U.S. patent application number 12/316385 was filed with the patent office on 2009-06-25 for transgenic plants with enhanced agronomic traits.
Invention is credited to Pranesh Badami, Stephen M. Duff, Steve S. He, Linda L. Lutfiyya, Savitha Madappa, Dhanalakshmi Ramachandra, S. Sangeetha, T. V. Venkatesh, K. R. Vidya.
Application Number | 20090165165 12/316385 |
Document ID | / |
Family ID | 40756046 |
Filed Date | 2009-06-25 |
United States Patent
Application |
20090165165 |
Kind Code |
A1 |
Madappa; Savitha ; et
al. |
June 25, 2009 |
Transgenic plants with enhanced agronomic traits
Abstract
This invention provides transgenic plant cells with recombinant
DNA for expression of proteins that are useful for imparting
enhanced agronomic trait(s) to transgenic crop plants. This
invention also provides transgenic plants and progeny seed
comprising the transgenic plant cells where the plants are selected
for having an enhanced trait selected from the group of traits
consisting of enhanced water use efficiency, enhanced cold
tolerance, increased yield, enhanced nitrogen use efficiency,
enhanced seed protein and enhanced seed oil. Also disclosed are
methods for manufacturing transgenic seed and plants with enhanced
traits.
Inventors: |
Madappa; Savitha;
(Bangalore, IN) ; Badami; Pranesh; (Bangalore,
IN) ; Ramachandra; Dhanalakshmi; (Bangalore, IN)
; Sangeetha; S.; (Bangalore, IN) ; Vidya; K.
R.; (Bangalore, IN) ; Duff; Stephen M.; (St.
Louis, MO) ; He; Steve S.; (St. Louis, MO) ;
Lutfiyya; Linda L.; (St. Louis, MO) ; Venkatesh; T.
V.; (St. Louis, MO) |
Correspondence
Address: |
MONSANTO COMPANY
800 N. LINDBERGH BLVD., ATTENTION: GAIL P. WUELLNER, IP PARALEGAL, (E2NA)
ST. LOUIS
MO
63167
US
|
Family ID: |
40756046 |
Appl. No.: |
12/316385 |
Filed: |
December 11, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61013179 |
Dec 12, 2007 |
|
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Current U.S.
Class: |
800/275 ;
435/320.1; 435/419; 800/298; 800/306; 800/312; 800/314; 800/320;
800/320.1; 800/320.2; 800/320.3 |
Current CPC
Class: |
C12N 15/8274 20130101;
C12N 15/8251 20130101; C12N 15/8218 20130101; C12N 15/8273
20130101; C12N 9/16 20130101; C12N 9/1096 20130101; C12Y 206/01
20130101; C07K 14/415 20130101; C12N 15/8241 20130101; C12N 15/8271
20130101; C12Y 204/00 20130101; C12N 15/8247 20130101; C12N 9/1048
20130101; C12Y 301/03012 20130101 |
Class at
Publication: |
800/275 ;
800/298; 800/320.1; 800/312; 800/314; 800/306; 800/320; 800/320.2;
800/320.3; 435/419; 435/320.1 |
International
Class: |
A01H 5/00 20060101
A01H005/00; A01H 1/02 20060101 A01H001/02; C12N 5/10 20060101
C12N005/10; C12N 15/63 20060101 C12N015/63 |
Claims
1. A transgenic plant cell having recombinant DNA in its
chromosomal DNA wherein said recombinant DNA comprises (a) a
promoter that is functional in a plant cell and that is operably
linked to a polynucleotide that encodes a protein having an amino
acid sequence having at least 95% identity over at least 95% of the
length of SEQ ID NO:13 and (b) a promoter that is functional in a
plant cell and that is operably linked to a polynucleotide that
encodes a protein having an amino acid sequence having at least 95%
identity over at least 95% of the length of SEQ ID NO:14.
2. A recombinant DNA construct comprising a promoter that is
functional in a plant cell and that is operably linked to a
polynucleotide that: (a) encodes a protein having an amino acid
sequence having at least 95% identity over at least 95% of the
length of a reference sequence selected from the group consisting
of SEQ ID NO: 12-16, and 20, when said amino acid sequence is
aligned to said reference sequence; or (b) is transcribed into an
RNA molecule that suppresses the level of an endogenous protein
that has an amino acid sequence that is at least 95% identical over
at least 95% of the length of a reference sequence of SEQ ID NO:
11, and 17-19, when said amino acid sequence is aligned to said
reference sequence; wherein said construct is stably integrated
into plant chromosomal DNA.
3. A transgenic plant cell comprising the recombinant DNA construct
of claim 2 wherein said plant cell is in a plant selected by
screening a population of transgenic plants that have been
transformed with said construct for an enhanced trait as compared
to control plants; and wherein said enhanced trait is enhanced
water use efficiency, enhanced cold tolerance, increased yield,
enhanced nitrogen use efficiency, enhanced seed protein or enhanced
seed oil.
4. A mixture comprising plant cells of claim 3 and an antibody to a
protein produced in said cells wherein said protein has an amino
acid sequence that has at least 95% identity over at least 95% of
the length of a reference sequence selected from the group
consisting of SEQ ID NO: 11-20 when said amino acid sequence is
aligned to said reference sequence.
5. The plant cell of claim 3 further comprising DNA expressing a
protein that provides tolerance from exposure to an herbicide
comprising an agent applied at levels that are lethal to a wild
type of said plant cell.
6. The plant cell of claim 5 wherein the agent of said herbicide is
a glyphosate, dicamba, or glufosinate compound.
7. A transgenic plant comprising a plurality of plant cells of
claim 3.
8. The transgenic plant of claim 7 which is homozygous for said
recombinant DNA.
9. A transgenic seed comprising a plurality of plant cells of claim
3.
10. The transgenic seed of claim 9 from a corn, soybean, cotton,
canola, alfalfa, wheat, rice, sugarcane, or sugar beet plant.
11. Grain comprising transgenic seed identifiable by the
recombinant DNA construct of claim 2.
12. Seed meal produced from transgenic seed identifiable by the
recombinant DNA construct of claim 2.
13. A transgenic pollen grain comprising a haploid derivative of a
plant cell nucleus having a chromosome comprising the recombinant
DNA construct of claim 2.
14. A method for manufacturing non-natural, transgenic seed that
can be used to produce a crop of transgenic plants with an enhanced
trait resulting from expression of the stably-integrated,
recombinant DNA construct of claim 2, said method comprising: (a)
screening a population of plants for said enhanced trait and said
recombinant DNA, wherein individual plants in said population
exhibit said trait at a level less than, essentially the same as or
greater than the level that said trait is exhibited in control
plants which do not contain said recombinant DNA, wherein said
enhanced trait is selected from the group of enhanced traits
consisting of enhanced water use efficiency, enhanced cold
tolerance, increased yield, enhanced nitrogen use efficiency,
enhanced seed protein and enhanced seed oil; (b) selecting from
said population one or more plants that exhibit said trait at a
level greater than the level that said trait is exhibited in
control plants, and (c) collecting seed from selected plant from
step b.
15. The method of claim 14 wherein said method for manufacturing
said transgenic seed further comprises: (a) verifying that said
recombinant DNA is stably integrated in said selected plants, and
(b) analyzing tissue of said selected plant to determine the
expression or suppression of a protein having the function of a
protein having an amino acid sequence selected from the group
consisting of one of SEQ ID NOs:11-20.
16. The method of claim 15 wherein said seed is corn, soybean,
cotton, canola, alfalfa, wheat, rice, sugarcane, or sugar beet
seed.
17. A method of producing hybrid corn seed comprising: (a)
acquiring hybrid corn seed from an herbicide tolerant corn plant
which also has the stably-integrated, recombinant DNA construct of
claim 2; (b) producing corn plants from said hybrid corn seed,
wherein a fraction of the plants produced from said hybrid corn
seed is homozygous for said recombinant DNA, a fraction of the
plants produced from said hybrid corn seed is hemizygous for said
recombinant DNA, and a fraction of the plants produced from said
hybrid corn seed has none of said recombinant DNA; (c) selecting
corn plants which are homozygous and hemizygous for said
recombinant DNA by treating with an herbicide; (d) collecting seed
from herbicide-treated-surviving corn plants and planting said seed
to produce further progeny corn plants; (e) repeating steps (c) and
(d) at least once to produce an inbred corn line; and (f) crossing
said inbred corn line with a second corn line to produce hybrid
seed.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 USC .sctn. 119(e)
of U.S. provisional application Ser. No. 61/013,179, filed Dec. 12,
2007, incorporated herein by reference in its entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] Two copies of the sequence listing (Copy 1 and Copy 2) and a
computer readable form (CRF) of the sequence listing, all on CD-Rs,
each containing the text file named "38-21-55579-B_seqListing.txt",
which is 5,484,544 bytes (measured in MS-WINDOWS), were created on
Dec. 8, 2008 and are incorporated herein by reference.
INCORPORATION OF LARGE TABLE
[0003] Two copies of a large table (Copy 1 and Copy 2) containing a
folder "pfamdir" on CD-Rs are incorporated herein by reference in
their entirety. Folder "pfamdir" contains 10 Pfam Hidden Markov
Models. The CD-Rs were created on Dec. 8, 2008, having a total size
of 864,256 bytes (measured in MS-WINDOWS).
FIELD OF THE INVENTION
[0004] Disclosed herein are recombinant DNA useful for providing
enhanced traits to transgenic plants, seeds, pollen, plant cells
and plant nuclei of such transgenic plants, methods of making and
using such recombinant DNA, plants, seeds, pollen, plant cells and
plant nuclei. Also disclosed are methods of producing hybrid corn
seed comprising such recombinant DNA.
SUMMARY OF THE INVENTION
[0005] An aspect of this invention provides recombinant DNA
constructs comprising polynucleotides characterized by an encoded
protein having amino acids representing a protein family domain
module as described in Table 10. Another aspect of this invention
provides recombinant DNA constructs comprising polynucleotides
characterized by an encoded protein with an amino acid sequence
that is at least 90% identical to a corresponding consensus
sequence defined in table 8. Yet another aspect of this invention
provides recombinant DNA constructs comprising polynucleotides
characterized by reference to SEQ ID NO:1-10 and the cognate
proteins with amino acid sequences having reference to SEQ ID
NO:11-20. The recombinant DNA constructs are useful for providing
enhanced traits when stably integrated into the chromosomes and
expressed in the nuclei of transgenic plants cells. In most aspects
of the invention the recombinant DNA constructs, when expressed in
a plant cell, provide for expression of cognate proteins. In
particular aspects of the invention the recombinant DNA constructs
for expressing cognate proteins are characterized by cognate amino
acid sequence that have at least 95% identity over at least 95% of
the length of a reference sequence in the group of SEQ ID NOs:
12-16, and 20 when the amino acid sequence is aligned to the
reference sequence. In particularly specific embodiments of the
invention, the recombinant DNA constructs comprise polynucleotide
stacks characterized by cognate proteins having amino acid
sequences that have at least 95% identity over at least 95% of the
length of reference sequences 13-14 when the amino acid sequences
are aligned to the reference sequences. In some aspects of the
invention, i.e. the recombinant DNA constructs are characterized as
being constructed with sense-oriented and anti-sense-oriented
polynucleotides from SEQ ID NOs: 1, and 7-9 which, when expressed
in a plant cell, provide for the suppression of cognate proteins
having amino acid sequences that have at least 95% identity over at
least 95% of the length of a reference sequence in the group
consisting of SEQ ID NOs: 11 and 17-19.
[0006] In practical aspects of this invention the recombinant DNA
constructs of the invention are stably integrated into the
chromosome of a plant cell nucleus.
[0007] This invention also provides transgenic plant cells
comprising the stably integrated recombinant DNA constructs of the
invention, transgenic plants and seeds comprising a plurality of
such transgenic plant cells and transgenic pollen of such plants.
Such transgenic plants are selected from a population of transgenic
plants regenerated from plant cells transformed with recombinant
DNA constructs by screening transgenic plants for an enhanced trait
as compared to control plants. The enhanced trait is one or more of
enhanced water use efficiency, enhanced cold tolerance, increased
yield, enhanced nitrogen use efficiency, enhanced seed protein and
enhanced seed oil.
[0008] In another aspect of the invention the plant cells, plants,
seeds, and pollen further comprise DNA expressing a protein that
provides tolerance from exposure to an herbicide applied at levels
that are lethal to a wild type plant cell.
[0009] This invention also provides methods for manufacturing
non-natural, transgenic seed that can be used to produce a crop of
transgenic plants with an enhanced trait resulting from expression
of a stably-integrated recombinant DNA construct. More
specifically, the method comprises (a) screening a population of
plants for an enhanced trait and a recombinant DNA construct, where
individual plants in the population can exhibit the trait at a
level less than, essentially the same as or greater than the level
that the trait is exhibited in control plants, (b) selecting from
the population one or more plants that exhibit the trait at a level
greater than the level that said trait is exhibited in control
plants, (c) collecting seed from a selected plant, (d) verifying
that the recombinant DNA is stably integrated in said selected
plants, (e) analyzing tissue of a selected plant to determine the
production or suppression of a protein having the function of a
protein encoded by nucleotides in a sequence of one of SEQ ID
NOs:1-10. In one aspect of the invention, the plants in the
population further comprise DNA expressing a protein that provides
tolerance to exposure to a herbicide applied at levels that are
lethal to wild type plant cells and the selecting is affected by
treating the population with the herbicide, e.g. a glyphosate,
dicamba, or glufosinate compound. In another aspect of the
invention the plants are selected by identifying plants with the
enhanced trait. The methods are especially useful for manufacturing
corn, soybean, cotton, canola, alfalfa, wheat, rice, sugarcane or
sugar beet seed.
[0010] Another aspect of the invention provides a method of
producing hybrid corn seed comprising acquiring hybrid corn seed
from a herbicide tolerant corn plant which also has
stably-integrated, recombinant DNA construct comprising a promoter
that is (a) functional in plant cells and (b) is operably linked to
DNA that encodes or suppresses a protein having the function of a
protein encoded by nucleotides in a sequence of one of SEQ ID
NOs:1-10. The methods further comprise producing corn plants from
said hybrid corn seed, wherein a fraction of the plants produced
from said hybrid corn seed is homozygous for said recombinant DNA,
a fraction of the plants produced from said hybrid corn seed is
hemizygous for said recombinant DNA, and a fraction of the plants
produced from said hybrid corn seed has none of said recombinant
DNA; selecting corn plants which are homozygous and hemizygous for
said recombinant DNA by treating with an herbicide; collecting seed
from herbicide-treated-surviving corn plants and planting said seed
to produce further progeny corn plants; repeating the selecting and
collecting steps at least once to produce an inbred corn line; and
crossing the inbred corn line with a second corn line to produce
hybrid seed.
[0011] Another aspect of the invention provides a method of
selecting a plant comprising plant cells of the invention by using
an immunoreactive antibody to detect the presence or absence of
protein expressed or suppressed by recombinant DNA in seed or plant
tissue. Yet another aspect of the invention provides
anti-counterfeit milled seed having, as an indication of origin,
plant cells of this invention.
[0012] Still other aspects of this invention relate to transgenic
plants with enhanced water use efficiency or enhanced nitrogen use
efficiency. For instance, this invention provides methods of
growing a corn, cotton, soybean, or canola crop without irrigation
water comprising planting seed having plant cells of the invention
which are selected for enhanced water use efficiency. Alternatively
methods comprise applying reduced irrigation water, e.g. providing
up to 300 millimeters of ground water during the production of a
corn crop. This invention also provides methods of growing a corn,
cotton, soybean or canola crop without added nitrogen fertilizer
comprising planting seed having plant cells of the invention which
are selected for enhanced nitrogen use efficiency.
[0013] Another aspect of the invention provides a mixture
comprising plants cells and an antibody to a protein produced in
the cells where the protein has an amino acid sequence that has at
least 95% identity over at least 95% of the length of a reference
sequence selected from the group consisting of SEQ ID NO: 11-20
when the sequence is aligned to the reference sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGS. 1-4 are plasmid maps.
DETAILED DESCRIPTION OF THE INVENTION
[0015] In the attached sequence listing:
[0016] SEQ ID NO:1-10 are nucleotide sequences of the coding strand
of DNA for "genes" used in the recombinant DNA imparting an
enhanced trait in plant cells, i.e. each represents a coding
sequence for a protein;
[0017] SEQ ID NO: 11-20 are amino acid sequences of the cognate
protein of the "genes" with nucleotide coding sequences 1-10;
[0018] SEQ ID NO: 21-1351 are amino acid sequences of homologous
proteins;
[0019] SEQ ID NO: 1352 is a nucleotide sequence of a base plasmid
vector useful for corn transformation;
[0020] SEQ ID NO: 1353 is a nucleotide sequence of a base plasmid
vector useful for soybean and canola transformation;
[0021] SEQ ID NO: 1354 is a nucleotide sequence of a base plasmid
vector useful for cotton transformation;
[0022] SEQ ID NO: 1355 is a nucleotide sequence of a base plasmid
vector useful for co-transformation to produce gene stacks in
corn;
[0023] SEQ ID NO: 1356-1358 are consensus sequences.
[0024] Table 8 lists the protein SEQ ID NOs and their corresponding
consensus SEQ ID NOs.
[0025] As used herein a "plant cell" means a plant cell that is
transformed with stably-integrated, non-natural, recombinant DNA,
e.g. by Agrobacterium-mediated transformation or by bombardment
using microparticles coated with recombinant DNA or other means. A
plant cell of this invention can be an originally-transformed plant
cell that exists as a microorganism or as a progeny plant cell that
is regenerated into differentiated tissue, e.g. into a transgenic
plant with stably-integrated, non-natural recombinant DNA, or seed
or pollen derived from a progeny transgenic plant.
[0026] As used herein a "transgenic plant" means a plant whose
genome has been altered by the stable integration of recombinant
DNA. A transgenic plant includes a plant regenerated from an
originally-transformed plant cell and progeny transgenic plants
from later generations or crosses of a transformed plant.
[0027] As used herein "recombinant DNA" means DNA which has been a
genetically engineered and constructed outside of a cell including
DNA containing naturally occurring DNA or cDNA or synthetic
DNA.
[0028] As used herein "consensus sequence" means an artificial
sequence of amino acids in a conserved region of an alignment of
amino acid sequences of homologous proteins, e.g. as determined by
a CLUSTALW alignment of amino acid sequence of homolog
proteins.
[0029] As used herein a "homolog" means a protein in a group of
proteins that perform the same biological function, e.g. proteins
that belong to the same Pfam protein family and that provide a
common enhanced trait in transgenic plants of this invention.
Homologs are expressed by homologous genes. With reference to
homologous genes, homologs include orthologs, i.e. genes expressed
in different species that evolved from a common ancestral genes by
speciation and encode proteins retain the same function, but do not
include paralogs, i.e. genes that are related by duplication but
have evolved to encode proteins with different functions.
Homologous genes include naturally occurring alleles and
artificially-created variants. 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. When optimally aligned, homolog proteins
have at least 60% identity, more preferably about 65% or higher,
more preferably about 70% or higher, more preferably at least 75%,
more preferably at least 80%, more preferably at least 85%, and
even more preferably at least 90% identity over the full length of
a protein identified as being associated with imparting an enhanced
trait when expressed in plant cells. In one aspect of the invention
homolog proteins have an amino acid sequence that has at least 90%
identity to a consensus amino acid sequence of proteins and
homologs disclosed herein.
[0030] Homologs are identified by comparison of amino acid
sequence, e.g. manually or by use of a computer-based tool using
known homology-based search algorithms such as those commonly known
and referred to as BLAST, FASTA, and Smith-Waterman. A local
sequence alignment program, e.g. BLAST, can be used to search a
database of sequences to find similar sequences, and the summary
Expectation value (E-value) used to measure the sequence base
similarity. Because a protein hit with the best E-value for a
particular organism may not necessarily be an ortholog, i.e. have
the same function, or be the only ortholog, a reciprocal query is
used to filter hit sequences with significant E-values for ortholog
identification. The reciprocal query entails search of the
significant hits against a database of amino acid sequences from
the base organism that are similar to the sequence of the query
protein. A hit can be identified as an ortholog, when the
reciprocal query's best hit is the query protein itself or a
protein encoded by a duplicated gene after speciation. A further
aspect of the homologs encoded by DNA useful in the transgenic
plants of the invention are those proteins that differ from a
disclosed protein as the result of deletion or insertion of one or
more amino acids in a native sequence.
[0031] "Percent identity" describes the extent to which the
sequences of DNA or protein segments are invariant throughout a
window of alignment of sequences, for example nucleotide sequences
or amino acid sequences. An "identity fraction" for a sequence
aligned with a reference sequence is the number of identical
components which are shared by the sequences, divided by the length
of the alignment not including gaps introduced by the alignment
algorithm. "Percent identity" ("% identity") is the identity
fraction times 100. Percent identity is calculated over the aligned
length preferably using a local alignment algorithm, such as
BLASTp. As used herein, sequences are "aligned" when the alignment
produced by BLASTp has a minimal e-value.
[0032] "Pfam" is a large collection of multiple sequence alignments
and hidden Markov models covering many common protein families,
e.g. Pfam version 19.0 (December 2005) contains alignments and
models for 8183 protein families and is based on the Swissprot 47.0
and SP-TrEMBL 30.0 protein sequence databases. See S. R. Eddy,
"Profile Hidden Markov Models", Bioinformatics 14:755-763, 1998.
The Pfam database is currently maintained and updated by the Pfam
Consortium. The alignments represent some evolutionary conserved
structure that has implications for the protein's function. Profile
hidden Markov models (profile HMMs) built from the protein family
alignments are useful for automatically recognizing that a new
protein belongs to an existing protein family even if the homology
by alignment appears to be low.
[0033] Protein domains are identified by querying the amino acid
sequence of a protein against Hidden Markov Models which
characterize protein family domains ("Pfam domains") using HMMER
software, which is available from the Pfam Consortium. The HMMER
software is also disclosed in patent application publication US
2008/0148432 A1 incorporated herein by reference. A protein domain
meeting the gathering cutoff for the alignment of a particular Pfam
domain is considered to contain the Pfam domain.
[0034] A "Pfam domain module" is a representation of Pfam domains
in a protein, in order from N terminus to C terminus. In a Pfam
domain module individual Pfam domains are separated by double
colons "::". The order and copy number of the Pfam domains from N
to C terminus are attributes of a Pfam domain module. Although the
copy number of repetitive domains is important, varying copy number
often enables a similar function. Thus, a Pfam domain module with
multiple copies of a domain should define an equivalent Pfam domain
module with variance in the number of multiple copies. A Pfam
domain module is not specific for distance between adjacent
domains, but contemplates natural distances and variations in
distance that provide equivalent function. The Pfam database
contains both narrowly- and broadly-defined domains, leading to
identification of overlapping domains on some proteins. A Pfam
domain module is characterized by non-overlapping domains. Where
there is overlap, the domain having a function that is more closely
associated with the function of the protein (based on the E value
of the Pfam match) is selected.
[0035] Once one DNA is identified as encoding a protein which
imparts an enhanced trait when expressed in transgenic plants,
other DNA encoding proteins with the same Pfam domain module are
identified by querying the amino acid sequence of protein encoded
by candidate DNA against the Hidden Markov Models which
characterizes the Pfam domains using HMMER software. Candidate
proteins meeting the same Pfam domain module are in the protein
family and have cognate DNA that is useful in constructing
recombinant DNA for the use in the plant cells of this invention.
Hidden Markov Model databases for use with HMMER software in
identifying DNA expressing protein with a common Pfam domain module
for recombinant DNA in the plant cells of this invention are
included in the large table incorporated into this application.
[0036] The HMMER software and Pfam databases (version 19.0) were
used to identify known domains in the proteins corresponding to
amino acid sequence of SEQ ID NO: 11 through SEQ ID NO: 16 and SEQ
ID NO: 20. All DNA encoding proteins that have scores higher than
the gathering cutoff disclosed in Table 11 by Pfam analysis
disclosed herein can be used in recombinant DNA of the plant cells
of this invention, e.g. for selecting transgenic plants having
enhanced agronomic traits. The relevant Pfams modules for use in
this invention, as more specifically disclosed below, are
Cu-oxidase.sub.--3::Cu-oxidase::Cu-oxidase.sub.--2,
Flavodoxin.sub.--1, Glyco_transf.sub.--20::Trehalose_PPase,
Aminotran.sub.--1.sub.--2, and B3::Auxin_resp::AUX_IAA, for which
the databases are included in the appended computer listing.
[0037] As used herein "promoter" means regulatory DNA for
initializing transcription. A "plant promoter" is a promoter
capable of initiating transcription in plant cells whether or not
its origin is a plant cell, e.g. is it well known that
Agrobacterium promoters are functional in plant cells. Thus, plant
promoters include promoter DNA obtained from plants, plant viruses
and bacteria such as Agrobacterium and Bradyrhizobium bacteria.
Examples of promoters under developmental control include promoters
that preferentially initiate transcription in certain tissues, such
as leaves, roots, or seeds. Such promoters are referred to as
"tissue preferred". Promoters that initiate transcription only in
certain tissues are referred to as "tissue specific". A "cell type"
specific promoter primarily drives expression in certain cell types
in one or more organs, for example, vascular cells in roots or
leaves. An "inducible" or "repressible" promoter is a promoter
which is under environmental control. Examples of environmental
conditions that may effect transcription by inducible promoters
include anaerobic conditions, or certain chemicals, or the presence
of light. Tissue specific, tissue preferred, cell type specific,
and inducible promoters constitute the class of "non-constitutive"
promoters. A "constitutive" promoter is a promoter which is active
under most conditions.
[0038] As used herein "operably linked" means the association of
two or more DNA fragments in a recombinant DNA construct so that
the function of one, e.g. protein-encoding DNA, is controlled by
the other, e.g. a promoter.
[0039] As used herein "expressed" means produced, e.g. a protein is
expressed in a plant cell when its cognate DNA is transcribed to
mRNA that is translated to the protein.
[0040] As used herein "suppressed" means decreased, e.g. a protein
is suppressed in a plant cell when there is a decrease in the
amount and/or activity of the protein in the plant cell. The
presence or activity of the protein can be decreased by any amount
up to and including a total loss of protein expression and/or
activity.
[0041] As used herein a "control plant" means a plant that does not
contain the recombinant DNA that imparts an enhanced trait. A
control plant is used to identify and select a transgenic plant
that has an enhanced trait. A suitable control plant can be a
non-transgenic plant of the parental line used to generate a
transgenic plant, i.e. devoid of recombinant DNA. A suitable
control plant may in some cases be a progeny of a hemizygous
transgenic plant line that does not contain the recombinant DNA,
known as a negative segregant.
[0042] As used herein an "enhanced trait" means a characteristic of
a transgenic plant that includes, but is not limited to, an enhance
agronomic trait characterized by enhanced plant morphology,
physiology, growth and development, yield, nutritional enhancement,
disease or pest resistance, or environmental or chemical tolerance.
In more specific aspects of this invention enhanced trait is
selected from group of enhanced traits consisting of enhanced water
use efficiency, enhanced cold tolerance, increased yield, enhanced
nitrogen use efficiency, enhanced seed protein and enhanced seed
oil. In an important aspect of the invention the enhanced trait is
enhanced yield including increased yield under non-stress
conditions and increased yield under environmental stress
conditions. Stress conditions may include, for example, drought,
shade, fungal disease, viral disease, bacterial disease, insect
infestation, nematode infestation, cold temperature exposure, heat
exposure, osmotic stress, reduced nitrogen nutrient availability,
reduced phosphorus nutrient availability and high plant density.
"Yield" can be affected by many properties including without
limitation, plant height, pod number, pod position on the plant,
number of internodes, incidence of pod shatter, grain size,
efficiency of nodulation and nitrogen fixation, efficiency of
nutrient assimilation, resistance to biotic and abiotic stress,
carbon assimilation, plant architecture, resistance to lodging,
percent seed germination, seedling vigor, and juvenile traits.
Yield can also be affected by efficiency of germination (including
germination in stressed conditions), growth rate (including growth
rate in stressed conditions), ear number, seed number per ear, seed
size, composition of seed (starch, oil, protein) and
characteristics of seed fill.
[0043] Increased yield of a transgenic plant of the present
invention can be measured in a number of ways, including test
weight, seed number per plant, seed weight, seed number per unit
area (i.e. seeds, or weight of seeds, per acre), bushels per acre,
tons per acre, or kilo per hectare. For example, corn yield may be
measured as production of shelled corn kernels per unit of
production area, for example in bushels per acre or metric tons per
hectare, often reported on a moisture adjusted basis, for example
at 15.5 percent moisture. Increased yield may result from improved
utilization of key biochemical compounds, such as nitrogen,
phosphorous and carbohydrate, or from improved responses to
environmental stresses, such as cold, heat, drought, salt, and
attack by pests or pathogens. Recombinant DNA used in this
invention can also be used to provide plants having improved growth
and development, and ultimately increased yield, as the result of
modified expression of plant growth regulators or modification of
cell cycle or photosynthesis pathways. Also of interest is the
generation of transgenic plants that demonstrate enhanced yield
with respect to a seed component that may or may not correspond to
an increase in overall plant yield. Such properties include
enhancements in seed oil, seed molecules such as protein and
starch, oil components as may be manifest by an alterations in the
ratios of seed components.
[0044] Recombinant DNA constructs are assembled using methods well
known to persons of ordinary skill in the art and typically
comprise a promoter operably linked to DNA, the expression of which
provides the enhanced agronomic trait. Other construct components
may include additional regulatory elements, such as 5' leaders and
introns for enhancing transcription, 3' untranslated regions (such
as polyadenylation signals and sites), DNA for transit or signal
peptides.
[0045] Numerous promoters that are active in plant cells have been
described in the literature. These include promoters present in
plant genomes as well as promoters from other sources, including
nopaline synthase (NOS) promoter and octopine synthase (OCS)
promoters carried on tumor-inducing plasmids of Agrobacterium
tumefaciens and the CaMV35S promoters from the cauliflower mosaic
virus as disclosed in U.S. Pat. Nos. 5,164,316 and 5,322,938.
Useful promoters derived from plant genes are found in U.S. Pat.
No. 5,641,876 which discloses a rice actin promoter, U.S. Pat. No.
7,151,204 which discloses a maize chloroplast aldolase promoter and
a maize aldolase (FDA) promoter, and US Patent Application
Publication 2003/0131377 A1 which discloses a maize nicotianamine
synthase promoter. These and numerous other promoters that function
in plant cells are known to those skilled in the art and available
for use in recombinant polynucleotides of the present invention to
provide for expression of desired genes in transgenic plant
cells.
[0046] Furthermore, the promoters may be altered to contain
multiple "enhancer sequences" to assist in elevating gene
expression. Such enhancers are known in the art. By including an
enhancer sequence with such constructs, the expression of the
selected protein may be enhanced. These enhancers often are found
5' to the start of transcription in a promoter that functions in
eukaryotic cells, but can often be inserted upstream (5') or
downstream (3') to the coding sequence. In some instances, these 5'
enhancing elements are introns. Particularly useful as enhancers
are the 5' introns of the rice actin 1 (see U.S. Pat. No.
5,641,876) and rice actin 2 genes, the maize alcohol dehydrogenase
gene intron, the maize heat shock protein 70 gene intron (U.S. Pat.
No. 5,593,874) and the maize shrunken 1 gene. See also US Patent
Application Publication 2002/0192813A1 which discloses 5', 3' and
intron elements useful in the design of effective plant expression
vectors.
[0047] In other aspects of the invention, sufficient expression in
plant seed tissues is desired to affect improvements in seed
composition. Exemplary promoters for use for seed composition
modification include promoters from seed genes such as napin as
disclosed in U.S. Pat. No. 5,420,034, maize L3 oleosin as disclosed
in U.S. Pat. No. 6,433,252), zein Z27 as disclosed by Russell et
al. (1997) Transgenic Res. 6 (2):157-166), globulin 1 as disclosed
by Belanger et al (1991) Genetics 129:863-872), glutelin 1 as
disclosed by Russell (1997) supra), and peroxiredoxin antioxidant
(Per1) as disclosed by Stacy et al. (1996) Plant Mol. Biol. 31 (6):
1205-1216.
[0048] Recombinant DNA constructs useful in this invention will
also generally include a 3' element that typically contains a
polyadenylation signal and site. Well-known 3' elements include
those from Agrobacterium tumefaciens genes such as nos 3', tml 3',
tmr 3', tms 3', ocs 3', tr7 3', for example disclosed in U.S. Pat.
No. 6,090,627; 3' elements from plant genes such as wheat (Triticum
aesevitum) heat shock protein 17 (Hsp17 3'), a wheat ubiquitin
gene, a wheat fructose-1,6-biphosphatase gene, a rice glutelin
gene, a rice lactate dehydrogenase gene and a rice beta-tubulin
gene, all of which are disclosed in US Patent Application
Publication 2002/0192813 A1; and the pea (Pisum sativum) ribulose
biphosphate carboxylase gene (rbs 3'), and 3' elements from the
genes within the host plant.
[0049] Constructs and vectors may also include a transit peptide
for targeting of a gene to a plant organelle, particularly to a
chloroplast, leucoplast or other plastid organelle. For
descriptions of the use of chloroplast transit peptides see U.S.
Pat. No. 5,188,642 and U.S. Pat. No. 5,728,925. For description of
the transit peptide region of an Arabidopsis EPSPS gene useful in
the present invention, see Klee, H. J. et al (MGG (1987)
210:437-442).
[0050] Recombinant DNA constructs for gene suppression can be
designed for any of a number the well-known methods for suppressing
transcription of a gene, the accumulation of the mRNA corresponding
to that gene or preventing translation of the transcript into
protein. Posttranscriptional gene suppression can be practically
effected by transcription of RNA that forms double-stranded RNA
(dsRNA) having homology to mRNA produced from a gene targeted for
suppression.
[0051] Gene suppression can also be achieved by insertion mutations
created by transposable elements may also prevent gene function.
For example, in many dicot plants, transformation with the T-DNA of
Agrobacterium may be readily achieved and large numbers of
transformants can be rapidly obtained. Also, some species have
lines with active transposable elements that can efficiently be
used for the generation of large numbers of insertion mutations,
while some other species lack such options. Mutant plants produced
by Agrobacterium or transposon mutagenesis and having altered
expression of a polypeptide of interest can be identified using the
polynucleotides of the present invention. For example, a large
population of mutated plants may be screened with polynucleotides
encoding the polypeptide of interest to detect mutated plants
having an insertion in the gene encoding the polypeptide of
interest.
[0052] Transgenic plants may comprise a stack of one or more
polynucleotides disclosed herein resulting in the production or
suppression of multiple polypeptide sequences. Transgenic plants
comprising stacks of polynucleotide sequences can be obtained by
either or both of traditional breeding methods or through genetic
engineering methods. These methods include, but are not limited to,
breeding individual lines each comprising a polynucleotide of
interest, transforming a transgenic plant comprising a gene
disclosed herein with a subsequent gene, and co-transformation of
genes into a single plant cell. Co-transformation of genes can be
carried out using single transformation vectors comprising multiple
genes or genes carried separately on multiple vectors.
[0053] Transgenic plants comprising or derived from plant cells of
this invention transformed with recombinant DNA can be further
enhanced with stacked traits, e.g. a crop plant having an enhanced
trait resulting from expression of DNA disclosed herein in
combination with herbicide and/or pest resistance traits. For
example, genes of the current invention can be stacked with other
traits of agronomic interest, such as a trait providing herbicide
resistance, or insect resistance, such as using a gene from
Bacillus thuringensis to provide resistance against lepidopteran,
coliopteran, homopteran, hemiopteran, and other insects. Herbicides
for which transgenic plant tolerance has been demonstrated and the
method of the present invention can be applied include, but are not
limited to, glyphosate, dicamba, glufosinate, sulfonylurea,
bromoxynil and norflurazon herbicides. Polynucleotide molecules
encoding proteins involved in herbicide tolerance are well-known in
the art and include, but are not limited to, a polynucleotide
molecule encoding 5-enolpyruvylshikimate-3-phosphate synthase
(EPSPS) disclosed in U.S. Pat. Nos. 5,094,945; 5,627,061; 5,633,435
and 6,040,497 for imparting glyphosate tolerance; polynucleotide
molecules encoding a glyphosate oxidoreductase (GOX) disclosed in
U.S. Pat. No. 5,463,175 and a glyphosate-N-acetyl transferase (GAT)
disclosed in US Patent Application Publication 2003/0083480 A1 also
for imparting glyphosate tolerance; dicamba monooxygenase disclosed
in US Patent Application Publication 2003/0135879 A1 for imparting
dicamba tolerance; a polynucleotide molecule encoding bromoxynil
nitrilase (Bxn) disclosed in U.S. Pat. No. 4,810,648 for imparting
bromoxynil tolerance; a polynucleotide molecule encoding phytoene
desaturase (crtI) described in Misawa et al, (1993) Plant J.
4:833-840 and in Misawa et al, (1994) Plant J. 6:481-489 for
norflurazon tolerance; a polynucleotide molecule encoding
acetohydroxyacid synthase (AHAS, aka ALS) described in Sathasiivan
et al. (1990) Nucl. Acids Res. 18:2188-2193 for imparting tolerance
to sulfonylurea herbicides; polynucleotide molecules known as bar
genes disclosed in DeBlock, et al. (1987) EMBO J. 6:2513-2519 for
imparting glufosinate and bialaphos tolerance; polynucleotide
molecules disclosed in US Patent Application Publication
2003/010609 A1 for imparting N-amino methyl phosphonic acid
tolerance; polynucleotide molecules disclosed in U.S. Pat. No.
6,107,549 for imparting pyridine herbicide resistance; molecules
and methods for imparting tolerance to multiple herbicides such as
glyphosate, atrazine, ALS inhibitors, isoxoflutole and glufosinate
herbicides are disclosed in U.S. Pat. No. 6,376,754 and US Patent
Application Publication 2002/0112260. Molecules and methods for
imparting insect/nematode/virus resistance are disclosed in U.S.
Pat. Nos. 5,250,515; 5,880,275; 6,506,599; 5,986,175 and US Patent
Application Publication 2003/0150017 A1.
Plant Cell Transformation Methods
[0054] Numerous methods for transforming chromosomes in a plant
cell nucleus with recombinant DNA are known in the art and are used
in methods of preparing a transgenic plant cell nucleus cell, and
plant. Two effective methods for such transformation are
Agrobacterium-mediated transformation and microprojectile
bombardment. Microprojectile bombardment methods are illustrated in
U.S. Pat. Nos. 5,015,580 (soybean); 5,550,318 (corn); 5,538,880
(corn); 5,914,451 (soybean); 6,160,208 (corn); 6,399,861 (corn);
6,153,812 (wheat) and 6,365,807 (rice) and Agrobacterium-mediated
transformation is described in U.S. Pat. Nos. 5,159,135 (cotton);
5,824,877 (soybean); 5,463,174 (canola); 5,591,616 (corn);
5,846,797 (cotton); 6,384,301 (soybean), 7,026,528 (wheat) and
6,329,571 (rice), US Patent Application Publication 2004/0087030 A1
(cotton), and US Patent Application Publication 2001/0042257 A1
(sugar beet), all of which are incorporated herein by reference for
enabling the production of transgenic plants. Transformation of
plant material is practiced in tissue culture on a nutrient media,
i.e. a mixture of nutrients that will allow cells to grow in vitro.
Recipient cell targets include, but are not limited to, meristem
cells, hypocotyls, calli, immature embryos and gametic cells such
as microspores, pollen, sperm and egg cells. Callus may be
initiated from tissue sources including, but not limited to,
immature embryos, hypocotyls, seedling apical meristems,
microspores and the like. Cells containing a transgenic nucleus are
grown into transgenic plants.
[0055] In addition to direct transformation of a plant material
with a recombinant DNA, a transgenic plant cell nucleus can be
prepared by crossing a first plant having cells with a transgenic
nucleus with recombinant DNA with a second plant lacking the
transgenic nucleus. For example, recombinant DNA can be introduced
into a nucleus from a first plant line that is amenable to
transformation to transgenic nucleus in cells that are grown into a
transgenic plant which can be crossed with a second plant line to
introgress the recombinant DNA into the second plant line. A
transgenic plant with recombinant DNA providing an enhanced trait,
e.g. enhanced yield, can be crossed with transgenic plant line
having other recombinant DNA that confers another trait, for
example herbicide resistance or pest resistance, to produce progeny
plants having recombinant DNA that confers both traits. Typically,
in such breeding for combining traits the transgenic plant donating
the additional trait is a male line and the transgenic plant
carrying the base traits is the female line. The progeny of this
cross will segregate such that some of the plants will carry the
DNA for both parental traits and some will carry DNA for one
parental trait; such plants can be identified by markers associated
with parental recombinant DNA, e.g. marker identification by
analysis for recombinant DNA or, in the case where a selectable
marker is linked to the recombinant, by application of the
selecting agent such as a herbicide for use with a herbicide
tolerance marker, or by selection for the enhanced trait. Progeny
plants carrying DNA for both parental traits can be crossed back
into the female parent line multiple times, for example usually 6
to 8 generations, to produce a progeny plant with substantially the
same genotype as one original transgenic parental line but for the
recombinant DNA of the other transgenic parental line
[0056] In the practice of transformation DNA is typically
introduced into only a small percentage of target plant cells in
any one transformation experiment. Marker genes are used to provide
an efficient system for identification of those cells that are
stably transformed by receiving and integrating a recombinant DNA
molecule into their genomes. Preferred marker genes provide
selective markers which confer resistance to a selective agent,
such as an antibiotic or a herbicide. Any of the herbicides to
which plants of this invention may be resistant are useful agents
for selective markers. Potentially transformed cells are exposed to
the selective agent. In the population of surviving cells will be
those cells where, generally, the resistance-conferring gene is
integrated and expressed at sufficient levels to permit cell
survival. Cells may be tested further to confirm stable integration
of the exogenous DNA. Commonly used selective marker genes include
those conferring resistance to antibiotics such as kanamycin and
paromomycin (nptII), hygromycin B (aph IV), spectinomycin (aadA)
and gentamycin (aac3 and aacC4) or resistance to herbicides such as
glufosinate (bar or pat), dicamba (DMO) and glyphosate (aroA or
EPSPS). Examples of such selectable markers are illustrated in U.S.
Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047. Markers
which provide an ability to visually screen transformants can also
be employed, for example, a gene expressing a colored or
fluorescent protein such as a luciferase or green fluorescent
protein (GFP) or a gene expressing a beta-glucuronidase or uidA
gene (GUS) for which various chromogenic substrates are known.
[0057] Plant cells that survive exposure to the selective agent, or
plant cells that have been scored positive in a screening assay,
may be cultured in regeneration media and allowed to mature into
plants. Developing plantlets regenerated from transformed plant
cells can be transferred to plant growth mix, and hardened off, for
example, in an environmentally controlled chamber at about 85%
relative humidity, 600 ppm CO.sub.2, and 25-250 microeinsteins
m.sup.-2 s.sup.-1 of light, prior to transfer to a greenhouse or
growth chamber for maturation. Plants are regenerated from about 6
weeks to 10 months after a transformant is identified, depending on
the initial tissue, and plant species. Plants may be pollinated
using conventional plant breeding methods known to those of skill
in the art and seed produced, for example self-pollination is
commonly used with transgenic corn. The regenerated transformed
plant or its progeny seed or plants can be tested for expression of
the recombinant DNA and selected for the presence of enhanced
agronomic trait.
Transgenic Plants and Seeds
[0058] Transgenic plants derived from transgenic plant cells having
a transgenic nucleus of this invention are grown to generate
transgenic plants having an enhanced trait as compared to a control
plant and produce transgenic seed and haploid pollen of this
invention. Such plants with enhanced traits are identified by
selection of transformed plants or progeny seed for the enhanced
trait. For efficiency a selection method is designed to evaluate
multiple transgenic plants (events) comprising the recombinant DNA,
for example multiple plants from 2 to 20 or more transgenic events.
Transgenic plants grown from transgenic seed provided herein
demonstrate improved agronomic traits that contribute to increased
yield or other trait that provides increased plant value,
including, for example, improved seed quality. Of particular
interest are plants having enhanced water use efficiency, enhanced
cold tolerance, increased yield, enhanced nitrogen use efficiency,
enhanced seed protein and enhanced seed oil. Table 1 provides a
list of protein encoding DNA ("genes") that are useful as
recombinant DNA for production of transgenic plants with enhanced
agronomic trait, the elements of Table 1 are described by reference
to:
"PEP SEQ ID NO" identifies an amino acid sequence from SEQ ID NO:
11 to 20. "NUC SEQ ID NO" identifies a DNA sequence from SEQ ID
NO:1 to 10. "Gene ID" refers to an arbitrary identifier. "Gene
Name" denotes a common name for protein encoded by the recombinant
DNA. "Annotation" refers to a description of the top hit protein
obtained from an amino acid sequence query of each PEP SEQ ID NO to
GENBANK database of the National Center for Biotechnology
Information (ncbi).
TABLE-US-00001 TABLE 1 NUC PEP SEQ ID SEQ ID NO NO Gene ID Gene
Name Annotation 1 11 Mnom000034 Corn ZmLac gb|AAX83113.1|laccase 1
[Zea mays] 2 12 Mnom000037 Anabaena FLD gb|AAB20462.1|flavodoxin
[Anabaena] 3 13 Mnom000048 Corn TPS1 gb|EAY98715.1|hypothetical
protein OsI_019948 [Oryza sativa (indica cultivar-group)] 4 14
Mnom000049 Corn TPP1 gb|EAY75823.1|hypothetical protein OsI_003670
[Oryza sativa (indica cultivar-group)] 5 15 Mnom000067 Lycopersicon
AlaT gb|AAZ43369.1|AlaT1 [Vitis vinifera] 6 16 Mnom000068
Lycopersicon AlaT gb|AAZ43369.1|AlaT1 [Vitis vinifera] 7 17
Mnom000090 Corn ZmGS3 gb|ABC84855.1|grain length and weight protein
[Oryza sativa (indica cultivar-group)] 8 18 Mnom000091 Corn ZmGS3
gb|ABC84855.1|grain length and weight protein [Oryza sativa (indica
cultivar-group)] 9 19 Mnom000092 Corn ZmBB
gb|EAZ25768.1|hypothetical protein OsJ_009251 [Oryza sativa
(japonica cultivar-group)] 10 20 Mnom000095 Corn
ref|NP_001052879.1|Os04g0442000 [Oryza sativa OSJNBa0064D (japonica
cultivar-group)] [Mendel G471-like ner] 20.11 Protein
Selection Methods for Transgenic Plants with Enhanced Agronomic
Trait
[0059] Within a population of transgenic plants each regenerated
from a plant cell having a nucleus with recombinant DNA many plants
that survive to fertile transgenic plants that produce seeds and
progeny plants will not exhibit an enhanced agronomic trait.
Selection from the population is necessary to identify one or more
transgenic plant cells having a transgenic nucleus that can provide
plants with the enhanced trait. Transgenic plants having enhanced
traits are selected from populations of plants regenerated or
derived from plant cells transformed as described herein by
evaluating the plants in a variety of assays to detect an enhanced
trait, e.g. enhanced water use efficiency, enhanced cold tolerance,
increased yield, enhanced nitrogen use efficiency, enhanced seed
protein and enhanced seed oil. These assays also may take many
forms including, but not limited to, direct screening for the trait
in a greenhouse or field trial or by screening for a surrogate
trait. Such analyses can be directed to detecting changes in the
chemical composition, biomass, physiological properties, morphology
of the plant. Changes in chemical compositions such as nutritional
composition of grain can be detected by analysis of the seed
composition and content of protein, free amino acids, oil, free
fatty acids, starch or tocopherols. Changes in biomass
characteristics can be made on greenhouse or field grown plants and
can include plant height, stem diameter, root and shoot dry
weights; and, for corn plants, ear length and diameter. Changes in
physiological properties can be identified by evaluating responses
to stress conditions, for example assays using imposed stress
conditions such as water deficit, nitrogen deficiency, cold growing
conditions, pathogen or insect attack or light deficiency, or
increased plant density. Changes in morphology can be measured by
visual observation of tendency of a transformed plant with an
enhanced agronomic trait to also appear to be a normal plant as
compared to changes toward bushy, taller, thicker, narrower leaves,
striped leaves, knotted trait, chlorosis, albino, anthocyanin
production, or altered tassels, ears or roots. Other selection
properties include days to pollen shed, days to silking, leaf
extension rate, chlorophyll content, leaf temperature, stand,
seedling vigor, internode length, plant height, leaf number, leaf
area, tillering, brace roots, stay green, stalk lodging, root
lodging, plant health, barreness/prolificacy, green snap, and pest
resistance. In addition, phenotypic characteristics of harvested
grain may be evaluated, including number of kernels per row on the
ear, number of rows of kernels on the ear, kernel abortion, kernel
weight, kernel size, kernel density and physical grain quality.
[0060] Assays for screening for a desired trait are readily
designed by those practicing in the art. The following illustrates
useful screening assays for corn traits using hybrid corn plants.
The assays can be readily adapted for screening other plants such
as canola, cotton and soybean either as hybrids or inbreds.
[0061] Transgenic corn plants having nitrogen use efficiency are
identified by screening in fields with three levels of nitrogen (N)
fertilizer being applied, e.g. low level (0 N), medium level (80
lb/ac) and high level (180 lb/ac). Plants with enhanced nitrogen
use efficiency provide higher yield as compared to control
plants.
[0062] Transgenic corn plants having enhanced yield are identified
by screening using progeny of the transgenic plants over multiple
locations with plants grown under optimal production management
practices and maximum weed and pest control. A useful target for
improved yield is a 5% to 10% increase in yield as compared to
yield produced by plants grown from seed for a control plant.
Selection methods may be applied in multiple and diverse geographic
locations, for example up to 16 or more locations, over one or more
planting seasons, for example at least two planting seasons, to
statistically distinguish yield improvement from natural
environmental effects.
[0063] Transgenic corn plants having enhanced water use efficiency
are identified by screening plants in an assay where water is
withheld for a period to induce stress followed by watering to
revive the plants. For example, a useful selection process imposes
3 drought/re-water cycles on plants over a total period of 15 days
after an initial stress free growth period of 11 days. Each cycle
consists of 5 days, with no water being applied for the first four
days and a water quenching on the 5th day of the cycle. The primary
phenotypes analyzed by the selection method are the changes in
plant growth rate as determined by height and biomass during a
vegetative drought treatment.
[0064] Transgenic corn plants having enhanced cold tolerance are
identified by screening plants in a cold germination assay and/or a
cold tolerance field trial. In a cold germination assay trays of
transgenic and control seeds are placed in a growth chamber at
9.7.degree. C. for 24 days (no light). Seeds having higher
germination rates as compared to the control are identified as
having enhanced cold tolerance. In a cold tolerance field trial
plants with enhanced cold tolerance are identified from field
planting at an earlier date than conventional Spring planting for
the field location. For example, seeds are planted into the ground
around two weeks before local farmers begin to plant corn so that a
significant cold stress is exerted onto the crop, named as cold
treatment. Seeds also are planted under local optimal planting
conditions such that the crop has little or no exposure to cold
condition, named as normal treatment. At each location, seeds are
planted under both cold and normal conditions preferably with
multiple repetitions per treatment.
[0065] Transgenic corn plants having seeds with increased protein
and/or oil levels are identified by analyzing progeny seed for
protein and/or oil. Near-infrared transmittance spectrometry is a
non-destructive, high-throughput method that is useful to determine
the composition of a bulk seed sample for properties listed in
table 2.
TABLE-US-00002 TABLE 2 Typical sample(s): Whole grain corn and
soybean seeds Typical analytical range: Corn - moisture 5-15%, oil
5-20%, protein 5-30%, starch 50-75%, and density 1.0-1.3%. Soybean
- moisture 5-15%, oil 15-25%, and protein 35-50%.
[0066] Although the plant cells and methods of this invention can
be applied to any plant cell, plant, seed or pollen, e.g. any
fruit, vegetable, grass, tree or ornamental plant, the various
aspects of the invention are preferably applied to corn, soybean,
cotton, canola, alfalfa, wheat, rice, sugarcane, and sugar beet
plants. In many cases the invention is applied to corn plants that
are inherently resistant to disease from the Mal de Rio Cuarto
virus or the Puccina sorghi fungus or both.
[0067] The following examples are included to demonstrate aspects
of the invention, those of skill in the art should, in light of the
present disclosure, appreciate that many changes can be made in the
specific aspects which are disclosed and still obtain a like or
similar results without departing from the spirit and scope of the
invention.
Example 1
Plant Expression Constructs
[0068] This example illustrates the construction of plasmids for
transferring recombinant DNA into a plant cell nucleus that can be
regenerated into transgenic plants.
A. Plant Expression Constructs for Corn Transformation
[0069] A base corn transformation vector pMON93039, as set forth in
SEQ ID NO:1352, illustrated in Table 3 and FIG. 1, is fabricated
for use in preparing recombinant DNA for Agrobacterium-mediated
transformation into corn tissue.
TABLE-US-00003 TABLE 3 Coordinates of Function Name Annotation SEQ
ID NO: 1352 Agrobacterium B-AGRtu.right border Agro right border
sequence, 11364-11720 T-DNA transfer essential for transfer of
T-DNA. Gene of interest E-Os.Act1 Upstream promoter region of the
19-775 expression rice actin 1 gene cassette E-CaMV.35S.2xA1-
Duplicated35S A1-B3 domain 788-1120 B3 without TATA box P-Os.Act1
Promoter region of the rice actin 1 1125-1204 gene L-Ta.Lhcb1 5'
untranslated leader of wheat 1210-1270 major chlorophyll a/b
binding protein I-Os.Act1 First intron and flanking UTR exon
1287-1766 sequences from the rice actin 1 gene T-St.Pis4 3'
non-translated region of the 1838-2780 potato proteinase inhibitor
II gene which functions to direct polyadenylation of the mRNA Plant
selectable P-Os.Act1 Promoter from the rice actin 1 gene 2830-3670
marker L-Os.Act1 First exon of the rice actin 1 gene 3671-3750
expression I-Os.Act1 First intron and flanking UTR exon 3751-4228
cassette sequences from the rice actin 1 gene TS-At.ShkG-CTP2
Transit peptide region of 4238-4465 Arabidopsis EPSPS
CR-AGRtu.aroA- Coding region for bacterial strain 4466-5833 CP4.nat
CP4 native aroA gene. T-AGRtu.nos A 3' non-translated region of the
5849-6101 nopaline synthase gene of Agrobacterium tumefaciens Ti
plasmid which functions to direct polyadenylation of the mRNA.
Agrobacterium B-AGRtu.left border Agro left border sequence,
essential 6168-6609 T-DNA transfer for transfer of T-DNA.
Maintenance in OR-Ec.oriV-RK2 The vegetative origin of replication
6696-7092 E. coli from plasmid RK2. CR-Ec.rop Coding region for
repressor of 8601-8792 primer from the ColE1 plasmid. Expression of
this gene product interferes with primer binding at the origin of
replication, keeping plasmid copy number low. OR-Ec.ori-ColE1 The
minimal origin of replication 9220-9808 from the E. coli plasmid
ColE1. P-Ec.aadA-SPC/STR Promoter for Tn7 10339-10380
adenylyltransferase (AAD(3'')) CR-Ec.aadA- Coding region for Tn7
10381-11169 SPC/STR adenylyltransferase (AAD(3'')) conferring
spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3' UTR
from the Tn7 11170-11227 adenylyltransferase (AAD(3'')) gene of E.
coli.
[0070] To construct transformation vectors for expressing a protein
identified in Table 1, primers for PCR amplification of the protein
coding nucleotides are designed at or near the start and stop
codons of the coding sequence, in order to eliminate most of the 5'
and 3' untranslated regions. The protein coding nucleotides are
inserted into the base vector in the gene of interest expression
cassette at an insertion site, i.e. between the intron element
(coordinates 1287-1766) and the polyadenylation element
(coordinates 1838-2780).
[0071] To construct transformation vectors for suppressing a
protein identified in Table 1, the amplified protein coding
nucleotides are assembled in a sense and antisense arrangement and
inserted into the base vector at the insertion site in the gene of
interest expression cassette to provide transcribed RNA that will
form a double-stranded RNA for RNA interference suppression of the
protein. In the embodiments of this invention the proteins that are
suppressed are ZmLac, ZmGS3, and ZmBB.
B. Plant Expression Constructs for Soy and Canola
Transformation
[0072] Vectors for use in transformation of soybean and canola
tissue are prepared having the elements of expression vector
pMON82053 (SEQ ID NO: 1353) as shown in Table 4 below and FIG.
2.
TABLE-US-00004 TABLE 4 Coordinates of Function Name Annotation SEQ
ID NO: 1353 Agrobacterium T- B-AGRtu.left border Agro left border
sequence, essential for 6144-6585 DNA transfer transfer of T-DNA.
Plant selectable P-At.Act7 Promoter from the Arabidopsis actin 7
gene 6624-7861 marker expression L-At.Act7 5'UTR of Arabidopsis
Act7 gene cassette I-At.Act7 Intron from the Arabidopsis actin7
gene TS-At.ShkG-CTP2 Transit peptide region of Arabidopsis
7864-8091 EPSPS CR-AGRtu.aroA- Synthetic CP4 coding region with
dicot 8092-9459 CP4.nno_At preferred codon usage. T-AGRtu.nos A 3'
non-translated region of the nopaline 9466-9718 synthase gene of
Agrobacterium tumefaciens Ti plasmid which functions to direct
polyadenylation of the mRNA. Gene of interest P-CaMV.35S-enh
Promoter for 35S RNA from CaMV 1-613 expression cassette containing
a duplication of the -90 to -350 region. T-Gb.E6-3b 3' untranslated
region from the fiber protein 688-1002 E6 gene of sea-island
cotton. Agrobacterium T- B-AGRtu.right Agro right border sequence,
essential for 1033-1389 DNA transfer border transfer of T-DNA.
Maintenance in E. coli OR-Ec.oriV-RK2 The vegetative origin of
replication from 5661-6057 plasmid RK2. CR-Ec.rop Coding region for
repressor of primer from 3961-4152 the ColE1 plasmid. Expression of
this gene product interferes with primer binding at the origin of
replication, keeping plasmid copy number low. OR-Ec.ori-ColE1 The
minimal origin of replication from the 2945-3533 E. coli plasmid
ColE1. P-Ec.aadA-SPC/STR Promoter for Tn7 adenylyltransferase
2373-2414 (AAD(3'')) CR-Ec.aadA- Coding region for Tn7
adenylyltransferase 1584-2372 SPC/STR (AAD(3'')) conferring
spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3' UTR
from the Tn7 adenylyltransferase 1526-1583 (AAD(3'')) gene of E.
coli.
[0073] To construct transformation vectors for expressing a protein
identified in Table 1, primers for PCR amplification of the protein
coding nucleotides are designed at or near the start and stop
codons of the coding sequence, in order to eliminate most of the 5'
and 3' untranslated regions. The protein coding nucleotides are
inserted into the base vector in the gene of interest expression
cassette at an insertion site, i.e. between the promoter element
(coordinates 1-613) and the polyadenylation element (coordinates
688-1002).
[0074] To construct transformation vectors for suppressing a
protein identified in Table 1, the amplified protein coding
nucleotides are assembled in a sense and antisense arrangement and
inserted into the base vector at the insertion site in the gene of
interest expression cassette to provide transcribed RNA that will
form a double-stranded RNA for RNA interference suppression of the
protein. In the embodiments of this invention the proteins that are
suppressed are ZmLac, ZmGS3, and ZmBB.
C. Cotton Transformation Vector
[0075] Plasmids for use in transformation of cotton tissue are
prepared with elements of expression vector pMON99053 (SEQ ID NO:
1354) as shown in Table 5 below and FIG. 3.
TABLE-US-00005 TABLE 5 Coordinates of SEQ ID NO: Function Name
Annotation 1354 Agrobacterium T- B-AGRtu.right border Agro right
border sequence, essential for 1-357 DNA transfer transfer of
T-DNA. Gene of interest Exp-CaMV.35S- Enhanced version of the 35S
RNA promoter 388-1091 expression cassette enh+Ph.DnaK from CaMV
plus the petunia hsp70 5' untranslated region T-Ps.RbcS2-E9 The 3'
non-translated region of the pea RbcS2 1165-1797 gene which
functions to direct polyadenylation of the mRNA. Plant selectable
Exp-CaMV.35S Promoter and 5' untranslated region from the 1828-2151
marker expression 35S RNA of CaMV cassette CR-Ec.nptII-Tn5 Coding
region for neomycin 2185-2979 phosphotransferase gene from
transposon Tn5 which confers resistance to neomycin and kanamycin.
T-AGRtu.nos A 3' non-translated region of the nopaline 3011-3263
synthase gene of Agrobacterium tumefaciens Ti plasmid which
functions to direct polyadenylation of the mRNA. Agrobacterium T-
B-AGRtu.left border Agro left border sequence, essential for
3309-3750 DNA transfer transfer of T-DNA. Maintenance in E. coli
OR-Ec.oriV-RK2 The vegetative origin of replication from 3837-4233
plasmid RK2. CR-Ec.rop Coding region for repressor of primer from
5742-5933 the ColE1 plasmid. Expression of this gene product
interferes with primer binding at the origin of replication,
keeping plasmid copy number low. OR-Ec.ori-ColE1 The minimal origin
of replication from the E. coli 6361-6949 plasmid ColE1.
P-Ec.aadA-SPC/STR Promoter for Tn7 adenylyltransferase 7480-7521
(AAD(3'')) CR-Ec.aadA-SPC/STR Coding region for Tn7
adenylyltransferase 7522-8310 (AAD(3'')) conferring spectinomycin
and streptomycin resistance. T-Ec.aadA-SPC/STR 3' UTR from the Tn7
adenylyltransferase 8311-8368 (AAD(3'')) gene of E. coli.
[0076] To construct transformation vectors for expressing a protein
identified in Table 1, primers for PCR amplification of the protein
coding nucleotides are designed at or near the start and stop
codons of the coding sequence, in order to eliminate most of the 5'
and 3' untranslated regions. The protein coding nucleotides are
inserted into the base vector in the gene of interest expression
cassette at an insertion site, i.e. between the promoter element
(coordinates 388-1091) and the polyadenylation element (coordinates
1165-1797).
[0077] To construct transformation vectors for suppressing a
protein identified in Table 1, the amplified protein coding
nucleotides are assembled in a sense and antisense arrangement and
inserted into the base vector at the insertion site in the gene of
interest expression cassette to provide transcribed RNA that will
form a double-stranded RNA for RNA interference suppression of the
protein. In the embodiments of this invention the proteins that are
suppressed are ZmLac, ZmGS3, and ZmBB.
D. Plant Expression Constructs for Gene Stacking in Corn.
[0078] A base corn transformation vector pMON96782, as set forth in
SEQ ID NO: 1355, illustrated in Table 6 and FIG. 4, is fabricated
for use in preparing recombinant DNA for Agrobacterium-mediated
transformation into corn tissue.
TABLE-US-00006 TABLE 6 Coordinates of Function Name Annotation SEQ
ID NO: 1355 Agrobacterium B-AGRtu.right border Agro right border
sequence, 1-357 T-DNA transfer essential for transfer of T-DNA.
Gene of interest P-Os.Act1 Promoter region of the rice actin 1
403-1243 expression gene cassette 1 L-Os.Act1 5' untranslated
leader of rice actin 1 1244-1323 gene I-Os.Act1 First intron and
flanking UTR exon 1324-1801 sequences from the rice actin 1 gene
T-Ta.Hsp17 3' un-translated region of wheat low 1834-2043 molecular
weight heat shock protein gene Gene of interest E-Os.Act1 Upstream
Promoter region of rice 2136-2892 expression actin 1 cassette 2
E-CaMV.35S.2xA1- 35S A1-B3 Domain 2905-2937 B3 P-Os.Act1 Promoter
from rice actin gene 3242-3321 L-Ta.Lhcb1 5' untranslated leader
from wheat 3327-3387 chlorophyll protein I-Os.Act1 Intron and 5'
untranslated region 3404-3883 from rice actin 1 gene T-AGRtu.tr7 3'
untranslated region from 3918-4425 "transcript 7" of Agrobacterium
Plant selectable P.Os.TubA Promoter of alpha-tubulin gene of
4452-5650 marker rice expression L.Os.TubA 5' untranslated region
of an alpha 5651-5736 cassette tubulin from rice I.Os.TubA Intron 1
of an alpha tubulin from 5737-6632 rice. Ts.Ta.waxy.nno_Zm
Chloroplast transit peptide from 6637-6846 wheat starch synthase
Cr.AGRtu.aroA- CP4 EPSPS gene 6847-8214 CP4.nno_Zm T.Os.TubA 3'
untranslated region of alpha 8219-8800 tubulin from rice
Agrobacterium B-AGRtu.left border Left border sequence for T-DNA
8828-9269 T-DNA transfer transfer Maintenance in OR-Ec.oriV-RK2
Origin of replication from the E. coli 9356-9752 E. coli plasmid
RK2. Cr-Ec.rop Coding region for repressor of 11261-11452 primer
from ColE1 plasmid OR-Ec.ori-ColE1 Minimum origin of replication
from 11880-12468 E. coli colE1 plasmid. P-Ec.aadA-SPC/STR Promoter
for Tn7 12999-13040 adenylyltransferase gene CR-Ec.aadA- Coding
region for Tn7 13041-13829 SPC/STR adenylyltransferase gene
T-Ec.aadA-SPC/STR 3' untranslated region from Tn7 13830-13887
adenylyltransferase gene
[0079] Primers for PCR amplification of protein coding nucleotides
of the genes of interest are designed at or near the start and stop
codons of the coding sequence, in order to eliminate most of the 5'
and 3' untranslated regions. Protein coding regions of genes
encoding a first and second protein of interest are amplified. The
amplified region from the first gene of interest is cloned between
nucleotides 1801 and 1834 of the base vector and the amplified
region from the second gene of interest is cloned between
nucleotides 3883 and 3918 of the base vector.
Example 2
Corn Transformation
[0080] This example illustrates transformation methods useful in
producing a transgenic nucleus in a corn plant cell, and the
plants, seeds and pollen produced from a transgenic cell with such
a nucleus having an enhanced trait, i.e. enhanced water use
efficiency, enhanced cold tolerance, increased yield, enhanced
nitrogen use efficiency, enhanced seed protein and enhanced seed
oil. A plasmid vector is prepared by cloning the DNA of SEQ ID NO:1
into the gene of interest expression cassette in the base vector
for use in corn transformation of corn tissue provided in Example
1, Table 3.
[0081] For Agrobacterium-mediated transformation of corn embryo
cells corn plants of a readily transformable line are grown in the
greenhouse and ears are harvested when the embryos are 1.5 to 2.0
mm in length. Ears are surface sterilized by spraying or soaking
the ears in 80% ethanol, followed by air drying. Immature embryos
are isolated from individual kernels on surface sterilized ears.
Prior to inoculation of maize cells, Agrobacterium cells are grown
overnight at room temperature. Immature maize embryo cells are
inoculated with Agrobacterium shortly after excision, and incubated
at room temperature with Agrobacterium for 5-20 minutes. Immature
embryo plant cells are then co-cultured with Agrobacterium for 1 to
3 days at 23.degree. C. in the dark. Co-cultured embryos are
transferred to selection media and cultured for approximately two
weeks to allow embryogenic callus to develop. Embryogenic callus is
transferred to culture medium containing 100 mg/L paromomycin and
subcultured at about two week intervals. Transformed plant cells
are recovered 6 to 8 weeks after initiation of selection.
[0082] For Agrobacterium-mediated transformation of maize callus
immature embryos are cultured for approximately 8-21 days after
excision to allow callus to develop. Callus is then incubated for
about 30 minutes at room temperature with the Agrobacterium
suspension, followed by removal of the liquid by aspiration. The
callus and Agrobacterium are co-cultured without selection for 3-6
days followed by selection on paromomycin for approximately 6
weeks, with biweekly transfers to fresh media. Paromomycin
resistant calli are identified about 6-8 weeks after initiation of
selection.
[0083] To regenerate transgenic corn plants a callus of transgenic
plant cells resulting from transformation and selection is placed
on media to initiate shoot development into plantlets which are
transferred to potting soil for initial growth in a growth chamber
at 26.degree. C. followed by a mist bench before transplanting to 5
inch pots where plants are grown to maturity. The regenerated
plants are self-fertilized and seed is harvested for use in one or
more methods to select seeds, seedlings or progeny second
generation transgenic plants (R2 plants) or hybrids, e.g. by
selecting transgenic plants exhibiting an enhanced trait as
compared to a control plant.
[0084] The above process is repeated to produce multiple events of
transgenic corn plant cells that are transformed with recombinant
DNA from each of the genes identified in Table 1. Events are
designed to produce in the transgenic cells one of the proteins
identified in Table 1, except the proteins of SEQ ID NOs: 11 and
17-19 which are suppressed. Progeny transgenic plants and seed of
the transformed plant cells are screened for enhanced water use
efficiency, enhanced cold tolerance, increased yield, enhanced
nitrogen use efficiency, enhanced seed protein and enhanced seed
oil. From each group of multiple events of transgenic plants with a
specific recombinant DNA from Table 1 the event that produces the
greatest enhancement in yield, water use efficiency, nitrogen use
efficiency, enhanced cold tolerance, enhanced seed protein and
enhanced seed oil is identified and progeny seed is selected for
commercial development.
Example 3
Soybean Transformation
[0085] This example illustrates plant transformation useful in
producing a transgenic nucleus in a soybean plant cell, and the
plants, seeds and pollen produced from a transgenic cell with such
a nucleus having an enhanced trait, i.e. enhanced water use
efficiency, enhanced cold tolerance, increased yield, enhanced
nitrogen use efficiency, enhanced seed protein and enhanced seed
oil.
[0086] For Agrobacterium mediated transformation, soybean seeds are
imbided overnight and the meristem explants excised. The explants
are placed in a wounding vessel. Soybean explants and induced
Agrobacterium cells from a strain containing plasmid DNA with the
gene of interest cassette and a plant selectable marker cassette
are mixed no later than 14 hours from the time of initiation of
seed imbibition, and wounded using sonication. Following wounding,
explants are placed in co-culture for 2-5 days at which point they
are transferred to selection media for 6-8 weeks to allow selection
and growth of transgenic shoots. Resistant shoots are harvested
approximately 6-8 weeks and placed into selective rooting media for
2-3 weeks. Shoots producing roots are transferred to the greenhouse
and potted in soil. Shoots that remain healthy on selection, but do
not produce roots are transferred to non-selective rooting media
for an additional two weeks. Roots from any shoots that produce
roots off selection are tested for expression of the plant
selectable marker before they are transferred to the greenhouse and
potted in soil.
[0087] The above process is repeated to produce multiple events of
transgenic soybean plant cells that are transformed with
recombinant DNA from each of the genes identified in Table 1.
Events are designed to produce in the transgenic cells one of the
proteins identified in Table 1, except the proteins of SEQ ID NOs:
11, and 17-19 which are suppressed. Progeny transgenic plants and
seed of the transformed plant cells are screened for enhanced water
use efficiency, enhanced cold tolerance, increased yield, enhanced
seed protein and enhanced seed oil. From each group of multiple
events of transgenic plants with a specific recombinant DNA from
Table 1 the event that produces the greatest enhancement in yield,
water use efficiency, nitrogen use efficiency, enhanced cold
tolerance, enhanced seed protein and enhanced seed oil is
identified and progeny seed is selected for commercial
development.
Example 4
Cotton Transgenic Plants with Enhanced Agronomic Traits
[0088] This example illustrates plant transformation useful in
producing a transgenic nucleus in a cotton plant cell, and the
plants, seeds and pollen produced from a transgenic cell with such
a nucleus having an enhanced trait, i.e. enhanced water use
efficiency, increased yield, enhanced nitrogen use efficiency and
enhanced seed oil.
[0089] Transgenic cotton plants containing each recombinant DNA
having a sequence of SEQ ID NO: 1 through SEQ ID NO: 10 are
obtained by transforming with recombinant DNA from each of the
genes identified in Table 1 using Agrobacterium-mediated
transformation. The above process is repeated to produce multiple
events of transgenic cotton plant cells that are transformed with
recombinant DNA from each of the genes identified in Table 1.
Events are designed to produce in the transgenic cells one of the
proteins identified in Table 1, except the proteins of SEQ ID NOs:
11, and 17-19 which are suppressed.
[0090] From each group of multiple events of transgenic plants with
a specific recombinant DNA from Table 1 the event that produces the
greatest enhancement in yield, water use efficiency, nitrogen use
efficiency, enhanced cold tolerance, enhanced seed protein and
enhanced seed oil is identified and progeny seed is selected for
commercial development.
[0091] Progeny transgenic plants are selected from a population of
transgenic cotton events under specified growing conditions and are
compared with control cotton plants. Control cotton plants are
substantially the same cotton genotype but without the recombinant
DNA, for example, either a parental cotton plant of the same
genotype that was not transformed with the identical recombinant
DNA or a negative isoline of the transformed plant. Additionally, a
commercial cotton cultivar adapted to the geographical region and
cultivation conditions, i.e. cotton variety ST474, cotton variety
FM 958, and cotton variety Siokra L-23, are used to compare the
relative performance of the transgenic cotton plants containing the
recombinant DNA.
[0092] Transgenic cotton plants with enhanced yield and water use
efficiency are identified by growing under variable water
conditions. Specific conditions for cotton include growing a first
set of transgenic and control plants under "wet" conditions, i.e.
irrigated in the range of 85 to 100 percent of evapotranspiration
to provide leaf water potential of -14 to -18 bars, and growing a
second set of transgenic and control plants under "dry" conditions,
i.e. irrigated in the range of 40 to 60 percent of
evapotranspiration to provide a leaf water potential of -21 to -25
bars. Pest control, such as weed and insect control is applied
equally to both wet and dry treatments as needed. Data gathered
during the trial includes weather records throughout the growing
season including detailed records of rainfall; soil
characterization information; any herbicide or insecticide
applications; any gross agronomic differences observed such as leaf
morphology, branching habit, leaf color, time to flowering, and
fruiting pattern; plant height at various points during the trial;
stand density; node and fruit number including node above white
flower and node above crack boll measurements; and visual wilt
scoring. Cotton boll samples are taken and analyzed for lint
fraction and fiber quality. The cotton is harvested at the normal
harvest timeframe for the trial area. Enhanced water use efficiency
is indicated by increased yield, improved relative water content,
enhanced leaf water potential, increased biomass, enhanced leaf
extension rates, and improved fiber parameters.
Example 5
Canola Transformation
[0093] This example illustrates plant transformation useful in
producing the transgenic canola plants of this invention and the
production and identification of transgenic seed for transgenic
canola having enhanced water use efficiency, enhanced cold
tolerance, increased yield, enhanced nitrogen use efficiency,
enhanced seed protein and enhanced seed oil.
[0094] Tissues from in vitro grown canola seedlings are prepared
and inoculated with overnight-grown Agrobacterium cells containing
plasmid DNA with the gene of interest cassette and a plant
selectable marker cassette. Following co-cultivation with
Agrobacterium, the infected tissues are allowed to grow on
selection to promote growth of transgenic shoots, followed by
growth of roots from the transgenic shoots. The selected plantlets
are then transferred to the greenhouse and potted in soil.
Molecular characterizations are performed to confirm the presence
of the gene of interest, and its expression in transgenic plants
and progenies. Progeny transgenic plants are selected from a
population of transgenic canola events under specified growing
conditions and are compared with control canola plants. Control
canola plants are substantially the same canola genotype but
without the recombinant DNA, for example, either a parental canola
plant of the same genotype that is not transformed with the
identical recombinant DNA or a negative isoline of the transformed
plant.
[0095] Transgenic canola plant cells are transformed with each of
the recombinant DNA identified in Table 1. The above process is
repeated to produce multiple events of transgenic canola plant
cells that are transformed with recombinant DNA from each of the
genes identified in Table 1. Events are designed to produce in the
transgenic cells one of the proteins identified in Table 1, except
the proteins of SEQ ID NOs: 11, and 17-19 which are suppressed.
Progeny transgenic plants and seed of the transformed plant cells
are screened for enhanced water use efficiency, enhanced cold
tolerance, increased yield, enhanced seed protein and enhanced seed
oil. From each group of multiple events of transgenic plants with a
specific recombinant DNA from Table 1 the event that produces the
greatest enhancement in yield, water use efficiency, nitrogen use
efficiency, enhanced cold tolerance, enhanced seed protein and
enhanced seed oil is identified and progeny seed is selected for
commercial development.
Example 6
Homolog Identification
[0096] This example illustrates the identification of homologs of
proteins encoded by the DNA identified in Table 1 which is used to
provide transgenic seed and plants having enhanced agronomic
traits. From the sequence of the homologs, homologous DNA sequence
can be identified for preparing additional transgenic seeds and
plants of this invention with enhanced agronomic traits.
[0097] An "All Protein Database" was constructed of known protein
sequences using a proprietary sequence database and the National
Center for Biotechnology Information (NCBI) non-redundant amino
acid database (nr.aa). For each organism from which a
polynucleotide sequence provided herein was obtained, an "Organism
Protein Database" was constructed of known protein sequences of the
organism; it is a subset of the All Protein Database based on the
NCBI taxonomy ID for the organism.
[0098] The All Protein Database was queried using amino acid
sequences provided herein as SEQ ID NO: 11 through SEQ ID NO: 20
using NCBI "blastp" program with E-value cutoff of 1e-8. Up to 1000
top hits were kept, and separated by organism names. For each
organism other than that of the query sequence, a list was kept for
hits from the query organism itself with a more significant E-value
than the best hit of the organism. The list contains likely
duplicated genes of the polynucleotides provided herein, and is
referred to as the Core List. Another list was kept for all the
hits from each organism, sorted by E-value, and referred to as the
Hit List.
[0099] The Organism Protein Database was queried using polypeptide
sequences provided herein as SEQ ID NO: 11 through SEQ ID NO: 20
using NCBI "blastp" program with E-value cutoff of 1e-4. Up to 1000
top hits were kept. A BLAST searchable database was constructed
based on these hits, and is referred to as "SubDB". SubDB is
queried with each sequence in the Hit List using NCBI "blastp"
program with E-value cutoff of 1e-8. The hit with the best E-value
was compared with the Core List from the corresponding organism.
The hit is deemed a likely ortholog if it belongs to the Core List,
otherwise it is deemed not a likely ortholog and there is no
further search of sequences in the Hit List for the same organism.
Homologs from a large number of distinct organisms were identified
and are reported below in table 7 with the SEQ ID NO of the
original query sequence and the identified homologs as [SEQ ID NO]:
[Homolog SEQ ID NOs].
TABLE-US-00007 TABLE 7 Protein Sequences and their Homologs 11: 24
28 32 34 40 44 46 47 49 50 51 52 53 60 68 71 72 74 75 80 81 85 87
90 118 120 121 129 135 143 153 154 157 159 164 168 173 176 182 184
194 197 200 202 203 213 214 228 237 246 248 249 251 253 255 258 261
268 274 277 292 294 299 303 306 313 314 320 325 326 328 333 347 348
349 354 355 357 360 361 362 363 364 365 366 370 374 383 389 390 391
395 397 425 426 431 434 436 456 457 459 463 467 469 470 471 472 473
474 477 480 488 490 492 499 508 515 522 529 530 532 540 541 555 568
574 577 578 579 581 585 587 588 589 595 596 598 600 610 616 618 621
623 626 633 638 639 640 643 644 645 651 652 658 661 662 663 666 674
677 691 692 693 694 695 709 717 718 720 728 729 745 746 753 756 757
774 775 784 787 791 793 799 801 810 825 831 837 850 851 856 857 865
866 868 871 873 875 876 879 881 886 888 891 892 896 899 904 905 906
908 916 917 919 929 935 940 943 946 948 950 962 963 964 969 974 982
993 997 1005 1009 1011 1013 1015 1027 1028 1031 1032 1035 1049 1050
1059 1066 1068 1069 1076 1083 1085 1088 1090 1093 1095 1097 1098
1100 1107 1108 1110 1111 1117 1118 1120 1131 1134 1136 1137 1145
1146 1147 1149 1150 1153 1155 1156 1158 1168 1169 1170 1176 1184
1188 1193 1196 1205 1207 1212 1216 1226 1229 1230 1232 1233 1236
1241 1242 1243 1244 1248 1249 1252 1254 1256 1257 1260 1262 1265
1267 1269 1280 1283 1287 1292 1306 1307 1334 1335 1345 1350 12: 36
38 43 45 48 56 61 62 65 73 78 79 82 83 84 89 100 107 109 124 126
131 132 142 144 146 148 152 167 171 196 204 206 212 215 218 219 224
233 235 242 243 254 260 264 265 267 279 290 291 295 297 305 311 316
321 322 324 331 332 340 342 358 369 375 398 410 413 414 417 435 437
438 441 462 468 475 485 493 496 498 500 502 504 509 511 516 518 521
525 531 534 538 539 542 546 556 563 580 606 607 611 620 629 632 636
656 664 665 678 683 705 706 710 711 725 738 742 743 762 788 795 798
802 803 812 822 835 838 839 845 846 852 860 863 869 872 877 883 887
894 930 931 936 939 942 944 971 977 979 983 992 999 1007 1010 1030
1046 1048 1065 1075 1087 1091 1112 1113 1119 1124 1133 1135 1181
1182 1192 1195 1197 1206 1211 1213 1217 1219 1220 1223 1234 1235
1237 1239 1246 1247 1251 1258 1259 1261 1266 1300 1310 1317 1321
1328 1331 13: 23 30 33 42 54 55 63 64 66 88 92 93 94 95 96 97 99
103 104 106 114 115 117 119 127 128 130 138 147 150 151 155 158 160
165 166 175 178 190 191 195 198 208 210 211 221 229 230 238 247 252
256 257 259 262 263 266 272 283 287 317 327 329 330 353 356 368 378
379 385 387 394 399 404 409 411 419 427 428 433 439 440 444 446 449
458 460 461 481 487 503 505 507 510 513 527 537 545 548 550 552 553
557 558 559 564 565 569 572 584 593 602 605 608 609 612 617 619 625
637 647 650 660 672 690 696 703 716 722 726 730 731 735 747 749 750
755 761 766 771 776 779 786 790 794 804 806 811 817 821 824 826 827
828 829 836 841 847 848 854 867 870 878 884 893 895 897 907 909 910
914 918 923 925 928 933 934 951 952 954 955 957 958 959 965 975 985
987 988 991 1012 1019 1020 1022 1029 1033 1034 1036 1039 1041 1043
1044 1052 1056 1057 1060 1061 1063 1071 1072 1073 1084 1086 1094
1102 1115 1122 1126 1128 1144 1148 1151 1159 1161 1162 1173 1174
1177 1179 1189 1190 1201 1202 1203 1208 1221 1225 1227 1228 1231
1255 1270 1273 1278 1279 1291 1296 1301 1302 1318 1322 1324 1330
1338 1346 1348 14: 23 30 33 54 55 63 64 66 88 91 92 93 94 95 96 97
99 103 104 106 112 115 117 119 127 128 130 137 138 147 150 151 158
160 162 165 174 175 178 181 186 189 190 191 198 201 217 221 229 230
238 244 247 252 256 257 259 262 263 266 269 272 276 283 286 317 318
319 327 329 330 344 353 356 368 379 385 387 393 394 399 404 408 409
411 419 423 427 440 445 446 449 450 458 460 461 482 487 489 503 505
507 510 513 526 527 535 537 545 548 550 551 552 553 557 558 559 569
572 575 584 590 593 603 605 608 609 612 619 625 628 631 637 642 647
650 653 660 668 672 675 688 690 696 700 703 707 716 722 726 730 731
732 735 747 749 750 754 755 761 766 771 776 778 779 780 781 782 786
790 794 804 806 811 817 820 821 824 826 828 836 841 847 848 854 867
870 878 882 884 893 895 897 907 909 910 913 914 918 920 921 923 924
932 933 934 949 951 952 954 955 957 959 960 965 975 985 987 988 991
1012 1019 1020 1022 1026 1029 1033 1034 1036 1039 1041 1043 1044
1052 1057 1060 1061 1063 1071 1072 1073 1077 1084 1086 1094 1102
1109 1126 1128 1132 1138 1144 1148 1157 1159 1161 1162 1173 1174
1177 1179 1189 1190 1202 1203 1208 1221 1222 1225 1228 1231 1255
1270 1271 1273 1276 1284 1289 1291 1293 1301 1302 1318 1322 1324
1346 1348 15: 21 26 27 31 35 37 41 57 58 59 69 70 76 77 86 98 101
102 108 110 111 113 116 122 123 125 133 136 139 140 145 149 156 161
163 169 170 172 177 180 183 185 187 188 192 193 199 209 216 220 222
223 225 226 227 231 232 234 236 239 240 241 245 250 270 271 273 275
278 280 281 282 284 285 288 289 293 296 298 300 301 302 307 308 309
310 312 315 323 335 336 337 338 339 341 343 345 346 351 352 359 367
371 372 373 376 377 380 381 382 384 386 392 396 400 401 402 403 406
407 412 415 416 418 420 421 422 424 429 430 432 442 443 447 448 452
453 455 464 465 466 476 478 479 483 484 486 491 494 495 497 501 506
517 519 520 523 524 528 533 536 543 547 549 554 560 561 562 566 567
570 571 573 576 582 583 594 597 599 601 604 613 614 615 622 624 627
630 634 635 641 646 648 649 654 655 657 659 667 669 670 671 673 679
681 682 684 685 686 687 689 697 698 699 701 702 704 708 712 714 715
719 721 723 724 727 733 734 736 739 741 744 748 751 752 758 759 760
763 764 765 767 768 769 770 772 773 783 785 789 792 796 800 805 807
808 809 813 814 815 816 818 823 830 832 833 834 840 842 843 844 849
853 855 858 859 861 862 864 874 880 889 890 898 900 901 903 911 915
922 926 927 937 945 947 953 956 961 966 967 968 970 972 973 976 980
981 984 986 989 990 994 995 996 998 1000 1001 1002 1003 1004 1006
1008 1014 1016 1017 1018 1021 1023 1024 1025 1037 1040 1042 1045
1047 1051 1053 1054 1055 1058 1067 1070 1074 1078 1079 1080 1081
1082 1092 1096 1099 1101 1103 1104 1105 1106 1114 1116 1123 1125
1127 1129 1130 1139 1141 1143 1152 1154 1160 1163 1164 1165 1166
1167 1171 1172 1175 1178 1180 1183 1185 1186 1187 1194 1198 1199
1204 1209 1210 1214 1215 1218 1224 1238 1240 1245 1250 1253 1263
1264 1268 1272 1274 1275 1277 1281 1282 1285 1288 1294 1295 1297
1298 1299 1303 1304 1305 1308 1309 1311 1312 1313 1314 1315 1316
1319 1320 1323 1325 1326 1327 1329 1332 1333 1336 1337 1339 1340
1341 1342 1343 1347 1349 1351 16: 21 26 27 31 35 37 41 57 58 59 69
70 76 77 86 98 101 102 108 110 111 113 116 122 123 125 133 136 139
140 145 149 156 161 163 169 170 172 177 180 183 185 187 188 192 193
199 209 216 220 222 223 225 226 227 231 232 234 236 239 240 241 245
250 270 271 273 275 278 280 281 282 284 285 288 289 293 296 298 300
301 302 307 308 309 310 312 315 323 335 336 337 338 339 341 343 345
346 351 352 359 367 371 372 373 376 377 380 381 382 384 386 392 396
400 401 402 403 406 407 412 415 416 418 420 421 422 424 429 430 432
442 443 447 448 452 453 455 464 465 466 476 478 479 483 484 486 491
494 495 497 501 506 517 519 520 523 524 528 533 536 543 547 549 554
560 561 562 566 567 570 571 573 576 582 583 594 597 599 601 604 613
614 615 622 624 627 630 634 635 641 646 648 649 654 655 657 659 667
669 670 671 673 679 681 682 684 685 686 687 689 697 698 699 701 702
704 708 712 714 715 719 721 723 724 727 733 734 736 739 741 744 748
751 752 758 759 760 763 764 765 767 768 769 770 772 773 783 785 789
792 796 800 805 807 808 809 813 814 815 816 818 823 830 832 833 834
840 842 843 844 849 853 855 858 859 861 862 864 874 880 889 890 898
900 901 903 911 915 922 926 927 937 945 947 953 956 961 966 967 968
970 972 973 976 980 981 984 986 989 990 994 995 996 998 1000 1001
1002 1003 1004 1006 1008 1014 1016 1017 1018 1021 1023 1024 1025
1037 1040 1042 1045 1047 1051 1053 1054 1055 1058 1067 1070 1074
1078 1079 1080 1081 1082 1092 1096 1099 1101 1103 1104 1105 1106
1114 1116 1123 1125 1127 1129 1130 1139 1141 1143 1152 1154 1160
1163 1164 1165 1166 1167 1171 1172 1175 1178 1180 1183 1185 1186
1187 1194 1198 1199 1204 1209 1210 1214 1215 1218 1224 1238 1240
1245 1250 1253 1263 1264 1268 1272 1274 1275 1277 1281 1282 1285
1288 1294 1295 1297 1298 1299 1303 1304 1305 1308 1309 1311 1312
1313 1314 1315 1316 1319 1320 1323 1325 1326 1327 1329 1332 1333
1336 1337 1339 1340 1341 1342 1343 1347 1349 1351 17: 179 207 514
586 591 902 1200 1286 18: 179 207 514 586 591 902 1200 1286 19: 22
29 141 592 20: 25 39 67 105 134 205 304 334 350 388 405 451 454 512
544 676 680 713 737 740 777 797 819 885 912 938 941 978 1038 1062
1064 1089 1121 1140 1142 1191 1290 1344
[0100] Recombinant DNA constructs are prepared using the DNA
encoding each of the identified homologs and the constructs are
used to prepare multiple events of transgenic corn, soybean, canola
and cotton plants as illustrated in Examples 2-5. Plants are
regenerated from the transformed plant cells and used to produce
progeny plants and seed that are screened for enhanced water use
efficiency, enhanced cold tolerance, increased yield, enhanced
nitrogen use efficiency, enhanced seed protein and enhanced seed
oil. From each group of multiple events of transgenic plants with a
specific recombinant DNA for a homolog the event that produces the
greatest enhancement in yield, water use efficiency, nitrogen use
efficiency, enhanced cold tolerance, enhanced seed protein and
enhanced seed oil is identified and progeny seed is selected for
commercial development.
Example 7
Consensus Sequence
[0101] This example illustrates the identification of consensus
amino acid sequence for the proteins and homologs encoded by DNA
that is used to prepare the transgenic seed and plants of this
invention having enhanced agronomic traits.
[0102] ClustalW program was selected for multiple sequence
alignments of the amino acid sequence of SEQ ID NO: 17-19 and their
homologs. Three major factors affecting the sequence alignments
dramatically are (1) protein weight matrices; (2) gap open penalty;
(3) gap extension penalty. Protein weight matrices available for
ClustalW program include Blosum, Pam and Gonnet series. Those
parameters with gap open penalty and gap extension penalty were
extensively tested. On the basis of the test results, Blosum weight
matrix, gap open penalty of 10 and gap extension penalty of 1 were
chosen for multiple sequence alignment.
[0103] The consensus amino acid sequence can be used to identify
DNA corresponding to the full scope of this invention that is
useful in providing transgenic plants, for example corn and soybean
plants with enhanced agronomic traits, for example improved
nitrogen use efficiency, improved yield, improved water use
efficiency and/or improved growth under cold stress, due to the
expression in the plants of DNA suppressing a protein with amino
acid sequence identical to the consensus amino acid sequence.
[0104] The SEQ ID NOs for the identified consensus sequences are
reported in table 8 below and the full consensus sequences are
provided in the attached sequence listing.
TABLE-US-00008 TABLE 8 Gene ID PEP SEQ ID NO Consensus SEQ ID NO
Mnom000090 17 1356 Mnom000091 18 1357 Mnom000092 19 1358
Example 9
Identification of Amino Acid Domain by Pfam Analysis
[0105] This example illustrates the identification of domain and
domain module by Pfam analysis.
[0106] The amino acid sequence of the expressed proteins that are
shown to be associated with an enhanced trait were analyzed for
Pfam protein family against the current Pfam collection of multiple
sequence alignments and hidden Markov models using the HMMER
software in the appended computer listing. The Pfam protein domains
and modules for the proteins of SEQ ID NO: 11 through 16 and 20 are
shown in Tables 9, 10 and 11. The Hidden Markov model databases for
the identified patent families are also in the appended computer
listing allowing identification of other homologous proteins and
their cognate encoding DNA to enable the full breadth of the
invention for a person of ordinary skill in the art. Certain
proteins are identified by a single Pfam domain and others by
multiple Pfam domains.
TABLE-US-00009 TABLE 9 Pfam annotation PEP SEQ ID NO Gene ID Pfam
domain name Begin Stop Score E-value 11 Mnom000034 Cu-oxidase_3 30
146 237 4.10E-68 11 Mnom000034 Cu-oxidase 156 310 158.7 1.50E-44 11
Mnom000034 Cu-oxidase_2 409 532 169.9 6.30E-48 12 Mnom000037
Flavodoxin_1 7 160 197.3 3.70E-56 13 Mnom000048 Glyco_transf_20 3
470 933.9 6.70E-278 13 Mnom000048 Trehalose_PPase 504 748 256.4
6.20E-74 14 Mnom000049 Glyco_transf_20 76 578 841.5 4.40E-250 14
Mnom000049 Trehalose_PPase 627 862 339.1 7.70E-99 15 Mnom000067
Aminotran_1_2 60 441 33.4 8.00E-09 16 Mnom000068 Aminotran_1_2 60
441 33.4 8.00E-09 20 Mnom000095 B3 312 417 118.5 2.00E-32 20
Mnom000095 Auxin_resp 439 524 160.7 3.80E-45 20 Mnom000095 AUX_IAA
681 826 -74.9 0.00043
TABLE-US-00010 TABLE 10 Pfam module annotation PEP SEQ ID NO Gene
ID Pfam domain module Position 11 Mnom000034 Cu-oxidase_3::Cu-
30-146::156-310::409-532 oxidase::Cu-oxidase_2 12 Mnom000037
Flavodoxin_1 7-160 13 Mnom000048 Glyco_transf_20::Trehalose_PPase
3-470::504-748 14 Mnom000049 Glyco_transf_20::Trehalose_PPase
76-578::627-862 15 Mnom000067 Aminotran_1_2 60-441 16 Mnom000068
Aminotran_1_2 60-441 20 Mnom000095 B3::Auxin_resp::AUX_IAA
312-417::439-524::681-826
TABLE-US-00011 TABLE 11 Description of Pfam domains Accession
Gathering Pfam domain name number cutoff Domain description AUX_IAA
PF02309.8 -83.0000; AUX/IAA family Aminotran_1_2 PF00155.13
-57.5000; Aminotransferase class I and II Auxin_resp PF06507.5
25.0000; Auxin response factor B3 PF02362.13 26.5000; B3 DNA
binding domain Cu-oxidase PF00394.14 -18.9000; Multicopper oxidase
Cu-oxidase_2 PF07731.6 -5.8000; Multicopper oxidase Cu-oxidase_3
PF07732.7 10.0000; Multicopper oxidase Flavodoxin_1 PF00258.17
6.3000; Flavodoxin Glyco_transf_20 PF00982.13 -243.6000;
Glycosyltransferase family 20 Trehalose_PPase PF02358.8 -49.4000;
Trehalose-phosphatase
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=US20090165165A1).
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=US20090165165A1).
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