerance: Polypeptides of interest for improving
plant tolerance to drought conditions include aquaporins,
polypeptides involved in biosynthesis of trehalose or wax, LEA
proteins and invertase.
[0040] Pathogen or pest tolerance: Polypeptides of interest for
improving plant tolerance to effects of plant pests or pathogens
include proteases, polypeptides involved in anthocyanin
biosynthesis, polypeptides involved in cell wall metabolism,
including cellulases, glucosidases, pectin methylesterase,
pectinase, polygalacturonase, chitinase, chitosanase, and cellulose
synthase, and polypeptides involved in biosynthesis of terpenoids
or indole for production of bioactive metabolites to provide
defense against herbivorous insects.
[0041] Cell cycle modification: Polypeptides encoding cell cycle
enzymes and regulators of the cell cycle pathway are useful for
manipulating growth rate in plants to provide early vigor and
accelerated maturation leading to improved yield. Improvements in
quality traits, such as seed oil content, may also be obtained by
expression of cell cycle enzymes and cell cycle regulators.
Polypeptides of interest for modification of cell cycle pathway
include cyclins and EIF5alpha pathway proteins, polypeptides
involved in polyamine metabolism, polypeptides which act as
regulators of the cell cycle pathway, including cyclin-dependent
kinases (CDKs), CDK-activating kinases, CDK-inhibitors, Rb and
Rb-binding proteins, and transcription factors that activate genes
involved in cell proliferation and division, such as the E2F family
of transcription factors, proteins involved in degradation of
cyclins, such as cullins, and plant homologs of tumor suppressor
polypeptides.
[0042] Seed protein yield/content: Polypeptides useful for
providing increased seed protein quantity and/or quality include
polypeptides involved in the metabolism of amino acids in plants,
particularly polypeptides involved in biosynthesis of
methionine/cystein and lysine, amino acid transporters, amino acid
efflux carriers, seed storage proteins, proteases, and polypeptides
involved in phytic acid metabolism.
[0043] Seed oil yield/content: Polypeptides useful for providing
increased seed oil quantity and/or quality include polypeptides
involved in fatty acid and glycerolipid biosynthesis,
beta-oxidation enzymes, enzymes involved in biosynthesis of
nutritional compounds, such as carotenoids and tocopherols, and
polypeptides that increase embryo size or number or thickness of
aleurone.
[0044] Disease response in plants: Polypeptides useful for
imparting improved disease responses to plants include polypeptides
encoded by cercosporin induced genes, antifungal proteins and
proteins encoded by R-genes or SAR genes. Expression of such
polypeptides in transgenic plants will provide an increase in
disease resistance ability of plants.
[0045] Galactomannanan biosynthesis: Polypeptides involved in
production of galactomannans are of interest for providing plants
having increased and/or modified reserve polysaccharides for use in
food, pharmaceutical, cosmetic, paper and paint industries.
[0046] Flavonoid/isoflavonoid metabolism in plants: Polypeptides of
interest for modification of flavonoid/isoflavonoid metabolism in
plants include cinnamate-4-hydroxylase, chalcone synthase and
flavonol synthase. Enhanced activity of such polypeptides in
transgenic plants will provide changes in the quantity and/or speed
of flavonoid metabolism in plants and may improve disease
resistance by enhancing synthesis of protective secondary
metabolites or improving signaling pathways governing disease
resistance.
[0047] Growth regulators: Polypeptides involved in production of
substances that regulate the growth of various plant tissues are of
interest in the present invention and may be used to provide
transgenic plants having altered morphologies and improved plant
growth and development profiles leading to improvements in yield
and stress response. Of particular interest are polypeptides
involved in the biosynthesis of plant growth hormones, such as
gibberellins, cytokinins, auxins, ethylene and abscisic acid, and
other proteins involved in the activity and/or transport of such
polypeptides, including for example, cytokinin oxidase,
cytokinin/purine permeases, F-box proteins, G-proteins and
phytosulfokines.
[0048] Herbicide tolerance: Polypeptides of interest for producing
plants having tolerance to plant herbicides include polypeptides
involved in the shikimate pathway, which are of interest for
providing glyphosate tolerant plants. Such polypeptides include
polypeptides involved in biosynthesis of chorismate, phenylalanine,
tyrosine and tryptophan.
[0049] Homologous recombination: Increasing the rate of homologous
recombination in plants is useful for accelerating the
introgression of transgenes into breeding varieties by
backcrossing, and to enhance the conventional breeding process by
allowing rare recombinants between closely linked genes in phase
repulsion to be identified more easily. Polypeptides useful for
expression in plants to provide increased homologous recombination
include polypeptides involved in mitosis and/or meiosis, including
for example, resolvases and polypeptide members of the RAD52
epistasis group. Lignin biosynthesis: Polypeptides involved in
lignin biosynthesis are of interest for increasing plants'
resistance to lodging and for increasing the usefulness of plant
materials as biofuels.
[0050] The function of polypeptides used in constructs of the
present invention may be known from previous experimental evidence,
or may be determined by comparison of the amino acid sequence of
the polypeptides to amino acid sequences of other polypeptides for
which a function is known. A variety of homology based search
algorithms are available to compare a query sequence to a protein
database, including for example, BLAST, FASTA, and Smith-Waterman.
In the present application, BLASTX and BLASTP algorithms are used
to provide protein function information. A number of values are
examined in order to assess the confidence of the function
assignment. Useful measurements include "E-value" (also shown as
"hit_p"), "percent identity", "percent query coverage", and
"percent hit coverage".
[0051] In BLAST, E-value, or expectation value, represents the
number of different alignments with scores equivalent to or better
than the raw alignment score, S, that are expected to occur in a
database search by chance. The lower the E value, the more
significant the match. Because database size is an element in
E-value calculations, E-values obtained by BLASTing against public
databases, such as GenBank, have generally increased over time for
any given query/entry match. In setting criteria for confidence of
polypeptide function prediction, a "high" BLAST match is considered
herein as having an E-value for the top BLAST hit provided in Table
1 of less than 1 E-30; a medium BLASTX E-value is 1E-30 to 1E-8;
and a low BLASTX E-value is greater than 1E-8.
[0052] Percent identity refers to the percentage of identically
matched amino acid residues that exist along the length of that
portion of the sequences which is aligned by the BLAST algorithm.
In setting criteria for confidence of polypeptide function
prediction, a "high" BLAST match is considered herein as having
percent identity for the top BLAST hit provided in Table 1 of at
least 70%; a medium percent identity value is 35% to 70%; and a low
percent identity is less than 35%.
[0053] Of particular interest in protein function assignment in the
present invention is the use of combinations of E-values, percent
identity, query coverage and hit coverage. Query coverage refers to
the percent of the query sequence that is represented in the
[0054] BLAST alignment. Hit coverage refers to the percent of the
database entry that is represented in the BLAST alignment. In the
present invention, function of a query polypeptide is inferred from
function of a protein homolog where either (1) (hit_p<le-30 or %
identity >35%) AND query_coverage>50% AND
hit_coverage>50%, or (2) hit_p <1 e-8 AND query_coverage
>70% AND hit_coverage >70%.
[0055] Functional homologs which differ in one or more amino acids
from those of a polypeptide described herein as the result of one
or more conservative amino acid substitutions are also of interest
for expression in plants using the constructs of the present
invention. It is well known in the art that one or more amino acids
in a native sequence can be substituted with at least one other
amino acid, the charge and polarity of which are similar to that of
the native amino acid, resulting in a silent change. For instance,
valine is a conservative substitute for alanine and threonine is a
conservative substitute for serine. Conservative substitutions for
an amino acid within the native polypeptide sequence can be
selected from other members of the class to which the naturally
occurring amino acid belongs. Amino acids can be divided into the
following four groups: (1) acidic amino acids, (2) basic amino
acids, (3) neutral polar amino acids, and (4) neutral nonpolar
amino acids. Representative amino acids within these various groups
include, but are not limited to: (1) acidic (negatively charged)
amino acids such as aspartic acid and glutamic acid; (2) basic
(positively charged) amino acids such as arginine, histidine, and
lysine; (3) neutral polar amino acids such as glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine; and (4)
neutral nonpolar (hydrophobic) amino acids such as alanine,
leucine, isoleucine, valine, proline, phenylalanine, tryptophan,
and methionine. Conserved substitutes for an amino acid within a
native amino acid sequence can be selected from other members of
the group to which the naturally occurring amino acid belongs. For
example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine; a group of amino
acids having aliphatic-hydroxyl side chains is serine and
threonine; a group of amino acids having amide-containing side
chains is asparagine and glutamine; a group of amino acids having
aromatic side chains is phenylalanine, tyrosine, and tryptophan; a
group of amino acids having basic side chains is lysine, arginine,
and histidine; and a group of amino acids having sulfur-containing
side chains is cysteine and methionine.
[0056] Naturally conservative amino acids substitution groups are:
valine-leucine, valine-isoleucine, phenylalanine-tyrosine,
lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and
asparagine-glutamine. Also of interest for expression in transgenic
plants using constructs as described herein are polypeptides which
differ in one or more amino acids from those of a microbial
polypeptide described herein as the result of deletion or insertion
of one or more amino acids in a native sequence.
[0057] Also of interest for use in constructs of the present
invention are functional homologs of the polypeptides described
herein which have the same function as a microbial polypeptide
provided herein, but with increased or decreased activity or
altered specificity. Such variations in protein activity may exist
naturally in polypeptides encoded by related genes, for example in
a related polypeptide encoded by a different allele or in a
different species, or can be achieved by mutagenesis. Naturally
occurring variant polypeptides may be obtained by well known
nucleic acid or protein screening methods using DNA or antibody
probes, for example by screening libraries for genes encoding
related polypeptides, or in the case of expression libraries, by
screening directly for variant polypeptides. Screening methods for
obtaining a modified protein or enzymatic activity of interest by
mutagenesis are disclosed in U.S. Pat. No. 5,939,250. An
alternative approach to the generation of variants uses random
recombination techniques such as "DNA shuffling" as disclosed in
U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721 and 5,837,458; and
International Applications WO 98/31837 and WO 99/65927, all of
which are incorporated herein by reference. An alternative method
of molecular evolution involves a staggered extension process
(StEP) for in vitro mutagenesis and recombination of nucleic acid
molecule sequences, as disclosed in U.S. Pat. No. 5,965,408 and
International Application WO 98/42832, both of which are
incorporated herein by reference.
[0058] Polypeptide variants useful for expression in transgenic
plants will generally demonstrate significant identity with the
polypeptides described herein. Of particular interest are
polypeptides having at least about 35% sequence identity, at least
about 50% sequence identity, at least about 60% sequence identity,
at least about 70% sequence identity, at least about 80% sequence
identity, and more preferably at least about 85%, 90%, 95% or even
greater, sequence identity with polypeptide sequences described
herein. Of particular interest in the present invention are
polypeptides having amino acid sequences provided herein (reference
polypeptides) and functional homologs of such reference
polypeptides, wherein such functional homologs comprises at least
50 consecutive amino acids having at least 80% identity to a 50
amino acid polypeptide fragment of said reference polypeptide.
[0059] Recombinant DNA Constructs--The present invention
encompasses the use of polynucleotides described herein in
recombinant constructs, i.e. constructs comprising polynucleotides
that are constructed or modified outside of cells and that join
nucleic acids that are not found joined in nature. Using methods
known to those of ordinary skill in the art, polypeptide encoding
sequences of this invention can be inserted into recombinant DNA
constructs that can be introduced into a host cell of choice for
expression of the encoded protein. Of particular interest in the
present invention is the use of the polynucleotides of the present
invention for preparation of constructs for use in plant
transformation.
[0060] In plant transformation, exogenous genetic material is
transferred into a plant cell. By "exogenous" it is meant that a
nucleic acid molecule, for example a recombinant DNA construct
comprising a polynucleotide of the present invention, is produced
outside the organism, e.g. plant, into which it is introduced. An
exogenous nucleic acid molecule can have a naturally occurring or
non-naturally occurring nucleotide sequence. One skilled in the art
recognizes that an exogenous nucleic acid molecule can be derived
from the same species into which it is introduced or from a
different species. Such exogenous genetic material may be
transferred into either monocot or dicot plants including, but not
limited to, soy, cotton, canola, maize, teosinte, wheat, rice and
Arabidopsis plants. Transformed plant cells comprising such
exogenous genetic material may be regenerated to produce whole
transformed plants.
[0061] Exogenous genetic material may be transferred into a plant
cell by the use of a
[0062] DNA vector or construct designed for such a purpose. A
construct can comprise a number of sequence elements, including
promoters, encoding regions, and selectable markers. Vectors are
available which have been designed to replicate in both E. coli and
A. tumefaciens and have all of the features required for
transferring large inserts of DNA into plant chromosomes. Design of
such vectors is generally within the skill of the art. See, for
example, Plant Molecular Biology: A Laboratory Manual, Clark (ed.),
Springier, New York (1997).
[0063] A construct will generally include a plant promoter to
direct transcription of the protein encoding region of choice.
Numerous promoters that are active in plant cells have been
described in the literature. These include the nopaline synthase
(NOS) promoter and octopine synthase (OCS) promoters carried on
tumor-inducing plasmids of Agrobacterium tumefaciens, caulimovirus
promoters such as the cauliflower mosaic virus (CaMV) 19S promoter
(Lawton et al., Plant Mol. Biol. 9:315-324 (1987) and 35S promoter
(Odell et al., Nature 313:810-812 (1985), CaMV enhanced 35s
promoter and the figwort mosaic virus 35S-promoter. Other desirable
promoters include the light-inducible promoter from the small
subunit of ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO), the
actin 1 promoter from rice (McElroy et al. (1991) Mol. Gen. Genet.
231:150-160) or maize (Wang et al. (1992) Molecular and Cellular
Biology 12:3399-3406), the Adh promoter (Walker et al., Proc. Natl.
Acad. Sci. (U.S.A.) 84:6624-6628 (1987), the sucrose synthase
promoter (Yang et al. (1990) Proc. Natl. Acad. Sci. (U.S.A.)
87:4144-4148), the R gene complex promoter (Chandler et al. (1989)
The Plant Cell 1:1175-1183), and the chlorophyll a/b binding
protein gene promoter. These promoters and numerous others have
been used to create DNA constructs for expression in plants. See,
for example, PCT publication WO 84/02913. Any promoter known or
found to cause transcription of DNA in plant cells can be used in
the invention. Other useful promoters are described, for example,
in U.S. Pat. Nos. 5,378,619; 5,391,725; 5,428,147; 5,447,858;
5,608,144; 5,608,144; 5,614,399; 5,633,441; 5,633,435; and
4,633,436, all of which are incorporated herein by reference.
[0064] In addition, promoter enhancers, such as the CaMV 35S
enhancer (Kay et al. (1987) Science 236:1299-1302) or a tissue
specific enhancer (Fromm et al. (1989) The Plant Cell 1:977-984),
may be used to enhance gene transcription levels. Enhancers often
are found 5' to the start of transcription in a promoter that
functions in eukaryotic cells, but can often be inserted in the
forward or reverse orientation 5' or 3' to the coding sequence. In
some instances, these 5' enhancing elements are introns. Deemed to
be particularly useful as enhancers are the 5' introns of the rice
actin 1 and rice actin 2 genes. Examples of other enhancers which
can be used in accordance with the invention include elements from
octopine synthase genes (Ellis et al. (1987) EMBO Journal
6:3203-3208), the maize alcohol dehydrogenase gene intron 1 (Callis
et al. (1987) Genes and Develop. 1:1183-1200), elements from the
maize shrunken 1 gene, the sucrose synthase intron (Vasil et aL
(1989) Plant Physiol. 91:1575-1579) and the TMV omega element
(Gallie et al. (1989) The Plant Cell 1:301-311), and promoters from
non-plant eukaryotes (e.g., yeast; Ruden et al. (1988) Proc Natl.
Acad. Sci. 85:4262-4266). DNA constructs can also contain one or
more 5' non-translated leader sequences which serve to enhance
polypeptide production from the resulting mRNA transcripts. Such
sequences may be derived from the promoter selected to express the
gene or can be specifically modified to increase translation of the
mRNA. Such regions may also be obtained from viral RNAs, from
suitable eukaryotic genes, or from a synthetic gene sequence. For a
review of optimizing expression of transgenes, see Koziel et aL
(1996) Plant Mol. Biol. 32:393-405).
[0065] Constructs and vectors may also include, with the coding
region of interest, a nucleic acid sequence that acts, in whole or
in part, to terminate transcription of that region. One type of 3'
untranslated sequence which may be used is a 3' UTR from the
nopaline synthase gene (nos 3') of Agrobacterium tumefaciens (Bevan
et al.(1983) Nucleic Acids Res. 11:369-385). Other 3' termination
regions of interest include those from a gene encoding the small
subunit of a ribulose-1,5-bisphosphate carboxylase-oxygenase
(rbcS), and more specifically, from a rice rbcS gene (PCT
Publication WO 00/70066), the 3' UTR for the T7 transcript of
Agrobacterium tumefaciens (Dhaese et al. (1983) EMBO J2:419-426),
the 3' end of the protease inhibitor I or II genes from potato (An
et al. (1989) Plant Cell 1:115-122) or tomato (Pearce et aL (1991)
Science 253:895-898), and the 3' region isolated from Cauliflower
Mosaic Virus (Timmermans et al. (1990), J Biotechnol 14:333-344).
Alternatively, one also can use a gamma coixin, oleosin 3 or other
3' UTRs from the genus Coix (PCT Publication WO 99/58659).
[0066] Constructs and vectors may also include a selectable marker.
Selectable markers may be used to select for plants or plant cells
that contain the exogenous genetic material. Examples of such
include, but are not limited to, a nptII gene (Potrykus et al.
(1985) Mol. Gen. Genet. 199:183-188) which codes for kanamycin
resistance and can be selected for using kanamycin, G418, etc.; a
bar gene which codes for bialaphos resistance; a mutant EPSP
synthase gene (Hinchee et al. (1988) Bio/Technology 6:915-922)
which encodes glyphosate resistance; a nitrilase gene which confers
resistance to bromoxynil (Stalker et al. (1988) J. Biol. Chem.
263:6310-6314); a mutant acetolactate synthase gene (ALS) which
confers imidazolinone or sulphonylurea resistance (European Patent
Application 154,204 (Sep. 11, 1985)); and a methotrexate resistant
DHFR gene (Thillet et al. (1988) J. Biol. Chem.
263:12500-12508.
[0067] Constructs and vectors may also include a screenable marker.
Screenable markers may be used to monitor transformation. Exemplary
screenable markers include a .beta.-glucuronidase or uidA gene
(GUS) which encodes an enzyme for which various chromogenic
substrates are known (Jefferson (1987) Plant Mol. Biol, Rep.
5:387-405); Jefferson et al. (1987) EMBO J. 6:3901-3907); an
R-locus gene, which encodes a product that regulates the production
of anthocyanin pigments (red color) in plant tissues (Dellaporta et
al. (1988) Stadler Symposium 11:263-282); Other possible selectable
and/or screenable marker genes will be apparent to those of skill
in the art.
[0068] Constructs and vectors may also include a transit peptide
for targeting of a gene target to a plant organelle, particularly
to a chloroplast, leucoplast or other plastid organelle (European
Patent Application Publication Number 0218571).
[0069] For use in Agrobacterium mediated transformation methods,
constructs of the present invention will also include T-DNA border
regions flanking the DNA to be inserted into the plant genome to
provide for transfer of the DNA into the plant host chromosome as
discussed in more detail below. An exemplary plasmid that finds use
in such transformation methods is pCGN8640, a T-DNA vector that can
be used to clone exogenous genes and transfer them into plants
using Agrobacterium-mediated transformation. pCGN8640 has the
restriction sites BamH1, Notl, HindIII, PstI, and SacI positioned
between a 35S promoter element and a transcription terminator.
Flanking this DNA are the left border and right border sequences
necessary for Agrobacterium transformation. The plasmid also has
origins of replication for maintaining the plasmid in both E. coli
and Agrobacterium tumefaciens strains. A spectinomycin resistance
gene on the plasmid can be used to select for the presence of the
plasmid in both E. coil and Agrobacterium tumefaciens.
[0070] A candidate gene is prepared for insertion into the T-DNA
vector, for example using well-known gene cloning techniques such
as PCR. Restriction sites may be introduced onto each end of the
gene to facilitate cloning. For example, candidate genes may be
amplified by PCR techniques using a set of primers. Both the
amplified DNA and the cloning vector are cut with-the same
restriction enzymes, for example, Nod and PstI. The resulting
fragments are gel-purified, ligated together, and transformed into
E. coli. Plasmid DNA containing the vector with inserted gene may
be isolated from E. coli cells selected for spectinomycin
resistance, and the presence to the desired insert in pCGN8640
verified by digestion with the appropriate restriction enzymes.
Undigested plasmid may then be transformed into Agrobacterium
tumefaciens using techniques well known to those in the art, and
transformed Agrobacterium cells containing the vector of interest
selected based on spectinomycih resistance. These and other similar
constructs useful for plant transformation may be readily prepared
by one skilled in the art.
[0071] Transformation Methods and Transgenic Plants--Methods and
compositions for transforming bacteria and other microorganisms are
known in the art. See for example Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.).
[0072] Technology for introduction of DNA into cells is well known
to those of skill in the art. Known methods for delivering a gene
into cells include: (a) chemical methods (Graham and van der Eb
(1973) Virology 54:536-539); (b) physical methods such as
microinjection (Capecchi (1980) Cell 22:479-488), electroporation
(Wong and Neumann (1982) Biochem. Biophys. Res. Commun.
107:584-587); Fromm et al. (1985) Proc. Natl. Acad. Sci. (U.S.A.)
82:5824-5828); U.S. Pat. No. 5,384,253); the gene gun (Johnston and
Tang (1994) Methods Cell Biol. 43:353-365); (c) viral vectors
(Clapp (1993) Clin. Perinatol. 20:155-168); Lu et al. (1993) J.
Exp. Med. 178:2089-2096); Eglitis and Anderson (1988) Biotechniques
6:608-614); (d) receptor-mediated mechanisms (Curiel et al. (1992)
Hum. Gen. Ther. 3:147-154), Wagner et al. (1992) Proc. Natl. Acad.
Sci. (USA) 89:6099-6103); and (e) Agrobacterium
tumefaciens-mediated transformation of plants (Fraley et al.,
Bio/Technology 3:629-635 (1985); and Rogers et al. (1987) Methods
Enzymol. 153:253-277). In addition, DNA constructs and methods for
stably transforming plant plastids have been described; see, for
example U.S. Pat. No. 5,877,402, incorporated herein by
reference.
[0073] After transformation, the transformed plant cells or tissues
may be grown in an appropriate medium to promote cell proliferation
and regeneration. In the case of protoplasts the cell wall will
first be allowed to reform under appropriate osmotic conditions,
and the resulting callus introduced into a nutrient regeneration
medium to promote the formation of shoots and roots. For gene gun
transformation of wheat and maize see U.S. Pat. Nos. 6,153,812 and
6,160,208, both of which are incorporated herein by reference. See
also, Chistou (1996) Particle Bombardment for Genetic Engineering
of Plants, Biotechnology Intelligence Unit, Academic Press, San
Diego, Calif.), and in 30-particular, pp. 63-69 (maize), and
pp50-60 (rice).
[0074] The use of Agrobacterium-mediated plant integrating vectors
to introduce DNA into plant cells for production of stably
transformed whole plants is well known in the art. The region of
DNA to be transferred into the host genome is defined by the tDNA
border sequences in Agrobacterium-inediated plant integrating
vectors and intervening DNA is usually inserted into the plant
genome as described (Spielmann et al. (1986) Mol. Gen. Genet.
205:34). See also U.S. Pat. Nos. 5,416,011; 5,463,174; and
5,959,179 for Agrobacterium mediated transformation of soy; U.S.
Pat. Nos. 5,591,616 and 5,731,179 for Agrobacterium mediated
transformation of monocots such as maize; and U.S. Pat. Nos.
6,037,527 for Agrobacterium mediated transformation of cotton, all
of which are incorporated herein by reference. Modem Agrobacterium
transformation vectors are capable of replication in E. coli as
well as Agrobacterium, allowing for convenient manipulations (Klee
et al. (1985) In: Plant DNA Infectious Agents, Hohn and Schell
(eds.), Springer-Verlag, New York, pp. 179-203).
[0075] Microprojectile bombardment techniques are also widely
applicable, and may be used to transform virtually any plant
species. Examples of species which have been transformed by
microprojectile bombardment include monocot species such as maize
(PCT Publication WO 95/06128), barley, wheat (U.S. Pat. No.
5,563,055), rice, oats, rye, sugarcane, and sorghum, and dicot
species including tobacco, soybean (U.S. Pat. No. 5,322,783),
sunflower, cotton, tomato, and legumes in general (U.S. Pat. No.
5,563,055).
[0076] Any of the constructs of the present invention may be
introduced into a plant cell in a permanent or transient manner in
combination with other genetic elements such as vectors, promoters
enhancers etc. Further any of the constructs of the present
invention may be introduced into a plant cell in a manner that
allows for production in the plant cell of one or more polypeptides
encoded by microbial polynucleotides in the construct. It is also
to be understood that two different transgenic plants can also be
mated to produce offspring that contain two independently
segregating added, exogenous genes. Selfing of appropriate progeny
can produce plants that are homozygous for both added, exogenous
genes that encode a polypeptide of interest. Back-crossing to a
parental plant and out-crossing with a non-transgenic plant are
also contemplated, as is vegetative propagation.
[0077] Expression of microbial polynucleotides using constructs of
the present invention and the concomitant production of
polypeptides encoded by the polynucleotides is of interest for
production of transgenic plants having improved properties,
particularly, improved properties which result in crop plant yield
improvement. Expression of polypeptides in plant cells may be
evaluated by specifically identifying the protein products of the
introduced genes or evaluating the phenotypic changes brought about
by their expression.
[0078] Assays for the production and identification of specific
proteins make use of various physical-chemical, structural,
functional, or other properties of the proteins. Unique
physical-chemical or structural properties allow the protein's to
be separated and identified by electrophoretic procedures, such as
native or denaturing gel electrophoresis or isoelectric focusing,
or by chromatographic techniques such as ion exchange or gel
exclusion chromatography. The unique structures of individual
proteins offer opportunities for use of specific antibodies to
detect their presence in formats such as an ELISA assay.
Combinations of approaches may be employed with even greater
specificity such as western blotting in which antibodies are used
to locate individual gene products that have been separated by
electrophoretic techniques. Additional techniques may be employed
to absolutely confirm the identity of the product of interest such
as evaluation by amino acid sequencing following purification.
Although these are among the most commonly employed, other
procedures may be additionally used.
[0079] Assay procedures may also be used to identify the expression
of proteins by their functionality, particularly where the
expressed protein is an enzyme capable of catalyzing chemical
reactions involving specific substrates and products. These
reactions may be measured, for example in plant extracts, by
providing and quantifying the loss of substrates or the generation
of products of the reactions by physical and/or chemical
procedures.
[0080] In many cases, the expression of a gene product is
determined by evaluating the phenotypic results of its expression.
Such evaluations may be simply as visual observations, or may
involve assays. Such assays may take many forms including but not
limited to analyzing changes in the chemical composition,
morphology, or physiological properties of the plant. Chemical
composition may be altered by expression of genes encoding enzymes
or storage proteins which change amino acid composition and may be
detected by amino acid analysis, or by enzymes which change starch
quantity which may be analyzed by near infrared reflectance
spectrometry. Morphological changes may include greater stature or
thicker stalks.
[0081] In addition to the above discussed procedures, practitioners
are familiar with the standard resource materials which describe
specific conditions and procedures for the construction,
manipulation and isolation of macromolecules (e.g., DNA molecules,
plasmids, etc.), generation of recombinant organisms and the
screening and isolating of clones, (see for example, Sambrook et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Press (1989); Mailga et al., Methods in Plant Molecular Biology,
Cold Spring Harbor Press (1995; Bin: en et al., Genome Analysis:
Analyzing DNA, 1, Cold Spring Harbor, N.Y. (1998)).
[0082] Having now generally described the invention, the same will
be more readily understood through reference to the following
example which is provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
EXAMPLE
[0083] Functions of polypeptides encoded by the microbial
polynucleotide sequences described herein are determined using a
hierarchical classification tool, termed FunCAT, for Functional
Categories Annotation Tool. Most categories collected in FunCAT are
classified by function, although other criteria are used, for
example, cellular localization or temporal process. The assignment
of a functional category to a query sequence is based on BLASTX
sequence search, which compares two protein sequences. FunCAT
assigns categories by iteratively scanning through all BLAST hits,
starting with the most significant match, and reporting the first
category assignment for each FunCAT source classification scheme.
In the present invention, function of a query polypeptide is
inferred from the function of a protein homolog where either (1)
(hit_p<le-30 or % identity>35%) AND query_coverage>50% AND
hit_coverage>50%, or (2) hit_p<le-8 AND query_coverage>70%
AND hit_coverage>70%.
[0084] Functional assignments from five public classification
schemes, GO_BP, GO_CC, GO_MF, KEGG and EC, and one internal
Monsanto classification scheme, POI, are provided in Table 1. The
column under the heading "cat_type" indicates the source of the
classification. GO_BP=Gene Ontology Consortium-biological process;
GO_CC=Gene Ontology Consortium-cellular component; GO_MF=Gene
Ontology Consortium -molecular function; KEGG=KEGG functional
hierarchy; EC=Enzyme Classification from ENZYME data bank release
25.0; POI=Pathways of Interest. The column under the heading
"cat_desc" provides the name of the subcategory into which the
query sequence was classified. The column under the heading
"hit_desc" provides a description of the BLAST hit to the query
sequences that led to the specific classification. The column under
the heading "hit_p" provides the e-value for the BLAST hit.
[0085] Table 2 provides the SEQ ID NO (SEQ NUM), sequence
designation (SEQ ID) of exemplary DNA sequence encoding the
polypeptides described in Table 1. Table 2 also provides the SEQ ID
NO (Prot Num) and sequence designation (Prot ID) of the proteins
encoded by the nucleotide sequences.
[0086] Sequences useful for producing transgenic plants having
improved biological properties are identified from their FunCAT
annotations and are provided in Tables 3-21.
TABLE-US-00001 Table Biological Property 3 Cold Tolerance 4 Disease
Control 5 Drought Tolerance 6 Plant Growth/Cell Cycle 7 Plant
Growth/Growth Regulators 8 Heat Tolerance 9 Herbicide Tolerance 10
Homologous Recombination 11 Osmotic Tolerance 12 Pathogen/Pest
Tolerance 13 Seed Oil Yield/Content 14 Seed Protein Yield/Content
15 Yield: Carbohydrate 16 Yield: Nitrogen 17 Yield: Phosphorus 18
Yield: Photosynthesis 19 Yield: Stress Tolerance 20 Lignin
Biosynthesis 21 Galactomannan Biosynthesis
[0087] A biological property of particular interest is plant yield.
Plant yield may be improved by alteration of a variety of plant
pathways, including those involving nitrogen, carbohydrate, or
phosphorus utilization and/or uptake. Plant yield may also be
improved by alteration of a plant's photosynthetic capacity or by
improving a plant's ability to tolerate a variety of environmental
stresses, including cold, heat, drought and osmotic stresses. Other
biological properties of interest that may be improved using
sequences of the present invention include pathogen or pest
tolerance, herbicide tolerance, disease resistance, growth rate
(for example by modification of cell cycle or expression of growth
regulators), seed oil and/or protein yield and quality, rate and
control of recombination, and lignin content.
TABLE-US-00002 TABLE 1 Column Descriptions Seq num provides the SEQ
ID NO for the listed polynucleotide sequences. SeqID provides an
arbitrary sequence name taken from the name of the clone from which
the cDNA sequence was obtained. cat_type indicates the
classification scheme used to classify the sequence. GO_BP = Gene
Ontology Consortium-biological process; GO_CC = Gene Ontology
Consortium- cellular component; GO_MF = Gene Ontology
Consortium-molecular function; KEGG = KEGG functional hierarchy
(KEGG = Kyoto Encyclopedia of Genes and Genomes); EC = Enzyme
Classification from ENZYME data bank release 25.0; POI = Pathways
of Interest. cat_desc provides the classification scheme
subcategory to which the query sequence was assigned. hit_desc
provides the description of the BLAST hit which resulted in
assignment of the sequence to the function category provided in the
cat_desc column. hit_p provides the E value for the BLAST hit in
the hit_desc column. pct_ ident refers to the percentage of
identically matched nucleotides (or residues) that exist along the
length of that portion of the sequences which is aligned in the
BLAST match provided in hit_desc. qry_range lists the range of the
query sequence aligned with the hit. hit_range lists the range of
the hit sequence aligned with the query. qry cvrg provides the
percent of query sequence length that matches to the hit (NCBI)
sequence in the BLAST match (% qry cvrg = (match length / query
total length) .times. 100). hit cvrg provides the percent of hit
sequence length that matches to the query sequence in the match
generated using BLAST (% hit cvrg = (match length/hit total length)
.times. 100). Species provides the name of the organism from which
the cDNA was isolated. Product _concept column provides the plant
biological properties that may be modified by expression of the
listed polypeptides.
[0088] All publications and patent applications cited herein are
incorporated by reference in their entirely to the same extent as
if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0089] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding; it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
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=US20150299720A1).
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=US20150299720A1).
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