U.S. patent application number 17/543811 was filed with the patent office on 2022-03-24 for compositions and methods for altering flowering and plant architecture to improve yield potential.
This patent application is currently assigned to Monsanto Technology LLC. The applicant listed for this patent is Monsanto Technology LLC. Invention is credited to Brent Brower-Toland, Rico A. Caldo, Shunhong Dai, Karen Gabbert, Alexander Goldshmidt, Miya D. Howell, Balasulojini Karunanandaa, Sivalinganna Manjunath, Bradley W. McDill, Daniel J. Ovadya, Sasha Preuss, Elena A. Rice, Beth Savidge, Vijay K. Sharma.
Application Number | 20220090105 17/543811 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-24 |
United States Patent
Application |
20220090105 |
Kind Code |
A1 |
Brower-Toland; Brent ; et
al. |
March 24, 2022 |
COMPOSITIONS AND METHODS FOR ALTERING FLOWERING AND PLANT
ARCHITECTURE TO IMPROVE YIELD POTENTIAL
Abstract
The present invention provides recombinant DNA constructs,
vectors and molecules comprising a polynucleotide sequence encoding
a florigenic FT protein operably linked to a vegetative stage
promoter, which may also be a meristem-preferred or
meristem-specific promoter. Transgenic plants, plant cells and
tissues, and plant parts are further provided comprising a
polynucleotide sequence encoding a florigenic FT protein.
Transgenic plants comprising a florigenic FT transgene may produce
more bolls, siliques, fruits, nuts, or pods per node on the
transgenic plant, particularly on the main stem of the plant,
relative to a control or wild type plant. Methods are further
provided for introducing a florigenic FT transgene into a plant,
and planting transgenic FT plants in the field including at higher
densities. Transgenic plants of the present invention may thus
provide greater yield potential than wild type plants and may be
planted at a higher density due to their altered plant
architecture.
Inventors: |
Brower-Toland; Brent; (St.
Louis, MO) ; Caldo; Rico A.; (Eureka, MO) ;
Dai; Shunhong; (Creve Coeur, MO) ; Gabbert;
Karen; (St. Louis, MO) ; Goldshmidt; Alexander;
(Davis, CA) ; Howell; Miya D.; (Ballwin, MO)
; Karunanandaa; Balasulojini; (Creve Coeur, MO) ;
Manjunath; Sivalinganna; (Chesterfield, MO) ; McDill;
Bradley W.; (Carlsbad, CA) ; Ovadya; Daniel J.;
(Davis, CA) ; Preuss; Sasha; (Webster Groves,
MO) ; Rice; Elena A.; (Olivette, MO) ;
Savidge; Beth; (Davis, CA) ; Sharma; Vijay K.;
(Wildwood, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Monsanto Technology LLC |
St. Louis |
MO |
US |
|
|
Assignee: |
Monsanto Technology LLC
St. Louis
MO
|
Appl. No.: |
17/543811 |
Filed: |
December 7, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16370546 |
Mar 29, 2019 |
11225671 |
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17543811 |
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15131987 |
Apr 18, 2016 |
10294486 |
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16370546 |
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62150142 |
Apr 20, 2015 |
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62233019 |
Sep 25, 2015 |
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International
Class: |
C12N 15/82 20060101
C12N015/82; C07K 14/415 20060101 C07K014/415 |
Claims
1. A recombinant DNA construct comprising a polynucleotide sequence
encoding a florigenic FT protein operably linked to a vegetative
stage promoter.
2. The recombinant DNA construct of claim 1, wherein the florigenic
FT protein comprises an amino acid sequence having at least 60%, at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or
least 99% identity to a sequence selected from the group consisting
of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20, or a
functional fragment thereof.
3. The recombinant DNA construct of claim 2, wherein the florigenic
FT protein further comprises one or more of the following amino
acids: a tyrosine or other uncharged polar or nonpolar residue at
the amino acid position of the florigenic FT protein corresponding
to amino acid position 85 of SEQ ID NO: 14; a leucine or other
nonpolar residue at the amino acid position of the florigenic FT
protein corresponding to amino acid position 128 of SEQ ID NO: 14;
and a tryptophan or other large nonpolar residue at the amino acid
position of the florigenic FT protein corresponding to amino acid
position 138 of SEQ ID NO: 14.
4. The recombinant DNA construct of claim 2, wherein the florigenic
FT protein does not have one or more of the following amino acids:
a histidine at the amino acid position corresponding to a lysine or
arginine at the amino acid position corresponding to position 85 of
SEQ ID NO: 14; a lysine or arginine at the amino acid position
corresponding to position 128 of SEQ ID NO: 14; and a serine,
aspartic acid, glutamic acid, lysine or arginine at the amino acid
position corresponding to position 138 of SEQ ID NO: 14.
5. The recombinant DNA construct of claim 2, wherein the florigenic
FT protein comprises an amino acid sequence selected from the group
consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20,
or a functional fragment thereof.
6. The recombinant DNA construct of claim 1, wherein the
polynucleotide sequence is at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identity
to a sequence selected from the group consisting of SEQ ID NOs: 1,
3, 5, 7, 9, 11, 13, 15, 17, and 19.
7. The recombinant DNA construct of claim 1, wherein the vegetative
stage promoter is a meristem-preferred or meristem-specific
promoter.
8. The recombinant DNA construct of claim 1, wherein the promoter
comprises a polynucleotide sequence that is at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, or least 99%
identical to a polynucleotide sequence selected from the group
consisting of SEQ ID NOs: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, or 35, or a functional portion thereof.
9. The recombinant DNA construct of claim 1, wherein the promoter
comprises the pAt.Erecta promoter of SEQ ID NO: 21, or a functional
portion thereof
10. The recombinant DNA construct of claim 9, wherein the promoter
comprises the truncated pAt.Erecta promoter of SEQ ID NO: 22 or SEQ
ID NO: 38.
11. The recombinant DNA construct of claim 1, wherein the promoter
comprises the pAt.Erl 1 promoter of SEQ ID NO: 34, or a functional
portion thereof.
12. A DNA molecule or vector comprising the recombinant DNA
construct of claim 1.
13. A plasmid vector for Agrobacterium-mediated transformation
comprising the recombinant DNA construct of claim 1.
14. A donor template molecule for site-directed integration
comprising the recombinant DNA construct of claim 1.
15. A transgenic plant comprising an insertion of the recombinant
DNA construct of claim 1 into the genome of the transgenic
plant.
16. The transgenic plant of claim 13, wherein the transgenic plant
is homozygous for the insertion of the recombinant DNA
construct.
17. The transgenic plant of claim 15, wherein the transgenic plant
is hemizygous for the insertion of the recombinant DNA
construct.
18. The transgenic plant of claim 15, wherein the transgenic plant
is a short day plant.
19. The transgenic plant of claim 15, wherein the transgenic plant
is a dicotyledonous plant.
20. The transgenic plant of claim 19, wherein the transgenic plant
is a leguminous plant.
21. The transgenic plant of claim 20, wherein the transgenic plant
is soybean.
22. The transgenic plant of claim 21, wherein the transgenic
soybean plant produces more pods per node than a control plant not
having the recombinant DNA construct.
23. The transgenic plant of claim 15, wherein the transgenic plant
produces more flowers per node than a control plant not having the
recombinant DNA construct.
24. The transgenic plant or part thereof of claim 15, wherein the
transgenic plant produces more bolls, siliques, fruits, nuts or
pods per node of the transgenic plant than a control plant not
having the recombinant DNA construct.
25. The transgenic plant or part thereof of claim 15, wherein the
transgenic plant flowers earlier than a control plant not having
the recombinant DNA construct.
26. The transgenic plant or part thereof of claim 15, wherein the
transgenic plant has more floral racemes per node than a control
plant not having the recombinant DNA construct.
27. A transgenic plant part comprising the recombinant DNA
construct of claim 1.
28. The transgenic plant part of claim 27, wherein the transgenic
plant part is one of the following: a seed, fruit, leaf, cotyledon,
hypocotyl, meristem, embryo, endosperm, root, shoot, stem, pod,
flower, infloresence, stalk, pedicel, style, stigma, receptacle,
petal, sepal, pollen, anther, filament, ovary, ovule, pericarp,
phloem, or vascular tissue.
29. A method for producing a transgenic plant, comprising (a)
transforming at least one cell of an explant with a recombinant DNA
construct comprising a polynucleotide sequence encoding a
florigenic FT protein operably linked to a vegetative stage
promoter; and (b) regenerating or developing the transgenic plant
from the transformed explant.
30. The method of claim 29, wherein the vegetative stage promoter
of the recombinant DNA construct is a meristem-preferred or
meristem-specific promoter.
31. The method of claim 29, further comprising: (c) selecting a
transgenic plant having one or more of the following traits or
phenotypes: earlier flowering, longer reproductive or flowering
duration, increased number of flowers per node, increased number of
floral racemes per node, increased number of pods, bolls, siliques,
fruits, or nuts per node, and increased number of seeds per node,
as compared to a control plant not having the recombinant DNA
construct.
32. The method of claim 29, wherein the transforming step (a) is
carried out via Agrobacterium-mediated transformation or
microprojectile bombardment of the explant.
33. The method of claim 29, wherein the transforming step (a)
comprises site-directed integration of the recombinant DNA
construct.
34. A method of planting a transgenic crop plant, comprising:
planting the transgenic crop plant at a higher density in the
field, wherein the transgenic crop plant is transformed with a
recombinant DNA construct comprising a polynucleotide sequence
encoding a florigenic FT protein operably linked to a vegetative
stage promoter.
35. The method of claim 34, wherein the vegetative stage promoter
is a meristem-preferred or meristem-specific promoter.
36. The method of claim 34, wherein the transgenic crop plant is
soybean, and wherein about 150,000 to 250,000 seeds of the
transgenic soybean plant are planted per acre.
37. The method of claim 34, wherein the transgenic crop plant is
cotton, and wherein about 48,000 to 60,000 seeds of the transgenic
cotton plant are planted per acre.
38. The method of claim 34, wherein the transgenic crop plant is
canola, and wherein about 450,000 to 680,000 seeds of the
transgenic canola plant are planted per acre.
Description
CROSS -REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 16/370,546, filed Mar. 29, 2019, which is a divisional of
U.S. patent application Ser. No. 15/131,987, filed Apr. 18, 2016
(now U.S. Pat. No. 10,294,486, issued May 21, 2019), which claims
benefit of priority to U.S. Provisional Patent Application Nos.
62/150,142 and 62/233,019, filed on Apr. 20, 2015 and Sep. 25,
2015, respectively, which are incorporated herein by reference in
their entireties.
INCORPORATION OF SEQUENCE LISTING
[0002] A computer readable form of a sequence listing is filed with
this application by electronic submission and is incorporated into
this application by reference in its entirety. The sequence listing
is contained in the file named P34317US05_SL.txt, which is 61,167
bytes in size (measured in operating system MS Windows) and created
on Dec. 3, 2021.
FIELD OF THE INVENTION
[0003] The present invention relates to compositions and methods
for modulating floral development and vegetative growth by genetic
modification of crop plants to increase yield.
BACKGROUND
[0004] The transition from vegetative growth to flowering is a
crucial process during plant development that is necessary for the
production of grain yield in crop plants. There are four major
pathways controlling flowering time in land plants that respond to
environmental or developmental cues, including photoperiodism
(i.e., day length), vernalization (i.e., response to winter cold),
and plant hormones (e.g., gibberellins or GA), in addition to the
autonomous (environmentally independent) pathways. Except for the
GA and autonomous pathways, regulation of flowering in plants
generally involves two central regulators of flowering time,
CONSTANS (CO) and FLOWERING LOCUS C (FLC). The FLC gene is a floral
repressor that regulates flowering in response to vernalization,
whereas the CO gene is a floral activator that responds to
photoperiod conditions. Under inductive photoperiodic conditions,
CO activity in source leaves increases expression of FLOWERING
LOCUS T (FT), which translocates to the meristem to trigger
expression of downstream floral activating genes, including LEAFY
(LFY), APETALA1 (AP1) and SUPPRESSOR OF OVEREXPRESSION OF CO 1
(SOCl). Other genes, such as FLOWERING LOCUS C (FLC) and TERMINAL
FLOWER 1 (TFL1), act to inhibit the expression or activity of these
genes.
[0005] Except for day length neutral plants, most flowering plants
respond to daily photoperiodic cycles and are classified as either
short day (SD) or long day (LD) plants based on the photoperiod
conditions required to induce flowering. The photoperiod refers to
the relative length or duration of light and dark periods within a
24-hour cycle. In general, long day plants tend to flower when the
day length exceeds a photoperiod threshold (e.g., as the days are
getting longer in the spring), whereas short day plants tend to
flower when the day length falls below a photoperiod threshold
(e.g., as the days are getting shorter after the summer solstice).
In other words, SD plants flower as the days are getting shorter,
while LD plants flower as the days are getting longer. Soybean is
an example of a short day (SD) plant in which flowering is induced
when plants are exposed to shorter daylight conditions.
[0006] Plant growers are always looking for new methods to
manipulate the yield of a plant, especially to enhance the seed
yield of agronomically important crops. Thus, there is a continuing
need in the art for improved compositions and methods for
increasing yields of various crop plants. It is presently proposed
that improved crop yields may be achieved by enhancing agronomic
traits related to flowering and reproductive development.
SUMMARY
[0007] According to a first aspect of the present invention, a
recombinant DNA construct is provided comprising a polynucleotide
sequence encoding a florigenic FT protein operably linked to a
vegetative stage promoter. The florigenic FT protein encoded by the
polynucleotide sequence may comprise an amino acid sequence having
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or least 99% identity to a sequence selected from the
group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and
20, or a functional fragment thereof. The polynucleotide sequence
may also be at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, or least 99% identity to a sequence
selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9,
11, 13, 15, 17, and 19. The vegetative stage promoter may further
be a meristem-preferred or meristem-specific promoter. DNA
molecules and vectors comprising such a recombinant DNA construct
are further provided.
[0008] According to a second aspect of the present invention,
transgenic plants, plant cells, plant tissues and plant parts are
further provided comprising an insertion of the recombinant DNA
construct of the present invention into the genome of such plants,
cells, tissues, and plant parts. A transgenic plant of the present
invention may be homozygous or hemizygous for an insertion of the
recombinant DNA construct. A transgenic plant may be a short day
plant and/or a dicotyledonous plant. Depending on the plant
species, transgenic plants of the present invention may produce
more bolls, siliques, fruits, nuts, or pods per node of the
transgenic plant, relative to a control or wild type plant not
having the recombinant DNA construct. Transgenic plants of the
present invention may also produce more flowers and/or floral
racemes per node relative to a control or wild type plant not
having the recombinant DNA construct.
[0009] According to a third aspect of the present invention,
methods for producing a transgenic plant having improved
yield-related traits or phenotypes are provided comprising (a)
transforming at least one cell of an explant with a recombinant DNA
construct comprising a polynucleotide sequence encoding a
florigenic FT protein operably linked to a vegetative stage
promoter; and (b) regenerating or developing the transgenic plant
from the transformed explant. Such methods may further comprise (c)
selecting a transgenic plant having one or more of the following
traits or phenotypes: earlier flowering, longer reproductive or
flowering duration, increased number of flowers per node, increased
number of floral racemes per node, increased number of pods, bolls,
siliques, fruits, or nuts per node, and increased number of seeds
per node, as compared to a control plant not having the recombinant
DNA construct.
[0010] According a fourth aspect of the present invention, methods
are provided for planting a transgenic crop plant of the present
invention at a normal or higher density in the field. According to
some embodiments, methods are provided comprising: planting a
transgenic crop plant at a higher density in the field, wherein the
transgenic crop plant is transformed with a recombinant DNA
construct comprising a polynucleotide sequence encoding a
florigenic FT protein operably linked to a vegetative stage
promoter. According to some of these embodiments, the vegetative
stage promoter may be a meristem-preferred or meristem-specific
promoter. For soybean, a higher density of about 150,000 to 250,000
seeds of the transgenic soybean plant may be planted per acre. For
cotton, a higher density of about 48,000 to 60,000 seeds of the
transgenic cotton plant may be planted per acre. For canola, a
higher density of about 450,000 to 680,000 seeds of the transgenic
canola plant may be planted per acre.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1A provides a matrix table showing a comparison of
nucleotide sequences for each combination of the various FT genes
including their percent identity.
[0012] FIG. 1B provides a matrix table showing a comparison of
protein sequences for each combination of the various FT proteins
including their percent identity.
[0013] FIG. 1C provides a CLUSTAL 2.0.9 multiple sequence alignment
of various FT proteins identified as Gm.FT2a with SEQ ID NO: 2,
Gm.FT2b with SEQ ID NO: 4, Le.FT with SEQ ID NO: 12, Pt.FT with SEQ
ID NO: 20, Os.HD3a with SEQ ID NO: 18, At.FT with SEQ ID NO: 14,
At. TSF with SEQ ID NO: 16, Nt.FT with SEQ ID NO: 10, Gm.FT5a with
SEQ ID NO: 6 and Zm.ZCN8 with SEQ ID NO: 8.
[0014] FIG. 2 shows the total FT transcript levels in soybean leaf
and apex tissues collected at 1, 3 and 5 days after either a short
day or long day light treatment.
[0015] FIGS. 3A to 3O and FIGS. 4A to 4O show the expression
pattern of the pAt.Erecta promoter by monitoring GUS activity
during early soybean development. FIGS. 3A to 3O are a set of black
and white images of stained tissues, and the images in FIGS. 4A to
4O correspond to FIGS. 3A to 3O but are filtered for blue GUS
staining. FIGS. 3A to 3C and 4A to 4C show expression in a
3-day-old germinating seedling; FIGS. 3D to 3I and 4D to 4I show
expression in a 10-day-old vegetative shoot (grown in 14 hour
light/10 hour dark photoperiod); FIGS. 3J to 3L and 4J to 4L show
expression in a 16-day-old reproductive shoot; and FIGS. 3M to 3O
and 4M to 4O show expression in the 30d old mature and immature
leaves of the reproductive shoot. Bars are 100.sub.11.m.
[0016] FIGS. 5A to 5F and FIGS. 6A to 6F show the GUS expression
pattern with the pAT.Erecta promoter during R1 and floral stages of
development (35-40 days after germination). FIGS. 5A to 5F are a
set of black and white images of stained tissues, and the images in
FIGS. 6A to 6F correspond to FIGS. 5A to 5F but are filtered for
blue GUS staining. FIGS. 5A and 6A show expression in the
inflorescence stems or pedicels (arrows), and FIGS. 5B and 6B show
expression in the floral peduncle (arrows). Expression is also
shown in the vasculature and parenchyma cells (FIGS. 5C and 6C), in
stamen filaments (FIGS. 5D and 6D; arrow), and unpollinated ovules
(FIGS. 5E, 5F, 6E and 6F; arrows). Bars are 1 mm.
[0017] FIG. 7 shows section imaging of the shoot apical meristem
(SAM) from wild type versus GmFT2a-expressing transgenic plants at
7 days after planting using scanning electron microscopy (eSEM)
analysis.
[0018] FIG. 8 shows scanning electron microscopy (eSEM) micrographs
of an axillary inflorescence primordia from a wild type plant
(collected at 27 days after planting), in comparison to an axillary
inflorescence primordia from a transgenic event expressing Gm.FT2a
(collected at 9 days after planting).
[0019] FIGS. 9A to 9C show the effects of Gm.FT2a expression driven
by the At.Erecta promoter in soybean. FIG. 9A depicts a null
segregant showing normal axillary buds, whereas FIG. 9B and FIG. 9C
(corresponding to plants homozygous or hemizygous for the Gm.FT2a
transgene, respectively) each show early flowering and increased
pods per node relative to the null segregant.
[0020] FIG. 10 shows a whole plant image of a wild type null
segregant next to plants hemizygous and homozygous for the Gm.FT2a
transgene as indicated.
[0021] FIG. 11 shows images of the main stem of plants that are
homozygous or hemizygous for the pAt.Erecta-Gm.FT2a transgene in
comparison to a null segregant as indicated.
[0022] FIG. 12A shows whole plant images of a wild type null
segregant and a plant homozygous for the pEr:Zm.ZCN8 transgene as
indicated.
[0023] FIG. 12B shows close up images of pods on the mainstem of a
wild type null segregant and a plant homozygous for the pEr:Zm.ZCN8
transgene as indicated.
[0024] FIG. 13A shows whole plant images of a wild type null
segregant and a plant homozygous for the pEr:Nt.FT-like transgene
as indicated.
[0025] FIG. 13B shows close up images of pods on the mainstem of a
wild type null segregant and a plant homozygous for the
pEr:Nt.FT-like transgene as indicated.
[0026] FIG. 14 shows whole plant images of a wild type null
segregant and a plant homozygous for the pEr:Gm.FT2b transgene as
indicated.
[0027] FIG. 15 shows whole plant images of a wild type null
segregant and a plant homozygous for the pEr:Le.SFT transgene as
indicated.
[0028] FIG. 16 shows whole plant images of a wild type null
segregant and a plant homozygous for the pEr:FT5a transgene as
indicated.
DETAILED DESCRIPTION
[0029] The goal of improving yield is common to all crops across
agriculture. The present invention includes methods and
compositions for improving yield in flowering (angiosperm) or
seed-bearing plants by modification of traits associated with
flowering time, reproductive development, and vegetative growth to
improve one or more flowering and/or yield-related traits or
phenotypes, such as the number of flowers, seeds and/or pods per
plant, and/or the number of flowers, seeds and/or pods per node
(and/or per main stem) of the plant. Without being bound by any
theory, compositions and methods of the present invention may
operate to improve yield of a plant by increasing the number of
floral meristems, increasing synchronization of lateral meristem
release, and/or extending the time period for pod or seed
development in the plant.
[0030] Previously, it was discovered that growing short day plants,
such as soybean, under long day conditions (e.g., about 14-16 hours
of light per day) and then briefly subjecting those plants to short
day growing conditions (e.g., about 9-11 hours of light per day for
about 3-21 days) before returning the plants to long day
(non-inductive) growing conditions, produced plants having
increased numbers of pods/seeds per plant (and pods/seeds per node
and/or per branch). See, e.g., U.S. Pat. No. 8,935,880 and U.S.
Patent Application Publication No. 2014/0259905, the entire
contents and disclosures of which are incorporated herein by
reference.
[0031] As described further below, this short day induction
phenotype in soybean was used to identify genes having altered
expression in these plants through transcriptional profiling. These
studies identified several genes with altered expression in these
treated soybean plants including an endogenous FT gene, Gm.FT2a,
having increased expression in response to the short day induction
treatment. Thus, it is presently proposed that transgenic FT
expression as described herein may be used in place of short day
induction to increase seed yield, alter reproductive traits or
phenotypes in plants, or both. According to an aspect of the
present invention, ectopic or transgenic expression of a Gm.FT2a
gene or other FT sequence, or a functional fragment, homolog or
ortholog thereof, in a flowering or seed-bearing plant may be used
to increase seed yield and/or alter one or more reproductive
phenotypes or traits, which may involve an increase in the number
of pods/seeds per plant (and/or the number of pods/seeds per node
or main stem of the plant). As explained further below and
depending on the particular plant species, these yield-related or
reproductive phenotypes or traits may also apply to other botanical
structures analogous to pods of leguminous plants, such as bolls,
siliques, fruits, nuts, tubers, etc. Thus, a plant ectopically
expressing a FT sequence may instead have an increased number of
bolls, siliques, fruits, nuts, tubers, etc., per node(s), main
stem, and/or branch(es) of the plant, and/or an increased number of
bolls, siliques, fruits, nuts, tubers, etc., per plant.
[0032] According to embodiments of the present invention, a
recombinant DNA molecule comprising an FT transgene is provided,
which may be used in transformation to generate a transgenic plant
expressing the FT transgene. The polynucleotide coding sequence of
the FT transgene may include Gm.FT2a (SEQ ID NO: 1), or any
polynucleotide sequence encoding the Gm.FT2a protein (SEQ ID NO:
2). The polynucleotide coding sequence of an FT transgene may also
correspond to other FT genes in soybean or other plants. For
example, other polynucleotide FT coding sequences from soybean that
may be used according to present embodiments include: Gm.FT5a (SEQ
ID NO: 3) or a polynucleotide encoding a Gm.FT5a protein (SEQ ID
NO: 4), or Gm.FT2b (SEQ ID NO: 5) or a polynucleotide encoding a
Gm.FT2b protein (SEQ ID NO: 6). In addition, examples of
polynucleotide FT coding sequences from other plant species that
may be used include: Zm.ZCN8 (SEQ ID NO: 7) from maize or a
polynucleotide encoding Zm.ZCN8 protein (SEQ ID NO: 8), Nt.FT-like
or Nt.FT4 (SEQ ID NO: 9) from tobacco or a polynucleotide encoding
Nt.FT-like or Nt.FT4 protein (SEQ ID NO: 10), Le.FT or SFT (SEQ ID
NO: 11) from tomato or a polynucleotide encoding Le.FT or SFT
protein (SEQ ID NO: 12), At.FT (SEQ ID NO: 13) from Arabidopsis or
a polynucleotide encoding At.FT protein (SEQ ID NO: 14), At. TSF
(SEQ ID NO: 15) from Arabidopsis or a polynucleotide encoding
At.TSF protein (SEQ ID NO: 16), Os.HD3a (SEQ ID NO: 17) from rice
or a polynucleotide encoding Os.HD3a protein (SEQ ID NO: 18), or
Pt.FT (SEQ ID NO: 19) from Populus trichocarpa or a polynucleotide
encoding Pt.FT protein (SEQ ID NO: 20).
[0033] Polynucleotide coding sequences for FT transgenes encoding
additional FT proteins from other species having known amino acid
sequences may also be used according to embodiments of the present
invention, which may, for example, include the following: Md.FT1
and Md.FT2 from apple (Malus domestica); Hv.FT2 and Hv.FT3 from
barley (Hordeum vulgare); Cs.FTL3 from Chrysanthemum; Ls.FT from
lettuce (Lactuca sativa); Pn.FT1 and Pn.FT2 from Lombardy poplar
(Populus nigra); Pa.FT from London plane tree (Platanus
acerifolia); Dl.FT1 from Longan (Dimocarpus longan); Ps.FTa1,
Ps.FTa2, Ps.FTb 1, Ps.FTb2, and Ps.FTc from pea (Pisum sativum);
Ac.FT from pineapple (Ananas comosus); Cm-FTL1 and Cm-FTL2 from
pumpkin (Cucurbita maxima); Ro.FT from rose; Cg.FT from spring
orchid (Cymbidium); Fv.FT1 from strawberry (Fragaria vesca); Bv.FT2
from sugar beet (Beta Vulgaris); Ha.FT4 from sunflower (Helianthus
annuus); and Ta.FT or TaFT1 from wheat (Triticum aestivum). See,
e.g., Wickland, DP et al., "The Flowering Locus T/Terminal Flower 1
Gene Family: Functional Evolution and Molecular Mechanisms",
Molecular Plant 8: 983-997 (2015), the content and disclosure of
which is incorporated herein by reference.
[0034] Unless otherwise stated, nucleic acid or polynucleotide
sequences described herein are provided (left-to-right) in the 5'
to 3' direction, and amino acid or protein sequences are provided
(left-to-right) in the N-terminus to C-terminus direction.
Additional known or later discovered FT genes and proteins from
these or other species may also be used according to embodiments of
the present invention. These FT genes may be known or inferred from
their nucleotide and/or protein sequences, which may be determined
by visual inspection or by use of a computer-based searching and
identification tool or software (and database) based on a
comparison algorithm with known FT sequences, structural domains,
etc., and according to any known sequence alignment technique, such
as BLAST, FASTA, etc.
[0035] According to embodiments of the present invention, an FT
transgene of a recombinant DNA molecule may comprise a
polynucleotide sequence that (when optimally aligned) is at least
60% identical, at least 65% identical, at least 70% identical, at
least 75% identical, at least 80% identical, at least 85%
identical, or at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, or least 99% identical to one or more of the
polynucleotide FT coding sequences listed above (e.g., SEQ ID NOS:
1, 3, 5, 7, 9, 11, 13, 15, 17, or 19), or to any other known
florigenic FT coding sequence. Sequence identity percentages among
polynucleotide sequences of the above listed full length FT genes
are presented in FIG. 1A. Each cell in the table in FIG. 1A shows
the percentage identity for the FT gene in the corresponding row
(query sequence) as compared to the FT gene in the corresponding
column (subject sequence) divided by the total length of the query
sequence, and the number in parenthesis is the total number of
identical bases between the query and subject sequences. As shown
in this figure, the percentage identities among polynucleotide
sequences for these sampled FT genes range from about 60% to about
90% identity. Thus, a polynucleotide sequence that is within one or
more of these sequence identity ranges or has a higher sequence
identity may be used according to embodiments of the present
invention to induce flowering, increase yield, and/or alter one or
more reproductive traits of a plant. Similar polynucleotide coding
sequences for FT may be designed or chosen based on known FT
protein sequences, conserved amino acid residues and domains, the
degeneracy of the genetic code, and any known codon optimizations
for the particular plant species to be transformed.
[0036] As described below, an FT transgene comprising any one of
the above coding sequences may further include one or more
expression and/or regulatory element(s), such as leader(s),
intron(s), etc. Indeed, an FT transgene may comprise a genomic
sequence encoding an FT protein or amino acid sequence, or a
fragment or portion thereof.
[0037] According to embodiments of the present invention, an FT
transgene of a recombinant DNA molecule may comprise a
polynucleotide sequence encoding an amino acid or protein sequence
that (when optimally aligned) is at least 60% identical, at least
65% identical, at least 70% identical, at least 75% identical, at
least 80% identical, at least 85% identical, or at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or least 99% identical to
any one or more of the FT protein or amino acid sequences listed
above (e.g., SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20) or
any other known florigenic FT protein sequence, or a functional
fragment thereof. Such a "functional fragment" is defined as a
protein having a polypeptide sequence that is identical or highly
similar to a full-length FT protein but lacking one or more amino
acid residues, portions, protein domains, etc., of the full-length
FT protein, as long as the fragment remains active in causing one
or more of the phenotypic effects or changes similar to the
full-length protein when transgenically expressed in a plant.
Sequence identity percentages among the above listed full length FT
proteins are presented in FIG. 1B. The percentages are calculated
as described above in reference to FIG. 1A based on the number of
identical amino acid residues (in parenthesis) between the query
and subject FT protein sequences. Multiple sequence alignment of
these FT proteins is also shown in FIG. 1C. As can be seen from
these figures, the percentage identity among protein sequences for
these FT genes ranges from about 60% to about 90% identity. Thus, a
polynucleotide sequence encoding an amino acid or protein sequence
that is within one or more of these sequence identity ranges or has
a higher sequence identity may be used according to embodiments of
the present invention to induce flowering, increase seed yield,
and/or alter one or more reproductive traits of a plant. These FT
protein sequences encoded by a polynucleotide sequence of the
present invention may be designed or chosen based on known FT
protein sequences and their conserved amino acid residues and
domains.
[0038] As used herein, the term "sequence identity" refers to the
extent to which two optimally aligned DNA sequences are identical.
Various pair-wise or multiple sequence alignment algorithms and
programs are known in the art, such as ClustalW, etc., that may be
used to compare the sequence identity or similarity between two or
more sequences, such as between two or more FT genes or protein
sequences, or an FT gene (nucleotide) or protein sequence and
another nucleotide or protein sequence. For example, the percentage
identity of one sequence (query) to another sequence (subject) may
be calculated as described above in reference to FIGS. 1A and 1B
(i.e., with the sequences optimally aligned, divide the number of
identical bases or residues by the total number of bases or
residues for the query sequence, and multiply by 100%). Although
other alignment and comparison methods are known in the art, the
alignment and percent identity between two sequences (including the
percent identity ranges described above) may be as determined by
the ClustalW algorithm, see, e.g., Chenna R. et al., "Multiple
sequence alignment with the Clustal series of programs," Nucleic
Acids Research 31: 3497-3500 (2003); Thompson J D et al., "Clustal
W: Improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, position-specific gap
penalties and weight matrix choice," Nucleic Acids Research 22:
4673-4680 (1994); and Larkin M A et al., "Clustal W and Clustal X
version 2.0," Bioinformatics 23: 2947-48 (2007), the entire
contents and disclosures of which are incorporated herein by
reference. For purposes of the present invention, when two
sequences are optimally aligned (with allowance for gaps in their
alignment), then the "percent identity" for the query sequence is
calculated as described above in reference to FIGS. 1A and
1B--i.e., Percent Identity=(Number of Identical Positions between
query and subject sequences/Total Number of Positions in the Query
Sequence).times.100%, with each sequence consisting of a series of
positions (nucleotide bases or amino acid residues).
[0039] An FT protein sequence encoded by a polynucleotide sequence
or transgene of the present invention may also be designed or
chosen to have one or more amino acid substitution(s) known to be
chemically and/or structurally conservative (e.g., replacing one
amino acid with another having similar chemical or physical
properties, such as hydrophobicity, polarity, charge, steric
effect, acid/base chemistry, similar side chain group, such as
hydroxyl, sulfhydryl, amino, etc.) to avoid or minimize structural
changes to the protein that might affect its function. For example,
valine is often a conservative substitute for alanine, and
threonine may be a conservative substitute for serine. Additional
examples of conservative amino acid substitutions in proteins
include: valine/leucine, valine/isoleucine, phenylalanine/tyrosine,
lysine/arginine, aspartic acid/glutamic acid, and
asparagine/glutamine. An FT protein sequence encoded by a
polynucleotide sequence or transgene of the present invention may
also include proteins that differ in one or more amino acids from
those of a known FT protein or similar sequence as a result of
deletion(s) and/or insertion(s) involving one or more amino
acids.
[0040] Various FT genes and proteins from different plant species
may be identified and considered FT homologs or orthologs for use
in the present invention if they have a similar nucleic acid and/or
protein sequence and share conserved amino acids and/or structural
domain(s) with at least one known FT gene or protein. As used
herein, the term "homolog" in reference to a FT gene or protein is
intended to collectively include any homologs, analogs, orthologs,
paralogs, etc., of the FT gene or protein, and the term
"homologous" in reference to polynucleotide or protein sequences is
intended to mean similar or identical sequences. Such a FT homolog
may also be defined as having the same or similar biological
function as known FT genes (e.g., acting to similarly influence
flowering and/or other reproductive or yield-related traits or
phenotypes when ectopically expressed in a plant).
[0041] Sequence analysis and alignment of FT protein sequences from
different plant species further reveals a number of conserved amino
acid residues and at least one conserved structural domain. By
subjecting the various aligned FT protein sequences (see, e.g.,
FIGS. 1B and 1C) to a protein domain identification tool using a
Pfam database (e.g., Pfam version 26.0, released November 2011, or
later version), these FT proteins have been found to contain and
share at least a portion of a putative phosphatidyl
ethanolamine-binding protein (PEBP) domain (Pfam domain name:
PBP_N; Accession number: PF01161). See, e.g., Banfield, M J et al.,
"The structure of Antirrhinum centroradialis protein (CEN) suggests
a role as a kinase inhibitor," Journal of Mol Biol., 297(5):
1159-1170 (2000), the entire contents and disclosure of which are
incorporated herein by reference. This PEBP domain was found to
correspond, for example, to amino acids 28 through 162 of the full
length Gm.FT2a protein (See Table 5 below). Thus, FT proteins
encompassed by embodiments of the present invention may include
those identified or characterized as having or containing at least
a PEBP domain (Accession number: PF01161) according to Pfam
analysis. Accordingly, the present invention may further include a
polynucleotide sequence(s) encoding an FT protein having at least a
PEBP domain. As known in the art, the "Pfam" database is a large
collection of multiple sequence alignments and hidden Markov models
covering many common protein families and containing information
about various protein families and their domain structure(s). By
identifying a putative Pfam structural domain(s) for a given
protein sequence, the classification and function of the protein
may be inferred or determined. See, e.g., Finn, R D et al., "The
Pfam protein families database," Nucleic Acids Research (Database
Issue), 42:D222-D230 (2014), the entire contents and disclosure of
which are incorporated herein by reference.
[0042] Embodiments of the present invention may further include
polynucleotide sequence(s) encoding inductive or florigenic FT
proteins. An FT protein encoded by a polynucleotide sequence may be
"inductive" or "florigenic" if the FT protein, when ectopically
expressed in a plant, is able to cause earlier flowering and/or an
increased prolificacy in the number of flowers, pods, and/or seeds
per one or more node(s) of the plant. Without being bound by any
theory, such increased prolificacy in the number of flowers, pods,
and/or seeds per node(s) of the plant may result from an increase
in the number of meristems at those node(s) that undergo a
vegetative to reproductive transition and produce flowers. Such an
increased prolificacy at each node due to ectopic expression of a
"florigenic" FT may be due to increased synchronization of the
release and floral development of early racemes and lateral
meristems at each node. Although a "florigenic" FT protein may
function to induce earlier flowering when ectopically expressed in
a plant, a transgenically expressed "florigenic" FT protein may
increase the number of flowers, pods, and/or seeds per node(s) of a
plant through one or more pathways or mechanisms that are
independent of, or in addition to, any florigenic effects related
to flowering time and/or reproductive duration.
[0043] Florigenic FT-like genes from various plant species are
generally well conserved. However, many proteins in the PEBP family
have amino acid sequences that are substantially similar to
florigenic FT proteins but do not behave as florigens. For example,
Terminal Flower (TFL) genes from various plant species have similar
protein sequences to florigenic FT genes but actually delay
flowering. Recent work has identified specific amino acid residues
that are generally not shared between florigenic FT proteins and
other PEBP proteins, such as TFLs, and substitutions at many of
these positions have been shown to convert florigenic FT proteins
into floral repressor proteins. See, e.g., Ho and Weigel, Plant
Cell 26: 552-564 (2014); Danilevskaya et al., Plant Physiology
146(1): 250-264 (2008); Harig et al., Plant Journal 72: 908-921
(2012); Hsu et al., Plant Cell 18: 1846-1861 (2006); Kojima et al.,
Plant Cell Physiology 43(10): 1096-1105 (2002); Kong et al., Plant
Physiology 154: 1220-1231 (2010); Molinero-Rosales et al., Planta
218: 427-434 (2004); Zhai et al., PLoS ONE, 9(2): e89030 (2014),
and Wickland D P et al. (2015), supra, the entire contents and
disclosures of which are incorporated herein by reference. Thus,
these amino acid residues can serve as signatures to further define
and distinguish florigenic FT proteins of the present
invention.
[0044] According to embodiments of the present invention, an
"inductive" or "florigenic" FT protein may be further defined or
characterized as comprising one or more of the following amino acid
residue(s) (amino acid positions refer to corresponding positions
of the full-length Arabidopsis FT protein, SEQ ID NO: 14): a
proline at amino acid position 21 (P21); an arginine or lysine at
amino acid position 44 (R44 or K44); a glycine at amino acid
position 57 (G57); a glutamic acid or an aspartic acid at amino
acid position 59 (E59 or D59); a tyrosine at amino acid position 85
(Y85); a leucine at amino acid position 128 (L128); a glycine at
amino acid position 129 (G129); a threonine at amino acid position
132 (T132); an alanine at amino acid position 135 (A135); a
tryptophan at amino acid position 138 (W138); a glutamic acid or an
aspartic acid at amino acid position 146 (E146 or D146); and/or a
cysteine at amino acid position 164 (C164). Corresponding amino
acid positions of other FT proteins can be determined by alignment
with the Arabidopsis FT sequence (see, e.g., FIG. 1C). One skilled
in the art would be able to identify corresponding amino acid
positions of other FT proteins based on their sequence alignment.
Several of these key residues fall within an external loop domain
of FT-like proteins, defined as amino acids 128 through 145 of the
Arabidopsis full-length FT sequence (SEQ ID NO: 14) and
corresponding sequences of other FT proteins (see, e.g., FIG. 1C).
Thus, polynucleotides of the present invention may encode
florigenic FT proteins having one or more of these conserved amino
acid residues.
[0045] Florigenic FT proteins of the present invention may also
have one or more other amino acids at one or more of the above
identified residue positions. For example, in reference to the
above amino acid positions of the Arabidopsis FT (At.FT) protein
sequence (SEQ ID NO: 14), a florigenic FT protein may alternatively
have one or more of the following amino acids: an alanine (in place
of proline) at the position corresponding to position 21 of the
At.FT protein sequence (P21A), or possibly other small, nonpolar
residues, such as glycine or valine, at this position; a histidine
(in place of lysine or arginine) at the amino acid position
corresponding to position 44 of the At.FT protein sequence, or
possibly other polar amino acids at this position; an alanine or
cysteine (in place of glycine) at the amino acid position
corresponding to position 57 of the At.FT protein sequence, or
possibly other small, nonpolar residues, such proline or valine, at
this position; an asparagine or serine (in place of glutamic acid
or aspartic acid) at the amino acid position corresponding to
position 59 of the At.FT protein sequence, or possibly other small,
polar residues, such as glutamine, cysteine, or threonine, at this
position; a variety of polar and nonpolar uncharged residues (other
than tyrosine) at the amino acid position corresponding to position
85 of the At.FT protein sequence; a nonpolar or hydrophobic
uncharged residue (other than leucine), such as isoleucine, valine,
or methionine, at the amino acid position corresponding to position
128 of the At.FT protein sequence; a variety of smaller nonpolar
and uncharged residues (other than glycine), such as alanine,
valine, leucine, isoleucine, methionine, etc., at the amino acid
position corresponding to position 129 of the At.FT protein
sequence, although some polar and charged residues may be tolerated
at this position; a polar uncharged residue (other than threonine)
at the amino acid position corresponding to position 132 of the
At.FT protein sequence; a variety of amino acids other than proline
, such as threonine, at the amino acid position corresponding to
position 135 of the At.FT protein sequence ; a variety of other
bulky nonpolar or hydrophobic amino acids (in place of tryptophan),
such as methionine or phenylalanine, at the amino acid position
corresponding to position 138 of the At.FT protein sequence; a
variety of other polar or non-positively charged amino acids , such
as asparagine or serine, at the amino acid position corresponding
to position 146 of the At.FT protein sequence; and/or a variety of
other polar or nonpolar amino acids (in place of cysteine, such as
isoleucine, at the amino acid position corresponding to position
164 of the At.FT protein sequence. One skilled in the art would be
able to identify corresponding amino acid positions and
substitutions of FT proteins based on their sequence alignment to
the Arabidopsis FT protein sequence. In addition, other chemically
conservative amino acid substitutions are also contemplated within
the scope of florigenic FT proteins based on the knowledge of one
skilled in the art of protein biochemistry. Accordingly,
polynucleotides of the present invention may further encode
florigenic FT proteins having one or more conservative amino acid
substitutions. Indeed, florigenic FT proteins encoded by
polynucleotides of the present invention include native sequences
and artificial sequences containing one or more conservative amino
acid substitutions, as well as functional fragments thereof.
[0046] Florigenic FT proteins of the present invention may also be
defined as excluding (i.e., not having) one or more amino acid
substitutions that may be characteristic of, or associated with,
TFL or other non-florigenic or anti-florigenic proteins. For
example, in reference to the amino acid positions of the
Arabidopsis FT protein sequence (SEQ ID NO: 14), a florigenic FT
protein may exclude one or more of the following amino acids: a
phenylalanine or serine at the position corresponding to position
21 of the At.FT protein sequence (e.g., in place of proline or
alanine); a phenylalanine at the position corresponding to position
44 of the At.FT protein sequence (e.g., in place of arginine or
lysine); a histidine, glutamic acid, or aspartic acid at the
position corresponding to position 57 of the At.FT protein sequence
(e.g., in place of glycine); a glycine or alanine at the position
corresponding to position 59 of the At.FT protein sequence (e.g.,
in place of glutamic acid or aspartic acid); a histidine at the
position corresponding to position 85 of the At.FT protein sequence
(e.g., in place of tyrosine); a lysine, arginine, alanine, or
methionine at the position corresponding to position 109 of the
At.FT protein sequence; a lysine or arginine at the position
corresponding to position 128 of the At.FT protein sequence (e.g.,
in place of leucine); a glutamine or asparagine at the position
corresponding to position 129 of the At.FT protein sequence (e.g.,
in place of glycine); a valine or cysteine at the position
corresponding to position 132 of the At.FT protein sequence (e.g.,
in place of threonine); a lysine, arginine, or alanine at the
position corresponding to position 134 of the At.FT protein
sequence (e.g., in place of tyrosine); a proline at the position
corresponding to position 135 of the At.FT protein sequence (e.g.,
in place of alanine or threonine); a serine, aspartic acid,
glutamic acid, alanine, lysine, or arginine at the position
corresponding to position 138 of the At.FT protein sequence (e.g.,
in place of tryptophan or methionine); a lysine or arginine at the
position corresponding to position 140 of the At.FT protein
sequence; a lysine or arginine at the position corresponding to
position 146 of the At.FT protein sequence (e.g., in place of
acidic or uncharged polar residues); a lysine or arginine at the
position corresponding to position 152 of the At.FT protein
sequence; and/or an alanine at the position corresponding to
position 164 of the At.FT protein sequence (e.g., in place of
cysteine or isoleucine). One skilled in the art would be able to
identify corresponding amino acid positions and substitutions of
other FT proteins based on their sequence alignment. Accordingly,
embodiments of the present invention may exclude polynucleotides
that encode FT-like proteins having one or more of the above amino
acid substitutions associated with TFL or other anti-florigens.
However, an FT protein may tolerate one or some of these amino acid
substitutions while still maintaining florigenic activity.
[0047] A florigenic FT protein of the present invention may also be
defined as being similar to a known FT protein in addition to
having one or more of the above signature amino acid residues. For
example, a florigenic protein may be defined as having at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, or least 99% identity to a sequence selected from the group
consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20,
or a functional fragment thereof, in addition to one or more of the
following signature residues: a tyrosine or other uncharged polar
or nonpolar residue (e.g., alanine, tryptophan, methionine,
leucine, threonine, cysteine, serine, or asparagine) at the amino
acid position corresponding to position 85 of the At.FT protein
sequence; a leucine or other nonpolar or hydrophobic residue (e.g.,
isoleucine, valine, or methionine) at the amino acid position
corresponding to position 128 of the At.FT protein sequence; and/or
a tryptophan or other large nonpolar or hydrophobic residue (e.g.,
methionine or phenylalanine) at the amino acid position
corresponding to position 138 of the At.FT protein sequence. Such a
florigenic FT protein may be further defined as having additional
signature amino acid residue(s), such as one or more of the
following: a glycine or other small nonpolar and uncharged residue
(e.g., alanine, valine, leucine, isoleucine, or methionine) at the
amino acid position corresponding to position 129 of the At.FT
protein sequence; and/or a threonine at the amino acid position
corresponding to position 132 of the At.FT protein sequence.
[0048] A florigenic FT protein of the present invention may also be
defined as having at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, or least 99% identity to a
sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6,
8, 10, 12, 14, 16, 18, and 20, or a functional fragment thereof,
but not having (i.e., excluding) one or more non-florigenic or
anti-florigenic residues, such as one or more of the following: a
histidine at the amino acid position corresponding to position 85
of the At.FT protein sequence; a lysine or arginine at the amino
acid position corresponding to position 128 of the At.FT protein
sequence; and/or a serine, aspartic acid, glutamic acid, lysine or
arginine at the amino acid position corresponding to position 138
of the At.FT protein sequence. Such a florigenic FT protein may be
further defined as not having (i.e., excluding) one or more
additional residues, such as one or more of the following: a
glutamine or asparagine at the amino acid position corresponding to
position 129 of the At.FT protein sequence; and/or a valine or
cysteine at the amino acid position corresponding to position 132
of the At.FT protein sequence.
[0049] Flowering Locus T (FT) genes play a key role in higher
plants and function to integrate floral pathways. FT proteins have
been shown to function as a mobile signal or florigen transported
from leaves to the shoot apical apex where it triggers initiation
of reproductive development in diverse species. See, e.g., Jaeger,
K. E. et al., "Interlocking feedback loops govern the dynamic
behavior of the floral transition in Arabidopsis," The Plant Cell,
25:820-833 (2013); Corbesier, L et al., "FT protein movement
contributes to long distance signaling in floral induction of
Arabidopsis," Science 316: 1030-1033 (2007); Jaeger, K E et al.,
"FT protein acts as a long range signal in Arabidopsis," Curr Biol
17: 1050-1054 (2007); and Amasino, R. M. et al., "The Timing of
Flowering," Plant Physiology, 154(2):516-520 (2010), the entire
contents and disclosures of which are incorporated herein by
reference. In Arabidopsis, FT protein binds to 14-3-3 and Flowering
Locus D (FD) proteins in the meristem to form a flowering complex
triggering activation of key floral meristem identity genes, such
as APETATALI (API) and SOCl at the shoot apex. See, e.g., Taoka, K.
et al., "14-3-3 protein act as intracellular receptors for rice
Hd3a florigen." Nature 476:332-335 (2011). The TERMINAL FLOWER 1
(TFL1) gene is a key repressor of FT targets that maintains the
center of the shoot apical meristem (SAM) in a vegetative state.
TFL1 acts by repressing the LEAFY (LFY) and API genes. Thus, the
relative concentrations of FT and TFL1 in the target tissues act
competitively to control the timing of the reproductive transition
of meristems from a vegetative state that may terminate further
vegetative growth. . See, e.g., Abe, M et al., Science
309:1052-1055 (2005); and McGarry, RC et al., Plant Science
188/189: 71-81 (2012).
[0050] FT genes have been identified from many diverse species, and
ectopic FT expression has been reported to induce early flowering.
See, e.g., Kong, F. et al., "Two Coordinately Regulated Homologs of
Flowering Locus T Are Involved in the Control of Photoperiodic
Flowering in Soybean," Plant Physiology 154: 1220-1231 (2010);
Turck, F. et al., "Regulation and identity of florigen: Flowering
Locus T moves center stage," Ann Rev Plant Biol 59: 573-594 (2008);
Blackman, B K et al., "The role of recently derived FT paralogs in
sunflower domestication," Curr Biol 20: 629-635 (2010); Lifschitz,
E. et al., "The tomato FT orthologs triggers systemic signals that
regulate growth and flowering and substitute for diverse
environmental stimuli," PNAS 103: 6398-6403 (2006); Trankner, C. et
al., "Over-expression of an FT-homologous gene of apple induces
early flowering in annual and perennial plants," Planta 232:
1309-1324 (2010); and Xiang, L. et al., "Functional analysis of
Flowering Locus T orthologs from spring orchid (Cymbidium goeringii
Rchb. f.) that regulates the vegetative to reproductive
transition," Plant Cell & Biochem 58: 98-105 (2012), the entire
contents and disclosures of which are incorporated herein by
reference. However, prior studies with expression of FT transgenes
used constitutive or tissue specific promoters that produced either
very severe phenotypes, non-cell autonomous (systemic) phenotypes,
or autonomous leaf specific phenotypes with plants or seedlings
flowering earlier than controls and terminating at early stages of
development. Given these findings, ectopic FT expression was
generally not seen as a viable approach to increasing yield in
plants by inducing flowers or altering flowering time.
[0051] According to embodiments of the present invention, a
recombinant DNA molecule or polynucleotide is provided comprising a
polynucleotide coding sequence encoding a FT protein that is
operably linked to one or more promoter(s) and/or other regulatory
element(s) that are operable in a plant cell to control or bias the
timing and/or location of FT expression when transformed into a
plant. As commonly understood in the art, the term "promoter" may
generally refer to a DNA sequence that contains an RNA polymerase
binding site, transcription start site, and/or TATA box and assists
or promotes the transcription and expression of an associated
transcribable polynucleotide sequence and/or gene (or transgene). A
promoter may be synthetically produced, varied or derived from a
known or naturally occurring promoter sequence or other promoter
sequence (e.g., as provided herein). A promoter may also include a
chimeric promoter comprising a combination of two or more
heterologous sequences. A promoter of the present invention may
thus include variants of promoter sequences that are similar in
composition, but not identical to, other promoter sequence(s) known
or provided herein. As used herein, the term "operably linked"
refers to a functional linkage between a promoter or other
regulatory element and an associated transcribable polynucleotide
sequence or coding sequence of a gene (or transgene), such that the
promoter, etc., operates to initiate, assist, affect, cause, and/or
promote the transcription and expression of the associated coding
or transcribable sequence, at least in particular tissue(s),
developmental stage(s), and/or under certain condition(s).
[0052] A promoter may be classified according to a variety of
criteria relating to the pattern of expression of a coding sequence
or gene (including a transgene) operably linked to the promoter,
such as constitutive, developmental, tissue-specific, inducible,
etc. Promoters that initiate transcription in all or most tissues
of the plant are referred to as "constitutive" promoters. Promoters
that initiate transcription during certain periods or stages of
development are referred to as "developmental" promoters. Promoters
whose expression is enhanced in certain tissues of the plant
relative to other plant tissues are referred to as
"tissue-enhanced" or "tissue-preferred" promoters. Thus, a
"tissue-preferred" promoter causes relatively higher or
preferential expression in a specific tissue(s) of the plant, but
with lower levels of expression in other tissue(s) of the plant.
Promoters that express within a specific tissue(s) of the plant,
with little or no expression in other plant tissues, are referred
to as "tissue-specific" promoters. A promoter that expresses in a
certain cell type of the plant is referred to as a "cell type
specific" promoter. An "inducible" promoter is a promoter that
initiates transcription in response to an environmental stimulus
such as cold, drought or light, or other stimuli, such as wounding
or chemical application. A promoter may also be classified in terms
of its origin, such as being heterologous, homologous, chimeric,
synthetic, etc. A "heterologous" promoter is a promoter sequence
having a different origin relative to its associated transcribable
sequence, coding sequence, or gene (or transgene), and/or not
naturally occurring in the plant species to be transformed. The
term "heterologous" may refer more broadly to a combination of two
or more DNA molecules or sequences when such a combination is not
normally found in nature.
[0053] According to embodiments of the present invention, a
recombinant DNA molecule or polynucleotide is provided comprising a
florigenic FT transgene or coding sequence operably linked to a
promoter that functions in a plant, which may be introduced or
transformed into a plant to cause the plant to have an altered
flowering, reproductive and/or yield-related trait or phenotype.
Since FT proteins are believed to operate in the meristems of a
plant, including the apical and/or axillary meristems, to trigger
floral transitioning of those meristems and induce flowering,
embodiments of the present invention provide a recombinant DNA
molecule comprising a FT transgene or coding sequence operably
linked to a "vegetative stage" promoter to cause, when introduced
or transformed into a plant, expression of the FT transgene earlier
in the development of the plant (i.e., during the vegetative growth
phase of the plant) to produce an increased level of FT in target
tissues than would otherwise occur in a wild type plant at the same
stage of development. Timing FT transgene expression during the
vegetative stage(s) of development may be important for affecting
one or more reproductive, flowering and/or yield-related traits or
phenotypes by providing a timely inductive signal for the
production of an increased number of floral meristems and
successful flowers at one or more node(s) of the plant. Without
being bound by any theory, vegetative stage expression of an FT
transgene in a plant may operate to synchronize and/or increase
early flowering at one or more node(s) to produce more flowers per
node of the plant. The promoters described below as a part of the
present invention provide options for timing FT expression.
[0054] As used herein, a "vegetative stage" promoter includes any
promoter that initiates, causes, drives, etc., transcription or
expression of its associated gene (or transgene) during one or more
vegetative stage(s) of plant development, such as during one or
more of Ve, Vc, V1, V2, V3, V4, etc., and/or any or all later
vegetative stages of development (e.g., up to V.sub.n stage). Such
a "vegetative stage" promoter may be further defined as a
"vegetative stage preferred" promoter that initiates, causes,
drives, etc., transcription or expression of its associated gene
(or transgene) at least preferably or mostly, if not exclusively,
during one or more vegetative stage(s) of plant development (as
opposed to reproductive stages). However, a "vegetative stage" and
a "vegetative stage preferred" promoter may each also permit,
allow, cause, etc., transcription or expression of its associated
gene (or transgene) during reproductive phase(s) of development in
one or more cells or tissues of the plant. The features and
characteristics of these vegetative stages for a given plant
species are known in the art. For dicot plants, vegetative
morphological features and characteristics of the plant during
vegetative stages of development may include cotyledon form,
vegetative meristems (apical, lateral/axillary, and root), leaf
arrangement, leaf shape, leaf margin, leaf venation, petioles,
stipules, ochrea, hypocotyl, and roots. According to embodiments of
the present invention, a "vegetative stage" promoter may also be
further defined by the particular vegetative stage during which
observable or pronounced transcription or expression of its
associated gene (or transgene) is first initiated. For example, a
vegetative stage promoter may be a Vc stage promoter, a V1 stage
promoter, a V2 stage promoter, a V3 stage promoter, etc. As such, a
"Vc stage" promoter is defined as a vegetative stage promoter that
first initiates transcription of its associated gene (or transgene)
during the Vc stage of plant development, a "V1 stage" promoter is
defined as a vegetative stage promoter that first initiates
transcription of its associated gene (or transgene) during the V1
stage of plant development, a "V2 stage" promoter is defined as a
vegetative stage promoter that first initiates transcription of its
associated gene (or transgene) during the V2 stage of plant
development, and so on, although expression of the associated gene
(or transgene) may be present continuously or discontinuously in
one or more tissues during later vegetative stage(s) of
development. One skilled in the art would be able to determine the
timing of expression of a given gene (or transgene) during plant
development using various molecular assays and techniques known in
the art.
[0055] According to embodiments of the present invention, a
"vegetative stage" promoter may include a constitutive,
tissue-preferred, or tissue-specific promoter. For example, a
vegetative stage promoter may drive FT expression in one or more
plant tissue(s), such as in one or more of the root(s), stem(s),
leaf/leaves, meristem(s), etc., during a vegetative stage(s) of
plant development. However, such a vegetative stage promoter may
preferably drive expression of its associated FT transgene or
coding sequence in one or more meristem(s) of the plant. According
to many embodiments, a "vegetative stage" promoter may preferably
be a "meristem-specific" or "meristem-preferred" promoter to cause
expression of the FT transgene or coding sequence in meristematic
tissue. FT proteins are known to operate in the meristems of a
plant to help trigger the transition from vegetative to
reproductive growth after translocation of the FT protein from the
leaves. In contrast, embodiments of the present invention provide
selective expression of an FT transgene directly in the meristem of
a plant to induce flowering and cause the plant to adopt an altered
reproductive and/or yield-related trait or phenotype. Thus,
according to embodiments of the present invention, a recombinant
DNA molecule is provided comprising an FT transgene or coding
sequence operably linked to a "meristem-specific" or
"meristem-preferred" promoter that drives expression of the FT
transgene at least preferentially in one or more meristematic
tissues of a plant when transformed into the plant. As used herein,
"meristem-preferred promoter" refers to promoters that
preferentially cause expression of an associated gene or transgene
in at least one meristematic tissue of a plant relative to other
plant tissues, whereas a "meristem-specific promoter" refers to
promoters that cause expression of an associated gene or transgene
exclusively (or almost exclusively) in at least one meristem of a
plant.
[0056] Recent work using artificial early "short day" light
treatments during vegetative stages of development revealed that
flowering time could be altered in a way that alters one or more
yield-related traits or phenotypes (e.g., by causing an increased
number of pods or seeds per node on a plant) and that the effect of
these treatments was dosage-dependent with the number of flowers,
seeds and/or pods per plant (and/or per node of the plant)
depending on (i) the duration of the short day exposure (i.e.,
floral induction signal dosage) and (ii) the length of the
post-short day photoperiods under long day conditions (i.e., the
dosage or length of the vegetative growth inducing signal after the
short day induction signal). See, e.g., U.S. Pat. No. 8,935,880 and
U.S. Patent Application Publication No. 2014/0259905, introduced
above. Soybean plants experiencing a lower or less prolonged early
short day induction (eSDI) treatment (prior to returning to long
day growing conditions) had more flowers, pods and seeds per plant
with more normal plant height and maturity, whereas soybean plants
exposed to a greater or more prolonged eSDI treatment produced
shorter, earlier-terminating plants with fewer pods and seeds per
plant (albeit perhaps with an increased number of pods and/or seeds
per node).
[0057] Without being bound by any theory, it is proposed that an
early florigenic signal (e.g., short days for soybean and other SD
plants) triggers an early vegetative to reproductive transition of
plants and even termination of a subset of its primary meristems.
However, by returning those plants to non-inductive growth
conditions (e.g., long days for SD plants) after the initial SD
signal, the remaining meristematic reserves of the plant may be
preserved and reproductive and/or flowering duration may be
extended or maintained, thus allowing for the successful
development of a greater number of productive flowers, pods and/or
seeds per node (and/or per plant) during the reproductive phase.
With early floral induction, a greater overlap may also be created
between reproductive development and vegetative growth of the
plant, which may further promote or coincide with an extended
reproductive and/or flowering duration. For purposes of the present
invention, "reproductive duration" refers to the length of time
from the initiation of flowering until the end of seed/pod
development and/or filling, whereas "flowering duration" or
"duration of flowering" refers the length of time from the
appearance of the first open flower until the last open flower
closes. By returning to non-inductive growth conditions after early
floral induction, more abundant resources may be available and
directed toward the production of an increased number of successful
(i.e., non-aborting) flowers, pods and/or seeds per plant, unlike
normal floral development in short day plants, which may coincide
with declining plant resources due to termination of meristematic
growth and maturation of the plant.
[0058] However, in addition to early flowering, a floral induction
signal (e.g., early short day conditions) also causes early
termination of the plant. Therefore, it is proposed that an optimal
dosage and timing of the floral induction signal may be needed to
maximize yield by balancing (i) the early vegetative to
reproductive transition and/or synchronization of flowering with
the early floral induction signal (leading to potential yield gains
at each node of the plant) against (ii) earlier growth termination
(leading to smaller plants with fewer internodes, less branching,
and fewer nodes and/or flowers per plant). It is believed that
lower dosages of a floral induction signal may be sufficient to
induce flowering while lessening or minimizing earlier termination
of the plant, such that larger plants are produced with increased
numbers of flowers, pods and/or seeds per node (and/or per plant).
On the other hand, higher dosages of a floral induction signal may
cause early termination of the plant (in addition to early
flowering) to produce smaller plants with relatively fewer numbers
of flowers, pods and/or seeds per plant due to the smaller plant
size with fewer internodes and/or branches per plant, despite
having perhaps a greater number of flowers, pods and/or seeds per
node (and/or per plant) relative to wild-type or control plants
under normal growth conditions. As stated above, these effects of
ectopic FT expression may also include an increased number of
bolls, siliques, fruits, nuts, tubers, etc., per node (and/or per
plant), depending on the particular plant species.
[0059] As mentioned above, the short day induction phenotype in
soybean was used to screen for genes having altered expression in
those plants through transcriptional profiling. These studies led
to the identification of an endogenous FT gene, Gm.FT2a, having
increased expression in response to the short day induction
treatment. Accordingly, it is presently proposed that expression of
a florigenic FT transgene can be used as a floral induction signal
to cause early flowering and increased flowers, pods and/or seeds
per node (and/or per plant) relative to a wild type or control
plant not having the FT transgene. According to embodiments of the
present invention, appropriate control of the timing, location and
dosage of florigenic FT expression during vegetative stages of
development can be used to induce flowering and produce plants
having increased flowers, pods and/or seeds per node relative to a
wild type or control plant not having the FT transgene. Rather than
attempting to transiently express FT and recapitulate the timing of
the eSDI treatments, it is proposed that FT could be weakly
expressed in the vegetative meristem to provide the early floral
induction signal. Accordingly, a promoter from the Erecta gene
(pErecta or pEr) having weak meristematic expression during
vegetative stages of development was selected for initial testing
with a Gm.FT2a transgene. However, given that prior studies showed
that constitutive FT expression produced plants having a severe,
early termination phenotype, and further that the site of action
for FT produced peripherally and translocated from the leaves is in
the meristem, it was possible that direct meristematic expression
of FT could produce even more potent and severe phenotypes (and/or
non-viable plants) relative to constitutive FT expression.
[0060] The effects of Gm.FT2a overexpression were immediately seen
in R.sub.0 transformed soybean plants, which had early flowering,
reduced seed yield (e.g., only about 8 seeds/plant), and very early
termination, suggesting that the balance between floral induction
and floral repression/vegetative growth was strongly in favor of
flowering and early termination. However, enough R1 seed was
salvaged from these plants to allow for additional experiments to
be performed. It was proposed that growing the Ri soybean seed
under long day (floral repressive) photoperiod conditions in the
greenhouse might delay the early flowering and termination
phenotypes observed in the Ro plants. Given the theorized dosage
response, it was further proposed that segregating FT2a homozygous,
hemizygous and null soybean plants could be tested together in the
greenhouse to evaluate the dosage response resulting from FT
overexpression. In these experiments (as described further below),
it was observed that segregating plants did have different
phenotypes: null plants were similar to wild-type plants in terms
of plant architecture and pods per node (and per plant), while
homozygous plants terminated early with a severe dwarf phenotype
(although possibly with an increased number of pods per node).
However, hemizygous plants were almost as large as null or
wild-type plants but exhibited the hyper-flowering phenotype with
an increased number of pods per node (and/or per plant). These
findings show that vegetative stage and/or meristematic expression
of a florigenic FT transgene may be used to produce a high yielding
plant (similar to the eSDI treatment), and that the effect of FT
expression is dosage-dependent since soybean plants hemizygous for
the FT2a transgene under the control of a weak meristematic
promoter displayed the high yield phenotype of increased pods per
node without the more severe early termination and short plant
height phenotypes observed with homozygous FT2a plants when grown
under long day (vegetative) conditions.
[0061] Interestingly, however, increased numbers of pods per node
was often observed independently of reproductive (R1-R7) duration
under greenhouse conditions and was not perfectly correlated with
early flowering among the FT transgenes tested, although it is
possible that the duration of flowering may still be prolonged in
some cases (at least during one or more reproductive stages) even
if the total reproductive duration is not significantly changed
relative to control plants. Without being bound by any theory, it
is believed that increased numbers of pods per node in transgenic
FT plants may result at least in part from an increase in the
number of inflorescence and floral meristems induced from
vegetative shoot apical and axillary meristems at each of the
affected node(s), which may give rise to a greater number of
flowers and/or released floral racemes at those node(s). Such an
increase in the number of floral meristems induced at each node of
the plant in response to FT overexpression may operate through one
or more mechanisms or pathways, which may be independent of
flowering time and/or reproductive duration. However, meristematic
changes may be microscopic at first, and thus not observed to cause
"early flowering" at such stage by simple visual inspection even
though reproductive changes to the meristem may have already begun
to occur.
[0062] Without being bound by any theory, early vegetative FT
expression may cause more reproductive meristems to form and
develop earlier than normal at one or more node(s) of the
transgenic plant. These reproductive meristems may then allow or
cause a greater number of floral racemes to form and elongate with
flowers at each node. On the other hand, it is further theorized
that later expression of FT during reproductive stages may function
to repress further floral development at each node. Thus, later
developing flowers within the respective raceme may become
terminated, and thus more of the plant's resources may be directed
to the earlier developing flowers within the raceme to more
effectively produce full-sized pods. Accordingly, it is
contemplated that by (i) forming a greater number of inflorescence
and floral meristems at each node by early FT induction, and then
(ii) directing more plant resources to the earlier, more developed
flowers within each of the racemes by termination of the
later-developing flowers, increased synchronization of floral
development may occur with a greater number of mature pods being
formed per node of the plant.
[0063] According to embodiments of the present invention, a
recombinant DNA molecule is provided comprising an FT coding
sequence operatively linked to a vegetative stage promoter, which
may also be a meristem-preferred and/or meristem-specific promoter.
For example, the promoter may include the pAt.Erecta promoter from
Arabidopsis (SEQ ID NO: 21), or a functional fragment or portion
thereof. Two examples of a truncated portion of the pAt.Erecta
promoter according to embodiments of the present invention are
provided as SEQ ID NO: 22 and SEQ ID NO: 38. See, e.g., Yokoyama,
R. et al., "The Arabidopsis ERECTA gene is expressed in the shoot
apical meristem and organ primordia," The Plant Journal 15(3):
301-310 (1998). pAt.Erecta is an example of a vegetative stage
promoter that is also meristem-preferred. Other vegetative stage,
meristem-preferred or meristem-specific promoters have been
identified based on their characterized expression profile (see,
e.g., Examples 4 and 7 below) that may also be used to drive FT
expression according to embodiments of the present invention. For
example, the following soybean receptor like kinase (RLK) genes
were identified that could be used as vegetative stage,
meristem-preferred promoters: Glyma10g38730 (SEQ ID NO: 23),
Glyma09g27950 (SEQ ID NO: 24), Glyma06g05900 (SEQ ID NO: 25), and
Glyma17g34380 (SEQ ID NO: 26). Vegetative stage, meristem-preferred
promoters according to embodiments of the present invention may
also include receptor like kinase (RLK) gene promoters from potato:
PGSC0003DMP400032802 (SEQ ID NO: 27) and PGSC0003DMP400054040 (SEQ
ID NO: 28). Given the characterization provided herein of the
pAt.Erecta promoter driving FT expression and the similar
expression profiles identified for other RLK, Erecta or Erecta-like
(Er1) genes, vegetative-stage, meristem-preferred or
meristem-specific promoters of the present invention may further
comprise any known or later identified promoter sequences of RLK,
Erecta and Erecta-like genes from other dicotyledonous species
having vegetative-stage pattern of expression in the meristems of
plants.
[0064] Additional examples of vegetative stage, meristem-preferred
or meristem-specific promoters may include those from the following
Arabidopsis genes: Pinhead (At.PNH) (SEQ ID NO: 29), Angustifolia 3
or At.AN3 (SEQ ID NO: 30), At.MYBI7 (At.LMI2 or Late Meristem
Identity 2; At3g61250) (SEQ ID NO: 31), Kinesin-like gene
(At5g55520) (SEQ ID NO: 32), AP2/B3-like genes, including ALREM17
(SEQ ID NO: 33) or ALREA119, and Erecta-like 1 and 2 genes, At.Erl1
(SEQ ID NO: 34) and At.Erl2 (SEQ ID NO: 35), and functional
portions thereof. The polynucleotide sequence of these promoters
(or a functional fragment thereof) may also have a relaxed sequence
identity while still maintaining a similar or identical pattern of
expression of an associated gene or transgene operably linked to
the promoter. For example, the promoter may comprise a
polynucleotide sequence that is at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or least 99% identical to
a polynucleotide sequence selected from the above SEQ ID NOs: 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35, or a
functional portion thereof. A "functional portion" of a known or
provided promoter sequence is defined as one or more continuous or
discontinuous portion(s) of the known or provided promoter sequence
that may functionally drive, cause, promote, etc., expression of
its associated gene (or transgene) in a manner that is identical or
similar to the known or provided promoter sequence. Based on the
present disclosure, one skilled in the art would be able to
determine if a promoter comprising one or more portion(s) of a
known or provided promoter sequence, and/or having a shorter
sequence and/or more relaxed sequence identity relative to a known
or provided promoter sequence, causes a similar pattern of
expression and/or similar phenotypes or effects when its associated
FT transgene is expressed in a plant as compared to the known or
provided promoter sequence.
[0065] As stated above, a recombinant DNA molecule of the present
invention may generally comprise an FT transgene or expression
cassette including a polynucleotide sequence encoding an FT protein
that is operatively linked to a vegetative stage promoter, which
may also be a meristem-preferred or meristem-specific promoter. The
polynucleotide coding sequence of the FT transgene or expression
cassette may also be operatively linked to one or more additional
regulatory element(s), such as an enhancer(s), leader,
transcription start site (TSS), linker, 5' and 3' untranslated
region(s), intron(s), polyadenylation signal, termination region or
sequence, etc., that are suitable or necessary for regulating or
allowing expression of the FT transgene or cassette to effectively
produce an FT protein in a plant cell. Such additional regulatory
element(s) may be optional and used to enhance or optimize
expression of the transgene. For purposes of the present invention,
an "enhancer" may be distinguished from a "promoter" in that an
enhancer typically lacks a transcription start site, TATA box, or
equivalent sequence and is thus insufficient alone to drive
transcription. As used herein, a "leader" may be defined generally
as the DNA sequence of the untranslated 5' region (5' UTR) of a
gene (or transgene) between the transcription start site (TSS) and
the protein coding sequence start site.
[0066] According to embodiments of the present invention, the term
"recombinant" in reference to a DNA molecule, construct, vector,
etc., refers to a DNA molecule or sequence that is not found in
nature and/or is present in a context in which it is not found in
nature, including a DNA molecule, construct, etc., comprising a
combination of DNA sequences that would not naturally occur
contiguously or in close proximity together without human
intervention, and/or a DNA molecule, construct, etc., comprising at
least two DNA sequences that are heterologous with respect to each
other. A recombinant DNA molecule, construct, etc., may comprise
DNA sequence(s) that is/are separated from other polynucleotide
sequence(s) that exist in proximity to such DNA sequence(s) in
nature, and/or a DNA sequence that is adjacent to (or contiguous
with) other polynucleotide sequence(s) that are not naturally in
proximity with each other. A recombinant DNA molecule, construct,
etc., may also refer to a DNA molecule or sequence that has been
genetically engineered and constructed outside of a cell. For
example, a recombinant DNA molecule may comprise any suitable
plasmid, vector, etc., and may include a linear or circular DNA
molecule. Such plasmids, vectors, etc., may contain various
maintenance elements including a prokaryotic origin of replication
and selectable marker, as well as a FT expressing transgene or
expression cassette perhaps in addition to a plant selectable
marker gene, etc.
[0067] As introduced above, the florigenic effects of FT expression
may be opposed by the activity of various other anti-florigenic (or
non-florigenic) proteins, such as Terminal Flower (TFL) genes, in
the meristem of a plant. Flowering time and duration may thus be
seen as a balance between florigenic and anti-florigenic signals
present within the meristem(s) of a plant. Accordingly, it is
further proposed that flowering time in a plant may be altered or
induced by suppression of one or more anti-florigenic genes during
vegetative stages of development to render the vegetative meristem
more responsive to a florigenic signal. In soybean, for example,
genes related to Arabidopsis TFL1 are primarily expressed in floral
meristems where they oppose the functions of florigenic signals to
regulate developmental decisions like stem growth habit. In
particular, the TFL1-like genes, TFL1a and TFL1b, in soybean are
expressed in the shoot apical meristem, and allelic diversity in
TFL1b may be largely responsible for changes in stem growth habit
in soy resulting in determinate or indeterminate growth. See, e.g.,
Liu, B. et al., "The soybean stem growth habit gene Dt1 is an
ortholog of Arabidopsis TERMINAL FLOWER 1." Plant Physiol
153(1):198-210 (2010). Accordingly, suppressing the expression of
TFL or another anti-florigenic protein in the meristem of a dicot
plant may result in increased sensitivity of those meristematic
tissues to florigenic signals, such as FT, resulting in early
flowering and increased pods per node, similarly to overexpression
of FT in these tissues.
[0068] Thus, it is contemplated that a recombinant DNA molecule of
the present invention may further comprise a suppression construct
having a transcribable DNA sequence operatively linked to a
vegetative stage promoter, which may also be a meristem-preferred
or meristem-specific promoter, wherein the transcribable DNA
sequence encodes an RNA molecule that causes targeted suppression
of an endogenous anti-florigenic gene, such as a TFL gene. Various
methods for suppressing the expression of an endogenous gene are
known in the art.
[0069] According to another broad aspect of the present invention,
methods are provided for transforming a plant cell, tissue or
explant with a recombinant DNA molecule or construct comprising an
FT transgene or expression cassette to produce a transgenic plant.
Numerous methods for transforming chromosomes in a plant cell with
a recombinant DNA molecule are known in the art, which may be used
according to methods of the present invention to produce a
transgenic plant cell and plant. Any suitable method or technique
for transformation of a plant cell known in the art may be used
according to present methods. Effective methods for transformation
of plants include bacterially mediated transformation, such as
Agrobacterium-mediated or Rhizhobium-mediated transformation, and
microprojectile bombardment-mediated transformation. A variety of
methods are known in the art for transforming explants with a
transformation vector via bacterially mediated transformation or
microprojectile bombardment and then subsequently culturing, etc,
those explants to regenerate or develop transgenic plants. Other
methods for plant transformation, such as microinjection,
electroporation, vacuum infiltration, pressure, sonication, silicon
carbide fiber agitation, PEG-mediated transformation, etc., are
also known in the art. Transgenic plants produced by these
transformation methods may be chimeric or non-chimeric for the
transformation event depending on the methods and explants used.
Methods are further provided for expressing an FT transgene in one
or more plant cells or tissues under the control of a
vegetative-stage promoter, which may also be a meristem-preferred
or meristem-specific promoter. Such methods may be used to alter
flowering time of a plant and/or the number of productive or
successful flowers, fruits, pods, and/or seeds per node of the
plant relative to a wild type or control plant not having the FT
transgene. Indeed, methods of the present invention may be used to
alter reproductive or yield-related phenotype(s) or trait(s) of the
transgenic plant.
[0070] Transformation of a target plant material or explant may be
practiced in tissue culture on nutrient media, for example a
mixture of nutrients that allow cells to grow in vitro. Recipient
cell targets or explants may include, but are not limited to,
meristems, shoot tips, protoplasts, hypocotyls, calli, immature or
mature embryos, shoots, buds, nodal sections, leaves, gametic cells
such as microspores, pollen, sperm and egg cells, etc., or any
suitable portions thereof. It is contemplated that any
transformable cell or tissue from which a fertile plant can be
regenerated or grown/developed may be used as a target for
transformation. Transformed explants, cells or tissues may be
subjected to additional culturing steps, such as callus induction,
selection, regeneration, etc., as known in the art. Transformed
cells, tissues or explants containing a recombinant DNA insertion
may be grown, developed or regenerated into transgenic plants in
culture, plugs or soil according to methods known in the art.
Transgenic plants may be further crossed to themselves or other
plants to produce transgenic seeds and progeny. A transgenic plant
may also be prepared by crossing a first plant comprising the
recombinant DNA sequence or transformation event with a second
plant lacking the insertion. For example, a recombinant DNA
sequence may be introduced into a first plant line that is amenable
to transformation, which may then be crossed with a second plant
line to introgress the recombinant DNA sequence into the second
plant line. Progeny of these crosses can be further back crossed
into the more desirable line multiple times, such as through 6 to 8
generations or back crosses, to produce a progeny plant with
substantially the same genotype as the original parental line but
for the introduction of the recombinant DNA sequence.
[0071] A recombinant DNA molecule or construct of the present
invention may be included within a DNA transformation vector for
use in transformation of a target plant cell, tissue or explant.
Such a transformation vector of the present invention may generally
comprise sequences or elements necessary or beneficial for
effective transformation in addition to the FT expressing transgene
or expression cassette. For Agrobacterium-mediated transformation,
the transformation vector may comprise an engineered transfer DNA
(or T-DNA) segment or region having two border sequences, a left
border (LB) and a right border (RB), flanking at least the FT
expressing transgene or expression cassette, such that insertion of
the T-DNA into the plant genome will create a transformation event
for the FT transgene or expression cassette. In other words, the FT
transgene or expression cassette would be located between the left
and right borders of the T-DNA, perhaps along with an additional
transgene(s) or expression cassette(s), such as a plant selectable
marker transgene and/or other gene(s) of agronomic interest that
may confer a trait or phenotype of agronomic interest to a plant.
In addition to protein encoding sequences, a gene of agronomic
interest may further comprise a polynucleotide sequence encoding a
RNA suppression element. According to alternative embodiments, the
FT transgene or expression cassette and the plant selectable marker
transgene (or other gene of agronomic interest) may be present in
separate T-DNA segments on the same or different recombinant DNA
molecule(s), such as for co-transformation. A transformation vector
or construct may further comprise prokaryotic maintenance elements,
which for Agrobacterium-mediated transformation may be located in
the vector backbone outside of the T-DNA region(s).
[0072] A plant selectable marker transgene in a transformation
vector or construct of the present invention may be used to assist
in the selection of transformed cells or tissue due to the presence
of a selection agent, such as an antibiotic or herbicide, wherein
the plant selectable marker transgene provides tolerance or
resistance to the selection agent. Thus, the selection agent may
bias or favor the survival, development, growth, proliferation,
etc., of transformed cells expressing the plant selectable marker
gene, such as to increase the proportion of transformed cells or
tissues in the Ro plant. Commonly used plant selectable marker
genes include, for example, those conferring tolerance or
resistance to antibiotics, such as kanamycin and paromomycin
(nptll), hygromycin B (aph IV), streptomycin or spectinomycin
(aadA) and gentamycin (aac3 and aacC4), or those conferring
tolerance or resistance to herbicides such as glufosinate (bar or
pat), dicamba (DMO) and glyphosate (aroA or EPSPS). Plant
screenable marker genes may also be used, which provide an ability
to visually screen for transformants, such as luciferase or green
fluorescent protein (GFP), or a gene expressing a beta
glucuronidase or uidA gene (GUS) for which various chromogenic
substrates are known.
[0073] According to embodiments of the present invention, methods
for transforming a plant cell, tissue or explant with a recombinant
DNA molecule or construct may further include site-directed or
targeted integration. According to these methods, a portion of a
recombinant DNA donor template molecule (i.e., an insertion
sequence) may be inserted or integrated at a desired site or locus
within the plant genome. The insertion sequence of the donor
template may comprise a transgene or construct, such as an FT
transgene or construct comprising a polynucleotide sequence
encoding a florigenic FT protein operatively linked to a
vegetative-stage promoter, which may also be a meristem-preferred
or meristem-specific promoter. The donor template may also have one
or two homology arms flanking the insertion sequence to promote the
targeted insertion event through homologous recombination and/or
homology-directed repair. Thus, a recombinant DNA molecule of the
present invention may further include a donor template for
site-directed or targeted integration of a transgene or construct,
such as an FT transgene or construct, into the genome of a
plant.
[0074] Any site or locus within the genome of a plant may
potentially be chosen for site-directed integration of a transgene
or construct of the present invention. For site-directed
integration, a double-strand break (DSB) or nick may first be made
at a selected genomic locus with a site-specific nuclease, such as,
for example, a zinc-finger nuclease, an engineered or native
meganuclease, a TALE-endonuclease, or an RNA-guided endonuclease
(e.g., Cas9 or Cpf1). Any method known in the art for site-directed
integration may be used. In the presence of a donor template
molecule, the DSB or nick may then be repaired by homologous
recombination between the homology arm(s) of the donor template and
the plant genome, or by non-homologous end joining (NHEJ),
resulting in site-directed integration of the insertion sequence
into the plant genome to create the targeted insertion event at the
site of the DSB or nick. Thus, site-specific insertion or
integration of a transgene or construct may be achieved.
[0075] According to embodiments of the present invention, a plant
that may be transformed with a recombinant DNA molecule or
transformation vector comprising an FT transgene may include a
variety of flowering plants or angiosperms, which may be further
defined as including various dicotyledonous (dicot) plant species,
such as soybean, cotton, alfalfa, canola, sugar beets, alfalfa and
other leguminous plants. A dicot plant could be a member of the
Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly
those Brassica species useful as sources of seed oil, alfalfa
(Medicago sativa), sunflower (Helianthus annuus), safflower
(Carthamus tinctorius), oil palm (Elaeis spp.), sesame (Sesamum
spp.), coconut (Cocos spp.), soybean (Glycine max), tobacco
(Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis
hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet
potato (Ipomoea batatus), cassava (Manihot esculenta), coffee
(Coffea spp.), tea (Camellia spp.), fruit trees, such as apple
(Mahis spp.), Prunus spp., such as plum, apricot, peach, cherry,
etc., pear (Pyrus spp.), fig (Ficus casica), banana (Musa spp.),
etc., citrus trees (Citrus spp.), cocoa (Theobroma cacao), avocado
(Persea americana), olive (Olea europaea), almond (Prunus
amygdalus), walnut (Juglans spp.), strawberry (Fragaria spp.),
watermelon (Citrullus lanatus), pepper (Capsicum spp.), sugar beet
(Beta vulgaris), grape (Vitis, Muscadinia), tomato (Lycopersicon
esculentum, Solanum lycopersicum), and cucumber (Cucumis sativis).
Leguminous plants include beans and peas. Beans include, for
example, guar, locust bean, fenugreek, soybean, garden beans,
cowpea, mungbean, lima bean, fava bean, lentils, and chickpea.
Given that the present invention may apply to a broad range of
plant species, the present invention further applies to other
botanical structures analogous to pods of leguminous plants, such
as bolls, siliques, fruits, nuts, tubers, etc. According to
embodiments of the present invention and depending on the
particular plant species transformed, a plant ectopically
expressing a florigenic FT sequence may have an altered or greater
number of bolls, siliques, fruits, nuts, tubers, etc., per node(s),
main stem, and/or branch(es) of the plant, and/or an altered or
greater number of bolls, siliques, fruits, nuts, tubers, etc., per
plant, relative to a wild type or control plant not having the FT
transgene.
[0076] According to another broad aspect of the present invention,
a transgenic plant(s), plant cell(s), seed(s), and plant part(s)
are provided comprising a transformation event or insertion into
the genome of at least one plant cell thereof, the transformation
event or insertion comprising a recombinant DNA sequence, construct
or polynucleotide including a Flowering Locus T (FT) transgene or
expression cassette, wherein the FT transgene or expression
cassette further comprises a polynucleotide sequence encoding an FT
protein operably linked to a vegetative stage promoter, which may
also be a meristem-preferred or meristem-specific promoter. The FT
protein encoded by the polynucleotide sequence may be native to the
transgenic plant transformed with the polynucleotide sequence, or
homologous or otherwise similar to a FT protein native to the
transgenic plant (i.e., not native to the transgenic plant). Such a
transgenic plant may be produced by any suitable transformation
method, which may be followed by selection, culturing,
regeneration, development, etc., as desired or needed to produce a
transgenic Ro plant, which may then be selfed or crossed to other
plants to generate R1 seed and subsequent progeny generations and
seed through additional crosses, etc. Similarly, embodiments of the
present invention further include a plant cell, tissue, explant,
etc., comprising one or more transgenic cells having a
transformation event or genomic insertion of a recombinant DNA or
polynucleotide sequence comprising an FT transgene.
[0077] Transgenic plants, plant cells, seeds, and plant parts of
the present invention may be homozygous or hemizygous for a
transgenic event or insertion of an FT transgene or expression
cassette into the genome of at least one plant cell thereof, or may
contain any number of copies of a transgenic event(s) or
insertion(s) comprising an FT transgene or expression cassette. The
dosage or amount of expression of an FT transgene or expression
cassette may be altered by its zygosity and/or number of copies,
which may affect the degree or extent of phenotypic changes in the
transgenic plant, etc. According to some embodiments, a transgenic
plant comprising an FT transgene may be further characterized as
having one or more altered flowering or reproductive phenotypes or
traits, which may include altered yield-related phenotypes or
traits, such as an increase in the number of flowers, pods, etc.,
and/or seeds per plant (and/or per node of the plant) relative to a
wild type or control plant not having the FT transgene. Such a
transgenic plant may be further characterized as having an altered
structure, morphology, and/or architecture due to altered plant
height, branching patterns, number of floral nodes, etc., relative
to a wild type or control plant. Indeed, yield-related phenotypes
or traits altered by FT overexpression in a transgenic plant may
include: flowering time, reproductive duration, flowering duration,
amount or timing of abscission of flowers, pods, siliques, bolls,
fruits, nuts, etc., number of flowers per node, number of racemes
per node, number of branches, number of nodes per plant, number of
nodes on the main stem, number of nodes on branches, number of
pods, bolls, siliques, fruits, nuts, etc., per plant, number of
pods, bolls, siliques, fruits, nuts, etc., per node, number of pods
on the main stem, number of pods, bolls, siliques, fruits, nuts,
etc., on branches, 1000 seed weight, number of seeds per plant,
number of seeds per node, and/or altered plant architecture, as
compared to a wild type or control plant not having the FT
transgene.
[0078] For purposes of the present invention, a "plant" may include
an explant, seedling, plantlet or whole plant at any stage of
regeneration or development. As used herein, a "transgenic plant"
refers to a plant whose genome has been altered by the integration
or insertion of a recombinant DNA molecule, construct or sequence.
A transgenic plant includes an Ro plant developed or regenerated
from an originally transformed plant cell(s) as well as progeny
transgenic plants in later generations or crosses from the Ro
transgenic plant. As used herein, a "plant part" may refer to any
organ or intact tissue of a plant, such as a meristem, shoot
organ/structure (e.g., leaf, stem and tuber), root, flower or
floral organ/structure (e.g., bract, sepal, petal, stamen, carpel,
anther and ovule), seed (e.g., embryo, endosperm, and seed coat),
fruit (e.g., the mature ovary), propagule, or other plant tissues
(e.g., vascular tissue, ground tissue, and the like), or any
portion thereof. Plant parts of the present invention may be
viable, nonviable, regenerable, and/or non-regenerable. A
"propagule" may include any plant part that is capable of growing
into an entire plant. For purposes of the present invention, a
plant cell transformed with an FT transgene according to
embodiments of the present invention may include any plant cell
that is competent for transformation as understood in the art based
on the method of transformation, such as a meristem cell, an
embryonic cell, a callus cell, etc. As used herein, a "transgenic
plant cell" simply refers to any plant cell that is transformed
with a stably-integrated recombinant DNA molecule or sequence. A
transgenic plant cell may include an originally-transformed plant
cell, a transgenic plant cell of a regenerated or developed Ro
plant, or a transgenic plant cell from any progeny plant or
offspring of the transformed Ro plant, including cell(s) of a plant
seed or embryo, or a cultured plant or callus cell, etc.
[0079] Embodiments of the present invention may further include
methods for making or producing transgenic plants having altered
reproductive and/or yield-related traits or phenotypes, such as by
transformation, crossing, etc., wherein the method comprises
introducing a recombinant DNA molecule or sequence comprising an FT
transgene into a plant cell, and then regenerating or developing
the transgenic plant from the transformed plant cell, which may be
performed under selection pressure favoring the transgenic event.
Such methods may comprise transforming a plant cell with a
recombinant DNA molecule or sequence comprising an FT transgene,
and selecting for a plant having one or more altered phenotypes or
traits, such as one or more of the following: flowering time,
reproductive duration, flowering duration, amount or timing of
abscission of flowers, pods, bolls, siliques, fruits, nuts, etc.,
number of flowers per node, number of racemes per node, number of
branches, number of nodes per plant, number of nodes on the main
stem, number of nodes on branches, number of pods, bolls, siliques,
fruits, or nuts per plant, number of pods, bolls, siliques, fruits,
nuts, etc., per node, number of pods, bolls, siliques, fruits,
nuts, etc., on the main stem, number of pods, bolls, siliques,
fruits, nuts, etc., on branches, 1000 seed weight, number of seeds
per plant, number of seeds per node, and altered plant
architecture, as compared to a wild type or control plant not
having the FT transgene. For example, embodiments of the present
invention may comprise methods for producing a transgenic plant
having an increased number of flowers, pods, and/or seeds per plant
(and/or an increased number of flowers, pods, and/or seeds per node
of the plant), wherein the method comprises introducing a
recombinant DNA molecule comprising an FT transgene into a plant
cell, and then regenerating or developing the transgenic plant from
the plant cell.
[0080] According to another broad aspect of the present invention,
methods are provided for planting a transgenic plant(s) of the
present invention at a normal or high density in field. According
to some embodiments, the yield of a crop plant per acre (or per
land area) may be increased by planting a transgenic plant(s) of
the present invention at a higher density in the field. As
described herein, transgenic plants of the present invention
expressing a florigenic FT protein during vegetative stage(s) of
development may exhibit increased pods per node (particularly on
the main stem), but may also have an altered plant architecture
with reduced branching and fewer nodes per branch. Thus, it is
proposed that transgenic plants of the present invention may be
planted at a higher density to increase yield per acre in the
field. For row crops, higher density may be achieved by planting a
greater number of seeds/plants per row length and/or by decreasing
the spacing between rows. According to some embodiments, a
transgenic crop plant of the present invention may be planted at a
density in the field (plants per land/field area) that is at least
5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%,
225%, or 250% higher than a normal planting density for that crop
plant according to standard agronomic practices.
[0081] For soybean, the typical planting density is in a range from
about 100,000 to 150,000 seeds per acre, and the typical row
spacing is in a range from about 26 to about 40 inches, such as 30
inch or 36 inch row spacing. Within a given row, about 6-8 soybean
seeds may typically be planted per foot. In contrast, high density
planting for soybean may include a range of approximately 150,000
to 250,000 seeds per acre, and the row spacing may be within a
range from about 10 inches or less to about 25 inches, such as 10
inch, 15 inch or 20 inch row spacing. For high density planting,
approximately 9-12 soybean seeds per foot may be planted within
each row, perhaps in combination with narrower row spacing.
However, high crop density may be achieved by narrow row spacing
without an increase in planting density within each row.
[0082] For cotton, the typical planting density is in a range from
about 28,000 to 45,000 seeds per acre, and the typical row spacing
is in a range from about 38 to about 40 inches, such as 38 inch or
40 inch row spacing. Within a given row, about 2-3 cotton seeds may
typically be planted per foot. In contrast, high density planting
for soybean may include a range of approximately 48,000 to 60,000
seeds per acre, and the row spacing may be within a range from
about 30 inches or less to about 36 inches. For high density
planting, approximately 3-5 cotton seeds per foot may be planted
within each row, perhaps in combination with narrower row spacing.
However, high crop density for cotton may be achieved by narrow row
spacing without an increase in planting density within each
row.
[0083] For canola, the typical planting density is in a range from
about 360,000 to 550,000 seeds per acre, and the typical row
spacing between openers is in a range from about 6 inches to about
16 inches. Within a given row, about 8-12 canola seeds may
typically be planted per foot. In contrast, high density planting
for soybean may include a range of approximately 450,000 to 680,000
seeds per acre, and the row spacing may be within a range from
about 5 inches or less to about 10 inches. For high density
planting, approximately 10-16 canola seeds per foot may be planted
within each row, perhaps in combination with the narrower row
spacing. However, high crop density for canola may be achieved by
narrow row spacing without an increase in planting density within
each row.
EXAMPLES
Example 1
Soybean Short Day Induction Treatment and Identification of
Flowering Locus T (FT) Genes by Transcriptional Profiling
[0084] Methods for the photoperiodic light treatment (i.e., short
day induction of flowering in plants) are described in U.S. Pat.
No. 8,935,880 and U.S. Patent Application Publication No.
2014/0259905, which are incorporated herein by reference in their
entirety. As described further therein, the early short day
induction treatment produced soybean plants having altered
reproductive traits including an increased number of pods/seeds per
plant. Transcriptional profiling experiments were performed using
gene expression microarrays to determine if particular transcripts
were up-regulated in these light-induced plants to identify genes
that may be responsible for mediating the short day induction
phenotypes. In these experiments, an analysis of transcripts was
conducted on soybean leaf and floral apex tissues collected after
1, 3 and 5 days from plants that received a short day inductive
light treatment (Short day) in comparison to tissues from plants
that did not receive the inductive treatment (Long day).
[0085] As shown in FIG. 2, an increased accumulation of transcripts
was observed for a particular Flowering Locus T gene, Gm.FT2a (SEQ
ID NO: 1), in leaf tissue harvested at 3 and 5 days after the early
short day induction (eSDI) treatment in comparison to samples taken
from either (i) floral apex tissues of the same short day induction
plants, or (ii) leaf tissues and floral apex tissues of soybean
plants that instead received the long day treatment. These data
support the conclusion that Gm.FT2a expression is induced in leaf
tissue of plants experiencing the eSDI treatment, which was not
seen in plants grown under long day conditions. Gm.FT2a expression
was also not observed in the floral apex of eSDI treated plants,
which is consistent with the model of FT protein expression being
induced in peripheral leafy tissues in response to inductive
photoperiod conditions and then migrating to its site of action in
the meristems to induce flowering. However, additional experiments
using a more sensitive RNA sequencing analysis of transcripts did
show some Gm.FT2a induction in the shoot apex and axillary buds in
response to the eSDI treatment (data not shown).
Example 2
Characterization of the pAtErecta Promoter Expression Patterns in
Soybean
[0086] Achieving desirable traits or phenotypes by transgenic
approaches may require control of the temporal and spatial patterns
of ectopic FT gene expression. Soy physiological experiments
identifying Gm.FT2a expression in vegetative tissues following the
short day induction treatment (see FIG. 2) indicated that achieving
yield positive traits may rely on earlier FT expression during the
vegetative stage. On the other hand, even though FT transcripts are
not detected in the vegetative apex, FT protein has been shown to
move long distance from the leaves to the apical tissue where it
triggers a vegetative to reproductive transition. See, e.g.,
Lifschitz, E. et al., (2006), supra; and Corbeiser, L. et al., "FT
Protein Movement Contributes to Long-Distance Signaling in Floral
Induction of Arabidopsis", Science 316: 1030-1033 (2007). Thus, in
light of our own observations, we proposed using a vegetative stage
promoter that is active in the meristem to control ectopic FT
expression in a plant. By expressing the morphogenetic FT signal
directly in the meristem at the desired developmental stage,
multiple endogenous pathways and regulatory feedbacks (e.g.,
control of FT transduction in the leaf and long distance
translocation of the FT signal) may be bypassed or avoided.
Previous experiments with the short day induction treatment
(described above in Example 1) revealed up-regulation of the
Gm.Erecta gene in the meristems of soybean plants. The pErecta
promoter (SEQ ID NO: 21) from Arabidopsis had been shown to have
weak expression in the meristem(s) of plants during vegetative
stages of development. Accordingly, the pAt.Erecta promoter was
selected for initial FT expression experiments.
[0087] Additional experiments were performed to further
characterize the expression patterns of pAt.Erecta fused to a GUS
reporter gene in vegetative and floral meristematic tissues.
Analysis of GUS expression patterns during the development of soy
seedlings indicated that the pAt.Erecta promoter exhibits a
temporal and spatial pattern of expression, preferably in the
meristematic tissues during the vegetative stage of development.
FIGS. 3A to 3O and 4A to 4O and FIGS. 5A to 5F and 6A to 6F include
two sets of images to show the pattern of GUS staining. FIGS. 3A to
3O and 5A to 5F provide black and white images of the stained
tissues, and FIGS. 4A to 4O and 6A to 6F provide black and white
images corresponding to FIGS. 3A to 3O and 5A to 5F, respectively,
but color filtered to show the pattern and intensity of blue GUS
staining. Thus, the GUS staining pattern of expression can be
viewed with these black and white images by comparing the
corresponding images of FIGS. 3A to 3O and 4A to 4O or FIGS. 5A to
5F and 6A to 6F. As shown in FIGS. 3A to 3O and 4A to 4O, GUS
staining was detected in the soy immature uni-foliate blade and
petiole (FIGS. 4A and 4B) at three days after sowing/germination.
pAt.Erecta:GUS expression was also broadly detected in the
trifoliate primordia, shoot apical meristem (SAM) and axillary
meristem sites at this early vegetative stage (FIG. 4C). GUS
activity was not detected in the fully expanded uni-foliate and
trifoliate leaves at ten days after germination or planting (FIGS.
4D and 4E). However, GUS activity was detected at the proximal part
of the immature, unexpanded, but fully developed trifoliate blade,
and at the adaxial side of the petiole (FIG. 4F). Detailed
observation of the developing apical tissue showed that broad
expression was retained in the developing immature leaf primordia,
axillary meristems and shoot apical meristems (FIGS. 4G-I).
[0088] At the early reproductive stage, pAt.Erecta promoter
activity was not detected in the mature blade and was reduced in
the developing leaf primordia. The GUS signal was not detected in
the indeterminate vegetative apex at the shoot apical meristem
(SAM) or in the axillary meristem (AM) once these tissues started
to form inflorescences (FIGS. 4J-4L). In all later stages, any
additional meristematic activity could not be detected in the apex
or in the axillaries or flower primordia. However, GUS expression
continued in the adaxial side of the petiole and proximal part of
the immature leaf blade (FIGS. 4M and 4N), but not in the fully
expanded leaf blade (FIG. 4O). GUS expression patterns with the
pAt.Erecta promoter were also analyzed at the later R1 stages of
development (35-40 days after germination). Similar to earlier
stages of development, no expression was detected in the mature
leaves or stems. However, strong promoter activity was detected in
the inflorescence stems (FIGS. 5A and 6A; see arrow) and floral
pedicels (FIGS. 5B and 6B; see arrow). In both tissues, expression
was detected in vasculature and parenchyma cells (FIGS. 5C and 6C).
At this stage, expression was also detected in the stamen filaments
(FIG. 5D and 6D; see arrows) and in the un-pollinated ovules (FIGS.
5E, 5F, 6E and 6F; see arrows in 6F).
[0089] Previously, the pAt.Erecta promoter was characterized in
Arabidopsis. Interestingly, pAt.Erecta expression patterns in
Arabidopsis were comparable to the patterns observed in soy during
the vegetative stage, but not during late reproductive stages. In
contrast, the pAt.Erecta expression pattern in soybean is
diminished in early reproductive tissues but remerges in some later
reproductive organs and tissues, including the inflorescence stems
and floral pedicels. See, e.g., Chen, M-K et al., FEBS Letters 588:
3912-17 (2014); Yokoyama, R et al.; Shpak, E D et al., Science 309:
290-293 (2005); and Yokoyama, Ret al., Plant J 15(3): 301-310
(1998), the entire contents and disclosures of which are
incorporated herein by reference. Thus, the pAt.Erecta promoter
provides a novel expression pattern in soybean.
Example 3
Expression of Flowering Locus T Gene, Gm.FT2a, Under Control of a
pAt Erecta Promoter Alters Flowering Time and Pods Per Node in
Soybean
[0090] Transgenic soybean plants were produced by transforming
soybean explants with a recombinant DNA molecule (i.e., a T-DNA
transformation vector) comprising the pAt.Erecta promoter operably
linked to the Gm.FT2a gene via Agrobacterium-mediated
transformation to generate four pErecta::Gm.FT2a events that were
carried forward for further testing. The effect of FT2a
overexpression was immediately seen in R.sub.0 plants, which had
very early flowering and termination with reduced seed yield (e.g.,
only about 8 seeds/plant). These transgenic Gm.FT2a plants also had
a short plant height and very few, if any, branches. Segregating R1
plants and their progeny were subsequently grown in the greenhouse
under long day conditions for initial study and characterization.
By growing these plants under long day conditions, the severe dwarf
phenotypes observed with Gm.FT2a transgenic Ro plants were
improved. In these experiments, both homozygous and hemizygous
plants grown in the greenhouse under 16-hour long day conditions
(i.e., 16/8 hours of day/night photoperiods) flowered much earlier
than wild type null segregants. Gm.FT2a transgenic plants flowered
at about 19-22 days after planting or seeding). (see, e.g., FIGS.
9A to 9C). Under these growth conditions, transgenic soybean plants
expressing Gm.FT2a further had an increased number of pods per node
on the main stem in comparison to wild type controls (see, e.g.,
FIGS. 10 and 11, discussed further below).
[0091] Plants containing one of the pEr::Gm.FT2a transgenic events
(Event 1) grown in controlled environment conditions were further
analyzed via scanning electron microscopy analysis (eSEM). Analysis
of the shoot apical meristem (SAM) of these transgenic plants
(collected at 7 days after planting) revealed an early transition
of the SAM into an inflorescence meristem (IM) and floral meristem
(FM) (FIG. 7). In contrast, the SAMs of wild type soybean plants
were not differentiated into IM at this growth stage. Similarly,
imaging of the axillary meristem of the FT2a transformants
(collected at 9 days after planting) indicated the development of
dormant inflorescence meristems (dIMs) (or lateral primordial
racemes) into IM and FM (FIG. 8), leading to more
earlier-developing floral branches (racemes) per node in these
transgenic plants. Additional phenotypic characterization revealed
early flowering at the V1 stage in Gm.FT2a expressing soybean
plants, which was well before the floral transition occurred in
null segregating plants (FIGS. 9A to 9C). These data in combination
with the pAt.Erecta:GUS expression pattern described above indicate
that early flowering, and more particularly the formation of
inflorescence and floral meristems, were induced by ectopic
expression of Gm.FT2a during the vegetative stage in leaf primordia
and the shoot apical and axillary meristems of seedlings. The
formation of a higher number of inflorescence and floral meristems
is believed to further cause earlier release and elongation of the
secondary and tertiary racemes, leading to a greater number of
productive flowers and pods being formed per node.
[0092] Not only did Gm.FT2a transgenic soybean plants experience
earlier flowering and produce more pods per node on the main stem
(relative to segregating null plants), the effects of ectopic
Gm.FT2a expression in transgenic plants were also found to be
dosage dependent. Although both homozygous and hemizygous plants
had a reduced height and less branching, plants homozygous for the
Gm.FT2a transgene were more severely affected than hemizygous
plants, presumably because homozygous plants contain two copies of
the transgene (i.e., a higher dosage), as opposed to only one copy
(i.e., a lower dosage) in hemizygous plants. Under long day growth
conditions, homozygous plants terminated earlier and had a shorter
overall height with fewer nodes and branches on the main stem in
comparison to plants hemizygous for the transgene (FIG. 10). Unlike
homozygous plants, which exhibited a number of sub-optimal dwarf
phenotypes including very few (if any) branches on the main stem,
hemizygous plants had an intermediate phenotype in terms of their
vegetative growth, plant height, and the number of nodes present on
the main stem relative to wild type and homozygous plants. Under
16-hour long day conditions, hemizygous plants had a more normal
plant height with some degree of branching and a more extended
duration of flowering, relative to homozygous plants (FIG. 10).
Hemizygous plants also flowered for 40-64 days after initiation of
R1, whereas homozygous plants flowered for only 16-24 days due to
their earlier termination.
[0093] Additional experiments were conducted with plants
transformed with the Gm.FT2a construct (3 events) in long day (16
hour) controlled environment conditions to further characterize the
dosage response between hemizygous and homozygous plants.
Differences in the number of nodes and pods on the main stem and
branches, as well as the average number of pods per node and the
average height per plant are shown in Table 1 for three homozygous
events (Homo-Event 2, Homo-Event 3, Homo-Event 4) and three
hemizygous events (Hemi-Event 2, Hemi-Event 3, Hemi-Event 4). These
events are distinguished from Event 1 above.
TABLE-US-00001 TABLE 1 Event level data for homozygous and
hemizygous Gm.FT2a transgenic plants. Avg. # MS Avg. # BR Avg. # MS
Avg. # BR Avg. Zygosity- nodes per nodes pods pods Avg. Pods Height
(in) Event # plant per plant per plant per plant per Node per plant
Homo- 11.8 6.9 46 9 2.9 17.5 Event 2 Homo- 12.3 6.5 66.4 9 4 21
Event 3 Homo- 12.5 6.8 49.6 9.1 3 19.5 Event 4 Hemi- 25.3 12.4
183.5 47.3 6.1 37.5 Event 2 Hemi- 23.9 13.2 200.3 28.8 6.1 40 Event
3 Hemi- 25.4 15.3 186.8 58 6 41.5 Event 4
[0094] As shown in Table 1, hemizygous plants consistently had a
higher number of nodes on the main stem (MS) and branches (BR) and
a greater plant height than homozygous plants. Thus, hemizygous
plants were generally less affected than homozygous plants and more
like wild type plants. Hemizygous plants also had an increased
number of pods per node and a higher number of pods on the main
stem and branches, relative to homozygous plants. Therefore,
hemizygous plants generally had a closer-to-normal plant
architecture with a greater number of pods per node (and per
plant), presumably due to their lower Gm.FT2a transgene dosage. The
relative dosage level of Gm.FT2a based on transgene zygosity was
further confirmed by additional experiments showing that Gm.FT2a
transcript levels were higher in tissues from homozygous plants,
than in tissues from hemizygous plants (data not shown).
[0095] The early induction of flowering in these Gm.FT2a transgenic
plants was associated with more pods (and seeds) per node on the
main stem in both hemizygous and homozygous plants. Homozygous and
hemizygous plants containing the Gm.FT2a transgene each had an
increased number of pods/seeds per node on the main stem of the
plant in comparison to wild type segregants (FIG. 11). The
distribution of pods on the main stem was also found to be
different between FT2a transgenic and wild type null plants. Both
homozygous and hemizygous plants grown under long day conditions
were found to have more pods on at least the lower nodes of the
main stem and more pods per node on average, in comparison to wild
type null plants (data not shown). Plants hemizygous for the
Gm.FT2a transgene contained the highest number of pods per node
over the length of the main stem. However, these effects were
dependent on the particular growing conditions including day
length, etc. In general, experiments performed with soybean under
longer day conditions tended to produce greater differences between
transgenic and null plants.
[0096] The dosage-dependent effects of transgenic Gm.FT2a
expression were also observed in field trial experiments. In a 2014
field trial, soybean plants hemizygous for two Gm.FT2a events
(Events 1 and 2 above) showed an average of about 2.68 pods per
node on the main stem, and plants homozygous for these events had
about 1.40 pods per node on average, whereas null segregating
plants had about 1.63 pods per node. In a 2013 field trial,
however, plants hemizygous for transgenic Gm.FT2a (Event 2) were
found to have an average number of about 3.21 pods per node, as
compared to an average of about 3.05 pods per node in homozygous
plants and about 2.19 pods per node in null segregating plants. In
another 2013 micro plot experiment conducted at a different field
location, plants hemizygous for the Gm.FT2a transgene (Event 1)
were found to have about 2.17 pods per node on average, as compared
to an average of about 2.05 pods per node in plants homozygous for
the Gm.FT2a transgene (Event 2) and about 1.30 pods per node in
null segregating plants. Thus, the number of pods per node on
plants containing the Gm.FT2a transgene may depend on a variety of
factors including dosage of the FT transgene, environmental and
field conditions, and perhaps differences in agronomic practices.
However, much like transgenic Gm.FT2a plants grown in the
greenhouse, homozygous and hemizygous Gm.FT2a transgenic plants
grown under field conditions often had fewer nodes on the main
stem, shorter overall plant height, and/or reduced branching in
transgenic plants. Indeed, wild type plants typically had more
branching and a greater number of total nodes per plant than
hemizygous and homozygous Gm.FT2a plants.
[0097] Additional physiological data was collected from homozygous
Gm.FT2a transgenic plants and wild type (WT) control plants grown
in the greenhouse under 14-hour long day conditions (see Table 2).
These data provide an average of measurements taken from six
Gm.FT2a transgenic plants for each event, or from eight wild type
plants. The following matrices were collected for phenotypic
characterization of these plants: Days to flowering at R1 (DOFR1);
Days to R7 (DOR7); reproductive duration in days from R1 to R7
(PDR1R7); number of branches per plant (BRPP); total fertile nodes
on branches (FNBR); total fertile nodes per plant (FNLP); total
fertile nodes on main stem (FNST); number of nodes on branches
(NDBR); number of nodes on main stem (NDMS); number of nodes/plant
(NDPL); percent fertile nodes on branches (PFNB); percent total
fertile nodes (PFNN); percent fertile nodes on main stem (PFNS);
number of pods per plant (PDPP); number of pods on main stem
(PODMS); number of pods on branches (PODBR); number of pods/node;
seeds per plant at R8 (SDPPR8); and weight of 1000 seeds (SW1000).
Each of these measurements was taken at harvest unless another time
point is specified.
TABLE-US-00002 TABLE 2 Construct level phenotypic data for
transgenic homozygous Gm.FT2a and WT plants. WT pErecta::Gm.FT2a
DOFR1 33.5 21.3 DOR7 106.9 92.9 PDR1R7 76.5 71.6 BRPP 20.1 1 FNBR
190.6 2 FNLP 214.6 15 FNST 24.0 14.3 NDBR 211.4 3 NDMS 33.4 15.3
NDPL 244.9 16.3 PDPP 575.8 61.2 PFNB 90.4 75 PFNN 87.8 92.0 PFNS
71.4 92.9 PODBR 487.3 3 PODMS 88.4 60.2 Pods/Node 2.4 3.8 SDPPR8
1319.6 122.1 SW1000 146 122.5 (grams)
[0098] Consistent with the observations noted above, homozygous
Gm.FT2a transgenic plants experienced earlier floral induction than
WT plants (DOFR1 about 21 days after planting, instead of about
33-34 days in wild type plants). These measurements further showed
that the number of branches (and other measurements related to
branching, such as the number of nodes or pods on branches) was
greatly reduced. Due to the transgenic plants having a shorter
stature with very little branching, the total numbers of nodes or
pods per plant were also greatly reduced. However, the number of
pods per node on the main stem was increased in transgenic plants
(e.g., about 3.8 average pods/node) relative to wild type null
plants (e.g., about 2.4 pods/node).
[0099] Without being bound by any theory, the larger number of pods
per node observed with transgenic soybean plants expressing FT2a in
the meristem during vegetative stages of development may be caused
at least in part by synchronization of early flowering with early
secondary and/or tertiary raceme release and/or better resource
utilization to produce more pod-producing flowers per node. Early
FT expression in the meristem (see, e.g., FIGS. 3 and 4) may cause
early release of the dormant inflorescence meristems to produce a
greater number of racemes per node of the plant, such that a
greater number of racemes produce mature flowers and fully
developed pods at each node. However, subsequent FT expression in
reproductive tissues (see, e.g., FIGS. 5 and 6) may terminate
floral development of later developing flowers at each node leading
to more efficient resource allocation to the earlier developing
racemes, flowers and pods. In wild-type soybean plants, a much
lower percentage of secondary and tertiary racemes produce flowers
and fully developed pods relative to primary racemes, and later
developing flowers of the primary raceme typically do not produce
mature flowers and/or full-sized pods prior to abscission. Thus, it
is theorized that more pods per node may be generated in plants
expressing FT proteins in the vegetative meristem by synchronizing
early flower development with early release of the lateral racemes
at one or more node(s) of the plant. With at least the pAt.Erecta
promoter driving FT expression, later developing flowers (that may
not otherwise produce fully developed or full-sized pods) may also
become terminated by later reproductive-stage expression of FT to
direct resources to the earlier developing flowers.
Example 4
Expression of Flowering Locus T Gene, Gm.FT2a, Under Control of
Alternative Vegetative Stage Promoters in Soybean
[0100] Based on the phenotypes observed in the preceding Example 3,
two promoters were also proposed to drive Gm.FT2a transgene
expression that were considered vegetative-stage, leaf-preferred
promoters: pAt.BLS (SEQ ID NO: 36) and pAt.ALMT6 (SEQ ID NO: 37).
As used herein, a "leaf-preferred" promoter refers to a promoter
that preferentially initiates transcription of its associated gene
in leaf tissues relative to other plant tissues. Since FT is
believed to function as a mobile florigen, early FT expression
during vegetative stages in peripheral tissues, such as in the leaf
with a leaf-preferred or leaf-specific promoter, may lead to
phenotypes similar to the meristem-preferred pAt.Erecta:Gm.FT2a
expression. It was further theorized that FT expression with a
vegetative leaf promoter might also attenuate the floral induction
signal, and thus mitigate the early termination phenotypes observed
with homozygous FT expression in the meristem, and increase plant
height and branching.
[0101] In these experiments, transformation vectors for
pAt.ALMT6::Gm.FT2a and pAt.BLS::Gm.FT2a were constructed and used
to transform a soybean line by Agrobacterium-mediated
transformation. Expression with the pAt.BLS promoter has been shown
to start in leaf primordia number 5 (p5) and is expressed in the
source leaf veins only until transition to flowering, and the
pAt.ALMT6 promoter is also a vegetative leaf promoter with
expression at later developmental stages relative to pAt.BLS. See,
e.g., Efroni et al., "A Protracted and Dynamic Maturation Schedule
Underlies Arabidopsis Leaf Development," The Plant Cell 20(9):
2293-2306 (2008); and Shani et al., "Stage-Specific Regulation of
Solanum lycopersicum Leaf Maturation by Class 1 KNOTTED1-LIKE
HOMEOBOX Proteins," The Plant Cell 21(10): 3078-3092 (2009).
Transgenic soybean plants were produced for each of these vector
constructs and characterized for phenotypes in growth chambers
under 14-hour photoperiod conditions in comparison to wild type
plants. For each of the pAt.BLS construct, six transgenic events
were tested (5 plants per event), and for the pAt.ALMT6 promoter,
seven transgenic events were tested (5 plants per event). For each
of these constructs, control data was collected from five wild type
plants.
[0102] The following matrices were collected for phenotypic
characterization of these transgenic plants (Tables 3 and 4). The
individual measurements are as defined above, and phenotypic
characterization was conducted on plants homozygous for the
transgene.
TABLE-US-00003 TABLE 3 Construct level phenotypic data for
pALMT6::Gm.FT2a and WT plants. WT pALMT6::FT2a DOFR1 35.2 38.8 DOR7
84.7 88.8 PDR1R7 49.5 50.0 BRPP 7.7 8.9 FNBR 57.8 73.3 FNLP 69.7
85.0 FNST 12.0 11.7 NDBR 78.9 96.0 NDMS 21.3 22.5 NDPL 100.2 118.5
PDPP 120.2 141.1 PFNB 73.2 76.8 PFNN 71.7 72.1 PFNS 57.9 51.7 PODBR
91.8 118.1 PODMS 28.3 22.9 Pods/Node 1.4 1.2
TABLE-US-00004 TABLE 4 Construct level phenotypic data for
pBLS::Gm.FT2a and WT plants. WT pBLS::FT2a DOFR1 31.3 35.2 DOR7
78.1 82.6 PDR1R7 46.9 47.5 BRPP 7.5 8.8 FNBR 65.7 81.2 FNLP 80.5
94.0 FNST 14.9 12.7 NDBR 72.2 95.6 NDMS 21.9 22.3 NDPL 94.0 117.9
PDPP 137.0 148.1 PFNB 92.3 85.3 PFNN 87.4 80.1 PFNS 68.1 57.3 PODBR
100.9 123.4 PODMS 36.1 24.8 Pods/Node 1.7 1.3
[0103] Transgenic plants expressing Gm.FT2a under the control of
the alternative pAt.ALMT6 and pAt.BLS promoters were phenotypically
more similar to wild type (WT) plants than pAT.Erecta::Gm.FT2a
transgenic plants. Plants transformed with the pAt.ALMT6::Gm.FT2a
and pAt.BLS:: Gm.FT2a constructs had flowering times and vegetative
growth traits similar to wild type control plants, perhaps with a
slightly increased number of nodes on branches as compared to wild
type plants (Tables 3 and 4). These data may be interpreted to
indicate that both the timing and location of transgenic FT
expression are important for producing reproductive and
yield-related traits or phenotypes that differ from wild-type
plants. Merely expressing a FT transgene during earlier vegetative
stages of development (e.g., in leaf tissues) may not be sufficient
to alter the reproductive or yield-related phenotypes of a plant
(e.g., pods per node). Thus, according to embodiments of the
present invention, a promoter operably linked to a florigenic FT
transgene may preferably be a meristem-specific or
meristem-preferred promoter in addition to driving expression
during the vegetative stages of plant development. However, when
the expression profiles for the above two leaf-preferred promoters
were tested in soybean plants, no GUS staining was observed in the
developing leaf with the pAt.BLS promoter, and the pAt.ALMT6
promoter did not produce detectable GUS expression in the leaf
until late vegetative stages with much higher expression during
early reproductive stages. Thus, it remains possible that
expression of FT transgenes in peripheral (leaf) tissues during
early vegetative stages using different tissue-specific promoters
may be sufficient in some cases to induce early flowering and/or
cause other reproductive or yield-related traits or phenotypes,
which may also depend on the particular plant species tested.
Example 5
Identification of Protein Domains of FT Homologs by Pfam
Analysis
[0104] Gm.FT2a orthologs were identified by sequence analysis and
literature review, and a few examples of these FT homologs are
listed in Table 5 along with Gm.FT2a. These included other soybean
FT genes as well as a few FT genes from other plant species. The
amino acid sequences of these FT proteins were analyzed to identify
any Pfam protein domains using the HMMER software and Pfam
databases (version 27.0). These FT protein sequences (SEQ ID NOs:
2, 4, 6, 8, 10 and 12) were found to have the same Pfam domain
identified as a phosphatidyl ethanolamine binding domain protein
(PEBP) having a Pfam domain name of "PBP N", and a Pfam accession
number of PF01161. The location of the PBP N domains in each of
these FT protein sequences are also listed in Table 5. The location
of the PBP N domain in other FT proteins can be determined by
sequence alignment. It is thus contemplated that any DNA sequence
encoding at least an FT protein comprising the PBP N domain may be
used in a recombinant DNA molecule of the present invention, as
long as the corresponding FT protein has florigenic activity when
ectopically expressed in the meristem of a plant.
TABLE-US-00005 TABLE 5 Location of PBP_N (Pfam) domain in FT
protein sequences. PROTEIN Domain SEQ ID NO. Gene Name location 2
Gm.FT2a 28-162 4 Gm.FT5a 26-157 6 Gm.FT2b 28-162 8 Zm.ZCN8 26-154
10 Nt.FT-like 25-159 12 Le.SFT 29-161
Example 6
Expression of FT Homologs Under Control of pAt.Erecta Promoter in
Soybean
[0105] Additional transformation vectors containing other FT
homologs (Table 6) under control of the pAt.Erecta promoter were
constructed and used to transform soybeans via
Agrobacterium-mediated transformation. Transgenic plants generated
from these events were characterized for their phenotypes in the
greenhouse with a 14 to 14.5 hour natural daylight photoperiod. For
each construct, six events were tested (6 plants per event). Six
plants were also tested and averaged for wild type (WT) control
plants. Different groups of experiments (A-E) were conducted as
shown in Table 6 with separate wild type controls.
TABLE-US-00006 TABLE 6 List of constructs for some Gm.FT2a and its
homologs with their protein sequences. Construct PROTEIN
Description Gene Name SEQ ID NO. Testing Group pErecta:Gm.FT2a
Gm.FT2a 2 A pErecta:Gm.FT2b Gm.FT2b 6 C pErecta:Gm.FT5a Gm.FT5a 4 E
pErecta:Zm.ZCN8 Zm.ZCN8 8 B pErecta:Nt.FT-like Nt.FT-like 10 B
pErecta:Le.SFT Le.SFT 12 D
[0106] The following matrices were collected for phenotypic
characterization of plants transformed with each of the constructs
listed in Table 6 for expressing other FT homologs with the
pAt.Erecta promoter, in addition to data collected for the Gm.FT2a
construct as described above. The individual measurements are as
defined above, and phenotypic characterization of transformants was
conducted on plants homozygous for the transgene.
[0107] Phenotypic data was collected for plants expressing the
Zm.ZCN8 and Nt.FT-like transgenes under the control of the
pAt.Erecta promoter (see Tables 7 and 8). Trait values for each
Event in Tables 7 and 8 are an average of all plants tested
containing the Event. A column is also provided with an average of
the Event values for each trait.
TABLE-US-00007 TABLE 7 Construct and event level phenotypic data
for Zm.ZCN8 and WT plants. Aver- Event Event Event Event Event
Event WT age 1 2 3 4 5 6 DOFR1 33.5 28.6 29 29.2 27.5 27 30.7 28
DOR7 106.9 93.5 97.3 89.2 88.2 93.5 100.3 92.8 PDR1R7 76.5 64.1
69.8 60 59 59.5 71.7 64.8 BRPP 20.1 3.2 2.8 1.3 1.5 1.3 9.5 3 FNBR
190.6 26.9 32 8 7 2.3 95.3 17 FNLP 214.6 54.9 67.5 28 40.5 20.3
132.5 40.5 FNST 24.0 28.3 35.5 22 33.5 18 37.3 23.5 NDBR 211.4 30.2
32.5 9 7.5 3.5 110.8 17.8 NDMS 33.4 30.5 36.3 24 34.3 20 44.8 24
NDPL 244.9 60.3 68.8 30.8 41.8 23.5 155.5 41.8 PDPP 575.8 317.5
498.3 144.8 319 76.3 658 208.8 PFNB 90.4 87.2 98.6 90.3 85.4 64.6
91.1 93.2 PFNN 87.8 93.1 98.1 92.3 97.0 86.5 88.5 96.4 PFNS 71.4
93.2 97.9 92.5 97.9 90.6 82.1 98.1 PODBR 487.3 105.4 162 19 18.5
3.3 384.5 45.3 PODMS 88.4 212.9 336.3 130.5 300.5 73 273.5 163.5
Pods/ 2.4 5.5 7.2 4.6 7.7 3.2 4.9 5.2 Node SDPP8 1319.6 564.7 961
200.5 562 136.8 1166.3 361.5 SW1000 146 108.9 102.9 127.4 105.3
82.9 116.6 117.9 (grams)
TABLE-US-00008 TABLE 8 Construct and event level phenotypic data
for Nt.FT-like and WT plants. Aver- Event Event Event Event Event
Event WT age 1 2 3 4 5 6 DOFR1 33.5 31.5 39.3 27.7 25.3 29 37.2
30.7 DOR7 106.9 93.9 115.8 90.7 80.7 83.7 102.2 90.2 PDR1R7 76.5
62.3 76.4 63 55.3 54.7 65 59.5 BRPP 20.1 9.8 20 8.3 2.3 5.3 17 6
FNBR 190.6 108.7 190.5 95.3 11 54.3 223 78.3 FNLP 214.6 131.4 212.3
118 29.5 77.8 248 103 FNST 24.0 23.2 21.8 22.8 21.3 23.5 25 24.8
NDBR 211.4 128.7 281.8 97 11 54.5 247.7 80.5 NDMS 33.4 28.9 33.8 27
23.3 24.8 35.7 28.8 NDPL 244.9 157.1 315.5 124 31.5 79.3 283.3
109.3 PDPP 575.8 462.1 638 511.3 150.8 296 745 431.5 PFNB 90.4 92.5
68.0 98.6 100 99.7 91.3 97.2 PFNN 87.8 89.6 67.6 95.4 93.3 98.3
88.4 94.2 PFNS 71.4 81.9 64.7 83.3 91.3 95.2 70.6 86.2 PODBR 487.3
326.3 529 342.3 22.7 147 633.3 283.5 PODMS 88.4 136.7 109 169 133.8
149 111.7 148 Pods/ 2.4 3.6 2.0 4.3 4.9 3.8 2.7 4.0 Node SDPPR8
1319.6 928.7 1359.8 965.5 382.7 591.5 1714.3 558.7 SW1000 146 149.0
143.7 121.2 133.8 179.3 142.2 174.0 (grams)
[0108] Transgenic soybean plants expressing the Zm.ZCN8 and
Nt.FT-like proteins flowered earlier than wild type control plants
and had an increased number of pods per node (similar to plants
expressing the Gm.FT2a transgene). Indeed, soybean plants
expressing the Zm.ZCN8 and Nt.FT-like transgenes had several
phenotypes similar to the Gm.FT2a transgenic plants, including
reduced number of days to flowering (DOFR1), reduced number of
branches (BRPP), fewer nodes per plant (NDPL), fewer nodes on
branches (NDBR), reduced number of pods per plant (PDPP), and fewer
pods on branches (PODBR), along with an increase in the number of
pods per node and a decrease in the number of seeds per plant
(Tables 7 and 8), relative to wild type controls. However, several
of the negative phenotypes observed in homozygous Gm.FT2a plants
were less pronounced in the Zm.ZCN8 and Nt.FT-like expressing
transgenic plants. Overall, plants expressing the Zm.ZCN8 transgene
had shorter plant height and less branching but more pods per node
on the main stem (FIGS. 12A and 12B). Similarly, plants expressing
the Nt.FT-like transgene had shorter plant height, reduced
branching and increased pods per node on the main stem (FIGS. 13A
and 13B), relative to wild type control plants.
[0109] Two transgenic Zm.ZCN8 events and four Nt.FT-like events
from above were also tested in 2015 field trials at two different
locations. Phenotypic data was collected for plants expressing
Zm.ZCN8 and Nt.FT-like transgenes under the control of the
pAt.Erecta promoter (Tables 9 and 10). Events 1 and 2 in Table 9
correspond to Events 2 and 3 in Table 7, and Events 1-4 in Table 10
correspond to Events 1-4 in Table 8, respectively. Except for days
to flowering at R1 (DOFR1) and reproductive duration in days from
R1 to R8 (PDR1R8), all phenotypic measurements were derived based
on data collected from two locations. Similar to the observations
in the greenhouse, transgenic soybean plants expressing Zm.ZCN8 and
Nt.FT-like proteins also flowered earlier than wild-type control
plants in the field. The Zm.ZCN8 transgenic plants had an increased
number of pods per node, while the Nt.FT-like plants did not
clearly show increased pods per node in the field trial.
TABLE-US-00009 TABLE 9 Phenotypic data from 2015 field trial for
Zm.ZCN8 and WT plants. WT Average Event 1 Event 2 DOFR1* 42.4 27.9
28.0 27.7 DOR8 110.7 95.0 92.0 98.0 PDR1R8* 65.7 67.1 63.5 70.7
BRPP 2.6 0.1 0.2 0.0 NDBR 9.7 0.3 0.5 0.1 NDMS 18.3 13.6 12.5 14.7
NDPL 28.0 13.9 13.0 14.8 PDPP 44.2 35.1 30.1 40.0 TPBR 9.5 0.3 0.5
0.1 PODMS 34.7 34.7 29.5 39.9 Pods/Node 1.6 2.5 2.3 2.6 SDPPR8 99.9
67.6 54.7 80.5 SW1000 5.1 4.1 3.8 4.3 (ounces) (*single location
data)
TABLE-US-00010 TABLE 10 Phenotypic data from 2015 field trial for
Nt.FT-like and WT plants. Aver- Event Event Event Event WT age 1 2
3 4 DOFR1* 42.4 38.0 42.5 26.8 26.8 25.8 DOR8 110.7 93.3 111.3 88.2
86.6 87.1 PDR1R8* 65.7 63.1 66.8 62.2 60.3 63.0 BRPP 2.6 0.7 2.4
0.1 0.2 0.1 NDBR 9.7 2.7 9.2 0.5 0.8 0.3 NDMS 18.3 11.5 18.3 9.9
7.6 10.1 NDPL 28.0 14.2 27.5 10.4 8.5 10.4 PDPP 44.2 23.5 43.0 18.9
11.6 20.3 TPBR 9.5 2.6 8.4 0.5 0.8 0.5 PODMS 34.7 20.9 34.6 18.5
10.8 19.8 Pods/ 1.6 1.6 1.6 1.8 1.4 1.7 Node SDPPR8 99.9 49.9 98.6
36.3 25.0 39.7 SW1000 5.1 4.5 5.1 4.4 4.6 4.0 (ounces) (*single
location data)
[0110] Additional phenotypic data was collected for plants
expressing the Gm.FT2b transgene under the control of the
pAt.Erecta promoter (Table 11).
TABLE-US-00011 TABLE 11 Construct and event level phenotypic data
for Gm.FT2b and WT plants. Aver- Event Event Event Event Event
Event WT age 1 2 3 4 5 6 DOFR1 43.7 34.6 41.2 34.3 22.7 33.2 37.2
39.3 DOR7 105.9 100.4 100.5 100.3 99.8 100.3 98.7 102.8 PDR1R7 62.2
65.8 59.3 66 77.2 67.2 61.5 63.5 BRPP 13.4 4.7 7 5 1.7 3.3 3.7 7.7
FNBR 103.8 32.4 52 29.7 12 30.7 21.7 48.7 FNLP 125.0 46.6 68.7 41.3
24.3 45 37.3 63 FNST 21.2 14.2 16.7 11.7 12.3 14.3 15.7 14.3 NDBR
108.4 34.2 54 30.3 12.7 33.7 24.7 50 NDMS 30.2 18.0 18.3 15.3 15 19
19.3 21 NDPL 138.7 52.2 72.3 45.7 27.7 52.7 44 71 PDPP 387.4 143.0
167 140 96 145.7 108.7 200.7 PFNB 95.5 94.6 96.4 97.7 96.8 91.0
87.7 97.7 PFNN 90.1 89.1 94.9 90.3 88.0 86.0 86.1 89.4 PFNS 69.7
79.2 91.5 74.5 82.5 77.0 81.1 68.7 PODBR 284.9 90.2 109.3 96 43
94.7 55.3 143 PODMS 102.5 52.7 57.7 44 53 51 53.3 57.7 Pods/ 2.8
2.7 2.3 3.1 3.5 2.8 2.5 2.8 Node SDPPR8 1159.3 322.3 411.3 292.3
195.3 346.7 245 443.3 SW1000 174.0 154.0 170.4 156.7 154.1 155
130.2 157.8 (grams)
[0111] Transgenic soybean plants expressing the Gm.FT2b transgene
flowered earlier and had less branching than wild type control
plants. Gm.FT2b expressing soybean plants had a reduced number of
days to flowering (DOFR1), reduced number of branches (BRPP), fewer
nodes per plant (NDPL), fewer nodes on branches (NDBR), reduced
number of pods per plant (PDPP), and fewer pods on branches (PODBR)
(Table 9). However, transgenic Gm.FT2b plants did not show an
increase in the number of pods per node. Overall, plants expressing
the Gm.FT2b transgene had shorter plant height and less branching
relative to wild type control plants (FIG. 14). Transgenic soybean
plants expressing four different events of the Gm.FT2b transgene
were also tested in 2015 field trials. Phenotypic data was
collected for plants expressing the Gm.FT2b transgene under the
control of the pAt.Erecta promoter (Table 12). Events 1-4 in Table
11 correspond to Events 3, 2, 1, and 4 in Table 12, respectively.
Similar to the observations in the greenhouse, Gm.FT2b expressing
soybean plants showed a reduced number of days to flowering (DOFR1)
in the field. The other phenotypic measurements also exhibited
similar traits as observed in the greenhouse relative to wild-type
control plants.
TABLE-US-00012 TABLE 12 Phenotypic data from 2015 field trial for
Gm.FT2b and WT plants. Aver- Event Event Event Event WT age 1 2 3 4
DOFR1 41.9 37.3 38.3 38.3 36.3 36.2 DOR8 115.4 109.2 111.1 110.6
110.6 104.6 PDR1R8 73.5 71.8 75.0 72.1 74.1 66.1 SDPPR8 188.5 95.5
99.4 81.1 117.7 83.6 SW1000 153.4 137.3 144.0 129.6 134.4 141.5
(grams)
[0112] Additional phenotypic data was collected from plants
expressing the Le.SFT transgene under the control of the pAt.Erecta
promoter (Table 13).
TABLE-US-00013 TABLE 13 Construct and event level phenotypic data
for Le.SFT and WT plants. Aver- Event Event Event Event Event Event
WT age 1 2 3 4 5 6 DOFR1 42.9 41.4 30 44.4 30.7 28 60.2 55 DOR7
108.6 103.8 90.7 106.2 99 91.5 116.5 119 PDR1R7 65.5 64.0 60.2 71
67 65 56.4 64.2 BRPP n/a n/a n/a n/a n/a n/a n/a n/a FNBR 131.5
37.1 2.7 125.2 4.7 1 47.7 41.3 FNLP 156.8 50.7 15.7 142.6 18.1 18.3
56.7 52.7 FNST 25.3 13.7 13 17.7 13.7 17.3 9 11.3 NDBR 140.2 38.9 3
129.4 5.4 1 51 43.7 NDMS 32.4 17.9 16 25.8 16.3 21 12.3 16.3 NDPL
172.4 56.7 19 154.7 21.2 22 63.3 60 PDPP 473.3 201.3 53.7 432.3
69.8 85.7 279.3 287 PFNB 94.1 94.0 100 96.9 83.3 100 92.9 90.7 PFNN
90.4 86.4 82.5 92.3 83.7 83.5 88.8 87.6 PFNS 77.1 76.5 81.2 69.5
84.0 82.8 72.7 68.6 PODBR 366.2 141.8 3.7 361.5 15.5 1.3 238.7
230.7 PODMS 114 60.3 50 73.6 57.1 84.3 40.7 56.3 Pods/ 2.7 3.6 2.8
2.8 3.3 3.9 4.4 4.8 Node SDPPR8 1247.4 476.0 136.7 1036 148.5 183
655 697 SW1000 167.7 153.2 170.0 182.8 157.5 148.5 131.5 128.8
(grams)
[0113] Overall, soybean plants expressing the Le.SFT transgene had
shorter plant height with less branching (FIG. 15) and an increased
number of pods per node on average relative to wild type plants
(Table 13). However, these effects were variable and
event-specific. For example, Events 1, 3 and 4 displayed early
flowering (DOFR1), while other events were neutral or actually had
delayed flowering. In addition, some of the Le.SFT transgenic
events showed increased pods per node on average to varying
extents, while a couple of the events were neutral in terms of the
average number of pods per node. Interestingly, two of the events
(Events 5 and 6) had the greatest number of pods per node on
average despite having a delay in flowering.
[0114] Additional phenotypic data was collected from plants
expressing the Gm.FT5a transgene under the control of the
pAt.Erecta promoter (Table 14).
TABLE-US-00014 TABLE 14 Construct and event level phenotypic data
for Gm.FT5a and WT plants. Aver- Event Event Event Event Event WT
age 1 2 3 4 5 DOFR1 48.2 29.9 32.2 29 28.6 29.2 30.5 DOR7 110 92.5
96.6 90.4 91 92.8 91.8 PDR1R7 61.8 62.7 64.4 61.4 62.4 63.6 61.3
BRPP 12.4 2.5 7 1.7 1 1.3 1.7 FNBR 105.6 7.3 20.3 4.7 3 4 4.3 FNLP
126.5 24.5 41.7 20 18.7 19.3 22.7 FNST 20.9 17.2 21.3 15.3 15.7
15.3 18.3 NDBR 108.6 7.5 21 5 3 4 4.3 NDMS 29 17.7 22 15.7 16.3 16
18.7 NDPL 137.6 25.2 43 20.7 19.3 20 23 PDPP 304.3 131.9 214.7 111
100.3 104.3 129.3 PFNB 97.2 98.0 97.3 93.3 100 100 100 PFNN 98.1
97.0 95.9 90 99.1 100 100 PFNS 72.1 97.0 97.1 98.1 96.1 95.8 97.9
PODBR 233.4 16.5 60.5 8 4 6 4 PODMS 75.1 108.6 159 98.5 95 92.5 98
Pods/ 2.2 5.2 5.0 5.4 5.2 5.2 5.6 Node SDPPR8 778.8 271.7 516 232.7
175.3 182.3 252 SW1000 151.6 126.0 143.7 122.4 122.2 121.8 116.8
(grams)
[0115] Transgenic soybean plants expressing the Gm.FT5a transgene
flowered significantly earlier than wild type control plants and
had an increased number of pods per node (similar to plants
expressing the Gm.FT2a transgene). Indeed, soybean plants
expressing the Gm.FT5a transgene had several phenotypes (similar to
the Gm.FT2a transgenic plants), including reduced number of days to
flowering (DOFR1), reduced number of branches (BRPP), fewer nodes
per plant (NDPL), fewer nodes on branches (NDBR), reduced number of
pods per plant (PDPP), and fewer pods on branches (PODBR), along
with an increase in the number of pods per node and a decrease in
the number of seeds per plant (Table 14). Overall, plants
expressing the Gm.FT2a transgene had shorter plant height and less
branching, but more pods per node (particularly on the main stem)
relative to wild type control plants (FIG. 16).
[0116] Without being bound by any theory, these data support a
model of FT overexpression acting in a dosage-dependent manner with
the degree or extent of associated phenotypes (e.g., early
flowering, increase in pods per node, and altered plant
architecture) depending on (i) the level and timing of FT
expression, (ii) tissue specificity of FT expression, and (iii) the
relative activity and target specificity of the particular FT
protein being expressed. For example, expression of the FT protein
orthologs from other plant species in soybean may produce a more
attenuated effect relative to overexpression of an endogenous FT
protein (Gm.FT2a) in soybean, which may result from the non-native
FT protein homologs having a lower activity in soybean. However,
expression of some native FT proteins may not produce significant
phenotypic effects if they have a different or specialized role in
their native state or context. Different FT proteins may also act
on different tissue targets and receptors and thus have
differential effects on the various plant architecture and
flowering traits and phenotypes.
[0117] Regardless of the activity level of the particular FT
homolog, altered reproductive and plant architecture phenotypes
appear to correlate with the timing and location of FT expression.
Vegetative-stage expression of FT transgenes may be necessary to
induce early flowering and/or cause increased numbers of floral
meristems, flowers, pods, etc., per node of the plant. Indeed, FT
expression in meristematic tissues during vegetative stages of
development is shown with proper dosing of the FT transgene to
cause reproductive changes in plants leading to increased numbers
of flowers, pods, and/or seeds per node. In contrast, expression of
a Gm.FT2a transgene under the control of leaf-preferred promoters
produced very little, if any, phenotypic changes, relative to wild
type plants. These data indicate that both the timing, and tissue
specificity (or tissue preference), of FT expression are important
factors that affect reproductive and/or yield-related phenotypic
changes in transgenic plants.
[0118] The present data suggest that different FT proteins may have
different activity levels and/or target specificities despite being
expressed using the same pErecta promoter. While several constructs
expressing Gm.FT2a, Zm.ZCN8, Nt.FT-like, and Gm.FT5a each caused
early flowering and termination in addition to an increased number
of pods per node, other constructs expressing Gm.FT2b and Le.SFT
had different correlative effects on flowering. Expression of
Gm.FT2b did cause early flowering and termination of plants but
without a significant increase in the number of pods per node. On
the other hand, Le.SFT expression showed increased pods per node
and early termination despite a delay in flowering. Interestingly,
increased numbers of pods per node in transgenic FT plants did not
correlate with an extended reproductive duration (PDR1R7) and was
not always aligned with early flowering (DOFR1) as noted above.
These data suggest that reproductive changes in response to
vegetative-stage expression of FT proteins in the meristem may
operate through one or more independent mechanisms or pathways.
Increased numbers of pods per node in transgenic FT plants may
depend on the number of inflorescent and floral meristems induced
from vegetative meristems at each node, which may occur
independently of flowering time and/or reproductive duration. As
noted above, however, reproductive duration may not necessarily
correlate with the duration of flowering.
Example 7
Identification of Additional Vegetative-Stage Meristem
Promoters
[0119] Having observed phenotypic effects with expression of
Gm.FT2a under the control of a vegetative-stage, meristem-preferred
promoter, pAt.Erecta, it is contemplated that other
vegetative-stage, meristem-preferred (or meristem-specific)
promoters may be used to drive expression of FT proteins to cause
reproductive or yield-related traits or phenotypes in plants, such
as increased number of pods per node (and/or per plant or main
stem). Using the characterized expression pattern of the pAt.Erecta
promoter (see Example 2), other vegetative-stage,
meristem-preferred (or meristem-specific) promoters were identified
from soybean, potato and Arabidopsis. Two bioinformatic approaches
were utilized to identify candidate genes from other dicotyledonous
species including, for example, Arabidopsis, soybean, Medicago,
potato and tomato, having similar expression profiles to
pAt.Erecta: BAR Espressolog and Expression Angler. See, e.g., BAR
expressolog identification: expression profile similarity ranking
of homologous genes in plant species, Plant J71(6): 1038-50 (2012);
and Toufighi, K et al., "The Botany Array Resource: e-Northerns,
Expression Angling, and promoter analyses," Plant J 43(1): 153-163
(2005). The promoter sequences from these genes are thus proposed
for use in expressing FT transgenes according to embodiments of the
present invention.
[0120] Examples of gene promoters identified by this analysis
include the following: four receptor like kinase (RLK) genes from
soybean, including Glyma10g38730 (SEQ ID NO: 23), Glyma09g27950
(SEQ ID NO: 24), Glyma06g05900 (SEQ ID NO: 25), and Glyma17g34380
(SEQ ID NO: 26). Additional examples include receptor like kinase
(RLK) gene promoters from potato, PGSC0003DMP400032802 (SEQ ID NO:
27) and PGSC0003DMP400054040 (SEQ ID NO: 28). It is possible that
these RLK genes may be related structurally and/or functionally to
Erecta and Erecta-like genes from Arabidopsis and other species
since they are also RLK genes. Other vegetative stage,
meristem-preferred promoters from Arabidopsis genes include the
following: At.MYB17 (At.LMI2; At3g61250) (SEQ ID NO: 31),
Kinesin-like gene (At5g55520) (SEQ ID NO: 32), AP2/B3-like genes
including ALREM17 (SEQ ID NO: 33) or ALREA119, and Erecta-like 1
and 2 genes, At.Erl1 (SEQ ID NO: 34) and At.Erl2 (SEQ ID NO: 35).
Each of these promoters and similar functional sequences may be
operatively linked to a FT gene to cause ectopic expression of FT
genes in one or more meristem(s) of plants at least during
vegetative stage(s) of development.
[0121] With regard to the At.MYB17 (At.LMI2) gene, see Pastore, J L
et al., "LATE MERISTEM IDENTITY 2 acts together with LEAFY to
activate APETALA1," Development 138: 3189-3198 (2011), the entire
contents and disclosure of which are incorporated herein by
reference. With regard to the Kinesin-like gene, see Fleury, D et
al., "The Arabidopsis thaliana Homolog of Yeast BREI Has a Function
in Cell Cycle Regulation during Early Leaf and Root Growth," Plant
Cell, 19(2): 417-432 (2007), the entire contents and disclosure of
which are incorporated herein by reference. With regard to the
REM17 and REM19 Arabidopsis genes, see Mantegazza, 0 et al.,
"Analysis of the Arabidopsis REM gene family predicts functions
during flower development," Ann Bot 114(7): 1507-1515 (2014), the
entire contents and disclosure of which are incorporated herein by
reference. Further, with regard to the At.Erl2 gene, see "Special
Issue: Receptor-like Kinases," JIPB 55(12): 1181-1286 (2013), and
particularly Shpak, E., "Diverse Roles of ERECTA Family Genes in
Plant Development," JIPB 55(12): 1251-1263 (2013), the entire
contents and disclosures of which are incorporated herein by
reference.
Example 8
Expression of Flowering Locus T Gene, Gm.FT2a, Under Control of a
Paterll Promoter Alters Flowering Time and Pods Per Node in
Soybean
[0122] A transformation vector containing Gm.FT2a under control of
the vegetative stage, meristem-preferred pAt.Erl1 promoter (SEQ ID
NO: 34) was constructed and used to transform soybeans via
Agrobacterium-mediated transformation. Transgenic plants generated
from these events were characterized for their phenotypes in the
greenhouse with a 14 to 14.5 hour natural daylight photoperiod. For
each pAt.Er11:Gm.FT2a construct, six events were tested (6 plants
per event). Six plants were also tested and averaged for wild type
(WT) control plants. The following matrices were collected for
phenotypic characterization of these plants and expressed as an
average for each Event as well as the wild type plants (see Table
15). A column providing an average for all the Events per trait is
further provided.
TABLE-US-00015 TABLE 15 Phenotypic data for pAt.Erl1:Gm.FT2a and WT
plants. Aver- Event Event Event Event Event Event WT age 1 2 3 4 5
6 DOFR1 46.1 32.6 40.0 32.3 34.0 29.3 28.2 31.8 DOR7 115.1 99.0
109.7 99.0 99.0 93.0 91.7 101.7 PDR1R7 69.0 66.4 69.7 66.7 65.0
63.7 63.5 69.8 BRPP 23.5 7.4 16.0 6.0 9.7 1.3 4.3 7.3 NDBR 277.6
80.8 215.7 51.7 139.3 3.3 18.0 56.7 NDMS 29.8 32.3 30.7 33.7 32.7
30.7 32.7 33.3 NDPL 307.4 113.0 246.3 85.3 172.0 34.0 50.7 90.0
PDPP 605.8 346.4 447.3 349.7 493.7 240.7 194.7 352.3 PODBR 503.1
173.7 332.3 164.7 323.3 8.3 42.7 171.0 PODMS 103.0 172.7 115.0
185.0 170.3 232.3 152.0 181.3 Pods/ 1.9 4.0 2.1 4.1 2.9 7.1 3.8 4.0
Node SDPPR8 1290.0 747.5 1129.0 603.5 881.0 577.0 432.3 862.3
SW1000 157.6 157.3 187.7 142.6 173.8 144.3 144.9 150.3 (grams)
[0123] Transgenic soybean plants expressing a pAt.Er11::Gm.FT2a
construct flowered earlier than wild type control plants and had an
increased number of pods per node (similar to plants expressing the
Gm.FT2a transgene under control of the pAt.Erecta promoter).
Indeed, soybean plants expressing pAt.Er11:Gm.FT2a had several
phenotypes similar to the pAt.Erecta:Gm.FT2a transgenic plants,
including reduced number of days to flowering (DOFR1), reduced
number of days to R7 (DOR7), reduced number of branches (BRPP),
fewer nodes per plant (NDPL), a reduced number of pods per plant
(PDPP), along with an increase in the number of pods per node
(Table 15), relative to wild type control plants. However, several
phenotypes observed in pAt.Erecta::Gm.FT2a plants, such as number
of pods on main stem (PODMS), number of pods on branches (PODBR),
and weight of 1000 seeds (SW1000), were less pronounced in
thepAtEr11::Gm.FT2a expressing transgenic plants.
[0124] The expression pattern for the Arabidopsis Erecta-like 1
promoter (pAt.Erl1) in soybean as measured by GUS staining is more
restricted than the expression pattern of pAt.Erecta in soybean as
described above. pAt.Erl1 drives GUS expression in vegetative
axillary meristems and in early floral meristems derived from
axillary tissue. However, GUS staining is not observed in the shoot
apical meristem at any stage where it can be distinguished from
other meristematic tissues of the developing plant. Expression of
the GUS reporter under the control of the pAt.Erl1 promoter is not
observed in leaf tissue, stem or root at any stage (data not
shown). Given that FT expression under the control of either the
pAt.Erecta or pAt.Erl1 promoter induced early flowering and
increased pods per node, vegetative expression of an FT transgene
at or near the meristem(s) of a plant may generally be sufficient
to induce these reproductive and yield-related phenotypes or
traits.
[0125] Having described the present disclosure in detail, it will
be apparent that modifications, variations, and equivalent
embodiments are possible without departing from the spirit and
scope of the present disclosure as described herein and in the
appended claims. Furthermore, it should be appreciated that all
examples in the present disclosure are provided as non-limiting
examples.
Sequence CWU 1
1
381531DNAGlycine max 1atgcctagtg gaagtaggga tcctctcgtt gttgggggag
taattgggga tgtattggat 60ccttttgaat attctattcc tatgagggtt acctacaata
acagagatgt cagcaatgga 120tgtgaattca aaccctcaca agttgtcaac
caaccaaggg taaatatcgg tggtgatgac 180ctcaggaact tctatacttt
gattgcggtt gatcccgatg cacctagccc aagtgacccc 240aatttgagag
aatacctcca ttggttggtg actgatatcc cagcaacaac aggggctagt
300ttcggccatg aggttgtaac atatgaaagt ccaagaccaa tgatggggat
tcatcgtttg 360gtgtttgtgt tatttcgtca actgggtagg gagaccgtgt
atgcaccagg atggcgccag 420aatttcaaca ctaaagaatt tgctgaactt
tacaaccttg gattgccagt tgctgctgtc 480tatttcaaca ttcagaggga
atctggttct ggtggaagga ggttatacta a 5312176PRTGlycine max 2Met Pro
Ser Gly Ser Arg Asp Pro Leu Val Val Gly Gly Val Ile Gly1 5 10 15Asp
Val Leu Asp Pro Phe Glu Tyr Ser Ile Pro Met Arg Val Thr Tyr 20 25
30Asn Asn Arg Asp Val Ser Asn Gly Cys Glu Phe Lys Pro Ser Gln Val
35 40 45Val Asn Gln Pro Arg Val Asn Ile Gly Gly Asp Asp Leu Arg Asn
Phe 50 55 60Tyr Thr Leu Ile Ala Val Asp Pro Asp Ala Pro Ser Pro Ser
Asp Pro65 70 75 80Asn Leu Arg Glu Tyr Leu His Trp Leu Val Thr Asp
Ile Pro Ala Thr 85 90 95Thr Gly Ala Ser Phe Gly His Glu Val Val Thr
Tyr Glu Ser Pro Arg 100 105 110Pro Met Met Gly Ile His Arg Leu Val
Phe Val Leu Phe Arg Gln Leu 115 120 125Gly Arg Glu Thr Val Tyr Ala
Pro Gly Trp Arg Gln Asn Phe Asn Thr 130 135 140Lys Glu Phe Ala Glu
Leu Tyr Asn Leu Gly Leu Pro Val Ala Ala Val145 150 155 160Tyr Phe
Asn Ile Gln Arg Glu Ser Gly Ser Gly Gly Arg Arg Leu Tyr 165 170
1753519DNAGlycine max 3atggcacggg agaaccctct tgttattggt ggtgtgattg
gggatgttct caaccctttt 60acaagctccg tttctttgac tgtttcaatc aataataggg
cgattagcaa tggcttggaa 120ctcaggccct ctcaagttgt taatcgccct
agggttactg ttggtggtga agacctaagg 180accttctaca ctctggttat
ggtggatgca gatgcaccta gccctagcaa ccctgtcttg 240agggaatacc
ttcactggat ggtgacagat attccagcta ccacaaatgc aagctttggg
300agagaggttg tgttttatga gagcccgaac ccttcagtag ggattcatcg
aatcgtgttc 360gtattgttcc agcaattggg cagagacact gtcatcaccc
cagaatggcg ccataatttc 420aattccagaa actttgctga aattaataac
cttgcacctg ttgcagcagc ttatgccaac 480tgccaaagag agcgtggttg
cggtggaagg agatattaa 5194172PRTGlycine max 4Met Ala Arg Glu Asn Pro
Leu Val Ile Gly Gly Val Ile Gly Asp Val1 5 10 15Leu Asn Pro Phe Thr
Ser Ser Val Ser Leu Thr Val Ser Ile Asn Asn 20 25 30Arg Ala Ile Ser
Asn Gly Leu Glu Leu Arg Pro Ser Gln Val Val Asn 35 40 45Arg Pro Arg
Val Thr Val Gly Gly Glu Asp Leu Arg Thr Phe Tyr Thr 50 55 60Leu Val
Met Val Asp Ala Asp Ala Pro Ser Pro Ser Asn Pro Val Leu65 70 75
80Arg Glu Tyr Leu His Trp Met Val Thr Asp Ile Pro Ala Thr Thr Asn
85 90 95Ala Ser Phe Gly Arg Glu Val Val Phe Tyr Glu Ser Pro Asn Pro
Ser 100 105 110Val Gly Ile His Arg Ile Val Phe Val Leu Phe Gln Gln
Leu Gly Arg 115 120 125Asp Thr Val Ile Thr Pro Glu Trp Arg His Asn
Phe Asn Ser Arg Asn 130 135 140Phe Ala Glu Ile Asn Asn Leu Ala Pro
Val Ala Ala Ala Tyr Ala Asn145 150 155 160Cys Gln Arg Glu Arg Gly
Cys Gly Gly Arg Arg Tyr 165 1705531DNAGlycine max 5atgcctcgtg
gaagtaggga ccctctagtt gttgggcgtg tgattgggga tgtattggac 60ccttttgaat
gttctattcc tatgagggtc acctacaata acaaagatgt cagcaatgga
120tgtgaattca aaccctcaca agttgtcaac caaccaagaa taaatatcgg
tggtgatgat 180ttcaggaact tctacacttt gatcgcggtt gatcctgatg
cacctagccc aagtgatccc 240aatttcagag aatacctcca ttggttagta
actgacattc cagcaacaac ggggcctact 300ttcggtcatg aggttgtaac
atatgaaaat ccacgaccca tgatggggat ccatcgtata 360gtctttgtgt
tatttcgtca acagggtaga gagacagtgt atgcaccagg atggcgccaa
420aatttcatta ctagagaatt tgctgaactt tacaatcttg gattgccagt
tgctgctgtc 480tattttaaca tccagagaga atctggttgt ggtggaagaa
ggctatgtta a 5316176PRTGlycine max 6Met Pro Arg Gly Ser Arg Asp Pro
Leu Val Val Gly Arg Val Ile Gly1 5 10 15Asp Val Leu Asp Pro Phe Glu
Cys Ser Ile Pro Met Arg Val Thr Tyr 20 25 30Asn Asn Lys Asp Val Ser
Asn Gly Cys Glu Phe Lys Pro Ser Gln Val 35 40 45Val Asn Gln Pro Arg
Ile Asn Ile Gly Gly Asp Asp Phe Arg Asn Phe 50 55 60Tyr Thr Leu Ile
Ala Val Asp Pro Asp Ala Pro Ser Pro Ser Asp Pro65 70 75 80Asn Phe
Arg Glu Tyr Leu His Trp Leu Val Thr Asp Ile Pro Ala Thr 85 90 95Thr
Gly Pro Thr Phe Gly His Glu Val Val Thr Tyr Glu Asn Pro Arg 100 105
110Pro Met Met Gly Ile His Arg Ile Val Phe Val Leu Phe Arg Gln Gln
115 120 125Gly Arg Glu Thr Val Tyr Ala Pro Gly Trp Arg Gln Asn Phe
Ile Thr 130 135 140Arg Glu Phe Ala Glu Leu Tyr Asn Leu Gly Leu Pro
Val Ala Ala Val145 150 155 160Tyr Phe Asn Ile Gln Arg Glu Ser Gly
Cys Gly Gly Arg Arg Leu Cys 165 170 1757528DNAZea mays 7atgtcagcaa
ccgatcattt ggttatggct cgtgtcatac aggatgtatt ggatcccttt 60acaccaacca
ttccactaag aataacgtac aacaataggc tacttctgcc aagtgctgag
120ctaaagccat ccgcggttgt aagtaaacca cgagtcgata tcggtggcag
tgacatgagg 180gctttctaca ccctggtact gattgacccg gatgccccaa
gtccaagcca tccatcacta 240agggagtact tgcactggat ggtgacagat
attccagaaa caactagtgt caactttggc 300caagagctaa tattttatga
gaggccggac ccaagatctg gcatccacag gctggtattt 360gtgctgttcc
gtcaacttgg cagggggaca gtttttgcac cagaaatgcg ccacaacttc
420aactgcagaa gctttgcacg gcaatatcac ctcagcattg ccaccgctac
acatttcaac 480tgtcaaaggg aaggtggatc cggcggaaga aggtttaggg aagagtag
5288175PRTZea mays 8Met Ser Ala Thr Asp His Leu Val Met Ala Arg Val
Ile Gln Asp Val1 5 10 15Leu Asp Pro Phe Thr Pro Thr Ile Pro Leu Arg
Ile Thr Tyr Asn Asn 20 25 30Arg Leu Leu Leu Pro Ser Ala Glu Leu Lys
Pro Ser Ala Val Val Ser 35 40 45Lys Pro Arg Val Asp Ile Gly Gly Ser
Asp Met Arg Ala Phe Tyr Thr 50 55 60Leu Val Leu Ile Asp Pro Asp Ala
Pro Ser Pro Ser His Pro Ser Leu65 70 75 80Arg Glu Tyr Leu His Trp
Met Val Thr Asp Ile Pro Glu Thr Thr Ser 85 90 95Val Asn Phe Gly Gln
Glu Leu Ile Phe Tyr Glu Arg Pro Asp Pro Arg 100 105 110Ser Gly Ile
His Arg Leu Val Phe Val Leu Phe Arg Gln Leu Gly Arg 115 120 125Gly
Thr Val Phe Ala Pro Glu Met Arg His Asn Phe Asn Cys Arg Ser 130 135
140Phe Ala Arg Gln Tyr His Leu Ser Ile Ala Thr Ala Thr His Phe
Asn145 150 155 160Cys Gln Arg Glu Gly Gly Ser Gly Gly Arg Arg Phe
Arg Glu Glu 165 170 1759525DNANicotiana tabacum 9atgccaagaa
tagatccttt gatagttggt cgtgtggtag gagatgtttt agatccattc 60acaaggtctg
ttgatcttag agtggtttac aataataggg aagtcaacaa tgcatgtggc
120ttgaaacctt ctcaaattgt tacgcaacct agggttcaaa ttggagggga
tgatcttcgc 180aacttttaca ctctggttat ggtggatcct gatgctccaa
gcccaagcaa ccctaacctg 240agggagtatc tacactggct ggtcacagat
atcccagcaa ctacagatac aagctttgga 300aatgaagtta tatgctacga
gaatccacaa ccatcattgg gaattcatcg ctttgttttc 360gtgttgtttc
gacaattggg tcgcgaaact gtgtatgcac caggttggcg tcagaatttc
420agcacaagag actttgcaga agtttacaat cttggtttgc ccgtttctgc
tgtttacttc 480aattgccata gggagagtgg tactggtggc cgccgcgcat attaa
52510174PRTNicotiana tabacum 10Met Pro Arg Ile Asp Pro Leu Ile Val
Gly Arg Val Val Gly Asp Val1 5 10 15Leu Asp Pro Phe Thr Arg Ser Val
Asp Leu Arg Val Val Tyr Asn Asn 20 25 30Arg Glu Val Asn Asn Ala Cys
Gly Leu Lys Pro Ser Gln Ile Val Thr 35 40 45Gln Pro Arg Val Gln Ile
Gly Gly Asp Asp Leu Arg Asn Phe Tyr Thr 50 55 60Leu Val Met Val Asp
Pro Asp Ala Pro Ser Pro Ser Asn Pro Asn Leu65 70 75 80Arg Glu Tyr
Leu His Trp Leu Val Thr Asp Ile Pro Ala Thr Thr Asp 85 90 95Thr Ser
Phe Gly Asn Glu Val Ile Cys Tyr Glu Asn Pro Gln Pro Ser 100 105
110Leu Gly Ile His Arg Phe Val Phe Val Leu Phe Arg Gln Leu Gly Arg
115 120 125Glu Thr Val Tyr Ala Pro Gly Trp Arg Gln Asn Phe Ser Thr
Arg Asp 130 135 140Phe Ala Glu Val Tyr Asn Leu Gly Leu Pro Val Ser
Ala Val Tyr Phe145 150 155 160Asn Cys His Arg Glu Ser Gly Thr Gly
Gly Arg Arg Ala Tyr 165 17011534DNASolanum lycopersicum
11atgcctagag aacgtgatcc tcttgttgtt ggtcgtgtgg taggggatgt attggaccct
60ttcacaagaa ctattggcct aagagttata tatagagata gagaagttaa taatggatgc
120gagcttaggc cttcccaagt tattaaccag ccaagggttg aagttggagg
agatgaccta 180cgtacctttt tcactttggt tatggtggac cctgatgctc
caagtccgag tgatccaaat 240ctgagagaat accttcactg gttggtcacc
gatattccag ctaccacagg ttcaagtttt 300gggcaagaaa tagtgagcta
tgaaagtcca agaccatcaa tgggaataca tcgatttgta 360tttgtattat
tcagacaatt aggtcggcaa acagtgtatg ctccaggatg gcgtcagaat
420ttcaacacaa gagattttgc agaactttat aatcttggtt tacctgttgc
tgctgtctat 480tttaattgtc aaagagagag tggcagtggt ggacgtagaa
gatctgctga ttga 53412177PRTSolanum lycopersicum 12Met Pro Arg Glu
Arg Asp Pro Leu Val Val Gly Arg Val Val Gly Asp1 5 10 15Val Leu Asp
Pro Phe Thr Arg Thr Ile Gly Leu Arg Val Ile Tyr Arg 20 25 30Asp Arg
Glu Val Asn Asn Gly Cys Glu Leu Arg Pro Ser Gln Val Ile 35 40 45Asn
Gln Pro Arg Val Glu Val Gly Gly Asp Asp Leu Arg Thr Phe Phe 50 55
60Thr Leu Val Met Val Asp Pro Asp Ala Pro Ser Pro Ser Asp Pro Asn65
70 75 80Leu Arg Glu Tyr Leu His Trp Leu Val Thr Asp Ile Pro Ala Thr
Thr 85 90 95Gly Ser Ser Phe Gly Gln Glu Ile Val Ser Tyr Glu Ser Pro
Arg Pro 100 105 110Ser Met Gly Ile His Arg Phe Val Phe Val Leu Phe
Arg Gln Leu Gly 115 120 125Arg Gln Thr Val Tyr Ala Pro Gly Trp Arg
Gln Asn Phe Asn Thr Arg 130 135 140Asp Phe Ala Glu Leu Tyr Asn Leu
Gly Leu Pro Val Ala Ala Val Tyr145 150 155 160Phe Asn Cys Gln Arg
Glu Ser Gly Ser Gly Gly Arg Arg Arg Ser Ala 165 170
175Asp13528DNAArabidopsis thaliana 13atgtctataa atataagaga
ccctcttata gtaagcagag ttgttggaga cgttcttgat 60ccgtttaata gatcaatcac
tctaaaggtt acttatggcc aaagagaggt gactaatggc 120ttggatctaa
ggccttctca ggttcaaaac aagccaagag ttgagattgg tggagaagac
180ctcaggaact tctatacttt ggttatggtg gatccagatg ttccaagtcc
tagcaaccct 240cacctccgag aatatctcca ttggttggtg actgatatcc
ctgctacaac tggaacaacc 300tttggcaatg agattgtgtg ttacgaaaat
ccaagtccca ctgcaggaat tcatcgtgtc 360gtgtttatat tgtttcgaca
gcttggcagg caaacagtgt atgcaccagg gtggcgccag 420aacttcaaca
ctcgcgagtt tgctgagatc tacaatctcg gccttcccgt ggccgcagtt
480ttctacaatt gtcagaggga gagtggctgc ggaggaagaa gactttag
52814175PRTArabidopsis thaliana 14Met Ser Ile Asn Ile Arg Asp Pro
Leu Ile Val Ser Arg Val Val Gly1 5 10 15Asp Val Leu Asp Pro Phe Asn
Arg Ser Ile Thr Leu Lys Val Thr Tyr 20 25 30Gly Gln Arg Glu Val Thr
Asn Gly Leu Asp Leu Arg Pro Ser Gln Val 35 40 45Gln Asn Lys Pro Arg
Val Glu Ile Gly Gly Glu Asp Leu Arg Asn Phe 50 55 60Tyr Thr Leu Val
Met Val Asp Pro Asp Val Pro Ser Pro Ser Asn Pro65 70 75 80His Leu
Arg Glu Tyr Leu His Trp Leu Val Thr Asp Ile Pro Ala Thr 85 90 95Thr
Gly Thr Thr Phe Gly Asn Glu Ile Val Cys Tyr Glu Asn Pro Ser 100 105
110Pro Thr Ala Gly Ile His Arg Val Val Phe Ile Leu Phe Arg Gln Leu
115 120 125Gly Arg Gln Thr Val Tyr Ala Pro Gly Trp Arg Gln Asn Phe
Asn Thr 130 135 140Arg Glu Phe Ala Glu Ile Tyr Asn Leu Gly Leu Pro
Val Ala Ala Val145 150 155 160Phe Tyr Asn Cys Gln Arg Glu Ser Gly
Cys Gly Gly Arg Arg Leu 165 170 17515528DNAArabidopsis thaliana
15atgtctttaa gtcgtagaga tcctcttgtg gtcggcagtg ttgttggaga tgttcttgat
60cctttcacga ggttggtctc tcttaaggtc acttatggcc atagagaggt tactaatggc
120ttggatctaa ggccttctca agttctgaac aaaccaatag tggagattgg
aggagacgac 180ttcagaaatt tctacacctt ggttatggtg gatccagatg
tgccgagtcc aagcaaccct 240caccaacgag aatatctcca ctggttggtg
actgatatac ctgccaccac tggaaatgcc 300tttggcaatg aggtggtgtg
ctacgagagt ccacgtcccc cctcgggaat tcatcgtatt 360gtgttggtat
tgttccggca actcggaaga caaacggttt atgcaccggg gtggcgccaa
420cagttcaaca ctcgtgagtt tgctgagatc tacaatcttg gtcttcctgt
ggctgcctct 480tacttcaact gccagaggga gaatggctgt gggggaagaa gaacgtag
52816175PRTArabidopsis thaliana 16Met Ser Leu Ser Arg Arg Asp Pro
Leu Val Val Gly Ser Val Val Gly1 5 10 15Asp Val Leu Asp Pro Phe Thr
Arg Leu Val Ser Leu Lys Val Thr Tyr 20 25 30Gly His Arg Glu Val Thr
Asn Gly Leu Asp Leu Arg Pro Ser Gln Val 35 40 45Leu Asn Lys Pro Ile
Val Glu Ile Gly Gly Asp Asp Phe Arg Asn Phe 50 55 60Tyr Thr Leu Val
Met Val Asp Pro Asp Val Pro Ser Pro Ser Asn Pro65 70 75 80His Gln
Arg Glu Tyr Leu His Trp Leu Val Thr Asp Ile Pro Ala Thr 85 90 95Thr
Gly Asn Ala Phe Gly Asn Glu Val Val Cys Tyr Glu Ser Pro Arg 100 105
110Pro Pro Ser Gly Ile His Arg Ile Val Leu Val Leu Phe Arg Gln Leu
115 120 125Gly Arg Gln Thr Val Tyr Ala Pro Gly Trp Arg Gln Gln Phe
Asn Thr 130 135 140Arg Glu Phe Ala Glu Ile Tyr Asn Leu Gly Leu Pro
Val Ala Ala Ser145 150 155 160Tyr Phe Asn Cys Gln Arg Glu Asn Gly
Cys Gly Gly Arg Arg Thr 165 170 17517540DNAOryza sativa
17atggccggaa gtggcaggga cagggaccct cttgtggttg gtagggttgt gggtgatgtg
60ctggacgcgt tcgtccggag caccaacctc aaggtcacct atggctccaa gaccgtgtcc
120aatggctgcg agctcaagcc gtccatggtc acccaccagc ctagggtcga
ggtcggcggc 180aatgacatga ggacattcta cacccttgtg atggtagacc
cagatgcacc aagcccaagt 240gaccctaacc ttagggagta tctacattgg
ttggtcactg atattcctgg tactactgca 300gcgtcatttg ggcaagaggt
gatgtgctac gagagcccaa ggccaaccat ggggatccac 360cggctggtgt
tcgtgctgtt ccagcagctg gggcgtcaga cagtgtacgc gcccgggtgg
420cgtcagaact tcaacaccaa ggacttcgcc gagctctaca acctcggctc
gccggtcgcc 480gccgtctact tcaactgcca gcgcgaggca ggctccggcg
gcaggagggt ctacccctag 54018179PRTOryza sativa 18Met Ala Gly Ser Gly
Arg Asp Arg Asp Pro Leu Val Val Gly Arg Val1 5 10 15Val Gly Asp Val
Leu Asp Ala Phe Val Arg Ser Thr Asn Leu Lys Val 20 25 30Thr Tyr Gly
Ser Lys Thr Val Ser Asn Gly Cys Glu Leu Lys Pro Ser 35 40 45Met Val
Thr His Gln Pro Arg Val Glu Val Gly Gly Asn Asp Met Arg 50 55 60Thr
Phe Tyr Thr Leu Val Met Val Asp Pro Asp Ala Pro Ser Pro Ser65 70 75
80Asp Pro Asn Leu Arg Glu Tyr Leu His Trp Leu Val Thr Asp Ile Pro
85 90 95Gly Thr Thr Ala Ala Ser Phe Gly Gln Glu Val Met Cys Tyr Glu
Ser 100 105 110Pro Arg Pro Thr Met Gly Ile His Arg Leu Val Phe Val
Leu Phe Gln 115 120 125Gln Leu Gly Arg Gln Thr Val Tyr Ala Pro Gly
Trp Arg Gln Asn Phe 130 135 140Asn Thr Lys Asp Phe Ala Glu Leu Tyr
Asn Leu Gly Ser Pro Val Ala145 150 155 160Ala Val Tyr Phe Asn Cys
Gln Arg Glu Ala Gly Ser Gly Gly Arg Arg 165 170 175Val Tyr
Pro19525DNAPopulus trichocarpa 19atgtcaaggg acagagatcc tctgagcgtt
ggccgtgtta taggggacgt gctggacccc 60ttcacaaagt ctatctccct cagggtcact
tacagctcca gagaggtcaa caatggttgc 120gagctcaagc cctctcaggt
tgccaaccag
cctagggttg atattggcgg ggaagatcta 180aggaccttct acactctggt
tatggtggac cctgatgcac ccagcccaag tgaccccagc 240ctaagagaat
atttgcattg gttggtgact gatattccag caacaactgg ggcaagcttt
300ggccatgaaa ctgtgtgcta tgagagcccg aggccgacaa tgggaattca
tcggtttgtt 360ttcgtcttgt ttcggcaact gggcaggcaa actgtgtatg
cccctgggtg gcgccagaac 420ttcaacacca gagactttgc tgaggtctac
aatcttggat cgccagtggc tgctgtttat 480ttcaactgcc agagggagag
tggctctggt ggtaggaggc gataa 52520174PRTPopulus trichocarpa 20Met
Ser Arg Asp Arg Asp Pro Leu Ser Val Gly Arg Val Ile Gly Asp1 5 10
15Val Leu Asp Pro Phe Thr Lys Ser Ile Ser Leu Arg Val Thr Tyr Ser
20 25 30Ser Arg Glu Val Asn Asn Gly Cys Glu Leu Lys Pro Ser Gln Val
Ala 35 40 45Asn Gln Pro Arg Val Asp Ile Gly Gly Glu Asp Leu Arg Thr
Phe Tyr 50 55 60Thr Leu Val Met Val Asp Pro Asp Ala Pro Ser Pro Ser
Asp Pro Ser65 70 75 80Leu Arg Glu Tyr Leu His Trp Leu Val Thr Asp
Ile Pro Ala Thr Thr 85 90 95Gly Ala Ser Phe Gly His Glu Thr Val Cys
Tyr Glu Ser Pro Arg Pro 100 105 110Thr Met Gly Ile His Arg Phe Val
Phe Val Leu Phe Arg Gln Leu Gly 115 120 125Arg Gln Thr Val Tyr Ala
Pro Gly Trp Arg Gln Asn Phe Asn Thr Arg 130 135 140Asp Phe Ala Glu
Val Tyr Asn Leu Gly Ser Pro Val Ala Ala Val Tyr145 150 155 160Phe
Asn Cys Gln Arg Glu Ser Gly Ser Gly Gly Arg Arg Arg 165
17021901DNAArabidopsis thaliana 21aaaccgaccg gagccaacca aaccggttaa
catcctaaaa ccaatcatat tttattaagt 60tttgtgttga tgctaaacca aaaatcattg
gcatgcatat ttctaaattt agtaataaac 120aaaaacactt agaaatcaca
cgttcactat actaaaaaac gttgacaaaa acacaacaac 180tatactaata
attaaagaag agaaaactga accaaacttt ttgtaaactc ctgaatttaa
240attagtaatt gaagtaagaa gatgaagaag aacatgttaa gcaaacaaaa
aaattacact 300aaaatcatat aaaaatacat aattacaaaa gtacccataa
gatggattta ttgatatggg 360tcatctgtga aacaagccac agagagacaa
agactcgtaa gtattgggca acgaaagcga 420cctcctttat tcaccactgc
cattaacatg ttcttcttct ccttcttctt ctacatttta 480tgaccgtttt
acccttcaag agagagaaac aaaatcactc cctctcactc actctatctc
540tctcttctgc aaagcttcag aactctggca gagagataaa agatgatggg
gtttttaact 600ttatcctccc caaataattc ttcttccctt catctctctc
tcttacacaa caggtcccta 660catttgtaca atctcctctc tttaaagact
ctctctcttt ctctctccat ctctatctta 720ctctgtattt ctgtcgtctg
agcactcaat gaaaccactg taaatttccg ccagaatttg 780atgtgatgga
acgataaaaa tcattttttc tcggttaaag taaaaaaaca aaaacaaatt
840tctgtagaaa tcataataaa agaaagaaaa aaaatctaat gtcggtacat
aatacggttc 900t 90122507DNAArabidopsis thaliana 22aaaccgaccg
gagccaacca aaccggttaa catcctaaaa ccaatcatat tttattaagt 60tttgtgttga
tgctaaacca aaaatcattg gcatgcatat ttctaaattt agtaataaac
120aaaaacactt agaaatcaca cgttcactat actaaaaaac gttgacaaaa
acacaacaac 180tatactaata attaaagaag agaaaactga accaaacttt
ttgtaaactc ctgaatttaa 240attagtaatt gcacaacagg tccctacatt
tgtacaatct cctctcttta aagactctct 300ctctttctct ctccatctct
atcttactct gtatttctgt cgtctgagca ctcaatgaaa 360ccactgtaaa
tttccgccag aatttgatgt gatggaacga taaaaatcat tttttctcgg
420ttaaagtaaa aaaacaaaaa caaatttctg tagaaatcat aataaaagaa
agaaaaaaaa 480tctaatgtcg gtacataata cggttct 507232000DNAGlycine max
23catgagcaag taagtaaaca ttttatctct gttacactcc aaacacatac actaacttaa
60gcagagtcct ttttcggcta ttcactccat caccaaagaa tgagctcatc cataaaaaca
120tatacatgtc aacatgtcga aagatgtaga acctggtaac aataataaag
aatgataatt 180tttttatttt taaaacgaag cactaaaata gatttaatta
tatttatcat ataaaattac 240caattaatca tatatctagt gaaatgatca
cttcaagtat aaaaaaagat tttatacttt 300tcgtctttct gtatttagaa
atattagcaa agtcttggat aaaaaagtgg tcgtagcctg 360tccctgtagt
ttccttgtta gtcttgatga acaagaagtt ctcagttctc cccaccctat
420tctgattcgg tttacatgga agtagtaagt aaccatacac cattatagaa
ataatacgat 480aaccacacgc catgtcttac ctcatgcgtt tactgaagtt
gttttcttct tttatttttc 540tttgatggag ttatggtatt aatattcaat
attagattgg aacttgcaga tcaacttcaa 600gaggcactct ttgataagga
taccccaggc attgcttttt gacataacgc caaaaccctc 660ctaaaaaacc
cttcattttc tatctcttag ttccatttta tgcaatgaaa taaaacttct
720caaatagtgt caaagcccga aaattgccta ccatatattt atcacgattc
atgaagggta 780tcttaattcc tttttttttt ttttcctgaa atgttttttt
tgaaggaatt ttcttgaaat 840gttttaatgc cttttttttt actcaagaaa
tgtcttaatg cttgtttact tacataaagt 900aatatcgttt gtcttttttt
acgcaatatt atattctagt actctgtctc tccaatctta 960ttatttttaa
aatttttctt ccttcctatc ctattatgca caaaaaggtg taattttaac
1020atttttctac tagtaaaaaa cctacaaact ttttctattg ttaaaattaa
atataaaagt 1080aattattttt attttatata aaaatacaaa gattttatgg
aaaaatatgt aagatataaa 1140aatatgatta attattttac tttcatctta
acttagcaaa tactttctga tcagtgcctt 1200atctcgcaca atccacaaac
attatctcgc acaagccaca aacacgcgct tcatgatcca 1260aaattgtacg
agcgacgctg tccatgtctc ctagaacgcg cgtagtaaga aataagtgtc
1320cctttgattt catgttgcat agttaatttt tagtttaaga ttaattttag
atagttttcc 1380atatttttaa ttttatatta aagataaagt caaaattaat
gtttagaatt aattgagttt 1440aagtcatttt aggtaccttt tggataaaga
aatttaaatt aaattttaat tcaaaattaa 1500tataaaccaa aattattaaa
acataaatca tattacttta aaattaattt ttcgaaaaag 1560cacattcaaa
gctccactaa aaattgtctt tgattaatag ccgtggatga gatttgattt
1620attagtgaga aaagacaaag aggtttaagc gcacgcgaag agaggcgcgt
aagtaaatag 1680gagaaacttt agctgtcaaa tatgctggga aacggcgagt
acgaatgacg gcggctacca 1740cccttatatt acagtgacag tctcactctc
acctatctag cctaacgtcg cttcaccgcc 1800gtttcccatt cttattctct
ctcttcataa cactcttcct atttacagtt cacgccaaat 1860gcctgcactc
tttctctact attaccaagc attggccaca ccaacaccaa cgaataacct
1920ttgttattgt aactaataac cactgcattt ttcccatact cgttgatctc
ttccactaag 1980tgctgtggtt ggtgaccgca 2000242479DNAGlycine max
24ttggattgtg tagaagaaaa aaattgaaaa aaaatatgac atccatgtta atttttgaaa
60ttttcttata ataattttcc gtcttcaacc aaaccagact cctattaagt atagtaaata
120ctattatatt tttgtggact tcttaataaa tagtagtaat attttgaaaa
gcttttttat 180ttttacgaaa gtcataataa attataaaca aacttataaa
aaatattaaa tcttatagta 240ctacttttat ataataataa taataataat
aataataata ataataataa taaaacgtgt 300tcaaaattat taatattcct
cttgaagttc cgtttcatat tctgtaaaaa aaagttgcgt 360ttgatattaa
aatataacag tactaaaaaa acaacataaa aaagaaaatc atgtttgatg
420aaagaaaata caaatatatt ttcataaaga gaacttcaca attactcgca
atgctgtgtg 480aaatagggat ataaccttta tccaagacac gttcccatca
ttgaagtata attaaatctt 540ttacggttaa ttataatgaa atcattttgg
atttgctttt gcctattatc acttttcaca 600cgatgatact taattattca
tagacctttt tgtcgagtaa gaggggaaat gctaaacttt 660tctgcttaga
ttttttggca tagttaatgg attttagcct ttttctttct tattaatttt
720tttctttcat aagcatagtc ccggtaaaat tctcactttc agttgatact
ttacctcctc 780cgaaaagttt cccatattag agactcaatg gcgtataaaa
tcatcttaaa catttactta 840taatgaatgg aaataaaatc taaaaagtta
gctactaatt ctttcacggc cattacgaag 900actttgctta aaaatggaaa
aaaagcaaaa tataaaagag tgtacattgt ctatttttat 960aattgacttg
gctctgtatg tattatgtaa ttaattttta atcttatatt ttgattattt
1020atagagatat aaaatgaatt tgatagttaa agaaagaaaa gaagagatga
aaaattgtgt 1080gttcgatcct ctaataaaac taacatttta ataaattaat
atttatcatt tttttaatta 1140tttgagtttt ggaagtgtaa tgagtcgagt
aattttattt gatggttgtt tggttcactt 1200atctatgttg atcaagtaat
cgatcaattt atctccatga ataatgatga tttttaagaa 1260tatttaacat
ttgaccatca attccttaaa tcatgtaatt atttttgtca accatgcaac
1320ctctataaat ataggtccta catatgtttg acattcatca tagtgtgtaa
tgtatttttt 1380ttattaaaaa aaacagagat gatgaactcg tgataaagaa
tcacctaaca cattactgat 1440actctctata aatatcacat gacaacctta
aacaaatacg cacaattcat atatcaatat 1500ccattacttt gtcatattct
aatttgagtg taaaagtctt tattattaca gtcttttaag 1560ttggtttaga
gcaatttgag ttaatatctt tatacaaaaa taacttaatt ttttaatatt
1620atttttaaga catatttctc ataaaaaatc acataattta gtttataatt
tttaatttaa 1680tattatcttc atttttattt ataaaattta attacctaat
tcatcaatat taaaaaaata 1740aattaattta attaatatca ataaatttat
cctaaactta taaacattat cattaatgct 1800cttctctctt aaatgtttat
ggatataact tcttttattt aattaaaatg tttattttaa 1860attaaattaa
tgtaagaaat aatacaaatt gaatattgta taaaggaaca aacataattt
1920tgttttgtat tagaccataa gtaatactcc atattagatt atatatataa
cttttatttt 1980aaaattatag agtatacttt ttttagagga aattatagag
caaactacat tcatatgatt 2040tctcttttat aaatattgaa aacaaaatag
ggatatgcaa cagcaaacga gggaggtttg 2100aggagagagg gagagagaga
gaatgtaggc gcgtgtggca cagttatgag ttaagactta 2160ggagaagtac
acattggcat aggcattgtt attggattat gtgtagagtc cgatagacta
2220gaatgacggc tactagttac tactctctct cttcataaac acaccattta
tgtttttccc 2280ttcccttcac gccaaacgcc tgcactctac actctactct
ctcgtgctct gtgactactg 2340tcactctctc ataaaccaaa catgccctta
atccattttc catagtagtt agtgttgtta 2400ctcatctctt ccatcttcaa
tctctcttct ttccttattg ttgctcacca aggtggggtt 2460ttttgtacgt
gtggtggca 2479252000DNAGlycine max 25cattaaataa ttctaaaaaa
gatataaatt tttgtataca aatctatatt taagaaactt 60ttaatctaga tgtcgatttt
aaaaaatatt atttaattaa aaaatattag atggtgtaat 120aattaatcaa
aattatatca agataatctg attcctttct atacacacat aatattattt
180catccttagt ccctaatatt ttcaattctc attttgttac cagacttgta
ccgaacaaaa 240acaaaatatc taatcatagt tttcattcaa caaaaatgat
ttttaactca atttgaaaca 300cttttcattc atttttaaaa ctaagaaaaa
atttgtgatt tatttttata attttgaaaa 360acattctacc atcattatta
ttgtttctac taccatcatt attaatgtta ctactatcac 420cttcctagtt
ataaccgtaa gcattatata tttttattat tattgttatt atgttatttt
480gttaatatat ttatttttgt tctaaaaaat tattattttt tcatatcttt
cactattttt 540gttattattt tagcaagttt gattattttt tattttaaat
atttttatgt gtcacttttt 600atatcacatt atttaacagt gtgaatcgat
aaaaaatata ataattatct ttaatttgta 660agaatttttt caaaattaaa
actgatttta gttcttgaaa aatgtaaaac taaaaatgaa 720aaccacctaa
cggggcctta gtaattagga catggtctcc ctggttaccc acgggatttt
780ttcacatcaa agaagacctg gtattttcat tttcatgaga tttttgcata
tcgaacaagg 840cattaagaca ggggttgtca ttgtcgtgat agtataattt
acatggtcga agtgatagaa 900actttaacca tcatttacct tgtaccttac
tataacacaa aatactacga tttccaaaca 960ctagatcgcg cgcttatgtt
ttcagacaca ttattcttct tcattcataa ataaatttgc 1020agctagtata
tgataattgt accaatttat gtaagttttt tacaaaggac attcttatct
1080caataaaaaa ctaaatgttt aaaatattct ctagcacatt ttttaaatac
attttgtcta 1140attaattaaa attaaaagag gatataaaaa atatgctgct
aacatcttga acatttccgc 1200aatcaataat ttctcaatct atctgaatat
ttttgcaact gtatacaaaa atctcagaac 1260agaaaattat tgattaaact
ggaagaattt aataacattt gattcacgtt tgtttagtga 1320ttaaaaaatc
ataacattac actatctaac aaatgcagca tccataacta ccaaacatta
1380aacaagagaa acagacaaag tccaataatc acagagacac gcagtgacaa
agaaaagaaa 1440gagggaacgg taaagagaaa ggtgtctctg tcatctcaaa
tagattgcca taactccctc 1500cttctctctc acaagctctt gcagagtgaa
agcgaccact ttccgatctc aattaaaagt 1560atggcataat ttgcaatggc
ggaactgaac gaataataat aagagatacc atagttaaga 1620gagagaaaca
caaacatgga aaaagctggg cctcactccc tgggtacaca tagatagaga
1680ctatggtgca gtgttgcagg ttgtagcaga agctctgcca aatagtgtta
actttattcg 1740agaaaattat tattattatt attattatta ttattctctc
tctctagtct attatcagtg 1800gtaattcagt aatgttgttg cattatagag
agagcgtggt ctatgtgcca gggtgatgtg 1860atgtcatttc actaccttca
aagccagaaa aatgcaacag aaaaagcttt catcccatca 1920catcatttga
accatgaatc atgaactagt tttctaaact aaaactataa caacaccttc
1980ggttgttgtt gttgttggct 2000262000DNAGlycine max 26aacatttaag
atcttaaaga tgccaagagc ttcatatgaa aatgtacaaa agagatttta 60aaggcaatat
caatgctgtg acgccatatt aaaataaaag ggatggtttc tcctgtatat
120tgagcaattt gtattactta tatacacaaa atctaaattg attcttaaca
aatatgtaaa 180gaaattaata atatgatcaa gttacctgaa gaagctaaaa
taaaatagaa aattaagtaa 240aagaaatgag gagtagaata taagataaca
tcaaaaaatt atttcagcat attttaagaa 300catcaaattt acctttcatc
aaaattaatc ttaaaagact aaaacattta attaagttta 360taaatactca
cacaaaatat taatttattt tgtaattatt attttttata tttttattta
420ctattgcctc aaaatttgca ctaaacaaga gaccctagag atttcgttag
aacaataata 480gacacggtat taaataatta aattaatacg aggatgcata
actaccaaac aaatgcgata 540aataaatgag acgacgagag agcacaacgc
gggaatgaga taattaagaa aaaaaatcta 600ataaattagg aaaaaaaaga
cataatatca taagcttgaa tccaatgtac aaagagaggt 660tggcaataaa
gagaaagaga aaagacgtcc ctgtcacctc aaatggattg cattactcat
720tgaaaaggac attattactt ccgacttttt atattaactt actaattata
aaatatataa 780aaaaatactt caaagatgca tatattttat tttattacat
aattacataa cagaataata 840taaaataatg taactacaca ttaaaacatt
aaaatagtga ttggagtagt ggtataagag 900gacgttgaat tcacgcggaa
gagaaggata tatttcatgt ttaatttgtt gtcatgccta 960gttcaatgta
atctaataag taaaaataaa atacaaacaa aataaagatt ttggtttctt
1020aacaaaagta cttttacttt aaatatatat ttttatctgg tttttaaaca
tgcacatatt 1080taacataaaa gttcatatta aactttttcc tacatacttg
gatcaaatag tcacgtattg 1140caggtaaaaa ataatagtgt agcttataga
aatcgtagaa ataagtctat aaaccagaag 1200aaaaaaaaca ttaaaataat
agtatagaaa tctatatcag tgtccccagt tcttacattc 1260atgacccatt
tccccataaa ctctttgcag ataatgcaat ggcaaaacca cacagaaagt
1320gacccctggg aatcaaaagt taaaaccaat ggcacagcat agcacagtgt
acagtgttta 1380tttactatat agcaaaacac tcactggcat aacactttag
ggagagagag agtgaaaaca 1440agtgtaaaaa gagagaaagt taggaggggg
atagagagtg tgtgtgtgtg cagagtttgc 1500aggcttgtag cagaaatggt
ggcagatggt tttaacttta tgtgtgaaat aattttcttc 1560tatctctttt
ctctttagtg ttttctctct ctctctctct cttctttttc ttcctgcatc
1620ttcttgtgtt tagggagtgt gatgttttgt ggcagaagaa cgatgtgatt
ggacacagcc 1680aaagctgtgg acttgttctg ttactacttt gtaattgtaa
tcacataaaa ggctagaggg 1740tatgaagagt gcacagaaaa atactagtac
tagtttcaaa caaaactcac cttactacta 1800cccttccatc tcaagccata
gttgagttga gtggtgcaca gtgtcactat acataccact 1860aacacccttt
tttggttctt gttctgtggc tccttgtgct ttgagcaaga gctttttgag
1920aaagagcttg gtggtggtgg ttgttgttga gtggtttcat ggttaggctg
ttgttaagtt 1980gaagttcatc agttgcagct 2000271399DNASolanum tuberosum
27aacgaaaaat ttagaaacta ttagtgatcc aaatgttcgt gattacctgc aacgagaaca
60acaacgaata cttgaaaaaa gaaatcgaca atcacaacca caaccataat cgcaacaatt
120ctcagaatca tatcctaatt ttttttcgaa tagtgctaaa tttgaaaacg
acctaccgaa 180tttctaaatt attgttgtga tcaattaatt attatgttat
gtattgtatt ttatcttgta 240tttaaattat tatgttatgt attgtattgt
tatcttgtat ttaaattatc atatcatgta 300ttgtattttt aaattaattt
tttttgcata ttctttataa tgaaaattaa taataaaaca 360attttattat
tcacgaaaat tagaaaaaaa gttaaaatac tattaatttg aaattaaaat
420agtatatatt aaataatatt tttaaaaata ttatattata tttaaaaaga
attatgaata 480ttagatattt aattaatgga attatatgta aaataatatg
ttaattagaa agtaatagaa 540aaataataaa ataatgaaaa agtagaaata
gagagtgtga atagtagaat ttggagaact 600attcaactct ctaaatttga
agaatatagg gtgatttgga ggtgggttgg agtgtccatt 660ctctatttta
ctctcaaaat atagagaatg gagagaaaaa tagaggtgga ttggagatgg
720tcttagtgac atttttgatt ccgccaatgc tcagttggcg tagtcgctgt
caaacttgag 780aaaggattac ccctttaggc ttgcacagac agtgacttat
gatgaaatga agccagagaa 840ggcactctgt tataacactt aaatgaaaat
acatgtgtat ggactagcaa taaaaggggc 900actagtaatt ttagtaattg
aaaagcaagt gtatagagag agataatgag agagaaagag 960taagtacact
actactgcta ctatcccata tagctgtaat gttgcaggtc tgatttttgc
1020agttgcagac ccccttcttg gcacaagctc ttttaacttt tatcttctca
aataattctc 1080tctctctctc tctctctttt ttctcttttt acattgtgag
gaaactgaat acccattgta 1140tgtattagtg tgaggcctat ctgccacaag
gatgtgatgg aacactatgc ttcctctgct 1200aaaaccccac aaccccaaaa
ctctttttca cttcacattt aatcacaatt cctcagtgaa 1260attattctgt
tgctctctct aatttcaatt tcaatgtcgg taagtccaag aactggtttt
1320tcaattcaaa ggagctgagt tagtgcaaac acttgaggtt ttgagttttg
acagagactt 1380gagtctcaga gaaactacc 1399282000DNASolanum tuberosum
28accttatata agttacaatt tagttatgta tataagttaa aattaaatta aaagacattt
60cgaaataata tgattatacc atttcgaaat taattagaga gagaaataag atctcgcaaa
120attaagtgtc ttcttgaaat taagaaccat ttttaggaga taattatgta
ttttttcatt 180tttaatttga cacgtatgca tatccactat tttgttttat
tccaaagtga cccctacttc 240ttttggtaat ttctttgagt attttaaact
ctagtccccc tttctcaagc aaaaaggctc 300actcgcgcac gcgcgaagag
acattgtgac gcgctggatg gaaaatccag aagcgtaact 360gtcaaaaaat
agaacaactt tgggaaacgg ggtgacggcc gctgccacca cttttttcat
420ttccaaacac tcattaacta acgtcgtttc accgccgttt actgcttaat
gagtatgaat 480tacactctaa tagtctattt ttacttattt ttaatgtgtt
tatcaaatta tatttttaaa 540tataatactt taaaaatatt atcatcaata
ataagagtaa attaaaaaat aaatgacaaa 600ttgtttctta aattgttaaa
ttaaacaatt aaaactgaat atttacaaaa tacctcttaa 660cttgctaaat
taaacaattg aaactatatt tatattaata aattgaactg acaaaaataa
720ataaaggaac tatatatttt ctcaattata tctttttact aaaatattat
ttttctaata 780ctagttaaac ttttaaaaaa catctaataa agaaaaagaa
tttgttcaat tatactttag 840aagcttttat tattattatt attattagta
gtagtagtag tagtaataaa ttagattaaa 900ttaaagagag aagtattcaa
aactcccaaa actattgtat tagttttatt tcagaactat 960tgacaatctt
aatttttttt tttttaattt gactaggtga acttaaatat acttcatttt
1020ttgcaaaaca agtgaagtac actcttaaat tttcatcaag tttagaaatg
ttttcaacaa 1080tttactagac tctttattaa gaacttcatg ttctttcaag
agtttatgag cacttgctat 1140gtcatgttac agatcaagaa tatctacaga
gtgtatctaa atttagtact agtaaagtag 1200aaaatgtatt acttatctct
caaacaatag gtattcatta tactattttg agatgtccaa 1260caattttttt
tcactttatg aaatcaatga ataatttaac acttagttcc taattcccag
1320taagcattaa ttatagttat ttacttatta tatttttcaa cacattatat
tgaaaaagtg 1380atatagtaaa tctatctttt tattttatta tttcttaaaa
tttgtacaaa cttaataata 1440gacaaatatt gttgaatagg aataataatt
tacattaaat ccaatatatt tttcaatagt 1500tgtcactaaa tgaaaatact
tcatctgttt caatttatgt gatagttttc atttttcaaa 1560agtcagacaa
ttatatattt ataaattaag taaaaaatat tataagtcac actaattaac
1620aattcgaaat attcggtacg gaggaactaa cacttatgtt tttagaccat
attagtcttt 1680tctctctatt tattatataa tattgagagg agagtgcaac
caccatggca actttctctg 1740tcttcataaa acgcagctga cattaaaaac
acagacacac acttcgcatt tcatatccct
1800ctcactacac gccaaatgcc tgctcttcct atttctcttc ttcttctttt
tcttcttctc 1860tctcattcac ataacacaca ttcttgtact aactctgcat
cataaactct accccacttt 1920cttcttcttc tccggtcata ttgctctgaa
actccactta ttgctctctc ccggcattta 1980tttttagttt ctcagaaata
200029471DNAArabidopsis thaliana 29taataagaga cgaaaaaaaa ataactaact
gatcattacc atccataaat aaatagttgc 60tgccataaac caaacacatt gtgcttatca
aaaagaagaa atttgtactt aatgaaacat 120tcattattag caaagtgtaa
aaccaaagaa aaacaaactt tatttctcat tttattagta 180aaagtgaaga
agagtaaaga aaaagagaga ctgagatgag gctgagagcc tgagtctgcg
240ggtggagaga gagagaaaga aagcctcttt acacgtgatt tttaaaagag
accaaaaccc 300caaaagcaaa cctcttttgc atgcgtcctt aaaagacata
aatttctctc aaaattttct 360acatcacaaa atcaatcttt ttctcttctt
cttcgtcttc atcatcatca tcatcatctt 420cctctttctc ttcctactga
gatattttct ccacattgag aggaaagcta t 471301231DNAArabidopsis thaliana
30aggataaatt tcatctatta agatatcagt caattataat gtgttacgtg attcgataaa
60aaaaaaagac caaaaaaaaa aagaagataa ctattggtaa gcgtaagaaa tgtgtttaca
120ttttggcatt ttgccaaaac acataaagat ggttagtgat gagacgagac
gagtcatgcg 180ctacttttaa aacaaaatga aaaacatcat taagctaaca
aaccaaacac acttgttttg 240ataacatgtt ctagggaact agttatgcca
aatctaatcc gcataagaag actaagtcac 300aacataattc agtaatttgg
ttgagattaa atcctataaa tatgatttta aggtataaga 360gagaagagac
tcttttgatc aacacaatca aacatctaca aagaaaatta tctcacatag
420ctacttctta atctaatttt ttcattaatc cattttattt taaatgtgaa
gaatcgcatc 480tagatgtgac ctctcatgat aaaaaattaa accattgtaa
aaaaaatgtt gtgtaaaact 540aaatataata aattattaaa aaaatacaaa
ttcaatccac taggttaaaa actcctatgt 600agaacatttt tttatattaa
aatgtaaata catgaatctt atttttcgaa aaactaaaga 660catctttttt
ttatatatta attaccaaaa caaaataaga cgacaaaaat attctttgat
720atagtaaaag aaaactagaa aactagaaaa caataaatta ccaaaacaat
ctagaaaaca 780ataaatccta ctttgcatta ctttattata aaatcccgaa
atgaatctat aaatgtagaa 840aatattatac aaaagttgta agagatttta
atatacataa ttacatatat atacaagtaa 900atacaccgta tatacatacg
aaggagtaaa cagtattatt tggtatatag ttacgtctct 960atatacgaag
ggttcaaact tcaaagtaat aatttaatca acaatgtgta catattgata
1020agtagtagta tatatgtaaa ggtctcacgt ctctataata aagtatgact
cgtcacgtga 1080cctcctcttc ttcgcagaga cagagatagg atgagacaga
aagaaaccaa caaaaccaaa 1140ccccaaaacc caagaaaaag agaaaaacac
tctcttctct tctctctctc tctttctatt 1200taagagactt cactgtctct
ctcagtcttt t 1231311548DNAArabidopsis thaliana 31ttcacgtgtt
tatttattta tttgggttat taaacataaa tcatgtaaat ctgaatcctg 60tggagatctc
tccctagttg atgaatagat atgatgaatt taattctttc atgaaataaa
120aatatatgaa acatatgtag cagaaaaaga agcatatcta tgaaacaaca
aacattcaaa 180aaaaaaagga aaacggaaaa ttattaatat gaaaactacg
gctttgactt gtagctgact 240acatttacga catatatata taaatggacc
ccactgagtg tctgcaaggt ctttacacaa 300cagtatcttc ttctgtttct
ttgactcttt gtgatcccta agcctaccca taatacgtgt 360ctacatttta
ttggattgtt tcgtgactct gtaatctttt ttataagaaa acaagtaata
420gtgaaattga agtaaatagc tcagcacaga aacttcgaca aaaataactc
acagattaga 480aaagaaaata tatgcataaa tagccatggt tcatttatga
acaatttatt cgttttttta 540gtttataatt tcattaaaac atgtttgtca
catcacattt catgtccttc ggctcctact 600acaacaacaa gtcactgtca
tctccattac ttccacttct gctcctttct ttattaactt 660gttcaaaaac
aattctaaga taaataacaa taaatgttgg tctctcttta ttatttcccg
720gctaaagaag gaggatgtct cgtattatcc gccatcaatg ctcttttgtt
tcctgtttct 780tgcaatttga atccctgaga atcctagccc acttatttac
tactttgcct tagctgtttt 840cgacatcaaa attttggtca tatgactcat
atcaatcttc aaatttgata aaatatgttc 900ccaattcaca aaaacaaaaa
agttttcgaa agctcaaaaa cctttaccat ttcaatagta 960gataggattc
ttttagattt gcatttcacg aaaagagaag aaaaaaaatc gaaaaatatt
1020tgcaatcatg attttttgtt tctgaaggag acctgtagtt gctgtcatga
acattaaata 1080caaatctaat aaatgttgta cgaattttgc gtgtaataaa
tggtcagggc cggctcgaag 1140ctcgctgatc gtcctttttt cgtgtctcta
tagcaacaca caatcgtatt tatttcaaac 1200tttttttact ttgtttccca
tccatcaaat ataagtataa aaatgtaaag aatcatcata 1260tatagatcgt
aaattcattg cttcctttgg ctttttattt catctagacg acgttaaaac
1320cagaccagac caaatacatt tatcattttt cccttttttc taaaattctc
tctttgattc 1380ctatcttctt ctctttattt tcactttgtg ctttctctgt
ctctcctatt atgagtctaa 1440aagtctacta gctgttcaat agttttgtct
ttctgtgttt cttcttcttc aaaaccgaaa 1500gaaattcaaa aagagtcttt
cgctgcttgt tagtggggtg aggaacaa 154832440DNAArabidopsis thaliana
32aaacccgaac ccgaaccaaa cccgaaccaa aatcttaaat tacccgaatg ggtcttaaat
60ttctaaatcc gaaaaacccg aacccaaaat acccaacccg aatctgaccc gaatatccga
120acgcctaatt tttctatgtt aatgaaatca attatatgac atgtttataa
agagaaataa 180attacggtga gaattaagcc catttacgtt acggaaataa
aacacccatt taaaaaagcc 240caacacgtga agcccatttc cgagtgcgtc
ccacatttac tccaacggtc gaatcgactc 300aaacattcaa aatacaaaaa
cgctatcttt atcgtcttcc tctgtctctc tctcacaaca 360cataacgttc
aaatcctctc tctctctatc tcgtctctta tctctagatc taaaaatctc
420ttctttcctc aatctctgtt 44033317DNAArabidopsis thaliana
33acagacattt acttatacgg ttattgaggt tgaactggac cggagtagca ataaattatc
60ggttcagttt gggagatcaa accgtttaaa agaaaataat ttgaaatggc cacgcagaat
120acgagggtct gaggattgta cctcctttct ctgcaaaaac ttaaacgttg
atttgactca 180agcgtcaagg taaggtactc tctcttcata caacatttta
gctttacttt ttctctttac 240tcttctctct ctctttctct ttctctttct
ctttcactcg ttctctctca ctcactctct 300tcacacacag atccaag
317343912DNAArabidopsis thaliana 34acaccaataa aaatacacag caataaaatc
gctacgtata tatatatata atatgtatta 60tctattacaa gatagtaata gagtatagca
agttgtatca tctaacaaac tatgcgaata 120aaatttgaac attgtgacat
gtagatgtag tgtaatttag ctaagtgctt atcatcagta 180acatagaccg
acttaacttt ttacgaaaaa aaaaaagtaa catagaccga aaaaatgcat
240atcgtaaatt taatggaaaa cacaatttac gataagtaaa aaacaaaaag
aaattacgat 300aagtcgagaa aaatgcaaca aattgagata aagtattgat
aaaaccatga aagtgtcggc 360gtatgtaaat gcggtgatta atgtgatcat
tagagcgtgt gtgttaaacg cggcggtttt 420agtggagatt gatcagctga
taacactctt accgggacga atctaattcc atattcatgg 480cttgttaaaa
cctaagacat acgcaatctc taatttgcta gtatagttag ttctatatta
540tttttcgact aataatgtaa acatatgatt attaagtcgc aaaaagagtg
cttaacaacc 600aaaaagtgga ttaattaact tggtgggaaa agttacaaaa
cctttaatga ttactctttg 660taccaagaat agtggcgaag cactataaga
gcagagaaaa gaagctcaat aatgtactaa 720aagttgtaga tttttacagc
ttaaatacac caaaattaat agaaaagttg gtaatttttt 780aattcatggc
tactgattta gattttagaa aacaatagta gtatcattgt cacatcttaa
840acacacaata ggtatgtttt aaatcaaagg ccgtagttaa tttgtcaaaa
atgtatgcat 900ttggtatttg gatgtctccg aaaggatgga tatatggact
tgttagataa tttcatacct 960cagtatcaat agtcatggag cccaaattgc
tcaaaaacat atttttaatt ccaagacttt 1020gatgaagacg taataatgag
tccaatgggc catcagatac aatgttcgga atttaacggg 1080tttgttagtt
ataagtattg ggcttgacct atctggttca atgatatgta ggaacaaccc
1140aatttgcaaa gctttattaa aagactcttt agttgtcgtc aaggtttaac
ttgtagtagt 1200tggtaagaaa ttctacgtga aataggcaac attacaaaaa
caaaaatcaa ttcgaaatca 1260tacaaaacga aaccaagtag taaccaacta
cactattatg acattaatga ttagacattc 1320ccaaatcata caagttcctg
tcatgaagga aacaatggtc cgtatttgca aacgattaca 1380aaaattcaaa
ccaaaaatga aaaaacgagt taaattattt ggtttataaa aatagtaatg
1440tcaacagaag actagattgg gaaacctgaa gcgaacagag cttttaaaaa
cgagtttgaa 1500cggctgggat catttggtac aatacccacc gtaagtttgt
ttaccctagg gatgcaagcc 1560aaaggcccaa atcagttact acttactgct
acaaccatcg tctcagcttt ttgtctcagc 1620tttttactaa tgaagcatac
aatttcttgg gcatgtcaca tctcgacacg tgtccactat 1680tctcttctct
tattggctac tcgttcgtag gcttctgtta atagatgatc tctctataac
1740tctaacagtc ttttctttct ctttatttcg ttttggtatt ttaagtttca
aattgaaaat 1800aataggagga aaagtctagt tttaaatatt gtttttttac
aagtgaacgt gaaccaattt 1860acctcttttt ttttatatat cctatcggct
aatctggtta gtatcggtag aaatgcaccg 1920aggtgctaca gagattaatg
ctagggatag tcagaccgct tgtatttctg actatcaagt 1980aaatctacgc
ccaactcaca tatttcccaa acaaatgtga tttttttttt tttttttttt
2040tttttttttt ttttgtaaca aatgtgattt tgttttcaag gaaaatagaa
cttacgtttg 2100ggaatttcac ccttcactaa agcttccttc tgccattaga
ccacaaaggc ttgggcaatt 2160taccattttt gtaaaagtag aaaacaaaat
gcctaaaatg ttcatacttc attacatcaa 2220caaggttatg cccacgatat
agaggcatgt aacatttata tatatagtgg aagaagccta 2280cgagctttat
taataagtat aaactctgat tattaggtaa ataaattact taaaacgatt
2340actcaactga caaaaccgta gttgaataat aaggttacta tgaataccga
ttgaatattg 2400caaagccgga attgaaaaat atataacaga tcaaatgttc
aagtgtggtc ataattctca 2460cataggtcat atagctgaac ccatgcatct
atttactagt ctatagaaag tactagagac 2520gcatacagct gaacctactc
tattctttta ttaattttgg ttctcgtgga tacaaaattc 2580ctccaacatt
tattagaacg aataaaacca atatgatgat gattagttat tggtaaacat
2640ataaacgttg agtaaacttc aaaatagatt gaagtactat taagacttgc
attttttccc 2700cttgggttat attcttgaat cgtttcgaag tattttaact
ttcaagaata gaaggttcct 2760caactataaa caattacatt aatcaaaacc
atttctatgt aaacaacata atttttgtat 2820attttagtct tccccaaaag
tttgaccgat agggcggttt agaccgtata gtacgactgt 2880acaacaaaaa
ggactctgga gacctaaaga tccaaaacta tgcaaaataa agatacggtc
2940ggaccaattt aatctaacaa aaccaaatcc ttatactaaa ctatttaccg
atacatttcc 3000atataacaca gtacacacaa ttaaatcaaa cattattgga
agaacaagat agaatattgg 3060cttaatctcg aacgattaga gttatcctag
agcctcggag cttttgtcac atataatata 3120aactatggta tatataaaca
tgactctcat ttgtatttat cgcaaggtac aattccacca 3180atttttttcg
tcccactcat acagctttaa ttgtgaaatc aatccataaa aaaccaacat
3240gtgacatggt ctctataact ataactataa gatagtaaaa aattcacatc
aacataaaag 3300aaaaccaatc atattggcta aaaaaaacta acggtcgaaa
aacgtataac cacaaaacca 3360aaccggtcca accggtgtcc ccaatcacta
tcaaagcatt aactaacttt cacaaggaaa 3420agcatagttc agtttctcta
catcgcttcc catcctctta accctgttta ctcgaatcat 3480ccaccgttgg
atcaaacacg cgctacaaat ctagcgcgtg accgaggttt ttacacagtg
3540gaatattacc atgcattgga aagcggcgtc tacaacaaac ggcgggtcat
gtcaccgtca 3600aaatcaacct ttcttaattc ctaacgccgt tacttatctc
cgtttactaa aaatgttaat 3660gcgtgtgaga gtgaagatca tatactaatt
agaagtggct aatgttttaa cgtgacatta 3720ttatcatagt taatggttcg
atcagagttt taagtagtaa atgatataag tgtgtgtata 3780taattgcata
catatatact ctcacactct gacagatttg tcgtggtctt agtattctct
3840ttcatggcta gttatatagg gctctagtac attatctctc tctccccatt
tctctgtctc 3900tctcttcttt aa 3912352000DNAArabidopsis thaliana
35atctttatgg tcaccgagtc tactgatata attttactgt cgcagtttgt ttccactact
60taagtttcta taatttcaca gtttgaaaga aaaattactg gttattcagc taaattacaa
120agattagttt aattagttta gccagtataa tgttttagta aagtattaaa
cggcattttt 180cgttgggaga attatgttat ggtataatct actaatacat
acttttacac atatatcaaa 240aagtttgacc atagtaggta gtacaacata
gaagaatcaa gatcggaacc agcaaggaag 300aagatacggt cggtccatat
taagctaaga ggaccaacgt aactcgatat atatttttct 360gttctctacg
ttaccaccat ataaatttta atattgaaaa aatcatcttt tggcattgtg
420tttgatgtcg gattcggaat atggaaagag gagagatatg agattttggc
acaaaggaag 480ctgccaaagc attagggcaa ccgagtagta acgagatcaa
acatcgtttc aatcggacgg 540tcggggtttg accaatattt ctcggatatc
ttttggaccc tacgttctga cttgaacttg 600atcagtcact tcagtacctt
agttttcatt ttcaatgtga tcatgagttt ttttttacat 660gttagcttca
aaacaaatac taatattcat taactatgga tcggcatagt tttcatgtaa
720tcagctgagc gtttatcata ttgattgaag ctaacatgta aaattctcat
gatcacattg 780acttttgcct acaaatttta aaagagtata caaataattg
cttaatgaag atagcttcca 840tagagaaaga gtaacagctt tatacggagg
catagcttta gacacgatct ctgctcttgt 900gttttttgtt taacactgaa
tccacagtga aattactgct catttttttt cattttttat 960tacatttttt
tttttacttt tttatttatt acaatctaca gttctaccaa cttattcaac
1020ctagtggtac catatcgacc ccaaaattaa tcaatctaat tacaaggtag
aaatagaaag 1080attttatcaa aggagacaac tctgatcgat aatatgttgc
aataaaacca tgaaaaactg 1140taaaaaatat tgaaagctga agaaaaattt
tcaaatcgat aaaaggatag tactaaacca 1200atccggtttg tggcatcttt
ttcacccaat cactatcaaa gcattaacta aaaatcacaa 1260ggaagagcat
aatttgattc tctacatcgc agtccacgat aggatttctc tatccaccgt
1320tggatcaaat ttaataatga tgcacgcgcg cgtcaacggg attttaccca
gcaaaggaat 1380gtcttttcac cggactctta aaagacgttc ttcttttttc
acctttgcat tggaaaacgg 1440cgtttcttct tagaaccgtc gccgtcaaat
catacggcct aataaatctc cgtttaacgc 1500cgttacttta ccgttaagta
ctaaaaaaac aaaaaaaaat catttcgatc actgtctcat 1560taagatgatc
ggagatgttt tagcagggtt taacaagtga tgatagtaat gtatgtatat
1620atgttactga cattattttg tcgttgtcta ataggagggt actaaagttt
ctctctctca 1680tggcgtcgga gctcagcctc tagtaatgta gactgtcctc
tctttctctc tctcttcttt 1740aaacatctct gctctgtttt ccttccagtt
cacgctaatc tcctgtgtcg gtcccctctc 1800tcttttcctt tggtctctcc
caacaatggc agaacgactt tgtacccttc ttttgctctt 1860tgtttgaatt
tcgtttcttg ctacaaagct tcaaaggatc tgacttttcc ctaaacagaa
1920aaagaggtct ttaaccaaaa aaggttgtta cttgttttct gggtttcgtg
gtgttactct 1980tgaggaagaa gaagaagaag 2000361470DNAArabidopsis
thaliana 36ctacctaata taactagcta gggatttcta ctcttgtttt cataatcgat
ctacggacat 60ttctcggaac gtggtcaaga ttcatgagtc ttctgttttt tatgtctctg
ttcaatttgg 120tttagagatt agtatgctta tttgtttatt tcatatatgg
ttatgagagg agaggctaat 180ggcatatact ctgatgtttg tgatggctgc
taatatcgtt gaggagttat tcacgttgtt 240tcatgcgcaa aaatcaacag
aaaaaattct gattatgagc caactctgtg aacccttata 300gtgcgcccag
aggtttgcga ggcaaaatcc cgatgaacca gaaggaattt tagatctcta
360tcaacaataa ctatgatgga gctcgtttaa attcatcaca gcgacaacat
cattaggctg 420cccaacgtct atgtctcctg gaggtgatgg tacttgatct
ctcaaccaat tttcttgaaa 480atatcatgcc ttgtgagcgc tttcatattg
cgcctaaaat acccaatacg caatgaacct 540acttccaaag gcatagaaaa
aaaactgata atgataatga gatttgtcac tatacttatc 600ctatccctac
ataggagccg tttgattgtt tagtccatgt tttcattttg tttagtctaa
660tgctatataa cttttcttta tcagtctatt gttatatgac ttatatatat
ctcaagagat 720aaggccaata aatcttcttc ttaattatat ctgaagactc
aaaacatatt ttgagtttaa 780taaaataaat aacgtccaaa tgctacatac
aaacggacca aattcatgga ggtataaatt 840taaattattt tttgttccaa
agtgtatgca gtgatttatt gatgaatgcg atagagcggc 900gaaagagaat
aatcgtcacc tagaagacaa attgatcggc cgtacatata tacataaata
960caaacctgcc acttcacatg tcacccacct ttaagcaccc ccttcacata
catactttct 1020ataacaaaaa tatcagcttc tagttcatat ttatgttaca
ataactcgag tgaatcatac 1080taaaaaaatg taatgctttc tctaaatagg
agataaaatg caccctccga cctaactaaa 1140gattccttat tttagctatt
taagacatat tgcacatgta tagagataca taaacacata 1200tgcaatatgc
acatcttcta tacattgaaa aaagctgatc ttgcaaatat ttgtcttaca
1260caacacaagc gaccaaagcg atgcgtttcc caatgataag gttacgacat
acttacacga 1320ctctctctat tgtctcgtct ctttctttcc tcatccctct
cctttgtctc ctttcactct 1380atttttcact tttcagaata cttttacgta
aaaatcatgg acatgtcatt gtctccaccc 1440tactatactc tttttttgtt
ctttttgttt 1470371793DNAArabidopsis thaliana 37ttggttgtct
ggcatcatca ttttgtaccg tttctcccaa agtaagaaac ggtacaatct 60tctcttatat
agatttcata ccccaaaacc ctaaattcat tagggttttc aaaaaaaaaa
120atcacgttta cctctaaacc aatcttctct tatatagata aatcataacg
tttgtttgat 180ttttcagttt tctacttaac caatattaaa ctaaagtcga
attgagatga gtggtagcaa 240accacagttt aattaagaag ttaattatag
gccacatgat tgagcaagcc tttttgtttt 300gtaacacatc ttatcagctg
cttaaaattt tggctgcctc ccattggcca actggtctaa 360acatcattgc
attggcattc tcataatcaa tcaatctaat gagaaacttt gaatatttat
420gaaaaactga ataacaacat aacataaacg aacaatgtaa aaaagaaaaa
cacaaaaaaa 480aaaacacttt aaaaaacaaa aaccaaaaac tcttaaacta
taaactcatg aacacttagt 540gatgaggtct gaaagggtgt aaccaccacc
tgttgtcaat aggtgacaac ttcttcttgg 600gaacattcgg gaaagtgaag
gcttaggtga cggttgtctt aaaagtctct tttagttaat 660tcatcgtatt
ttcgatgggc attacgtttg atgcataaag gcccatatgg gctatacatg
720tactgcgttt gagtggcttt ctaaggattg atgtattgtc tctatgagag
tattcgttta 780actcatggag atctactctc cacgatattt tctgtaaact
tttctttttg ttgattagat 840aaatagaaaa ttgtgtagag cgaaactttt
aatgaattaa aatgcggaag cgattaaagc 900atgaatagat aaattggaca
agagattaaa cgagggatca tctagttttt acactgatca 960ctagtcatct
gcttgcagaa gaagtatatc attaatcaag caaaacgagg gcataaattt
1020cttacaaata acttttacag taggttaatg attttttaat aacttgtcca
tttcacatgc 1080atgtgtatct ttgtactata catgctaagt gtttcattaa
tcaagataaa cgtgtctacg 1140aataacttaa aacagtacaa cttccctaaa
aaattcatta aatgaaaggt ttttatagat 1200tatacattgc acggtacggt
tcggttacca ttcgaagtct aaaaagagaa tgacggttct 1260gataatgctt
ttaatcgctt ttgtattgta aatcattaaa acagtaagcc ggataccgaa
1320ttactaatca gacccaaaaa agaatctata ggaaaaatat caactgaaga
gcgggtaggc 1380ttgaccttga aaggaagaat ggtgagcgag cggtggatag
atatgtaata aattgtaacg 1440ctttcaaaat gtcaaagtca caagtcacat
tactcacgag ccaacactaa ccatgcaact 1500tttgttttga cattttccta
aactttaggt ataaaatacc cgcgtaataa ataacctctt 1560cataattggg
tccacccact cacaggtcca cacataagga accgaaaaag gtaaaattca
1620aaaacttaca aagtttttta gagatgatgt ggtgaagtat tgcattaatg
gaataatggg 1680aaaagaaagt aattgcaacg tacgtataga ttaatccatt
gacacaaatg aaaagtttct 1740ttctatttaa tgtacacaac aaaggttctc
ttcagagtaa tttaggggaa aaa 179338257DNAArabidopsis thaliana
38acacaacagg tccctacatt tgtacaatct cctctcttta aagactctct ctctttctct
60ctccatctct atcttactct gtatttctgt cgtctgagca ctcaatgaaa ccactgtaaa
120tttccgccag aatttgatgt gatggaacga taaaaatcat tttttctcgg
ttaaagtaaa 180aaaacaaaaa caaatttctg tagaaatcat aataaaagaa
agaaaaaaaa tctaatgtcg 240gtacataata cggttct 257
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