U.S. patent application number 13/909978 was filed with the patent office on 2014-03-27 for transgenic plants with enhanced agronomic traits.
The applicant listed for this patent is MONSANTO TECHNOLOGY LLC. Invention is credited to Maureen Daley, Jason Fenner, Beth Savidge, Dale Val, Wei Zheng.
Application Number | 20140090101 13/909978 |
Document ID | / |
Family ID | 43606379 |
Filed Date | 2014-03-27 |
United States Patent
Application |
20140090101 |
Kind Code |
A1 |
Daley; Maureen ; et
al. |
March 27, 2014 |
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, enhanced seed oil and modified oil
composition. Also disclosed are methods for manufacturing
transgenic seed and plants with enhanced traits.
Inventors: |
Daley; Maureen; (Sacramento,
CA) ; Fenner; Jason; (Sacramento, CA) ;
Savidge; Beth; (Davis, CA) ; Val; Dale;
(Zamora, CA) ; Zheng; Wei; (Davis, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MONSANTO TECHNOLOGY LLC |
St. Loius |
MO |
US |
|
|
Family ID: |
43606379 |
Appl. No.: |
13/909978 |
Filed: |
June 4, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12860188 |
Aug 20, 2010 |
8502026 |
|
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13909978 |
|
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61236306 |
Aug 24, 2009 |
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Current U.S.
Class: |
800/278 ;
435/320.1; 435/415; 435/419; 554/1 |
Current CPC
Class: |
C12N 15/8218 20130101;
Y02A 40/146 20180101; C11B 1/00 20130101; C12N 15/8261 20130101;
C12N 15/8243 20130101 |
Class at
Publication: |
800/278 ;
435/320.1; 435/419; 435/415; 554/1 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C11B 1/00 20060101 C11B001/00 |
Claims
1. A recombinant DNA molecule comprising a promoter that is
functional in a plant cell and that is operably linked to a
polynucleotide that, when expressed in a plant cell: (a) encodes a
protein: i) having the amino acid sequence of SEQ ID NO:4; or ii)
having an amino acid sequence having at least 90% identity to SEQ
ID NO:4 over its full length, wherein the sequence exhibits
diacylglycerol acyltransferase activity; or (b) is transcribed into
an RNA molecule that suppresses the level of an endogenous protein
in said plant cell through inhibition of transcription or
translation of a transcript encoding the amino acid sequence of SEQ
ID NO:4.
2. The recombinant DNA molecule of claim 1 wherein said molecule
further comprises at least one regulatory element selected from the
group consisting of a 5' untranslated region, intron, 3'
untranslated region, and transit peptide region.
3. A transgenic plant cell comprising a recombinant DNA molecule
comprising a promoter that is functional in a plant cell and that
is operably linked to a polynucleotide that, when expressed in a
plant cell: (a) encodes a protein: i) having the amino acid
sequence of SEQ ID NO:4; ii) having an amino acid sequence having
at least 90% identity to SEQ ID NO:4 over its full length, wherein
the sequence exhibits diacylglycerol acyltransferase activity; or
(b) is transcribed into an RNA molecule that suppresses the level
of an endogenous protein in said plant cell through inhibition of
transcription or translation of the transcript encoding the amino
acid sequence of SEQ ID NO:4.
4. The transgenic plant cell of claim 3 wherein said recombinant
DNA molecule is stably integrated into a chromosome in a plant cell
nucleus.
5. The transgenic plant cell of claim 3 wherein said plant cell is
selected by screening a population of transgenic plant cells that
have been transformed with said molecule for an enhanced trait as
compared to control plant cells; and wherein said enhanced trait is
increased yield, enhanced seed oil, or modified oil
composition.
6. The transgenic plant cell of claim 3 further comprising a DNA
molecule expressing a protein that provides tolerance from exposure
to a herbicide that is lethal to a wild type of said plant
cell.
7. The transgenic plant cell of claim 6 wherein said herbicide
comprises a glyphosate, dicamba, or glufosinate compound.
8. The transgenic plant cell of claim 3 wherein said plant cell is
part of a transgenic plant.
9. The transgenic plant cell of claim 3 wherein said plant cell is
in a plant seed.
10. The transgenic plant cell of claim 9 wherein said seed is from
a corn, soybean, cotton, canola, alfalfa, wheat, rice, sugarcane,
or sugar beet plant.
11. The transgenic plant cell of claim 3 further comprising at
least one DNA molecule expressing a protein that provides an
enhanced trait as compared to control plant cells; and wherein the
enhanced trait is increased yield, enhanced seed oil, or modified
oil composition.
12. The transgenic plant cell of claim 6 further comprising at
least one DNA molecule expressing a protein that provides an
enhanced trait as compared to control plant cells; and wherein the
enhanced trait is increased yield, enhanced seed oil, or modified
oil composition.
13. The transgenic plant cell of claim 7 further comprising at
least one DNA molecule expressing a protein that provides an
enhanced trait as compared to control plant cells; and wherein the
enhanced trait is increased yield, enhanced seed oil, or modified
oil composition.
14. Oil derived from the transgenic plant cell of claim 3, that
comprises said recombinant DNA molecule.
15. Oil derived from the transgenic plant cell of claim 6, that
comprises said recombinant DNA molecule.
16. Oil derived from the transgenic plant cell of claim 7, that
comprises said recombinant DNA molecule.
17. Oil derived from the transgenic plant cell of claim 11, that
comprises said recombinant DNA molecule.
18. Oil derived from the transgenic plant cell of claim 12, that
comprises said recombinant DNA molecule.
19. Oil derived from the transgenic plant cell of claim 13, that
comprises said recombinant DNA molecule.
20. The recombinant DNA construct of claim 2 wherein said
recombinant DNA construct is in a transgenic pollen grain
comprising a haploid derivative of said plant cell nucleus.
21. A method for manufacturing non-natural, transgenic plants that
can be used to produce a crop of transgenic plants with an enhanced
trait resulting from expression of a stably-integrated, recombinant
DNA molecule comprising a promoter that is functional in a plant
and that is operably linked to a polynucleotide that, when
expressed in a plant: (a) encodes a protein: i) having the amino
acid sequence of SEQ ID NO:4; or ii) having an amino acid sequence
having at least 90% identity to SEQ ID NO:4, wherein the sequence
exhibits diacylglycerol acyltransferase activity; or (b) is
transcribed into an RNA molecule that suppresses the level of an
endogenous protein in said plant through inhibition of
transcription or translation of the transcript encoding the amino
acid sequence of SEQ ID NO:4; said method comprising: (a) obtaining
a population of plants transformed with said recombinant DNA
molecule; (b) screening a population of plants for said enhanced
trait and said recombinant DNA molecule, 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 molecule, 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, enhanced seed oil,
and modified oil composition; (c) 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 (d)
collecting seed from the selected plant or plants from step c.
22. The method of claim 21 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 the amino acid sequence of SEQ ID NO:4.
23. The method of claim 22 wherein said seed is corn, soybean,
cotton, canola, alfalfa, wheat, rice, sugarcane, or sugar beet
seed.
24. A recombinant nucleic acid molecule comprising a nucleic acid
sequence encoding a polypeptide having diacylglycerol
acyltransferase activity, wherein the nucleic acid molecule is
selected from the group consisting of: (a) a nucleic acid sequence
that encodes a polypeptide comprising a sequence that is at least
95% identical to SEQ ID NO: 5 over its full length, and (b) a
nucleic acid sequence comprising a sequence that is at least 90%
identical to SEQ ID NO: 2.
25. The recombinant nucleic acid molecule of claim 24, wherein the
polypeptide comprises the sequence of SEQ ID NO: 5.
26. The recombinant nucleic acid molecule of claim 24, defined as
comprising the nucleic acid sequence of SEQ ID NO: 2.
27. The transgenic plant cell of claim 3 wherein said plant cell is
in an algae.
28. Oil derived from the transgenic plant cell of claim 27, that
comprises said recombinant DNA molecule.
Description
[0001] This application claims the priority of U.S. Provisional
application Ser. No. 61/236,306, filed Aug. 24, 2009, the entire
disclosure of which is incorporated herein by reference.
INCORPORATION OF SEQUENCE LISTING
[0002] The sequence listing that is contained in the file named
"MONS239US_seq.txt", which is 57,344 bytes (measured in
MS-WINDOWS), created on Aug. 18, 2010 is filed herewith by
electronic submission and incorporated herein by reference.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] 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. Also disclosed are plants and
seeds having an increased oil content or modified oil composition.
All genetic resources disclosed herein were directly obtained from
sources that are currently common to the United States; the
ancestral sources of each specific genetic material is unknown.
SUMMARY OF THE INVENTION
[0004] This invention provides recombinant DNA encoding or
suppressing proteins with at least 95% identity to at least 95% of
a sequence selected from SEQ ID NOs: 4-6. The invention further
provides isolated polypeptides with at least 95% identity to at
least 95% of a sequence selected from SEQ ID NOs: 4-6.
[0005] Another aspect of the invention further employs recombinant
DNA for expression or suppression of proteins thereby imparting
enhanced agronomic traits to the transgenic plants. Recombinant DNA
in this invention is provided in a construct comprising a promoter
that is functional in plant cells and that is operably linked to
DNA that encodes or suppresses a protein having at least 95%
identity to at least 95% of a sequence selected from the group
consisting of SEQ ID NOs 4-6.
[0006] Other aspects of the invention are specifically directed to
transgenic plant cells comprising the recombinant DNA of the
invention, transgenic plants comprising a plurality of such plant
cells, progeny transgenic seed, embryo and transgenic pollen from
such plants. Such transgenic plants are selected from a population
of transgenic plants regenerated from plant cells transformed with
recombinant DNA and expressing or suppressing the protein(s) by
screening transgenic plants in the population for an enhanced trait
as compared to control plants that do not have said recombinant
DNA, where the 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, enhanced seed oil and modified oil
composition. In further embodiments, such transgenic plant cells
include polynucleotide stacks which express or suppress multiple
proteins of the invention. In a particularly specific embodiment of
the invention, such transgenic plants comprise polynucleotide
stacks encoding proteins which are at least 95% identical to at
least 95% of SEQ ID NOs: 4-6.
[0007] In yet another aspect of the invention the plant cells,
plants, seeds, embryo 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. Such
tolerance is especially useful not only as an advantageous trait in
such plants but is also useful in a selection step in the methods
of the invention. In aspects of the invention the agent of such
herbicide is a glyphosate, dicamba, or glufosinate compound.
[0008] Yet other aspects of the invention provide transgenic plants
which are homozygous for the recombinant DNA and transgenic seed of
the invention from corn, soybean, cotton, canola, alfalfa, wheat or
rice plants. In certain embodiments, for instance for practice of
various aspects of the invention in Argentina, the recombinant DNA
is provided in plant cells derived from corn lines that are and
maintain resistance to the Mal de Rio Cuarto virus or the Puccinia
sorghi fungus or both.
[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:
1-3. 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:
1-3. 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 transgenic plants
with enhanced oil levels, including algae. In a particular
embodiment, the invention provides transgenic seeds with seed
composition improvement including enhanced protein, oil or starch
levels.
[0014] Some of the recombinant DNA provided by this invention are
transcription factors. This invention provides a method of
producing a transgenic plant having an enhanced agronomic trait
produced by expression of a transcription factor. This method
includes identifying target genes of a transcription factor, which
includes the steps of assessing a dataset of expression profiles of
a transcription factor gene and other genes and analyzing said
dataset to determine a subset of genes that are regulated by said
transcription factor, and cloning the coding sequence of at least
one of the subset of genes into a plant transformation vector and
transforming a plant with such vector.
[0015] Furthermore, this invention provides novel genes of Glycine
max GLABRA2 (Gm.GL2) and Rhodosporidium toruloides DGAT2
(Rt.DGAT2); and an Arabidopsis/Brassica chimeric DNA construct
(At.Bn.Otf1) of an oil transcription factor, coding for proteins as
set forth in SEQ ID NO: 4 through SEQ ID NO: 6, respectively, which
are particularly useful for generating transgenic crop plants
having seeds with enhanced oil levels.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIGS. 1-3 are plasmid maps of base vectors for corn, soybean
and cotton transformation.
DETAILED DESCRIPTION OF THE INVENTION
[0017] In the attached sequence listing:
[0018] SEQ ID NO: 1-3 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;
[0019] SEQ ID NO: 4-6 are amino acid sequences of the cognate
protein of the "genes" with nucleotide coding sequences 1-3;
[0020] SEQ ID NO: 7 is a nucleotide sequence of a base plasmid
vector useful for corn transformation;
[0021] SEQ ID NO: 8 is a nucleotide sequence of a base plasmid
vector useful for soybean and canola transformation;
[0022] SEQ ID NO: 9 is a nucleotide sequence of a base plasmid
vector useful for cotton transformation;
[0023] 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.
[0024] As used herein a "transgenic plant" includes a plant, plant
part, plant cells or seed 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.
[0025] As used herein "recombinant DNA" means DNA which has been a
genetically engineered and constructed outside of a cell.
[0026] 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%, more
preferably at least 90% identity, more preferably at least 95, 96,
97, 98, or 99% 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.
[0027] 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 the suite of BLAST
programs available from NCBI. 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.
[0028] Percent identity describes the extent to which the sequences
of DNA or protein segments are invariant in an alignment of
sequences, for example nucleotide sequences or amino acid
sequences. An alignment of sequences is created by manually
aligning two sequences, e.g. a stated sequence, as provided herein,
as a reference, and another sequence, to produce the highest number
of matching elements, e.g. individual nucleotides or amino acids,
while allowing for the introduction of gaps into either sequence.
An "identity fraction" for a sequence aligned with a reference
sequence is the number of matching elements, divided by the full
length of the reference sequence, not including gaps introduced by
the alignment process into the reference sequence. "Percent
identity" ("% identity") as used herein is the identity fraction
times 100.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] As used herein an "enhanced trait" means a characteristic of
a transgenic plant that includes, but is not limited to, an
enhanced 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 an
enhanced trait is selected from a group of enhanced traits
consisting of enhanced water use efficiency, enhanced cold
tolerance, increased yield, enhanced nitrogen use efficiency,
enhanced seed protein, enhanced oil production in the seed or other
tissue and modified oil composition. 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.
[0035] 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; and oil components as may be manifest by alterations in the
ratios of seed components.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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
aestivum) 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.
[0041] 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).
[0042] 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.
[0043] Gene suppression can also be achieved by insertion mutations
created by transposable elements which 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.
[0044] 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,
coleopteran, 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. (Nucl. Acids Res. 18:2188-2193 (1990)) for imparting
tolerance to sulfonylurea herbicides; polynucleotide molecules
known as bar genes disclosed in DeBlock, et al. (EMBO J.
6:2513-2519, 1987) for imparting glufosinate and bialaphos
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; 5,986,175 and US Patent Application
Publication 2003/0150017 A1. Methods and tools for utilization of
the current gene sequences for enhanced oil production in algae can
be found in U.S. Pat. No. 6,027,900.
[0045] Plant Cell Transformation Methods
[0046] 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. No. 5,015,580 (soybean); U.S. Pat. No. 5,550,318 (corn);
U.S. Pat. No. 5,538,880 (corn); U.S. Pat. No. 5,914,451 (soybean);
U.S. Pat. No. 6,160,208 (corn); U.S. Pat. No. 6,399,861 (corn);
U.S. Pat. No. 6,153,812 (wheat) and U.S. Pat. No. 6,365,807 (rice);
Agrobacterium-mediated transformation is described in U.S. Pat. No.
5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S. Pat.
No. 5,463,174 (canola); U.S. Pat. No. 5,591,616 (corn); U.S. Pat.
No. 5,846,797 (cotton); U.S. Pat. No. 6,384,301 (soybean), U.S.
Pat. No. 7,026,528 (wheat) and U.S. Pat. No. 6,329,571 (rice), US
Patent Application Publication 2004/0087030 A1 (cotton), and US
Patent Application Publication 2001/0042257 A1 (sugar beet); and
algal transformation can be demonstrated in Kumar et al., Genetic
Transformation of the Green Alga--Chlamydomonas reinhardtii by
Agrobacterium tumefaciens, PLANT SCI. 166(3) pp 731-38 (2004), 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.
[0047] 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
[0048] 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.
[0049] 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.
[0050] Transgenic Plants and Seeds
[0051] 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 another 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, enhanced seed oil and modified oil
composition.
[0052] Table 1 provides a list of protein encoding DNA ("genes")
that are useful as recombinant DNA for production of transgenic
plants with enhanced agronomic traits; the elements of Table 1 are
described by reference to:
[0053] "PEP SEQ ID NO" identifies an amino acid sequence from SEQ
ID NO: 4 to 6.
[0054] "NUC SEQ ID NO" identifies a DNA sequence from SEQ ID NO:1
to 3.
[0055] "Gene ID" refers to an arbitrary identifier.
[0056] "Gene Name" denotes a common name for the protein encoded by
the recombinant DNA preceded by the abbreviated genus and species
as fully defined in the sequence listing. The + or - preceding the
gene name indicates whether the protein is expressed (+) or
suppressed (-) in plants to provide an enhanced trait.
[0057] "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 List of certain genes useful as recombinant
DNA for production of transgenic plants with enhanced agronomic
traits. NUC PEP SEQ SEQ ID NO ID NO Gene ID Gene Name Annotation 1
4 GLABRA2 +Gm.GL2 BNLGHi8377 [Gossypium hirsutum] 2 5
Diacylglycerol +Rt.DGAT2 Diacylglycerol acyltransferase
acyltransferase- [Rhodosporidium toruloides] 2a 3 6 Oil +At.Bn.Otf1
activator of sporamin LUC 1 transcription chimera [Arabidopsis
thaliana] factor-1 WRINKLED 1 [Arabidopsis thaliana]
[0058] Selection Methods for Transgenic Plants with Enhanced
Agronomic Traits
[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, enhanced seed oil and modified oil composition. 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, or 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 Composition of bulk seed samples. 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.
EXAMPLES
[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:7, 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 Components of exemplary plant transformation
vector. Coordinates of Function Name Annotation SEQ ID NO: 7
Agrobacterium B-AGRtu.right Agro right border sequence, 11364-11720
T-DNA border essential for transfer of T- transfer DNA. Gene of
E-Os.Act1 Upstream promoter region 19-775 interest of the rice
actin 1 gene expression E- Duplicated35S A1-B3 788-1120 cassette
CaMV.35S.2xA1- domain without TATA box B3 P-Os.Act1 Promoter region
of the rice 1125-1204 actin 1 gene L-Ta.Lhcb1 5' untranslated
leader of 1210-1270 wheat major chlorophyll a/b binding protein
I-Os.Act1 First intron and flanking 1287-1766 UTR exon sequences
from the rice actin 1 gene T-St.Pis4 3' non-translated region of
1838-2780 the potato proteinase inhibitor II gene which functions
to direct polyadenylation of the mRNA Plant P-Os.Act1 Promoter from
the rice actin 2830-3670 selectable 1 gene marker L-Os.Act1 First
exon of the rice actin 1 3671-3750 expression gene cassette
I-Os.Act1 First intron and flanking 3751-4228 UTR exon 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 4466-5833 CP4.nat strain CP4 native aroA gene.
T-AGRtu.nos A 3' non-translated region of 5849-6101 the nopaline
synthase gene of Agrobacterium tumefaciens Ti plasmid which
functions to direct polyadenylation of the mRNA. Agrobacterium
B-AGRtu.left Agro left border sequence, 6168-6609 T-DNA border
essential for transfer of T- transfer DNA. Maintenance
OR-Ec.oriV-RK2 The vegetative origin of 6696-7092 in E. coli
replication from plasmid RK2. CR-Ec.rop Coding region for repressor
8601-8792 of 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 9220-9808 replication from the E. coli plasmid
ColE1. P-Ec.aadA- Promoter for Tn7 10339-10380 SPC/STR
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- 3' UTR from
the Tn7 11170-11227 SPC/STR 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.
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: 8) as shown in Table 4 below and FIG. 2.
TABLE-US-00004 TABLE 4 Genetic components of exemplary plant
expression vector. Coordinates of Function Name Annotation SEQ ID
NO: 8 Agrobacterium B-AGRtu.left Agro left border sequence,
essential 6144-6585 T-DNA transfer border for transfer of T-DNA.
Plant selectable P-At.Act7 Promoter from the Arabidopsis actin
6624-7861 marker 7 gene expression L-At.Act7 5'UTR of Arabidopsis
Act7 gene cassette I-At.Act7 Intron from the Arabidopsis actin7
gene TS-At.ShkG- Transit peptide region of 7864-8091 CTP2
Arabidopsis EPSPS CR-AGRtu.aroA- Synthetic CP4 coding region with
8092-9459 CP4.nno_At dicot preferred codon usage. T-AGRtu.nos A 3'
non-translated region of the 9466-9718 nopaline 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 containing a
duplication of the -90 to cassette -350 region. T-Gb.E6-3b 3'
untranslated region from the fiber 688-1002 protein E6 gene of
sea-island cotton. Agrobacterium B-AGRtu.right Agro right border
sequence, 1033-1389 T-DNA transfer border essential for transfer of
T-DNA. Maintenance in OR-Ec.oriV-RK2 The vegetative origin of
replication 5661-6057 E. coli from plasmid RK2. CR-Ec.rop Coding
region for repressor of 3961-4152 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 2945-3533 from
the E. coli plasmid ColE1. P-Ec.aadA- Promoter for Tn7 2373-2414
SPC/STR adenylyltransferase (AAD(3'')) CR-Ec.aadA- Coding region
for Tn7 1584-2372 SPC/STR adenylyltransferase (AAD(3'')) conferring
spectinomycin and streptomycin resistance. T-Ec.aadA- 3' UTR from
the Tn7 1526-1583 SPC/STR adenylyltransferase (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.
C. Cotton Transformation Vector
[0075] Plasmids for use in transformation of cotton tissue are
prepared with elements of expression vector pMON99053 (SEQ ID NO:
9) as shown in Table 5 below and FIG. 3.
TABLE-US-00005 TABLE 5 Genetic components of exemplary plant
expression vector. Coordinates of SEQ ID Function Name Annotation
NO: 9 Agrobacterium B-AGRtu.right border Agro right border 1-357
T-DNA transfer sequence, essential for transfer of T-DNA. Gene of
interest Exp-CaMV.35S- Enhanced version of the 388-1091 expression
enh + Ph.DnaK 35S RNA promoter from cassette CaMV plus the petunia
hsp70 5' untranslated region T-Ps.RbcS2-E9 The 3' non-translated
1165-1797 region of the pea RbcS2 gene which functions to direct
polyadenylation of the mRNA. Plant selectable Exp-CaMV.35S Promoter
and 5' 1828-2151 marker untranslated region from expression the 35S
RNA of CaMV cassette CR-Ec.nptII-Tn5 Coding region for 2185-2979
neomycin phosphotransferase gene from transposon Tn5 which confers
resistance to neomycin and kanamycin. T-AGRtu.nos A 3'
non-translated region 3011-3263 of the 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, 3309-3750 T-DNA transfer essential for
transfer of T- DNA. Maintenance in OR-Ec.oriV-RK2 The vegetative
origin of 3837-4233 E. coli replication from plasmid RK2. CR-Ec.rop
Coding region for repressor 5742-5933 of 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 6361-6949 replication
from the E. coli plasmid ColE1. P-Ec.aadA-SPC/STR Promoter for Tn7
7480-7521 adenylyltransferase (AAD(3'')) CR-Ec.aadA-SPC/STR Coding
region for Tn7 7522-8310 adenylyltransferase (AAD(3'')) conferring
spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3' UTR
from the Tn7 8311-8368 adenylyltransferase (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.
Example 2
Corn Transformation
[0078] 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, enhanced seed oil
and modified oil composition. A plasmid vector is prepared by
cloning DNA from SEQ ID NO:1, SEQ ID NO: 2 or SEQ ID NO: 3 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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. 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, enhanced seed oil
and modified oil composition. 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, enhanced seed oil and modified oil
composition is identified and progeny seed is selected for
commercial development.
Example 3
Soybean Transformation
[0083] 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, enhanced seed oil
and modified oil composition.
[0084] For Agrobacterium mediated transformation, soybean seeds are
imbibed 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 at
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.
[0085] 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. 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, enhanced seed oil and modified oil composition. 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,
enhanced seed oil and modified oil composition is identified and
progeny seed is selected for commercial development.
Example 4
Cotton Transgenic Plants with Enhanced Agronomic Traits
[0086] 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,
enhanced seed oil and modified oil composition.
[0087] Transgenic cotton plants containing each recombinant DNA
having a sequence from SEQ ID NO: 1 through SEQ ID NO: 3 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.
[0088] 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,
enhanced seed oil and modified oil composition is identified and
progeny seed is selected for commercial development.
[0089] 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.
[0090] 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
[0091] 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, enhanced seed oil and modified oil
composition.
[0092] 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 media 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.
[0093] 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. 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, enhanced seed
oil and modified oil composition. 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, enhanced seed oil and modified
oil composition is identified and progeny seed is selected for
commercial development.
[0094] Similarly such transformation can be done for various algae,
with progeny selected for heightened oil production levels.
Example 6
Homolog Identification
[0095] 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.
[0096] An "All Protein Database" is 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" is 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.
[0097] The All Protein Database is queried using amino acid
sequences provided herein as SEQ ID NO: 4 through SEQ ID NO: 6
using NCBI "blastp" program with E-value cutoff of 1e-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 contains 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.
[0098] The Organism Protein Database is queried using polypeptide
sequences provided herein as SEQ ID NO: 4 through SEQ ID NO: 6
using NCBI "blastp" program with E-value cutoff of 1e-4. Up to 1000
top hits are kept. A BLAST searchable database is 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 is 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 can be identified and
reported.
[0099] 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, enhanced seed oil
and modified oil composition. 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, enhanced seed oil and modified oil
composition is identified and progeny seed is selected for
commercial development.
Sequence CWU 1
1
912133DNAGlycine max 1ggctcgagtc cgagcagttt tagggaaata cgcaccaggg
tcaacgtccc cttcatgttc 60ttctggccat gaccaagaga atagaagctc tttggatttt
tacactggaa tttttggact 120tgataagtca aggataatgg atacagtaaa
ccaagctatg gaggagctca tcaagatggc 180taccgtgggg gaaccattat
ggcttcgtag cttcgagact ggtcgcgaaa ttcttaacta 240tgatgaatat
gttagggagt ttgcagttga aaattcaagc agtggaaagc caaggagatc
300cattgaagcc tcaagagaca ctgcagttgt ttttgtggat ctccctcggc
ttgtccaaag 360ttttctagat gtgaatcagt ggaaggaaat gtttccgtgt
ttaatatcta aggcggccac 420tgttgatgta atatgcaatg gagagggtcc
tggcaggaat ggtgcagtgc aactgatgtt 480tgctgagctg caaatgctca
ctcctatggt gcccacaaga gaagtatatt ttgtccgttt 540ctgcaagcag
ttgagtgctg aacagtgggc aatcgttgat gtatccatag acaaagtaga
600agacaacatt gacgcgtccc tcgtgaaatg cagaaaacgc ccttccggtt
gcattattga 660ggacaagtcc aatggccatt gcaaagtaat atgggtggag
cacttggaat gccagaagag 720tgcagtccat tcaatgtatc gcaccattgt
gaacagtggc ctagcttttg gggccaggca 780ttggattgcg actctacaac
ttcaatgtga acgtctagtt ttcttcatgg caacaaatgt 840tcccatgaag
gattcaaccg gtgttgccac gttggccggg agaaaaagca ttttgaagtt
900ggcacaaaga atgacatgga gtttctgcca tgcaattggc gcgtcaagct
tccacacatg 960gactaagttt acaagtaaaa ctggagaaga cataaggata
agttctagaa agaacttgaa 1020cgatcctggt gaacctcttg ggttgatatt
gtgcgctgtt tgttctgtat ggttgcctgt 1080ctcacctaat gttctgtttg
atttcctgag ggatgaaacg cgacgaactg aagtaccact 1140ctcttgtctc
ctttccttct caatcctttt gtcaattttg gttaataatt tgaatggtta
1200aatgtcctga tacgtatcac attatcttag tgggacatca tgtcaagtgg
tgggacagtg 1260cagtccattg caaatttagc caaaggacaa gaccgaggaa
atgccgtagc cattcaagta 1320agttacttcg gttcaaattt caatacctta
aaatgaaaat gcaaagtgca ttgaggtaaa 1380aaagacatgg ttctaagtga
aatatggtat tcttctgatt tggcagacaa ttaaatcgaa 1440agaaaacagt
gtgtggatac tgcaagatag ctacacaaac ccttatgagt caatggtggt
1500atatgcttct gtggacatta ctggcactca gtctgtgatg acaggatgtg
attcgagcaa 1560tcttgccata ctgccctcag gattctctat tattcctgat
ggtcttgagt caaggccatt 1620ggtgattagt tcaaggcagg aagaaaaaaa
taccgaggga ggatctttgt ttacaatggc 1680attccagatt cttaccaatg
cttctcccgc tgccaagtta acaatggagt ctgtggactc 1740ggtcaacact
cttgtatctt gtacattgag aaatatccga acgagtctac aatgtgaaga
1800tggctagtca aaatcagata atcacttgat agagagtcat agactttaat
tagctgtgat 1860aatcttaggc tctctattcc cttttttgga tggtattcgg
tttggcaaaa atagcttgct 1920tttgtcccct ttctccgttt ctgggttagt
tttactcatt attccactac caaggagggt 1980tggttgagtt tgtatatctg
tatctgcagt tctgtaggta gaaagataga caaaaagctt 2040ttagaaccta
gagtattaag tatggcgtgt atagaattat catttgttaa ttccttatgc
2100agttggtttt ttggtaaaaa aaaaaaaaaa agg 213321047DNARhodosporidium
toruloides 2atgggccagc aggcgacgcc cgaggagcta tacacacgct cagagatctc
caagatcaaa 60ttcgcaccct ttggcgtccc gcggtcgcgc cggctgcaga ccttctccgt
ctttgcctgg 120acgacggcac tgcccatcct actcggcgtc ttcttcctcc
tctgctcgtt cccaccgctc 180tggccggctg tcattgccta cctcacctgg
gtctttttca ttgaccaggc gccgattcac 240ggtggacggg cgcagtcttg
gctgcggaag agtcggatat gggtctggtt tgcaggatac 300tatcccgtca
gcttgatcaa gagcgccgac ttgccgcctg accggaagta cgtctttggc
360taccacccgc acggcgtcat aggcatgggc gccatcgcca acttcgcgac
cgacgcaacc 420ggcttctcga cactcttccc cggcttgaac cctcacctcc
tcaccctcca aagcaacttc 480aagctcccgc tctaccgcga gttgctgctc
gctctcggca tatgctccgt ctcgatgaag 540agctgtcaga acattctgcg
gcaaggtcct ggctcggctc tcactatcgt cgtcggtggc 600gccgccgaga
gcttgagtgc gcatcccgga accgccgatc ttacgctcaa gcgacgaaaa
660ggcttcatca aactcgcgat ccggcaaggc gccgaccttg tgcccgtctt
ttcgttcggc 720gagaacgaca tctttggcca gctgcgaaac gagcgaggaa
cgcggctgta caagttgcag 780aagcgtttcc aaggcgtgtt tggcttcacc
ctccctctct tctacggccg gggactcttc 840aactacaacg tcggattgat
gccgtatcgc catccgatcg tctctgtcgt cggtcgacca 900atctcggtag
agcagaagga ccacccgacc acggcggacc tcgaagaagt tcaggcgcgg
960tatatcgcag aactcaagcg catctgggaa gaatacaagg acgcctacgc
caaaagtcgc 1020acgcgggagc tcaatattat cgcctga 104731317DNAArtificial
sequencePlant 3atgaaacgcc cacttacgac aagtccgtca agctctagcc
caagctcctc tgtttcttct 60tctactacta cttcctctcc tattcagtcg gaggctccaa
ggcctaaacg agccaaaagg 120gctaagaaat cttctccttc tggtgataaa
tctcataacc cgacaagccc tgcttctacc 180cgacgcagct ctatctacag
aggagtcact agacatagat ggactgggag attcgaggct 240catctttggg
acaaaagctc ttggaattcg attcagaaca agaaaggcaa acaagtttat
300ctgggagcat atgacagtga agaagcagca gcacatacgt acgatctggc
tgctctcaag 360tactggggac ccgacaccat cttgaatttt ccggcagaga
cgtacacaaa ggaattggaa 420gaaatgcaga gagtgacaaa ggaagaatat
ttggcttctc tccgccgcca gagcagtggt 480ttctccagag gcgtctctaa
atatcgcggc gtcgctaggc atcaccacaa cggaagatgg 540gaggctcgga
tcggaagagt gtttgggaac aagtacttgt acctcggcac ctataatacg
600caggaggaag ctgctgcagc atatgacatg gctgcgattg agtatcgagg
cgcaaacgcg 660gttactaatt tcgacattag taattacatt gaccggttaa
agaagaaagg tgttttcccg 720ttccctgtga accaagctaa ccatcaagag
ggtattcttg ttgaagccaa acaagaagtt 780gaaacgagag aagcgaagga
agagcctaga gaagaagtga aacaacagta cgtggaagaa 840ccaccgcaag
aagaagaaga gaaggaagaa gagaaagcag agcaacaaga agcagagatt
900gtaggatatt cagttgaaga agcagtgatt acctgctgca tagacagctc
aaccataatg 960gaaatggata ggtgtgggga gtcaaatgag ctcgcttggg
acttctgtat gatggattca 1020gggtttgctc cgtttttgac tgattcaaat
ctctcgagtg agaatcccat tgagtatcct 1080gagcttttca atgagatggg
ttttgaggat aacattgact tcatgttcga ggaagggaag 1140caagactgct
tgagcttgga gaatcttgat tgttgcgatg gtgttgttgt ggtgggaaga
1200gagagcccaa cttcattgtc gtcttctccg ttgtcctgct tgtctactga
ctctgcttca 1260tcaacaacaa caacagcaac aacagtaacc tctgttagct
ggaactatag cgtctag 13174354PRTGlycine max 4Met Asp Thr Val Asn Gln
Ala Met Glu Glu Leu Ile Lys Met Ala Thr 1 5 10 15 Val Gly Glu Pro
Leu Trp Leu Arg Ser Phe Glu Thr Gly Arg Glu Ile 20 25 30 Leu Asn
Tyr Asp Glu Tyr Val Arg Glu Phe Ala Val Glu Asn Ser Ser 35 40 45
Ser Gly Lys Pro Arg Arg Ser Ile Glu Ala Ser Arg Asp Thr Ala Val 50
55 60 Val Phe Val Asp Leu Pro Arg Leu Val Gln Ser Phe Leu Asp Val
Asn 65 70 75 80 Gln Trp Lys Glu Met Phe Pro Cys Leu Ile Ser Lys Ala
Ala Thr Val 85 90 95 Asp Val Ile Cys Asn Gly Glu Gly Pro Gly Arg
Asn Gly Ala Val Gln 100 105 110 Leu Met Phe Ala Glu Leu Gln Met Leu
Thr Pro Met Val Pro Thr Arg 115 120 125 Glu Val Tyr Phe Val Arg Phe
Cys Lys Gln Leu Ser Ala Glu Gln Trp 130 135 140 Ala Ile Val Asp Val
Ser Ile Asp Lys Val Glu Asp Asn Ile Asp Ala 145 150 155 160 Ser Leu
Val Lys Cys Arg Lys Arg Pro Ser Gly Cys Ile Ile Glu Asp 165 170 175
Lys Ser Asn Gly His Cys Lys Val Ile Trp Val Glu His Leu Glu Cys 180
185 190 Gln Lys Ser Ala Val His Ser Met Tyr Arg Thr Ile Val Asn Ser
Gly 195 200 205 Leu Ala Phe Gly Ala Arg His Trp Ile Ala Thr Leu Gln
Leu Gln Cys 210 215 220 Glu Arg Leu Val Phe Phe Met Ala Thr Asn Val
Pro Met Lys Asp Ser 225 230 235 240 Thr Gly Val Ala Thr Leu Ala Gly
Arg Lys Ser Ile Leu Lys Leu Ala 245 250 255 Gln Arg Met Thr Trp Ser
Phe Cys His Ala Ile Gly Ala Ser Ser Phe 260 265 270 His Thr Trp Thr
Lys Phe Thr Ser Lys Thr Gly Glu Asp Ile Arg Ile 275 280 285 Ser Ser
Arg Lys Asn Leu Asn Asp Pro Gly Glu Pro Leu Gly Leu Ile 290 295 300
Leu Cys Ala Val Cys Ser Val Trp Leu Pro Val Ser Pro Asn Val Leu 305
310 315 320 Phe Asp Phe Leu Arg Asp Glu Thr Arg Arg Thr Glu Val Pro
Leu Ser 325 330 335 Cys Leu Leu Ser Phe Ser Ile Leu Leu Ser Ile Leu
Val Asn Asn Leu 340 345 350 Asn Gly 5348PRTRhodosporidium
toruloides 5Met Gly Gln Gln Ala Thr Pro Glu Glu Leu Tyr Thr Arg Ser
Glu Ile 1 5 10 15 Ser Lys Ile Lys Phe Ala Pro Phe Gly Val Pro Arg
Ser Arg Arg Leu 20 25 30 Gln Thr Phe Ser Val Phe Ala Trp Thr Thr
Ala Leu Pro Ile Leu Leu 35 40 45 Gly Val Phe Phe Leu Leu Cys Ser
Phe Pro Pro Leu Trp Pro Ala Val 50 55 60 Ile Ala Tyr Leu Thr Trp
Val Phe Phe Ile Asp Gln Ala Pro Ile His 65 70 75 80 Gly Gly Arg Ala
Gln Ser Trp Leu Arg Lys Ser Arg Ile Trp Val Trp 85 90 95 Phe Ala
Gly Tyr Tyr Pro Val Ser Leu Ile Lys Ser Ala Asp Leu Pro 100 105 110
Pro Asp Arg Lys Tyr Val Phe Gly Tyr His Pro His Gly Val Ile Gly 115
120 125 Met Gly Ala Ile Ala Asn Phe Ala Thr Asp Ala Thr Gly Phe Ser
Thr 130 135 140 Leu Phe Pro Gly Leu Asn Pro His Leu Leu Thr Leu Gln
Ser Asn Phe 145 150 155 160 Lys Leu Pro Leu Tyr Arg Glu Leu Leu Leu
Ala Leu Gly Ile Cys Ser 165 170 175 Val Ser Met Lys Ser Cys Gln Asn
Ile Leu Arg Gln Gly Pro Gly Ser 180 185 190 Ala Leu Thr Ile Val Val
Gly Gly Ala Ala Glu Ser Leu Ser Ala His 195 200 205 Pro Gly Thr Ala
Asp Leu Thr Leu Lys Arg Arg Lys Gly Phe Ile Lys 210 215 220 Leu Ala
Ile Arg Gln Gly Ala Asp Leu Val Pro Val Phe Ser Phe Gly 225 230 235
240 Glu Asn Asp Ile Phe Gly Gln Leu Arg Asn Glu Arg Gly Thr Arg Leu
245 250 255 Tyr Lys Leu Gln Lys Arg Phe Gln Gly Val Phe Gly Phe Thr
Leu Pro 260 265 270 Leu Phe Tyr Gly Arg Gly Leu Phe Asn Tyr Asn Val
Gly Leu Met Pro 275 280 285 Tyr Arg His Pro Ile Val Ser Val Val Gly
Arg Pro Ile Ser Val Glu 290 295 300 Gln Lys Asp His Pro Thr Thr Ala
Asp Leu Glu Glu Val Gln Ala Arg 305 310 315 320 Tyr Ile Ala Glu Leu
Lys Arg Ile Trp Glu Glu Tyr Lys Asp Ala Tyr 325 330 335 Ala Lys Ser
Arg Thr Arg Glu Leu Asn Ile Ile Ala 340 345 6438PRTArtificial
SequencePlant 6Met Lys Arg Pro Leu Thr Thr Ser Pro Ser Ser Ser Ser
Pro Ser Ser 1 5 10 15 Ser Val Ser Ser Ser Thr Thr Thr Ser Ser Pro
Ile Gln Ser Glu Ala 20 25 30 Pro Arg Pro Lys Arg Ala Lys Arg Ala
Lys Lys Ser Ser Pro Ser Gly 35 40 45 Asp Lys Ser His Asn Pro Thr
Ser Pro Ala Ser Thr Arg Arg Ser Ser 50 55 60 Ile Tyr Arg Gly Val
Thr Arg His Arg Trp Thr Gly Arg Phe Glu Ala 65 70 75 80 His Leu Trp
Asp Lys Ser Ser Trp Asn Ser Ile Gln Asn Lys Lys Gly 85 90 95 Lys
Gln Val Tyr Leu Gly Ala Tyr Asp Ser Glu Glu Ala Ala Ala His 100 105
110 Thr Tyr Asp Leu Ala Ala Leu Lys Tyr Trp Gly Pro Asp Thr Ile Leu
115 120 125 Asn Phe Pro Ala Glu Thr Tyr Thr Lys Glu Leu Glu Glu Met
Gln Arg 130 135 140 Val Thr Lys Glu Glu Tyr Leu Ala Ser Leu Arg Arg
Gln Ser Ser Gly 145 150 155 160 Phe Ser Arg Gly Val Ser Lys Tyr Arg
Gly Val Ala Arg His His His 165 170 175 Asn Gly Arg Trp Glu Ala Arg
Ile Gly Arg Val Phe Gly Asn Lys Tyr 180 185 190 Leu Tyr Leu Gly Thr
Tyr Asn Thr Gln Glu Glu Ala Ala Ala Ala Tyr 195 200 205 Asp Met Ala
Ala Ile Glu Tyr Arg Gly Ala Asn Ala Val Thr Asn Phe 210 215 220 Asp
Ile Ser Asn Tyr Ile Asp Arg Leu Lys Lys Lys Gly Val Phe Pro 225 230
235 240 Phe Pro Val Asn Gln Ala Asn His Gln Glu Gly Ile Leu Val Glu
Ala 245 250 255 Lys Gln Glu Val Glu Thr Arg Glu Ala Lys Glu Glu Pro
Arg Glu Glu 260 265 270 Val Lys Gln Gln Tyr Val Glu Glu Pro Pro Gln
Glu Glu Glu Glu Lys 275 280 285 Glu Glu Glu Lys Ala Glu Gln Gln Glu
Ala Glu Ile Val Gly Tyr Ser 290 295 300 Val Glu Glu Ala Val Ile Thr
Cys Cys Ile Asp Ser Ser Thr Ile Met 305 310 315 320 Glu Met Asp Arg
Cys Gly Glu Ser Asn Glu Leu Ala Trp Asp Phe Cys 325 330 335 Met Met
Asp Ser Gly Phe Ala Pro Phe Leu Thr Asp Ser Asn Leu Ser 340 345 350
Ser Glu Asn Pro Ile Glu Tyr Pro Glu Leu Phe Asn Glu Met Gly Phe 355
360 365 Glu Asp Asn Ile Asp Phe Met Phe Glu Glu Gly Lys Gln Asp Cys
Leu 370 375 380 Ser Leu Glu Asn Leu Asp Cys Cys Asp Gly Val Val Val
Val Gly Arg 385 390 395 400 Glu Ser Pro Thr Ser Leu Ser Ser Ser Pro
Leu Ser Cys Leu Ser Thr 405 410 415 Asp Ser Ala Ser Ser Thr Thr Thr
Thr Ala Thr Thr Val Thr Ser Val 420 425 430 Ser Trp Asn Tyr Ser Val
435 711722DNAArtificial SequenceSynthetic Construct 7cgcgcctgcc
tgcaggtact cgaggtcatt catatgcttg agaagagagt cgggatagtc 60caaaataaaa
caaaggtaag attacctggt caaaagtgaa aacatcagtt aaaaggtggt
120ataaagtaaa atatcggtaa taaaaggtgg cccaaagtga aatttactct
tttctactat 180tataaaaatt gaggatgttt ttgtcggtac tttgatacgt
catttttgta tgaattggtt 240tttaagttta ttcgcttttg gaaatgcata
tctgtatttg agtcgggttt taagttcgtt 300tgcttttgta aatacagagg
gatttgtata agaaatatct ttagaaaaac ccatatgcta 360atttgacata
atttttgaga aaaatatata ttcaggcgaa ttctcacaat gaacaataat
420aagattaaaa tagctttccc ccgttgcagc gcatgggtat tttttctagt
aaaaataaaa 480gataaactta gactcaaaac atttacaaaa acaaccccta
aagttcctaa agcccaaagt 540gctatccacg atccatagca agcccagccc
aacccaaccc aacccaaccc accccagtcc 600agccaactgg acaatagtct
ccacaccccc ccactatcac cgtgagttgt ccgcacgcac 660cgcacgtctc
gcagccaaaa aaaaaaagaa agaaaaaaaa gaaaaagaaa aaacagcagg
720tgggtccggg tcgtgggggc cggaaacgcg aggaggatcg cgagccagcg
acgaggagct 780taggcctcat cgttgaagat gcctctgccg acagtggtcc
caaagatgga cccccaccca 840cgaggagcat cgtggaaaaa gaagacgttc
caaccacgtc ttcaaagcaa gtggattgat 900gtgatatctc cactgacgta
agggatgacg cacaatccca ctatccttcg aggcctcatc 960gttgaagatg
cctctgccga cagtggtccc aaagatggac ccccacccac gaggagcatc
1020gtggaaaaag aagacgttcc aaccacgtct tcaaagcaag tggattgatg
tgatatctcc 1080actgacgtaa gggatgacgc acaatcccac tatccttcga
agctccctcc ctccgcttcc 1140aaagaaacgc cccccatcgc cactatatac
ataccccccc ctctcctccc atccccccaa 1200cccttctaga accatcttcc
acacactcaa gccacactat tggagaacac acagggacaa 1260cacaccataa
gatccaaggg aggcctccgc cgccgccggt aaccaccccg cccctctcct
1320ctttctttct ccgttttttt ttccgtctcg gtctcgatct ttggccttgg
tagtttgggt 1380gggcgagagg cggcttcgtg cgcgcccaga tcggtgcgcg
ggaggggcgg gatctcgcgg 1440ctggggctct cgccggcgtg gatccggccc
ggatctcgcg gggaatgggg ctctcggatg 1500tagatctgcg atccgccgtt
gttgggggag atgatggggg gtttaaaatt tccgccgtgc 1560taaacaagat
caggaagagg ggaaaagggc actatggttt atatttttat atatttctgc
1620tgcttcgtca ggcttagatg tgctagatct ttctttcttc tttttgtggg
tagaatttga 1680atccctcagc attgttcatc ggtagttttt cttttcatga
tttgtgacaa atgcagcctc 1740gtgcggagct tttttgtagg tagaagcgga
ccggtcgcgc ctcagcagtc gctgtcgtta 1800acccagcggt actcgctgag
gcgatcgcgg gcccggtacc ctgcaatgtg accctagact 1860tgtccatctt
ctggattggc caacttaatt aatgtatgaa ataaaaggat gcacacatag
1920tgacatgcta atcactataa tgtgggcatc aaagttgtgt gttatgtgta
attactaatt 1980atctgaataa gagaaagaga tcatccatat ttcttatcct
aaatgaatgt cacgtgtctt 2040tataattctt tgatgaacca gatgcatttt
attaaccaat tccatataca tataaatatt 2100aatcatatat aattaatatc
aattgggtta gcaaaacaaa tctagtctag gtgtgttttg 2160ctaattattg
ggggatagtg caaaaagaaa tctacgttct caataattca gatagaaaac
2220ttaataaagt gagataattt acatagattg cttttatcct ttgatatatg
tgaaaccatg 2280catgatataa ggaaaataga tagagaaata attttttaca
tcgttgaata tgtaaacaat 2340ttaattcaag aagctaggaa tataaatatt
gaggagttta tgattattat tattattttg 2400atgttcaatg aagttttttt
taatttcata tgaagtatac aaaaattctt catagatttt 2460tgtttctatg
ccgtagttat ctttaatata tttgtggttg aagaaattta ttgctagaaa
2520cgaatggatt gtcaattttt ttttaaagca aatatatatg aaattatact
gtatattatt 2580ttagtcatga ttaaaatgtg gccttaattg aatcatcttt
ctcattcatt ttttcaaaag 2640catatcagga tgattgatat ttatctattt
taaaaattaa tttaagggtt caaattaaat 2700ttaacttaaa agtgtcctaa
ccgtagttaa aggtttactt taaaaaaata ctatgaaaaa 2760tctaatcttc
tatgaatcga cctgcaggat ttaaatccat cgttctgggg cctaacgggc
2820caagcttact cgaggtcatt catatgcttg agaagagagt
cgggatagtc caaaataaaa 2880caaaggtaag attacctggt caaaagtgaa
aacatcagtt aaaaggtggt ataaagtaaa 2940atatcggtaa taaaaggtgg
cccaaagtga aatttactct tttctactat tataaaaatt 3000gaggatgttt
ttgtcggtac tttgatacgt catttttgta tgaattggtt tttaagttta
3060ttcgcttttg gaaatgcata tctgtatttg agtcgggttt taagttcgtt
tgcttttgta 3120aatacagagg gatttgtata agaaatatct ttagaaaaac
ccatatgcta atttgacata 3180atttttgaga aaaatatata ttcaggcgaa
ttctcacaat gaacaataat aagattaaaa 3240tagctttccc ccgttgcagc
gcatgggtat tttttctagt aaaaataaaa gataaactta 3300gactcaaaac
atttacaaaa acaaccccta aagttcctaa agcccaaagt gctatccacg
3360atccatagca agcccagccc aacccaaccc aacccaaccc accccagtcc
agccaactgg 3420acaatagtct ccacaccccc ccactatcac cgtgagttgt
ccgcacgcac cgcacgtctc 3480gcagccaaaa aaaaaaagaa agaaaaaaaa
gaaaaagaaa aaacagcagg tgggtccggg 3540tcgtgggggc cggaaacgcg
aggaggatcg cgagccagcg acgaggccgg ccctccctcc 3600gcttccaaag
aaacgccccc catcgccact atatacatac ccccccctct cctcccatcc
3660ccccaaccct accaccacca ccaccaccac ctccacctcc tcccccctcg
ctgccggacg 3720acgagctcct cccccctccc cctccgccgc cgccgcgccg
gtaaccaccc cgcccctctc 3780ctctttcttt ctccgttttt ttttccgtct
cggtctcgat ctttggcctt ggtagtttgg 3840gtgggcgaga ggcggcttcg
tgcgcgccca gatcggtgcg cgggaggggc gggatctcgc 3900ggctggggct
ctcgccggcg tggatccggc ccggatctcg cggggaatgg ggctctcgga
3960tgtagatctg cgatccgccg ttgttggggg agatgatggg gggtttaaaa
tttccgccgt 4020gctaaacaag atcaggaaga ggggaaaagg gcactatggt
ttatattttt atatatttct 4080gctgcttcgt caggcttaga tgtgctagat
ctttctttct tctttttgtg ggtagaattt 4140gaatccctca gcattgttca
tcggtagttt ttcttttcat gatttgtgac aaatgcagcc 4200tcgtgcggag
cttttttgta ggtagaagtg atcaaccatg gcgcaagtta gcagaatctg
4260caatggtgtg cagaacccat ctcttatctc caatctctcg aaatccagtc
aacgcaaatc 4320tcccttatcg gtttctctga agacgcagca gcatccacga
gcttatccga tttcgtcgtc 4380gtggggattg aagaagagtg ggatgacgtt
aattggctct gagcttcgtc ctcttaaggt 4440catgtcttct gtttccacgg
cgtgcatgct tcacggtgca agcagccggc ccgcaaccgc 4500ccgcaaatcc
tctggccttt ccggaaccgt ccgcattccc ggcgacaagt cgatctccca
4560ccggtccttc atgttcggcg gtctcgcgag cggtgaaacg cgcatcaccg
gccttctgga 4620aggcgaggac gtcatcaata cgggcaaggc catgcaggcg
atgggcgccc gcatccgtaa 4680ggaaggcgac acctggatca tcgatggcgt
cggcaatggc ggcctcctgg cgcctgaggc 4740gccgctcgat ttcggcaatg
ccgccacggg ctgccgcctg acgatgggcc tcgtcggggt 4800ctacgatttc
gacagcacct tcatcggcga cgcctcgctc acaaagcgcc cgatgggccg
4860cgtgttgaac ccgctgcgcg aaatgggcgt gcaggtgaaa tcggaagacg
gtgaccgtct 4920tcccgttacc ttgcgcgggc cgaagacgcc gacgccgatc
acctaccgcg tgccgatggc 4980ctccgcacag gtgaagtccg ccgtgctgct
cgccggcctc aacacgcccg gcatcacgac 5040ggtcatcgag ccgatcatga
cgcgcgatca tacggaaaag atgctgcagg gctttggcgc 5100caaccttacc
gtcgagacgg atgcggacgg cgtgcgcacc atccgcctgg aaggccgcgg
5160caagctcacc ggccaagtca tcgacgtgcc gggcgacccg tcctcgacgg
ccttcccgct 5220ggttgcggcc ctgcttgttc cgggctccga cgtcaccatc
ctcaacgtgc tgatgaaccc 5280cacccgcacc ggcctcatcc tgacgctgca
ggaaatgggc gccgacatcg aagtcatcaa 5340cccgcgcctt gccggcggcg
aagacgtggc ggacctgcgc gttcgctcct ccacgctgaa 5400gggcgtcacg
gtgccggaag accgcgcgcc ttcgatgatc gacgaatatc cgattctcgc
5460tgtcgccgcc gccttcgcgg aaggggcgac cgtgatgaac ggtctggaag
aactccgcgt 5520caaggaaagc gaccgcctct cggccgtcgc caatggcctc
aagctcaatg gcgtggattg 5580cgatgagggc gagacgtcgc tcgtcgtgcg
tggccgccct gacggcaagg ggctcggcaa 5640cgcctcgggc gccgccgtcg
ccacccatct cgatcaccgc atcgccatga gcttcctcgt 5700catgggcctc
gtgtcggaaa accctgtcac ggtggacgat gccacgatga tcgccacgag
5760cttcccggag ttcatggacc tgatggccgg gctgggcgcg aagatcgaac
tctccgatac 5820gaaggctgcc tgatgagctc gaattcccga tcgttcaaac
atttggcaat aaagtttctt 5880aagattgaat cctgttgccg gtcttgcgat
gattatcata taatttctgt tgaattacgt 5940taagcatgta ataattaaca
tgtaatgcat gacgttattt atgagatggg tttttatgat 6000tagagtcccg
caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta
6060ggataaatta tcgcgcgcgg tgtcatctat gttactagat cggggatggg
ggatccacta 6120gtgatatccg tcgactggta cctacgcgta gctagcccgt
gaagtttctc atctaagccc 6180ccatttggac gtgaatgtag acacgtcgaa
ataaagattt ccgaattaga ataatttgtt 6240tattgctttc gcctataaat
acgacggatc gtaatttgtc gttttatcaa aatgtacttt 6300cattttataa
taacgctgcg gacatctaca tttttgaatt gaaaaaaaat tggtaattac
6360tctttctttt tctccatatt gaccatcata ctcattgctg atccatgtag
atttcccgga 6420catgaagcca tttacaattg aatatatcct gccgccgctg
ccgctttgca cccggtggag 6480cttgcatgtt ggtttctacg cagaactgag
ccggttaggc agataatttc cattgagaac 6540tgagccatgt gcaccttccc
cccaacacgg tgagcgacgg ggcaacggag tgatccacat 6600gggacttttc
ctagcttggc tgccattttt ggggtgaggc cgttcgcggc cgaggggcgc
6660agcccctggg gggatgggag gcccgcgtta gcgggccggg agggttcgag
aagggggggc 6720accccccttc ggcgtgcgcg gtcacgcgca cagggcgcag
ccctggttaa aaacaaggtt 6780tataaatatt ggtttaaaag caggttaaaa
gacaggttag cggtggccga aaaacgggcg 6840gaaacccttg caaatgctgg
attttctgcc tgtggacagc ccctcaaatg tcaataggtg 6900cgcccctcat
ctgtcagcac tctgcccctc aagtgtcaag gatcgcgccc ctcatctgtc
6960agtagtcgcg cccctcaagt gtcaataccg cagggcactt atccccaggc
ttgtccacat 7020catctgtggg aaactcgcgt aaaatcaggc gttttcgccg
atttgcgagg ctggccagct 7080ccacgtcgcc ggccgaaatc gagcctgccc
ctcatctgtc aacgccgcgc cgggtgagtc 7140ggcccctcaa gtgtcaacgt
ccgcccctca tctgtcagtg agggccaagt tttccgcgag 7200gtatccacaa
cgccggcggc cggccgcggt gtctcgcaca cggcttcgac ggcgtttctg
7260gcgcgtttgc agggccatag acggccgcca gcccagcggc gagggcaacc
agcccggtga 7320gcgtcggaaa gggtcgatcg accgatgccc ttgagagcct
tcaacccagt cagctccttc 7380cggtgggcgc ggggcatgac tatcgtcgcc
gcacttatga ctgtcttctt tatcatgcaa 7440ctcgtaggac aggtgccggc
agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg 7500agcgcgacga
tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa
7560gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat
tatcgccggc 7620atggcggccg acgcgctggg ctacgtcttg ctggcgttcg
cgacgcgagg ctggatggcc 7680ttccccatta tgattcttct cgcttccggc
ggcatcggga tgcccgcgtt gcaggccatg 7740ctgtccaggc aggtagatga
cgaccatcag ggacagcttc aaggatcgct cgcggctctt 7800accagcctaa
cttcgatcat tggaccgctg atcgtcacgg cgatttatgc cgcctcggcg
7860agcacatgga acgggttggc atggattgta ggcgccgccc tataccttgt
ctgcctcccc 7920gcgttgcgtc gcggtgcatg gagccgggcc acctcgacct
gaatggaagc cggcggcacc 7980tcgctaacgg attcaccact ccaagaattg
gagccaatca attcttgcgg agaactgtga 8040atgcgcaaac caacccttgg
cagaacatat ccatcgcgtc cgccatctcc agcagccgca 8100cgcggcgcat
ctcgggcagc gttgggtcct ggccacgggt gcgcatgatc gtgctcctgt
8160cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat
gaatcaccga 8220tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc
tgcgacctga gcaacaacat 8280gaatggtctt cggtttccgt gtttcgtaaa
gtctggaaac gcggaagtca gcgccctgca 8340ccattatgtt ccggatctgc
atcgcaggat gctgctggct accctgtgga acacctacat 8400ctgtattaac
gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc
8460ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca
tcatcagtaa 8520cccgtatcgt gagcatcctc tctcgtttca tcggtatcat
tacccccatg aacagaaatc 8580ccccttacac ggaggcatca gtgaccaaac
aggaaaaaac cgcccttaac atggcccgct 8640ttatcagaag ccagacatta
acgcttctgg agaaactcaa cgagctggac gcggatgaac 8700aggcagacat
ctgtgaatcg cttcacgacc acgctgatga gctttaccgc agctgcctcg
8760cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
acggtcacag 8820cttgtctgta agcggatgcc gggagcagac aagcccgtca
gggcgcgtca gcgggtgttg 8880gcgggtgtcg gggcgcagcc atgacccagt
cacgtagcga tagcggagtg tatactggct 8940taactatgcg gcatcagagc
agattgtact gagagtgcac catatgcggt gtgaaatacc 9000gcacagatgc
gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga
9060ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
aggcggtaat 9120acggttatcc acagaatcag gggataacgc aggaaagaac
atgtgagcaa aaggccagca 9180aaaggccagg aaccgtaaaa aggccgcgtt
gctggcgttt ttccataggc tccgcccccc 9240tgacgagcat cacaaaaatc
gacgctcaag tcagaggtgg cgaaacccga caggactata 9300aagataccag
gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
9360gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
ctcatagctc 9420acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
aagctgggct gtgtgcacga 9480accccccgtt cagcccgacc gctgcgcctt
atccggtaac tatcgtcttg agtccaaccc 9540ggtaagacac gacttatcgc
cactggcagc agccactggt aacaggatta gcagagcgag 9600gtatgtaggc
ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
9660gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
gagttggtag 9720ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
ttttttgttt gcaagcagca 9780gattacgcgc agaaaaaaag gatctcaaga
agatcctttg atcttttcta cggggtctga 9840cgctcagtgg aacgaaaact
cacgttaagg gattttggtc atgagattat caaaaaggat 9900cttcacctag
atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
9960gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
cagcgatctg 10020tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
tagataacta cgatacggga 10080gggcttacca tctggcccca gtgctgcaat
gataccgcga gacccacgct caccggctcc 10140agatttatca gcaataaacc
agccagccgg aagggccgag cgcagaagtg gtcctgcaac 10200tttatccgcc
tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
10260agttaatagt ttgcgcaacg ttgttgccat tgctgcaggt cgggagcaca
ggatgacgcc 10320taacaattca ttcaagccga caccgcttcg cggcgcggct
taattcagga gttaaacatc 10380atgagggaag cggtgatcgc cgaagtatcg
actcaactat cagaggtagt tggcgtcatc 10440gagcgccatc tcgaaccgac
gttgctggcc gtacatttgt acggctccgc agtggatggc 10500ggcctgaagc
cacacagtga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa
10560acaacgcggc gagctttgat caacgacctt ttggaaactt cggcttcccc
tggagagagc 10620gagattctcc gcgctgtaga agtcaccatt gttgtgcacg
acgacatcat tccgtggcgt 10680tatccagcta agcgcgaact gcaatttgga
gaatggcagc gcaatgacat tcttgcaggt 10740atcttcgagc cagccacgat
cgacattgat ctggctatct tgctgacaaa agcaagagaa 10800catagcgttg
ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag
10860gatctatttg aggcgctaaa tgaaacctta acgctatgga actcgccgcc
cgactgggct 10920ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt
ggtacagcgc agtaaccggc 10980aaaatcgcgc cgaaggatgt cgctgccgac
tgggcaatgg agcgcctgcc ggcccagtat 11040cagcccgtca tacttgaagc
taggcaggct tatcttggac aagaagatcg cttggcctcg 11100cgcgcagatc
agttggaaga atttgttcac tacgtgaaag gcgagatcac caaggtagtc
11160ggcaaataat gtctaacaat tcgttcaagc cgacgccgct tcgcggcgcg
gcttaactca 11220agcgttagat gctgcaggca tcgtggtgtc acgctcgtcg
tttggtatgg cttcattcag 11280ctccggttcc caacgatcaa ggcgagttac
atgatccccc atgttgtgca aaaaagcggt 11340tagctccttc ggtcctccga
tcgaggattt ttcggcgctg cgctacgtcc gcgaccgcgt 11400tgagggatca
agccacagca gcccactcga ccttctagcc gacccagacg agccaaggga
11460tctttttgga atgctgctcc gtcgtcaggc tttccgacgt ttgggtggtt
gaacagaagt 11520cattatcgca cggaatgcca agcactcccg aggggaaccc
tgtggttggc atgcacatac 11580aaatggacga acggataaac cttttcacgc
ccttttaaat atccgattat tctaataaac 11640gctcttttct cttaggttta
cccgccaata tatcctgtca aacactgata gtttaaactg 11700aaggcgggaa
acgacaatct gg 1172289769DNAArtificial SequenceSynthetic Construct
8ggtccgatgt gagacttttc aacaaagggt aatatccgga aacctcctcg gattccattg
60cccagctatc tgtcacttta ttgtgaagat agtggaaaag gaaggtggct cctacaaatg
120ccatcattgc gataaaggaa aggccatcgt tgaagatgcc tctgccgaca
gtggtcccaa 180agatggaccc ccacccacga ggagcatcgt ggaaaaagaa
gacgttccaa ccacgtcttc 240aaagcaagtg gattgatgtg atggtccgat
tgagactttt caacaaaggg taatatccgg 300aaacctcctc ggattccatt
gcccagctat ctgtcacttt attgtgaaga tagtggaaaa 360ggaaggtggc
tcctacaaat gccatcattg cgataaagga aaggccatcg ttgaagatgc
420ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg
tggaaaaaga 480agacgttcca accacgtctt caaagcaagt ggattgatgt
gatatctcca ctgacgtaag 540ggatgacgca caatcccact atccttcgca
agacccttcc tctatataag gaagttcatt 600tcatttggag aggaccaggt
ggtaccggcg cgcctcagca gtcgctgtcg ttaacccagc 660ggtactcgct
gaggcgatcg cgggccctga tcacctgtcg tacagtattt ctacatttga
720tgtgtgattt gtgaagaaca tcaaacaaaa caagcactgg ctttaatatg
atgataagta 780ttatggtaat taattaattg gcaaaaacaa caatgaagct
aaaattttat ttattgagcc 840ttgcggttaa tttcttgtga tgatcttttt
ttttattttc taattatata tagtttcctt 900tgctttgaaa tgctaaaggt
ttgagagagt tatgctcttt ttttcttcct ctttcttttt 960taactttatc
atacaaattt tgaataaaaa tgtgagtaca ttgagctcat ttaaataagc
1020ttgatgggga tcagattgtc gtttcccgcc ttcagtttaa actatcagtg
tttgacagga 1080tatattggcg ggtaaaccta agagaaaaga gcgtttatta
gaataatcgg atatttaaaa 1140gggcgtgaaa aggtttatcc gttcgtccat
ttgtatgtgc atgccaacca cagggttccc 1200ctcgggagtg cttggcattc
cgtgcgataa tgacttctgt tcaaccaccc aaacgtcgga 1260aagcctgacg
acggagcagc attccaaaaa gatcccttgg ctcgtctggg tcggctagaa
1320ggtcgagtgg gctgctgtgg cttgatccct caacgcggtc gcggacgtag
cgcagcgccg 1380aaaaatcctc gatcggagga ccgaaggagc taaccgcttt
tttgcacaac atgggggatc 1440atgtaactcg ccttgatcgt tgggaaccgg
agctgaatga agccatacca aacgacgagc 1500gtgacaccac gatgcctgca
gcatctaacg cttgagttaa gccgcgccgc gaagcggcgt 1560cggcttgaac
gaattgttag acattatttg ccgactacct tggtgatctc gcctttcacg
1620tagtgaacaa attcttccaa ctgatctgcg cgcgaggcca agcgatcttc
ttgtccaaga 1680taagcctgcc tagcttcaag tatgacgggc tgatactggg
ccggcaggcg ctccattgcc 1740cagtcggcag cgacatcctt cggcgcgatt
ttgccggtta ctgcgctgta ccaaatgcgg 1800gacaacgtaa gcactacatt
tcgctcatcg ccagcccagt cgggcggcga gttccatagc 1860gttaaggttt
catttagcgc ctcaaataga tcctgttcag gaaccggatc aaagagttcc
1920tccgccgctg gacctaccaa ggcaacgcta tgttctcttg cttttgtcag
caagatagcc 1980agatcaatgt cgatcgtggc tggctcgaag atacctgcaa
gaatgtcatt gcgctgccat 2040tctccaaatt gcagttcgcg cttagctgga
taacgccacg gaatgatgtc gtcgtgcaca 2100acaatggtga cttctacagc
gcggagaatc tcgctctctc caggggaagc cgaagtttcc 2160aaaaggtcgt
tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt caccgtaacc
2220agcaaatcaa tatcactgtg tggcttcagg ccgccatcca ctgcggagcc
gtacaaatgt 2280acggccagca acgtcggttc gagatggcgc tcgatgacgc
caactacctc tgatagttga 2340gtcgatactt cggcgatcac cgcttccctc
atgatgttta actcctgaat taagccgcgc 2400cgcgaagcgg tgtcggcttg
aatgaattgt taggcgtcat cctgtgctcc cgacctgcag 2460caatggcaac
aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
2520aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
cgctcggccc 2580ttccggctgg ctggtttatt gctgataaat ctggagccgg
tgagcgtggg tctcgcggta 2640tcattgcagc actggggcca gatggtaagc
cctcccgtat cgtagttatc tacacgacgg 2700ggagtcaggc aactatggat
gaacgaaata gacagatcgc tgagataggt gcctcactga 2760ttaagcattg
gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
2820ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
atgaccaaaa 2880tcccttaacg tgagttttcg ttccactgag cgtcagaccc
cgtagaaaag atcaaaggat 2940cttcttgaga tccttttttt ctgcgcgtaa
tctgctgctt gcaaacaaaa aaaccaccgc 3000taccagcggt ggtttgtttg
ccggatcaag agctaccaac tctttttccg aaggtaactg 3060gcttcagcag
agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
3120acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
ttaccagtgg 3180ctgctgccag tggcgataag tcgtgtctta ccgggttgga
ctcaagacga tagttaccgg 3240ataaggcgca gcggtcgggc tgaacggggg
gttcgtgcac acagcccagc ttggagcgaa 3300cgacctacac cgaactgaga
tacctacagc gtgagctatg agaaagcgcc acgcttcccg 3360aagggagaaa
ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
3420gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
cgccacctct 3480gacttgagcg tcgatttttg tgatgctcgt caggggggcg
gagcctatgg aaaaacgcca 3540gcaacgcggc ctttttacgg ttcctggcct
tttgctggcc ttttgctcac atgttctttc 3600ctgcgttatc ccctgattct
gtggataacc gtattaccgc ctttgagtga gctgataccg 3660ctcgccgcag
ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
3720tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
tggtgcactc 3780tcagtacaat ctgctctgat gccgcatagt taagccagta
tacactccgc tatcgctacg 3840tgactgggtc atggctgcgc cccgacaccc
gccaacaccc gctgacgcgc cctgacgggc 3900ttgtctgctc ccggcatccg
cttacagaca agctgtgacc gtctccggga gctgcatgtg 3960tcagaggttt
tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
4020gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
cgttgagttt 4080ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
atgttaaggg cggttttttc 4140ctgtttggtc actgatgcct ccgtgtaagg
gggatttctg ttcatggggg taatgatacc 4200gatgaaacga gagaggatgc
tcacgatacg ggttactgat gatgaacatg cccggttact 4260ggaacgttgt
gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
4320tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
gtagccagca 4380gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
gctgacttcc gcgtttccag 4440actttacgaa acacggaaac cgaagaccat
tcatgttgtt gctcaggtcg cagacgtttt 4500gcagcagcag tcgcttcacg
ttcgctcgcg tatcggtgat tcattctgct aaccagtaag 4560gcaaccccgc
cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
4620ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg
cggacgcgat 4680ggatatgttc tgccaagggt tggtttgcgc attcacagtt
ctccgcaaga attgattggc 4740tccaattctt ggagtggtga atccgttagc
gaggtgccgc cggcttccat tcaggtcgag 4800gtggcccggc tccatgcacc
gcgacgcaac gcggggaggc agacaaggta tagggcggcg 4860cctacaatcc
atgccaaccc gttccatgtg ctcgccgagg cggcataaat cgccgtgacg
4920atcagcggtc caatgatcga agttaggctg gtaagagccg cgagcgatcc
ttgaagctgt 4980ccctgatggt cgtcatctac ctgcctggac agcatggcct
gcaacgcggg catcccgatg 5040ccgccggaag cgagaagaat cataatgggg
aaggccatcc agcctcgcgt cgcgaacgcc 5100agcaagacgt agcccagcgc
gtcggccgcc atgccggcga taatggcctg cttctcgccg 5160aaacgtttgg
tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
5220accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
gccgaaaatg 5280acccagagcg ctgccggcac ctgtcctacg agttgcatga
taaagaagac agtcataagt 5340gcggcgacga tagtcatgcc ccgcgcccac
cggaaggagc tgactgggtt gaaggctctc 5400aagggcatcg gtcgatcgac
cctttccgac gctcaccggg ctggttgccc tcgccgctgg 5460gctggcggcc
gtctatggcc ctgcaaacgc gccagaaacg ccgtcgaagc cgtgtgcgag
5520acaccgcggc cggccgccgg cgttgtggat acctcgcgga aaacttggcc
ctcactgaca 5580gatgaggggc ggacgttgac acttgagggg ccgactcacc
cggcgcggcg ttgacagatg 5640aggggcaggc tcgatttcgg ccggcgacgt
ggagctggcc agcctcgcaa atcggcgaaa 5700acgcctgatt ttacgcgagt
ttcccacaga tgatgtggac aagcctgggg ataagtgccc 5760tgcggtattg
acacttgagg ggcgcgacta ctgacagatg aggggcgcga tccttgacac
5820ttgaggggca gagtgctgac agatgagggg cgcacctatt gacatttgag
gggctgtcca 5880caggcagaaa atccagcatt tgcaagggtt tccgcccgtt
tttcggccac cgctaacctg 5940tcttttaacc tgcttttaaa ccaatattta
taaaccttgt ttttaaccag ggctgcgccc 6000tgtgcgcgtg accgcgcacg
ccgaaggggg gtgccccccc ttctcgaacc ctcccggccc 6060gctaacgcgg
gcctcccatc cccccagggg ctgcgcccct cggccgcgaa cggcctcacc
6120ccaaaaatgg cagccaagct aggaaaagtc ccatgtggat cactccgttg
ccccgtcgct 6180caccgtgttg gggggaaggt gcacatggct cagttctcaa
tggaaattat ctgcctaacc 6240ggctcagttc tgcgtagaaa ccaacatgca
agctccaccg ggtgcaaagc ggcagcggcg 6300gcaggatata ttcaattgta
aatggcttca tgtccgggaa atctacatgg atcagcaatg 6360agtatgatgg
tcaatatgga gaaaaagaaa gagtaattac caattttttt tcaattcaaa
6420aatgtagatg tccgcagcgt tattataaaa tgaaagtaca ttttgataaa
acgacaaatt 6480acgatccgtc gtatttatag gcgaaagcaa taaacaaatt
attctaattc ggaaatcttt 6540atttcgacgt gtctacattc acgtccaaat
gggggcttag atgagaaact tcacgatcga 6600tgcggccacc actcgagaag
cttactagtc aacaattggc caatctttgt tctaaattgc 6660taataaacga
ccatttccgt caattctcct tggttgcaac agtctacccg tcaaatgttt
6720actaatttat aagtgtgaag tttgaattat gaaagacgaa atcgtattaa
aaattcacaa 6780gaataaacaa ctccatagat tttcaaaaaa acagtcacga
gaaaaaaacc acagtccgtt 6840tgtctgctct tctagttttt attatttttc
tattaatagt tttttgttat ttcgagaata 6900aaatttgaac gatgtccgaa
ccacaaaagc cgagccgata aatcctaagc cgagcctaac 6960tttagccgta
accatcagtc acggctcccg ggctaattca tttgaaccga atcataatca
7020acggtttaga tcaaactcaa aacaatctaa cggcaacata gacgcgtcgg
tgagctaaaa 7080agagtgtgaa agccaggtca ccatagcatt gtctctccca
gattttttat ttgggaaata 7140atagaagaaa tagaaaaaaa taaaagagtg
agaaaaatcg tagagctata tattcgcaca 7200tgtactcgtt tcgctttcct
tagtgttagc tgctgccgct gttgtttctc ctccatttct 7260ctatctttct
ctctcgctgc ttctcgaatc ttctgtatca tcttcttctt cttcaaggtg
7320agtctctaga tccgttcgct tgattttgct gctcgttagt cgttattgtt
gattctctat 7380gccgatttcg ctagatctgt ttagcatgcg ttgtggtttt
atgagaaaat ctttgttttg 7440ggggttgctt gttatgtgat tcgatccgtg
cttgttggat cgatctgagt taattcttaa 7500ggtttatgtg ttagatctat
ggagtttgag gattcttctc gcttctgtcg atctctcgct 7560gttatttttg
tttttttcag tgaagtgaag ttgtttagtt cgaaatgact tcgtgtatgc
7620tcgattgatc tggttttaat cttcgatctg ttaggtgttg atgtttacaa
gtgaattcta 7680gtgttttctc gttgagatct gtgaagtttg aacctagttt
tctcaataat caacatatga 7740agcgatgttt gagtttcaat aaacgctgct
aatcttcgaa actaagttgt gatctgattc 7800gtgtttactt catgagctta
tccaattcat ttcggtttca ttttactttt tttttagtga 7860accatggcgc
aagttagcag aatctgcaat ggtgtgcaga acccatctct tatctccaat
7920ctctcgaaat ccagtcaacg caaatctccc ttatcggttt ctctgaagac
gcagcagcat 7980ccacgagctt atccgatttc gtcgtcgtgg ggattgaaga
agagtgggat gacgttaatt 8040ggctctgagc ttcgtcctct taaggtcatg
tcttctgttt ccacggcgtg catgcttcat 8100ggagcttcat ctaggccagc
tactgccagg aagtctagcg ggctcagtgg caccgtgcgc 8160atccctggcg
ataaaagtat ttcacacagg agcttcatgt tcggaggact tgctagtgga
8220gagacgagaa tcactggttt gcttgagggc gaagatgtta tcaacaccgg
taaggcgatg 8280caagcaatgg gtgccagaat ccgaaaagag ggcgatacgt
ggatcatcga cggtgttggt 8340aacggaggat tgctcgctcc cgaagcgcca
cttgactttg ggaacgcagc tacggggtgc 8400cgtcttacta tgggactggt
aggcgtgtat gactttgact ctaccttcat cggtgacgcg 8460agcctcacta
agagaccaat gggacgagtg ctgaatcccc tgagggagat gggtgtccag
8520gtgaaatctg aggatggtga tcgtcttccg gttactctgc gaggccccaa
gacccccacg 8580ccaatcacgt acagggttcc gatggcgtca gcacaggtca
agtcagcggt actcctggcg 8640ggcctcaaca cacctggaat cacaaccgtg
attgaaccca tcatgactag agaccacacg 8700gagaagatgt tgcagggttt
cggcgctaat ctaacggtcg aaaccgacgc cgacggcgtg 8760aggacaatcc
gcttggaggg cagaggtaaa ctgactggcc aagtcatcga tgtgcctgga
8820gatccctcgt ccacagcgtt tcccctcgta gctgcgttgc tcgtccctgg
atctgatgtg 8880acgatcctga atgtcctcat gaatccaact agaaccggcc
tcatcctcac attgcaggag 8940atgggtgctg acatcgaggt tatcaatcct
aggttggcag gtggagagga tgtggccgat 9000ctgcgcgtgc gttctagtac
actcaaaggc gtgaccgtcc ctgaggatcg cgctccatcc 9060atgatcgacg
agtaccccat tctcgccgtt gctgctgcgt ttgccgaggg cgcaactgta
9120atgaacggcc ttgaggagtt gagggttaag gagagtgaca ggctgtccgc
ggtggcgaat 9180ggcctgaagc taaacggcgt ggactgcgac gaaggtgaaa
cgtcccttgt agtccgtggt 9240cgcccagacg ggaaggggtt ggggaatgct
tcgggagctg ctgtggcgac gcaccttgat 9300catagaatcg ccatgtcatt
tctggtgatg ggacttgtct ccgagaatcc ggtgaccgtt 9360gacgatgcta
ccatgatcgc cacctccttt cctgagttca tggacctcat ggcaggcttg
9420ggggccaaga tcgagctgtc tgatactaag gccgcttgaa ttcccgatcg
ttcaaacatt 9480tggcaataaa gtttcttaag attgaatcct gttgccggtc
ttgcgatgat tatcatataa 9540tttctgttga attacgttaa gcatgtaata
attaacatgt aatgcatgac gttatttatg 9600agatgggttt ttatgattag
agtcccgcaa ttatacattt aatacgcgat agaaaacaaa 9660atatagcgcg
caaactagga taaattatcg cgcgcggtgt catctatgtt actagatcgg
9720ggatcccacg tgcggaccgc ctgcaggccg cgttatcaag ctaactgca
976998504DNAArtificial SequenceSynthetic Construct 9aggatttttc
ggcgctgcgc tacgtccgcg accgcgttga gggatcaagc cacagcagcc 60cactcgacct
tctagccgac ccagacgagc caagggatct ttttggaatg ctgctccgtc
120gtcaggcttt ccgacgtttg ggtggttgaa cagaagtcat tatcgcacgg
aatgccaagc 180actcccgagg ggaaccctgt ggttggcatg cacatacaaa
tggacgaacg gataaacctt 240ttcacgccct tttaaatatc cgattattct
aataaacgct cttttctctt aggtttaccc 300gccaatatat cctgtcaaac
actgatagtt taaactgaag gcgggaaacg acaatctgat 360ccccatcaag
cttggccagc ttctgcaggt ccgattgaga cttttcaaca aagggtaata
420tccggaaacc tcctcggatt ccattgccca gctatctgtc actttattgt
gaagatagtg 480gaaaaggaag gtggctccta caaatgccat cattgcgata
aaggaaaggc catcgttgaa 540gatgcctctg ccgacagtgg tcccaaagat
ggacccccac ccacgaggag catcgtggaa 600aaagaagacg ttccaaccac
gtcttcaaag caagtggatt gatgtgatgg tccgattgag 660acttttcaac
aaagggtaat atccggaaac ctcctcggat tccattgccc agctatctgt
720cactttattg tgaagatagt ggaaaaggaa ggtggctcct acaaatgcca
tcattgcgat 780aaaggaaagg ccatcgttga agatgcctct gccgacagtg
gtcccaaaga tggaccccca 840cccacgagga gcatcgtgga aaaagaagac
gttccaacca cgtcttcaaa gcaagtggat 900tgatgtgata tctccactga
cgtaagggat gacgcacaat cccactatcc ttcgcaagac 960ccttcctcta
tataaggaag ttcatttcat ttggagagga cacagaaaaa tttgctacat
1020tgtttcacaa acttcaaata ttattcattt atttgtcagc tttcaaactc
tttgtttctt 1080gtttgttgat tagatctggt accctcagca gtcgctgtgc
gatcgccagc ggtactcgct 1140gaggtcgacg tagttagtta attcagcttt
cgttcgtatc atcggtttcg acaacgttcg 1200tcaagttcaa tgcatcagtt
tcattgcgca cacaccagaa tcctactgag tttgagtatt 1260atggcattgg
gaaaactgtt tttcttgtac catttgttgt gcttgtaatt tactgtgttt
1320tttattcggt tttcgctatc gaactgtgaa atggaaatgg atggagaaga
gttaatgaat 1380gatatggtcc ttttgttcat tctcaaatta atattatttg
ttttttctct tatttgttgt 1440gtgttgaatt tgaaattata agagatatgc
aaacattttg ttttgagtaa aaatgtgtca 1500aatcgtggcc tctaatgacc
gaagttaata tgaggagtaa aacacttgta gttgtaccat 1560tatgcttatt
cactaggcaa caaatatatt ttcagaccta gaaaagctgc aaatgttact
1620gaatacaagt atgtcctctt gtgttttaga catttatgaa ctttccttta
tgtaattttc 1680cagaatcctt gtcagattct aatcattgct ttataattat
agttatactc atggatttgt 1740agttgagtat gaaaatattt tttaatgcat
tttatgactt gccaattgat tgacaacgcg 1800gccgccactc gagtggaagc
tagctttccg atcctacctg tcacttcatc aaaaggacag 1860tagaaaagga
aggtggcacc tacaaatgcc atcattgcga taaaggaaag gctatcattc
1920aagatgcctc tgccgacagt ggtcccaaag atggaccccc acccacgagg
agcatcgtgg 1980aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga
ttgatgtgat acttccactg 2040acgtaaggga tgacgcacaa tcccactatc
cttcgcaaga cccttcctct atataaggaa 2100gttcatttca tttggagagg
acacgctgaa atcaccagtc tctctctaca agatcgggga 2160tctctagcta
gacgatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
2220gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
cggctgctct 2280gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
ttctttttgt caagaccgac 2340ctgtccggtg ccctgaatga actgcaggac
gaggcagcgc ggctatcgtg gctggccacg 2400acgggcgttc cttgcgcagc
tgtgctcgac gttgtcactg aagcgggaag ggactggctg 2460ctattgggcg
aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
2520gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
tacctgccca 2580ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
ctcggatgga agccggtctt 2640gtcgatcagg atgatctgga cgaagagcat
caggggctcg cgccagccga actgttcgcc 2700aggctcaagg cgcgcatgcc
cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc 2760ttgccgaata
tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
2820ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
tgaagagctt 2880ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
tcgccgctcc cgattcgcag 2940cgcatcgcct tctatcgcct tcttgacgag
ttcttctgag cgggactctg gggttcgatc 3000cccaattccc gatcgttcaa
acatttggca ataaagtttc ttaagattga atcctgttgc 3060cggtcttgcg
atgattatca tataatttct gttgaattac gttaagcatg taataattaa
3120catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc
cgcaattata 3180catttaatac gcgatagaaa acaaaatata gcgcgcaaac
taggataaat tatcgcgcgc 3240ggtgtcatct atgttactag atcggggatc
gggccactcg agtggtggcc gcatcgatcg 3300tgaagtttct catctaagcc
cccatttgga cgtgaatgta gacacgtcga aataaagatt 3360tccgaattag
aataatttgt ttattgcttt cgcctataaa tacgacggat cgtaatttgt
3420cgttttatca aaatgtactt tcattttata ataacgctgc ggacatctac
atttttgaat 3480tgaaaaaaaa ttggtaatta ctctttcttt ttctccatat
tgaccatcat actcattgct 3540gatccatgta gatttcccgg acatgaagcc
atttacaatt gaatatatcc tgccgccgct 3600gccgctttgc acccggtgga
gcttgcatgt tggtttctac gcagaactga gccggttagg 3660cagataattt
ccattgagaa ctgagccatg tgcaccttcc ccccaacacg gtgagcgacg
3720gggcaacgga gtgatccaca tgggactttt cctagcttgg ctgccatttt
tggggtgagg 3780ccgttcgcgg ccgaggggcg cagcccctgg ggggatggga
ggcccgcgtt agcgggccgg 3840gagggttcga gaaggggggg cacccccctt
cggcgtgcgc ggtcacgcgc acagggcgca 3900gccctggtta aaaacaaggt
ttataaatat tggtttaaaa gcaggttaaa agacaggtta 3960gcggtggccg
aaaaacgggc ggaaaccctt gcaaatgctg gattttctgc ctgtggacag
4020cccctcaaat gtcaataggt gcgcccctca tctgtcagca ctctgcccct
caagtgtcaa 4080ggatcgcgcc cctcatctgt cagtagtcgc gcccctcaag
tgtcaatacc gcagggcact 4140tatccccagg cttgtccaca tcatctgtgg
gaaactcgcg taaaatcagg cgttttcgcc 4200gatttgcgag gctggccagc
tccacgtcgc cggccgaaat cgagcctgcc cctcatctgt 4260caacgccgcg
ccgggtgagt cggcccctca agtgtcaacg tccgcccctc atctgtcagt
4320gagggccaag ttttccgcga ggtatccaca acgccggcgg ccggccgcgg
tgtctcgcac 4380acggcttcga cggcgtttct ggcgcgtttg cagggccata
gacggccgcc agcccagcgg 4440cgagggcaac cagcccggtg agcgtcggaa
agggtcgatc gaccgatgcc cttgagagcc 4500ttcaacccag tcagctcctt
ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg 4560actgtcttct
ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcattttc
4620ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc
ggtattcgga 4680atcttgcacg ccctcgctca agccttcgtc actggtcccg
ccaccaaacg tttcggcgag 4740aagcaggcca ttatcgccgg catggcggcc
gacgcgctgg gctacgtctt gctggcgttc 4800gcgacgcgag gctggatggc
cttccccatt atgattcttc tcgcttccgg cggcatcggg 4860atgcccgcgt
tgcaggccat gctgtccagg caggtagatg acgaccatca gggacagctt
4920caaggatcgc tcgcggctct taccagccta acttcgatca ttggaccgct
gatcgtcacg 4980gcgatttatg ccgcctcggc gagcacatgg aacgggttgg
catggattgt aggcgccgcc 5040ctataccttg tctgcctccc cgcgttgcgt
cgcggtgcat ggagccgggc cacctcgacc 5100tgaatggaag ccggcggcac
ctcgctaacg gattcaccac tccaagaatt ggagccaatc 5160aattcttgcg
gagaactgtg aatgcgcaaa ccaacccttg gcagaacata tccatcgcgt
5220ccgccatctc cagcagccgc acgcggcgca tctcgggcag cgttgggtcc
tggccacggg 5280tgcgcatgat cgtgctcctg tcgttgagga cccggctagg
ctggcggggt tgccttactg 5340gttagcagaa tgaatcaccg atacgcgagc
gaacgtgaag cgactgctgc tgcaaaacgt 5400ctgcgacctg agcaacaaca
tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa 5460cgcggaagtc
agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc
5520taccctgtgg aacacctaca tctgtattaa cgaagcgctg gcattgaccc
tgagtgattt 5580ttctctggtc ccgccgcatc cataccgcca gttgtttacc
ctcacaacgt tccagtaacc 5640gggcatgttc atcatcagta acccgtatcg
tgagcatcct ctctcgtttc atcggtatca 5700ttacccccat gaacagaaat
cccccttaca cggaggcatc agtgaccaaa caggaaaaaa 5760ccgcccttaa
catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca
5820acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac
cacgctgatg 5880agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg
tgaaaacctc tgacacatgc 5940agctcccgga gacggtcaca gcttgtctgt
aagcggatgc cgggagcaga caagcccgtc 6000agggcgcgtc agcgggtgtt
ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg 6060atagcggagt
gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
6120ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
tcaggcgctc 6180ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
cggctgcggc gagcggtatc 6240agctcactca aaggcggtaa tacggttatc
cacagaatca ggggataacg caggaaagaa 6300catgtgagca aaaggccagc
aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 6360tttccatagg
ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
6420gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
ccctcgtgcg 6480ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
gcctttctcc cttcgggaag 6540cgtggcgctt tctcatagct cacgctgtag
gtatctcagt tcggtgtagg tcgttcgctc 6600caagctgggc tgtgtgcacg
aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 6660ctatcgtctt
gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
6720taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
agtggtggcc 6780taactacggc tacactagaa ggacagtatt tggtatctgc
gctctgctga agccagttac 6840cttcggaaaa agagttggta gctcttgatc
cggcaaacaa accaccgctg gtagcggtgg 6900tttttttgtt tgcaagcagc
agattacgcg cagaaaaaaa ggatctcaag aagatccttt 6960gatcttttct
acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
7020catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
gaagttttaa 7080atcaatctaa agtatatatg agtaaacttg gtctgacagt
taccaatgct taatcagtga 7140ggcacctatc tcagcgatct gtctatttcg
ttcatccata gttgcctgac tccccgtcgt 7200gtagataact acgatacggg
agggcttacc atctggcccc agtgctgcaa tgataccgcg 7260agacccacgc
tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
7320gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
gttgccggga 7380agctagagta agtagttcgc cagttaatag tttgcgcaac
gttgttgcca ttgctgcagg 7440tcgggagcac aggatgacgc ctaacaattc
attcaagccg acaccgcttc gcggcgcggc 7500ttaattcagg agttaaacat
catgagggaa gcggtgatcg ccgaagtatc gactcaacta 7560tcagaggtag
ttggcgtcat cgagcgccat ctcgaaccga cgttgctggc cgtacatttg
7620tacggctccg cagtggatgg cggcctgaag ccacacagtg atattgattt
gctggttacg 7680gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga
tcaacgacct tttggaaact 7740tcggcttccc ctggagagag cgagattctc
cgcgctgtag aagtcaccat tgttgtgcac 7800gacgacatca ttccgtggcg
ttatccagct aagcgcgaac tgcaatttgg agaatggcag 7860cgcaatgaca
ttcttgcagg tatcttcgag ccagccacga tcgacattga tctggctatc
7920ttgctgacaa aagcaagaga acatagcgtt gccttggtag gtccagcggc
ggaggaactc 7980tttgatccgg ttcctgaaca ggatctattt gaggcgctaa
atgaaacctt aacgctatgg 8040aactcgccgc ccgactgggc tggcgatgag
cgaaatgtag tgcttacgtt gtcccgcatt 8100tggtacagcg cagtaaccgg
caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg 8160gagcgcctgc
cggcccagta tcagcccgtc atacttgaag ctaggcaggc ttatcttgga
8220caagaagatc gcttggcctc gcgcgcagat cagttggaag aatttgttca
ctacgtgaaa 8280ggcgagatca ccaaggtagt cggcaaataa tgtctaacaa
ttcgttcaag ccgacgccgc 8340ttcgcggcgc ggcttaactc aagcgttaga
tgctgcaggc atcgtggtgt cacgctcgtc 8400gtttggtatg gcttcattca
gctccggttc ccaacgatca aggcgagtta catgatcccc 8460catgttgtgc
aaaaaagcgg ttagctcctt cggtcctccg atcg 8504
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