U.S. patent application number 15/881270 was filed with the patent office on 2018-06-07 for isolated novel nucleic acid and protein molecules from soy and methods of using those molecules to generate transgenic plants with enhanced agronomic traits.
This patent application is currently assigned to Monsanto Technology LLC. The applicant listed for this patent is Monsanto Technology LLC. Invention is credited to Liang Guo, David Kovalic, Bo-Xing Qiu, Jack Tabaska, Wei Wu.
Application Number | 20180155734 15/881270 |
Document ID | / |
Family ID | 40952601 |
Filed Date | 2018-06-07 |
United States Patent
Application |
20180155734 |
Kind Code |
A1 |
Guo; Liang ; et al. |
June 7, 2018 |
Isolated Novel Nucleic Acid and Protein Molecules From Soy and
Methods of Using Those Molecules to Generate Transgenic Plants With
Enhanced Agronomic Traits
Abstract
This disclosure provides purified nucleic acids and
polypeptides. Also provided are transgenic plants, seeds, and plant
cells containing DNA for expression of the proteins that are useful
for imparting enhanced agronomic trait(s) to transgenic crop
plants, methods of making such plants and methods of making
agricultural commodity including seeds and hybrid seeds from such
plants.
Inventors: |
Guo; Liang; (St. Louis,
MO) ; Kovalic; David; (Clayton, MO) ; Qiu;
Bo-Xing; (Chesterfield, MO) ; Tabaska; Jack;
(O'Fallon, MO) ; Wu; Wei; (Chesterfield,
MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Monsanto Technology LLC |
St. Louis |
MO |
US |
|
|
Assignee: |
Monsanto Technology LLC
St. Louis
MO
|
Family ID: |
40952601 |
Appl. No.: |
15/881270 |
Filed: |
January 26, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14685063 |
Apr 13, 2015 |
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15881270 |
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12864690 |
Jul 27, 2010 |
9029636 |
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PCT/US2009/000655 |
Feb 2, 2009 |
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14685063 |
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61063633 |
Feb 5, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8275 20130101; C12N 15/8273 20130101; C12N 15/8278
20130101; C12N 15/8274 20130101; C12N 15/8218 20130101; A01H 5/10
20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; A01H 5/10 20180101 A01H005/10; C07K 14/415 20060101
C07K014/415 |
Claims
1. A recombinant DNA construct comprising a polynucleotide encoding
a protein that has 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: 116779-233556 when
said amino acid sequence is aligned with said reference
sequence.
2. (canceled)
3. 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: 116779-233556, 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:
116779-233556, when said amino acid sequence is aligned to said
reference sequence; and wherein said construct is stably integrated
into a chromosome in a plant cell nucleus.
4. A transgenic plant cell comprising the recombinant DNA construct
of claim 3 wherein said DNA construct provides 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.
5. The plant cell of claim 4 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 nucleus.
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 4.
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
4.
10. The transgenic seed of claim 9 from a corn, soybean, cotton,
canola, alfalfa, wheat, rice, sugarcane, or sugar beet plant.
11. A transgenic pollen grain comprising a haploid derivative of a
plant cell nucleus having a chromosome comprising the recombinant
DNA construct of claim 3.
12. 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 a stably-integrated, recombinant
DNA construct of claim 3, 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.
13. The method of claim 12 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:116779-233556.
14. The method of claim 13 wherein said seed is corn, soybean,
cotton, canola, alfalfa, wheat, rice, sugarcane, or sugar beet
seed.
15. A method of producing hybrid corn seed comprising: (a)
acquiring hybrid corn seed from an herbicide tolerant corn plant
which also has a stably-integrated, recombinant DNA construct of
claim 3; (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.
16. A substantially purified nucleic acid molecule comprising a
nucleic acid sequence wherein said nucleic acid sequence exhibits
95% or greater identity to a nucleic acid sequence selected from
the group consisting of SEQ ID NO: 1 through SEQ ID NO: 116778 and
sequences complementary to SEQ ID NO: 1 through SEQ ID NO: 116778.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. provisional
application Ser. No. 61/063,633 filed on Feb. 5, 2008 which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] Disclosed herein are inventions in the field of plant
genetics and developmental biology. More specifically, this
invention provides novel compositions of soy DNA and peptide
molecules. Also disclosed are plants comprising recombinant DNA
providing one or more enhanced traits in a transgenic plant,
including cells, seed, and pollen derived from such a plant, as
well as methods of making and using such plant.
INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC
[0003] A sequence listing having the file name
"38-21(55306)B-PCT_SeqListing.txt" which has 370,353,632 bytes
(measured in MS-WINDOWS) was created on Feb. 2, 2009 an has 233556
sequences is enclosed herewith in computer readable format and is
herein incorporated by reference.
SUMMARY OF THE INVENTION
[0004] Certain embodiments of the disclosed invention provide
recombinant DNA constructs having polynucleotides characterized by
reference to SEQ ID NO:1-116778 and the cognate proteins with amino
acid sequences having reference to SEQ ID NO:116779-233556. The
recombinant DNA constructs are used in aspects of the various
embodiments of the invention to provide enhanced traits when stably
integrated into the chromosomes and expressed in the nuclei of
transgenic plants cells. In many aspects of these embodiments, 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 selected from the group consisting of SEQ ID
NOs: 116779-233556 when the amino acid sequence is aligned to the
reference sequence. In some aspects of the invention, the
recombinant DNA constructs are characterized as being constructed
with sense-oriented and/or anti-sense-oriented polynucleotides from
the group consisting of SEQ ID NOs: 1-116778 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 selected from the
group consisting of SEQ ID NOs: 116779-233556.
[0005] In certain aspects of this invention the recombinant DNA
constructs of the invention are stably integrated into the
chromosome of a plant cell nucleus.
[0006] Certain aspects of this embodiment of the invention provide
transgenic plant cells having stably integrated recombinant DNA
constructs, transgenic plants and seeds comprising a plurality of
such transgenic plant cells and transgenic pollen of such plants.
Such transgenic plants can be 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
provided may include, but is not limited to, enhanced water use
efficiency, enhanced cold tolerance, increased yield, enhanced
nitrogen use efficiency, altered seed protein composition, altered
seed oil composition, or any combinations thereof.
[0007] Other embodiments of the invention provide for plant cells,
plants, seeds, and pollen that can 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.
[0008] Embodiments of the invention also provide 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. The methods may comprise one or more of the following
steps: (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
at least one sequence selected from SEQ ID NOs:1-116778. In certain
embodiments of the invention, the plants in the population further
include 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 embodiment of the invention, the
plants are selected by identifying plants with the enhanced trait.
The methods can be used for the manufacturing corn, soybean,
cotton, canola, alfalfa, wheat, rice, sugarcane or sugar beet seed.
In other embodiments of the present invention, the methods can also
be used for manufacture transgenic plants including, but are not
limited to, millet, barley, peanut, pigeon pea, sorghum, vegetables
(including but not limited to Broccoli, Cauliflower, Cabbage,
Radish, Chinese cabbage, Melons, Watermelons, Cucumber, Gourds,
Pumpkin, Squash, Pepper, Tomato, Eggplant, Onion, Carrot, Garden
Bean, Sweet Corn, Pea, Dry Bean, Okra, Spinach, Leek, Lettuce, and
Fennel), grape, berries (including blue, black, raspberry,
mullberry, boisenberry, etc), cherry and related fruit trees
(including but not limited to plum, peach, apricot, kiwi,
pomegranate, mango, fig), fruit trees (including but not limited to
orange, lemon, lime, blood orange, grapefruit, and the like), and
nut trees (including but not limited to coconut, walnut (English
and black), pecan, almond, hazelnut, brazil nut, hickory nut,
acorn, and the like) and sunflower, other oilseed producing plants
or any combinations thereof.
[0009] Other embodiments of the invention provide a methods for
producing hybrid corn seed by acquiring hybrid corn seed from a
herbicide tolerant corn plant which also has stably-integrated,
recombinant DNA construct having 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 at least one sequence selected from the group
consisting of SEQ ID NOs:1-116778. The methods of these embodiments
may further include 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.
[0010] Other embodiments of the invention provide methods for
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. Another embodiment of the invention provides
anti-counterfeit milled seed having, as an indication of origin,
plant cells of this invention.
[0011] Still other embodiments of this invention provide for
transgenic plants with enhanced water use efficiency or enhanced
nitrogen use efficiency. For example, this invention provides
methods of growing a corn, cotton, soybean, or canola crop without
irrigation water by planting seed having plant cells of the
invention which are selected for enhanced water use efficiency.
Alternatively embodiments of these methods include 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 by planting seed having plant
cells of the invention which are selected for enhanced nitrogen use
efficiency.
[0012] Other embodiments of the invention provide mixtures
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:
116779-233556 when the sequence is aligned to the reference
sequence.
ILLUSTRATIVE EMBODIMENTS OF THE INVENTION
[0013] While the making and using of various embodiments of the
present invention are discussed in detail below, it should be
appreciated that the present invention provides many applicable
inventive concepts that can be embodied in a wide variety of
specific contexts. The specific embodiments discussed herein are
merely illustrative of specific ways to make and use the invention
and do not delimit the scope of the invention.
[0014] To facilitate the understanding of this invention, a number
of terms are defined below. Terms defined herein have meanings as
commonly understood by a person of ordinary skill in the areas
relevant to the present invention. Terms such as "a", "an" and
"the" are not intended to refer to only a singular entity, but
include the general class of which a specific example may be used
for illustration. The terminology herein is used to describe
specific embodiments of the invention, but their usage does not
delimit the invention, except as outlined in the claims.
[0015] In the attached sequence listing:
[0016] SEQ ID NO:1-116778 are nucleotide sequences of the coding
strand of DNA for "genes" used in the recombinant DNA imparting an
enhanced trait in plant cells, e.g. each comprises a coding
sequence for a protein;
[0017] SEQ ID NO: 116779-233556 are amino acid sequences of the
cognate protein of the "genes" with nucleotide coding sequences
provided by SEQ ID NO: 1-116778;
[0018] 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 duplicated by regeneration 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.
[0019] 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.
[0020] A "consensus amino acid sequence" means an artificial, amino
acid sequence indicating conserved amino acids in the sequence of
homologous proteins as determined by statistical analysis of an
optimal alignment, e.g. CLUSTALW, of amino acid sequence of homolog
proteins. The consensus sequences listed in the sequence listing
were created by identifying the most frequent amino acid at each
position in a set of aligned protein sequences. When there was 100%
identity in an alignment the amino acid is indicated by a capital
letter. When the occurrence of an amino acid is at least about 70%
in an alignment, the amino acid is indicated by a lower case
letter. When there is no amino acid occurrence of at least about
70%, e.g. due to diversity or gaps, the amino acid is indicated by
an "x". When used to defined embodiments of the invention, a
consensus amino acid sequence will be aligned with a query protein
amino acid sequence in an optimal alignment, e.g. CLUSTALW. An
embodiment of the invention will have identity to the conserved
amino acids indicated in the consensus amino acid sequence.
[0021] As used herein, "control plant" means a plant that does not
contain the recombinant DNA that expressed a protein which imparts
an enhanced trait. A control plant is to identify and select a
transgenic plant that has an enhance trait. A suitable control
plant can be a non-transgenic plant of the parental line used to
generate a transgenic plant, e.g., devoid of recombinant DNA. A
suitable control plant can in some cases be a progeny of a
hemizygous transgenic plant line that is does not contain the
recombinant DNA, known as a negative segregant.
[0022] 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 can 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.
[0023] 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 (e.g., seeds, or weight of seeds, per acre), bushels per acre,
tonnes per acre, tons per acre, kilo per hectare. For example,
maize yield can be measured as production of shelled corn kernels
per unit of production area in bushels per acre or metric tons per
hectare, often reported on a moisture adjusted basis at about 15.5
percent moisture. Increased yield can 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 can correspond to an increase
in overall plant yield. Such properties include enhancements in
seed oil, seed molecules such as tocopherol, protein and starch, or
oil, particular oil components as can be manifest by alterations in
the ratios of seed components.
[0024] Seed according to the present invention may be planted,
grown and harvested to produce a crop or terminal crop. As used
herein, a "crop" is a plant or plant product that is grown and
harvested, such plant or plant product including but not limited to
plants or plant parts such as leaf, root, shoot, fruit, seed,
grain, or the like. A "terminal crop" is a crop grown for uses
other than for use as planting seed to produce subsequent
generations of plants. In some crop plants, such as grain produced
from hybrid corn, the crop is not very suitable for planting
because it does not breed true and the crop can then be
conveniently referred to as "hybrid grain." In other crop plants,
where the crop does breed true, such as soybean, whether a crop is
planting seed or a terminal crop will depend on the uses and
marketing channels of the crop. If used or marketed for planting,
it will be a crop of planting seed; if used or marketed for other
purposes it will be a terminal crop.
[0025] As used herein, "exogenous promoter region" refers to a
sequence, capable of promoting mRNA transcription, that does not
naturally occur in the plant at the same site and/or linked to the
nucleic acids. The promoter can be from a different plant species
or it can be from the same plant species, but naturally found in a
different location in a non-genetically modified plant. Moreover,
the promoter region can be found in the same genetic locus as is
present a native plant, but linked to different sequence(s), than
are native.
[0026] 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 can 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.
[0027] 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.
[0028] As used herein, an "expression cassette of a DNA construct"
is capable of integrating in a plant genome, expressing a
functional polypeptide and providing a transgenic plant expressing
polypeptides of the invention.
[0029] 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.
[0030] As used herein, a "functional fragment" refers to a portion
of a polypeptide provided herein which retains full or partial
molecular, physiological or biochemical function of the full length
polypeptide. A functional fragment often contains the domain(s),
such as Pfam domain, identified in the polypeptide provided in the
sequence listing.
[0031] 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, e.g., 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, e.g., 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 typically at least about 60% identity, in some instances at
least about 70%, for example about 80% and even at least about 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.
[0032] 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, e.g., 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.
[0033] Other functional homolog proteins differ in one or more
amino acids from those of a trait-improving protein disclosed
herein as the result of one or more of the well-known conservative
amino acid substitutions, e.g., valine is a conservative substitute
for alanine and threonine is a conservative substitute for serine.
Conservative substitutions for an amino acid within the native
sequence can be selected from other members of a class to which the
naturally occurring amino acid belongs. Representative amino acids
within these various classes include, but are not limited to: (1)
acidic (negatively charged) amino acids such as aspartic acid and
glutamic acid; (2) basic (positively charged) amino acids such as
arginine, histidine, and lysine; (3) neutral polar amino acids such
as glycine, serine, threonine, cysteine, tyrosine, asparagine, and
glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such
as alanine, leucine, isoleucine, valine, proline, phenylalanine,
tryptophan, and methionine. Conserved substitutes for an amino acid
within a native amino acid sequence can be selected from other
members of the group to which the naturally occurring amino acid
belongs. For example, a group of amino acids having aliphatic side
chains is glycine, alanine, valine, leucine, and isoleucine; a
group of amino acids having aliphatic-hydroxyl side chains is
serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having basic side
chains is lysine, arginine, and histidine; and a group of amino
acids having sulfur-containing side chains is cysteine and
methionine. Naturally conservative amino acids substitution groups
are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine,
lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and
asparagine-glutamine. A further aspect of the invention includes
proteins that differ in one or more amino acids from those of a
described protein sequence as the result of deletion or insertion
of one or more amino acids in a native sequence.
[0034] Genes that are homologous to each other can be grouped into
families and included in multiple sequence alignments. Then a
consensus sequence for each group can be derived. This analysis
enables the derivation of conserved and class-(family) specific
residues or motifs that are functionally important. These conserved
residues and motifs can be further validated with 3D protein
structure if available. The consensus sequence can be used to
define the full scope of the invention, e.g., to identify proteins
with a homolog relationship. Thus, the present invention
contemplates that protein homologs include proteins with an amino
acid sequence that has at least 90% identity to such a consensus
amino acid sequence sequences.
[0035] As used herein, "operably linked" refers to the association
of two or more nucleic acid elements in a recombinant DNA
construct, e.g. as when a promoter is operably linked with DNA that
is transcribed to RNA whether for expressing or suppressing a
protein. Recombinant DNA constructs can be designed to express a
protein which can be an endogenous protein, an exogenous homologue
of an endogenous protein or an exogenous protein with no native
homologue. Alternatively, recombinant DNA constructs can be
designed to suppress the level of an endogenous protein, e.g. by
suppression of the native gene. Such gene suppression can be
effectively employed through a native RNA interference (RNAi)
mechanism in which recombinant DNA comprises both sense and
anti-sense oriented DNA matched to the gene targeted for
suppression where the recombinant DNA is transcribed into RNA that
can form a double-strand to initiate an RNAi mechanism. Gene
suppression can also be effected by recombinant DNA that comprises
anti-sense oriented DNA matched to the gene targeted for
suppression. Gene suppression can also be effected by recombinant
DNA that comprises DNA that is transcribed to a microRNA matched to
the gene targeted for suppression. In the examples illustrating the
invention recombinant DNA for effecting gene suppression that
imparts is identified by the term "antisense". It will be
understood by a person of ordinary skill in the art that any of the
ways of effecting gene suppression are contemplated and enabled by
a showing of one approach to gene suppression.
[0036] As used herein, "percent identity" means the extent to which
two optimally aligned DNA or protein segments are invariant
throughout a window of alignment of components, for example
nucleotide sequence or amino acid sequence. An "identity fraction"
for aligned segments of a test sequence and a reference sequence is
the number of identical components that are shared by sequences of
the two aligned segments divided by the total number of sequence
components in the reference segment over a window of alignment
which is the smaller of the full test sequence or the full
reference sequence. "Percent identity" ("% identity") is the
identity fraction times 100. Such optimal alignment is understood
to be deemed as local alignment of DNA sequences. For protein
alignment, a local alignment of protein sequences should allow
introduction of gaps to achieve optimal alignment. Percent identity
is calculated over the aligned length not including the gaps
introduced by the alignment per se.
[0037] As used herein, a "plant by-product" includes any product
that is made from a plant or plant product, for example, by
dehulling, crushing, milling, extraction, hydrogenation, and other
processes. A plant by-products in accordance with the invention,
therefore, will include such as, for example, dehulled soybeans,
crushed corn, soybean meal, soy milk, paper made from corn stalks,
and a wide range of other useful products of processing based on
plant vitamins, minerals, lipids, proteins and carbohydrates and
their constituents that can be characterized as being produced from
crops or terminal crops in accordance with the invention.
[0038] As used herein, a "plant cell" means a plant cell that is
transformed with stably-integrated, 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.
[0039] As used herein, the term "polypeptide" or "polypeptide
molecule" means a chain of amino acids. Polypeptide is also
commonly referred as "protein".
[0040] As used herein, "polyadenylated ribonucleotides" refers to
the series of adenosines at the 3' end of a polyribonucleotide
commonly referred to as a "poly-A tail".
[0041] 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.
[0042] As used herein, the term "structural nucleic acid molecule"
refers to a molecule having sequence that encodes a protein,
functional peptide fragment, or any other molecule that has
biological activity (including, but not limited to, mRNA and
bioactive RNA molecules, including antisense RNA).
[0043] As used herein, the term "substantially purified nucleic
acid" or polypeptide means nucleic acid or protein separated from
substantially all other molecules normally associated with it in
its native state. A substantially purified nucleic acid can be
greater than about 60% free from the other molecules (exclusive of
solvent) present in the natural mixture. The term "substantially
purified" is not intended to encompass molecules present in their
native state.
[0044] "Pfam" database 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.
Recombinant DNA Constructs
[0045] 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
can 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.
[0046] 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 U.S. 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.
[0047] Furthermore, the promoters can 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 can 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 US Patent 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 U.S. Patent
Application Publication 2002/0192813A1 which discloses 5', 3' and
intron elements useful in the design of effective plant expression
vectors.
[0048] 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.
[0049] Recombinant DNA constructs in this invention will 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 U.S. Patent Application
Publication Number 2002/0192813 A1; and the pea (Pisum sativum)
ribulose biphosphate carboxylase gene (rbs 3'), and 3' elements
from the genes within the host plant.
[0050] Constructs and vectors can 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).
[0051] 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.
[0052] Gene suppression can also be achieved by insertion mutations
created by transposable elements can also prevent gene function.
For example, in many dicot plants, transformation with the T-DNA of
Agrobacterium can 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 can be screened with polynucleotides
encoding the polypeptide of interest to detect mutated plants
having an insertion in the gene encoding the polypeptide of
interest.
[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 U.S. Patent Application Publication Number
2003/0083480 A1 also for imparting glyphosate tolerance; dicamba
monooxygenase disclosed in U.S. Patent Application Publication
Number 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
(crtl) 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 U.S.
Patent Application Publication Number 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 U.S. Patent Application Publication
Number 2002/0112260. Molecules and methods for imparting
insect/nematode/virus resistance are disclosed in U.S. Pat. No.
5,250,515; 5,880,275; 6,506,599; 5,986,175 and U.S. Patent
Application Publication Number 2003/0150017 A1.
[0054] Embodiments of the invention provide molecules of that
include "fragments" of the disclosed recombinant DNA molecules;
including oligonucleotides of at least 15, at least 16 or 17, at
least 18 or 19, and at least 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30 or more, consecutive nucleotides of any of the sequences
provided. Such oligonucleotides are fragments of the larger
molecules having a sequence selected from the group consisting of
SEQ ID NO: 1 through SEQ ID NO: 116778, and find use, for example
as probes and primers for detection of the polynucleotides of the
present invention.
[0055] Aspects of the various embodiments of the invention also
provide for nucleic acid fragments of SEQ ID NO: 1 through SEQ ID
NO: 116778 that are at least about 50, 100, 150, 200, 400, 500 or
more nucleotides in length. Some aspects of these embodiments
provide for nucleic acid molecules that encode functional fragments
of any of the polypeptide sequences provided in SEQ ID NO: 116779
to SEQ ID NO: 233556.
[0056] Other embodiments of the present invention provide for one
or more polypeptides having at least about 10 contiguous peptide
residues of one or more of the peptide sequences provided in SEQ ID
NO: 116779 to SEQ ID NO: 233556. In other aspects of these
embodiment, the polypeptide(s) comprises at least 15, 20, 25, 30,
35, 50, 75, 100 or more contiguous peptide residues from one or
more of the sequences provided in SEQ ID NO: 116779 to SEQ ID NO:
233556. In particularly aspects, these embodiments the poly peptide
includes a functional fragment of one or more of the polypeptides
provided in the Sequence Listing.
[0057] Other aspects of the various embodiments of the invention
includes, transgenic plants, transgenic plant seeds, transgenic
crops, plant products and byproducts having any of the nucleic acid
or protein fragments described above. The invention also provides
for various methods that use such fragments.
[0058] Embodiments of the present invention also contemplate that
the trait-improving recombinant DNA provided herein can be used in
combination with other recombinant DNA to create plants with
multiple desired traits or a further enhanced trait. The
combinations generated can include multiple copies of any one or
more of the recombinant DNA constructs. These stacked combinations
can be created by any method, including but not limited to cross
breeding of transgenic plants, or multiple genetic
transformation.
Plant Cell Transformation Methods
[0059] 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 U.S. 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,
e.g., 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 can
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.
[0060] 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.
[0061] In certain embodiments of the invention the recombinant DNA
insertion is "targeted" in order to achieve site-specific
integration, for example to replace an existing gene in the genome,
to use an existing promoter in the plant genome, or to insert a
recombinant polynucleotide at a predetermined site known to be
active for gene expression. Several site specific recombination
systems exist which are known to function implants include cre-lox
as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in
U.S. Pat. No. 5,527,695, both incorporated herein by reference.
[0062] Transformation methods of this invention can be practiced in
tissue culture on media and in a controlled environment. "Media"
refers to the numerous nutrient mixtures that are used to grow
cells in vitro, that is, outside of the intact living organism.
Recipient cell targets include, but are not limited to, meristem
cells, callus, immature embryos and gametic cells such as
microspores, pollen, sperm and egg cells. It is contemplated that
any cell from which a fertile plant can be regenerated is useful as
a recipient cell. Callus can be initiated from tissue sources
including, but not limited to, immature embryos, seedling apical
meristems, microspores and the like. Cells capable of proliferating
as callus are also recipient cells for genetic transformation.
Practical transformation methods and materials for making
transgenic plants of this invention, for example, various media and
recipient target cells, transformation of immature embryo cells and
subsequent regeneration of fertile transgenic plants are disclosed
in U.S. Pat. No. 6,194,636 and 6,232,526, which are incorporated
herein by reference.
[0063] The seeds of transgenic plants can be harvested from fertile
transgenic plants and be used to grow progeny generations of
transformed plants of this invention including hybrid plants line
for selection of plants having an enhanced trait. In addition to
direct transformation of a plant with a recombinant DNA, transgenic
plants can be prepared by crossing a first plant having a
recombinant DNA with a second plant lacking the DNA. For example,
recombinant DNA can be introduced into first plant line that is
amenable to transformation to produce 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.
[0064] Descriptions of commonly used breeding terms such as
"crossing", "hybrids" and methods for crossing and producing hybrid
that are used to describe present invention can be found in one of
several reference books (Allard, "Principles of Plant Breeding,"
John Wiley & Sons, NY, U. of Calif., Davis, Calif., 50-98,
1960; Simmonds, "Principles of crop improvement," Longman, Inc.,
N.Y., 369-399, 1979; Sneep and Hendriksen, "Plant breeding
perspectives," Wageningen (ed), Center for Agricultural Publishing
and Documentation, 1979; Fehr, In: Soybeans: Improvement,
Production and Uses, 2nd Edition, Monograph., 16:249, 1987; Fehr,
"Principles of variety development," Theory and Technique, (Vol. 1)
and Crop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub.
Co., N.Y., 360-376, 1987).
[0065] In some embodiments of the invention, during transformation,
DNA is introduced into only a small percentage of target plant
cells in any one transformation. Marker genes are used to provide
an efficient system for identification of those cells that are
stably transformed by receiving and integrating a transgenic DNA
construct into their genomes. Marker genes provide selective
markers which confer resistance to a selective agent, such as an
antibiotic or herbicide. Any of the herbicides to which plants of
this invention can 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 can
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) and gentamycin (aac3 and aacC4) or
resistance to herbicides such as glufosinate (bar or pat) and
glyphosate (aroA or EPSPS). Examples of such selectable are
illustrated in U.S. Pat. No. 5,550,318; 5,633,435; 5,780,708 and
6,118,047, all of which are incorporated herein by reference.
Selectable markers which provide an ability to visually identify
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.
[0066] Plant cells that survive exposure to the selective agent, or
plant cells that have been scored positive in a screening assay,
can 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.-2s.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. Plants can be pollinated using conventional
plant breeding methods known to those of skill in the art and seed
produced. 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.
[0067] Progeny can be recovered from transformed plants and tested
for expression of the exogenous recombinant polynucleotide. Useful
assays include, for example, "molecular biological" assays, such as
Southern and Northern blotting and PCR; "biochemical" assays, such
as detecting the presence of RNA, e.g., double stranded RNA, or a
protein product, e.g., by immunological means (ELISAs and Western
blots) or by enzymatic function; plant part assays, such as leaf or
root assays; and also, by analyzing the phenotype of the whole
regenerated plant.
Transgenic Plants and Seeds
[0068] Transgenic plants derived from the plant cells 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) including 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. Transgenic plants of the present invention
include, but are not limited to, corn, soybean, cotton, canola,
alfalfa, wheat, rice, sugarcane, sugar beet seed, millet, barley,
peanut, pigeon pea, sorghum, vegetables (including but not limited
to Broccoli, Cauliflower, Cabbage, Radish, Chinese cabbage, Melons,
Watermelons, Cucumber, Gourds, Pumpkin, Squash, Pepper, Tomato,
Eggplant, Onion, Carrot, Garden Bean, Sweet Corn, Pea, Dry Bean,
Okra, Spinach, Leek, Lettuce, and Fennel), grape, berries
(including blue, black, raspberry, mullberry, boysenberry . . .
etc), cherry and related fruit trees (including but not limited to
plum, peach, apricot, kiwi, pomegranate, mango, fig), fruit trees
(including but not limited to orange, lemon, lime, blood orange,
grapefruit, and the like), nut trees (including but not limited to
coconut, walnut (English and black), pecan, almond, hazelnut,
brazil nut, hickory nut, acorn, and the like), sunflower, other
oilseed producing plants or any combinations thereof.
Selection Methods for Transgenic Plants with Enhanced Agronomic
Trait
[0069] 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 may not exhibit an enhanced agronomic trait.
Selection from such 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. These assays also can 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 can 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.
[0070] Assays for screening for a desired trait are readily
designed by those practicing in the art. The following illustrates
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.
[0071] In certain embodiments of the invention transgenic corn
plants having nitrogen use efficiency can be 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.
[0072] In other embodiments, transgenic corn plants having enhanced
yield can be 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 can 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.
[0073] In other embodiments, transgenic corn plants having enhanced
water use efficiency can be 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.
[0074] In other embodiments, transgenic corn plants having enhanced
cold tolerance can be 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). For
example, seeds having higher germination rates as compared to the
control can be identified as having enhanced cold tolerance. In a
cold tolerance field trial plants with enhanced cold tolerance can
be 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 can be
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 may be can be planted under both
cold and normal conditions preferably with multiple repetitions per
treatment.
[0075] In other embodiments, transgenic corn plants having seeds
with increased protein and/or oil levels can be 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 1.
TABLE-US-00001 TABLE 1 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%.
[0076] 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.
Homolog Identification
[0077] In certain embodiment, the present invention also includes
identification of homologs of proteins encoded by the DNA
identified in the sequence listing which is used to provide
transgenic seed and plants having enhanced agronomic traits. From
the sequence of the homologs, homologous DNA sequence are
identified for preparing additional transgenic seeds and plants of
this invention with enhanced agronomic traits.
[0078] An "All Protein Database" are 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 can be obtained, an
"Organism Protein Database" are 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.
[0079] The All Protein Database are queried using amino acid
sequences provided herein as SEQ ID NO: 116779 through SEQ ID NO:
233556 using NCBI "blastp" program with E-value cutoff of 1
e.sup.-8. Up to 1000 top hits are kept, and separated by organism
names. For each organism other than that of the query sequence, a
list is kept for hits from the query organism itself with a more
significant E-value than the best hit of the organism. The list
contain likely duplicated genes of the polynucleotides provided
herein, and is referred to as the Core List. Another list is kept
for all the hits from each organism, sorted by E-value, and
referred to as the Hit List.
[0080] The Organism Protein Database are queried using polypeptide
sequences provided herein as SEQ ID NO: 116779 through SEQ ID NO:
233556 using NCBI "blastp" program with E-value cutoff of le
.sup.4. Up to 1000 top hits are kept. A BLAST searchable database
is constructed based on these hits, and are referred to as "SubDB".
SubDB are queried with each sequence in the Hit List using NCBI
"blastp" program with E-value cutoff of 1 e.sup.-8. The hit with
the best E-value are 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 are identified and reported.
[0081] Recombinant DNA constructs can be prepared using the DNA
encoding each of the identified homologs and the constructs can be
used to prepare multiple events of transgenic corn, soybean,
canola, cotton and other transgenic plants mentioned. Plants can be
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 can be selected
for commercial development.
Pfam Module Annotation
[0082] The amino acid sequence of the expressed proteins shown to
be associated with an enhanced trait are 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 domain modules and individual
protein domain for the proteins shown in the sequence listing. The
Hidden Markov model databases for the identified patent families
are known to a skilled artisan 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.
[0083] In one aspect, the present invention includes recombinant
DNA constructs having a polynucleotide encoding a protein that has
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: 116779-233556 when said amino acid
sequence is aligned with said reference sequence.
[0084] In another aspect, the present invention includes a mixture
having plant cells, 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:
116779-233556 when said amino acid sequence is aligned to said
reference sequence.
[0085] Yet another aspect of the present invention includes
recombinant DNA constructs having 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:
116779-233556, when said amino acid sequence is aligned to said
reference sequence; or 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: 116779-233556,
when said amino acid sequence is aligned to said reference
sequence; and wherein said construct is stably integrated into a
chromosome in a plant cell nucleus. In some aspect, this invention
includes a transgenic plant cell having the recombinant DNA
construct wherein said DNA construct provides 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. The DNA construct further can express a protein
that provides tolerance from exposure to an herbicide (e.g., a
glyphosate, dicamba, or glufosinate compound) having an agent
applied at levels that are lethal to a wild type of said plant cell
nucleus.
[0086] In some aspects, this invention provides transgenic plants
(e.g., corn, soybean, cotton, canola, alfalfa, wheat, rice,
sugarcane, or sugar beet plant), that is homozygous for said
recombinant DNA and have a plurality of plant cells.
[0087] Yet in another aspect, this invention includes transgenic
pollen grains having a haploid derivative of a plant cell nucleus
having a chromosome comprising the recombinant DNA construct.
[0088] In one aspect, this inventions provides a method for
manufacturing non-natural, transgenic seed (e.g., corn, soybean,
cotton, canola, alfalfa, wheat, rice, sugarcane, or sugar beet
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, said method includes (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.
[0089] Yet in another aspect, the method of this invention further
includes: (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:
116779-233556.
[0090] In one aspect, this invention provides a method of producing
hybrid corn seed including: (a) acquiring hybrid corn seed from an
herbicide tolerant corn plant which also has a stably-integrated,
recombinant DNA construct; (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.
[0091] In some aspect, this invention provides substantially
purified nucleic acid molecule having a nucleic acid sequence
wherein said nucleic acid sequence exhibits 95% or greater identity
to a nucleic acid sequence selected from the group consisting of
SEQ ID NO: 1 through SEQ ID NO: 116778 and sequences complementary
to SEQ ID NO: 1 through SEQ ID NO: 116778.
[0092] It is contemplated that any embodiment discussed in this
specification can be implemented with respect to any method, kit,
reagent, or composition of the invention, and vice versa.
Furthermore, compositions of the invention can be used to achieve
methods of the invention.
[0093] It will be understood that particular embodiments described
herein are shown by way of illustration and not as limitations of
the invention. The principal features of this invention can be
employed in various embodiments without departing from the scope of
the invention. Those skilled in the art will recognize, or be able
to ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of this invention
and are covered by the claims.
[0094] All publications and patent applications mentioned in the
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
[0095] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one." The use of
the term "or" in the claims is used to mean "and/or" unless
explicitly indicated to refer to alternatives only or the
alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0096] As used in this specification and claim(s), the words
"comprising" (and any form of comprising, such as "comprise" and
"comprises"), "having" (and any form of having, such as "have" and
"has"), "including" (and any form of including, such as "includes"
and "include") or "containing" (and any form of containing, such as
"contains" and "contain") are inclusive or open-ended and do not
exclude additional, unrecited elements or method steps.
[0097] The term "or combinations thereof" as used herein refers to
all permutations and combinations of the listed items preceding the
term. For example, "A, B, C, or combinations thereof" is intended
to include at least one of: A, B, C, AB, AC, BC, or ABC, and if
order is important in a particular context, also BA, CA, CB, CBA,
BCA, ACB, BAC, or CAB. Continuing with this example, expressly
included are combinations that contain repeats of one or more item
or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so
forth. The skilled artisan will understand that typically there is
no limit on the number of items or terms in any combination, unless
otherwise apparent from the context.
[0098] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the invention as defined by the appended
claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20180155734A1).
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=US20180155734A1).
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