U.S. patent application number 14/647536 was filed with the patent office on 2015-10-29 for transgenic plants with enhanced traits.
The applicant listed for this patent is MONSANTO TECHNOLOGY LLC. Invention is credited to Mark S. Abad, Monnanda S. Rajani, Tyamagondlu V. Venkatesh, Kammaradi R. Vidya.
Application Number | 20150307894 14/647536 |
Document ID | / |
Family ID | 50828328 |
Filed Date | 2015-10-29 |
United States Patent
Application |
20150307894 |
Kind Code |
A1 |
Abad; Mark S. ; et
al. |
October 29, 2015 |
Transgenic Plants With Enhanced Traits
Abstract
This disclosure provides plants having enhanced traits such as
increased yield, increased nitrogen use efficiency and increased
water use efficiency; propagules, progeny and field crops of such
plants; and methods of making and using such plants. This
disclosure also provides methods of producing seed from such
plants, growing such seed and selecting progeny plants with the
composition, or with enhanced traits. Also disclosed are plants
with altered phenotypes which are useful for screening and
selecting events for the desired enhanced trait.
Inventors: |
Abad; Mark S.; (Webster
Groves, MO) ; Rajani; Monnanda S.; (Chesterfield,
MO) ; Venkatesh; Tyamagondlu V.; (St. Louis, MO)
; Vidya; Kammaradi R.; (Bangalore, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MONSANTO TECHNOLOGY LLC |
St. Louis |
MO |
US |
|
|
Family ID: |
50828328 |
Appl. No.: |
14/647536 |
Filed: |
March 6, 2013 |
PCT Filed: |
March 6, 2013 |
PCT NO: |
PCT/US13/29245 |
371 Date: |
May 27, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61730765 |
Nov 28, 2012 |
|
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Current U.S.
Class: |
800/260 ;
435/419; 800/278; 800/290; 800/298; 800/305; 800/306; 800/307;
800/308; 800/309; 800/310; 800/312; 800/313; 800/314; 800/315;
800/316; 800/317; 800/317.1; 800/317.2; 800/317.3; 800/317.4;
800/319; 800/320; 800/320.1; 800/320.2; 800/320.3; 800/322 |
Current CPC
Class: |
C12N 15/8271 20130101;
C12N 15/825 20130101; C12N 15/8269 20130101; C12N 15/8261 20130101;
C12N 15/8273 20130101; Y02A 40/146 20180101; C12N 15/8218 20130101;
C07K 14/415 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Claims
1. A plant comprising a recombinant DNA molecule comprising a
polynucleotide encoding a polypeptide, wherein the nucleotide
sequence of the polynucleotide is selected from the group
consisting of: a) a nucleotide sequence encoding a protein having
the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,
23, 24, 25, 26, 27, 28, or 29; and b) a nucleotide sequence
encoding a protein with 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%, at least 99% identity to SEQ ID NO: 2, 4, 6, 8,
10, 12, 14, 16, 23, 24, 25, 26, 27, 28 or 29; wherein said plant
has at least one enhanced trait as compared to a control plant.
2. A plant comprising a recombinant DNA molecule comprising a
polynucleotide, wherein the nucleotide sequence of the
polynucleotide is selected from the group consisting of: a) a
nucleotide sequence set forth as SEQ ID NO: 17, or 20; b) a
nucleotide sequence that suppresses at least one target gene set
forth as SEQ ID NO: 18, or 21; c) a nucleotide sequence that
expresses a RNA that suppresses the expression of a protein having
the amino acid sequence of SEQ ID NO: 19 or 22; d) a nucleotide
sequence with 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%, at least 99% identity to SEQ ID NO: 17 or 20; e) a
nucleotide sequence with 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%, at least 99% identity to SEQ ID NO: 18 or 21;
and f) a nucleotide sequence encoding a protein with 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%, at least 99%
identity to SEQ ID NO: 19 or 22; wherein said plant has at least
one enhanced trait as compared to a control plant.
3. The plant of claim 1, wherein said enhanced trait is selected
from the group consisting of increased yield, increased nitrogen
use efficiency, and increased water use efficiency.
4. The plant of claim 1, wherein said plant is a monocot plant or
is a member of the family Poaceae, wheat plant, maize plant, sweet
corn plant, rice plant, wild rice plant, barley plant, rye, millet
plant, sorghum plant, sugar cane plant, turfgrass plant, bamboo
plant, oat plant, brome-grass plant, Miscanthus plant, pampas grass
plant, switchgrass (Panicum) plant, and/or teosinte plant, or is a
member of the family Alliaceae, onion plant, leek plant, garlic
plant, or a member of the family Musaceae, banana plant; or wherein
the plant is a dicot plant or is a member of the family
Amaranthaceae, spinach plant, quinoa plant, a member of the family
Anacardiaceae, mango plant, a member of the family Asteraceae,
sunflower plant, endive plant, lettuce plant, artichoke plant, a
member of the family Bras sicaceae, Arabidopsis thaliana plant,
rape plant, oilseed rape plant, broccoli plant, Brussels sprouts
plant, cabbage plant, canola plant, cauliflower plant, kohlrabi
plant, turnip plant, radish plant, a member of the family
Bromeliaceae, pineapple plant, a member of the family Caricaceae,
papaya plant, a member of the family Chenopodiaceae, beet plant, a
member of the family Curcurbitaceae, melon plant, cantaloupe plant,
squash plant, watermelon plant, honeydew plant, cucumber plant,
pumpkin plant, a member of the family Dioscoreaceae, yam plant, a
member of the family Ericaceae, blueberry plant, a member of the
family Euphorbiaceae, cassava plant, a member of the family
Fabaceae, alfalfa plant, clover plant, peanut plant, a member of
the family Grossulariaceae, currant plant, a member of the family
Juglandaceae, walnut plant, a member of the family Lamiaceae, mint
plant, a member of the family Lauraceae, avocado plant, a member of
the family Leguminosae, soybean plant, bean plant, pea plant, a
member of the family Malvaceae, cotton plant, a member of the
family Marantaceae, arrowroot plant, a member of the family
Myrtaceae, guava plant, eucalyptus plant, a member of the family
Rosaceae, peach plant, apple plant, cherry plant, plum plant, pear
plant, prune plant, blackberry plant, raspberry plant, strawberry
plant, a member of the family Rubiaceae, coffee plant, a member of
the family Rutaceae, citrus plant, orange plant, lemon plant,
grapefruit plant, tangerine plant, a member of the family
Salicaceae, poplar plant, willow plant, a member of the family
Solanaceae, potato plant, sweet potato plant, tomato plant,
Capsicum plant, tobacco plant, tomatillo plant, eggplant plant,
Atropa belladona plant, Datura stramonium plant, a member of the
family Vitaceae, grape plant, a member of the family Umbelliferae,
carrot plant; or wherein the plant is a member of the family
Pinaceae, cedar plant, fir plant, hemlock plant, larch plant, pine
plant, or spruce plant.
5. The plant of claim 2, wherein said enhanced trait is selected
from the group consisting of increased yield, increased nitrogen
use efficiency, and increased water use efficiency.
6. The plant of claim 2, wherein said plant is a monocot plant or
is a member of the family Poaceae, wheat plant, maize plant, sweet
corn plant, rice plant, wild rice plant, barley plant, rye, millet
plant, sorghum plant, sugar cane plant, turfgrass plant, bamboo
plant, oats plant, brome-grass plant, Miscanthus plant, pampas
grass plant, switchgrass (Panicum) plant, and/or teosinte plant, or
is a member of the family Alliaceae, onion plant, leek plant,
garlic plant, or a member of the family Musaceae, banana plant; or
wherein the plant is a dicot plant or is a member of the family
Amaranthaceae, spinach plant, quinoa plant, a member of the family
Anacardiaceae, mango plant, a member of the family Asteraceae,
sunflower plant, endive plant, lettuce plant, artichoke plant, a
member of the family Brassicaceae, Arabidopsis thaliana plant, rape
plant, oilseed rape plant, broccoli plant, Brussels sprouts plant,
cabbage plant, canola plant, cauliflower plant, kohlrabi plant,
turnip plant, radish plant, a member of the family Bromeliaceae,
pineapple plant, a member of the family Caricaceae, papaya plant, a
member of the family Chenopodiaceae, beet plant, a member of the
family Curcurbitaceae, melon plant, cantaloupe plant, squash plant,
watermelon plant, honeydew plant, cucumber plant, pumpkin plant, a
member of the family Dioscoreaceae, yam plant, a member of the
family Ericaceae, blueberry plant, a member of the family
Euphorbiaceae, cassava plant, a member of the family Fabaceae,
alfalfa plant, clover plant, peanut plant, a member of the family
Grossulariaceae, currant plant, a member of the family
Juglandaceae, walnut plant, a member of the family Lamiaceae, mint
plant, a member of the family Lauraceae, avocado plant, a member of
the family Leguminosae, soybean plant, bean plant, pea plant, a
member of the family Malvaceae, cotton plant, a member of the
family Marantaceae, arrowroot plant, a member of the family
Myrtaceae, guava plant, eucalyptus plant, a member of the family
Rosaceae, peach plant, apple plant, cherry plant, plum plant, pear
plant, prune plant, blackberry plant, raspberry plant, strawberry
plant, a member of the family Rubiaceae, coffee plant, a member of
the family Rutaceae, citrus plant, orange plant, lemon plant,
grapefruit plant, tangerine plant, a member of the family
Salicaceae, poplar plant, willow plant, a member of the family
Solanaceae, potato plant, sweet potato plant, tomato plant,
Capsicum plant, tobacco plant, tomatillo plant, eggplant plant,
Atropa belladona plant, Datura stramonium plant, a member of the
family Vitaceae, grape plant, a member of the family Umbelliferae,
carrot plant; or wherein the plant is a member of the family
Pinaceae, cedar plant, fir plant, hemlock plant, larch plant, pine
plant, or spruce plant.
7. The plant of claim 1, wherein the recombinant DNA molecule
further comprises a promoter that is operably linked to the
polynucleotide encoding a polypeptide, wherein said promoter is
selected from the group consisting of a constitutive, inducible,
tissue specific, diurnally regulated, tissue enhanced, and cell
specific promoter.
8. The plant of claim 2, wherein the recombinant DNA molecule
further comprises a promoter that is operably linked to the
polynucleotide that suppresses at least one target gene, wherein
said promoter is selected from the group consisting of a
constitutive, inducible, tissue specific, diurnally regulated,
tissue enhanced, and cell specific promoter.
9. The plant of claim 1, wherein said plant is selected from the
group consisting of corn, soybean, cotton, canola, rice, barley,
oats, wheat, turf grass, alfalfa, sugar beet, sunflower, quinoa and
sugar cane.
10. The plant of claim 1, wherein said plant is a propagule
selected from the group consisting of a cell, pollen, ovule,
flower, embryo, leaf, root, stem, shoot, meristem, grain and
seed.
11. The plant of claim 2, wherein said plant is selected from the
group consisting of corn, soybean, cotton, canola, rice, barley,
oats, wheat, turf grass, alfalfa, sugar beet, sunflower, quinoa and
sugar cane.
12. The plant of claim 2, wherein said plant is a propagule
selected from the group consisting of a cell, pollen, ovule,
flower, embryo, leaf, root, stem, shoot, meristem, grain and
seed.
13. A method for producing a plant comprising: introducing into a
plant cell a recombinant DNA molecule comprising a polynucleotide
encoding a polypeptide, wherein the nucleotide sequence of the
polynucleotide is selected from the group consisting of: a) a
nucleotide sequence encoding a protein having the amino acid
sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 23, 24, 25, 26,
27, 28, or 29; and b) a nucleotide sequence encoding a protein with
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%, at
least 99% identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 23,
24, 25, 26, 27, 28, or 29; and growing a plant from said plant
cell.
14. A method for producing a plant comprising: introducing into a
plant cell a recombinant DNA molecule comprising a nucleotide
sequence, wherein said nucleotide sequence suppresses at least one
target gene encoding at least one target protein, and wherein said
nucleotide sequence is selected from the group consisting of: a) a
nucleotide sequence set forth as SEQ ID NO: 17, or 20; b) a
nucleotide sequence that suppresses at least one target gene set
forth as SEQ ID NO: 18, or 21; and c) a nucleotide sequence that
suppresses at least one target gene encoding a target protein set
forth as SEQ ID NO: 19, or 22; and growing a plant from said plant
cell.
15. The method of claim 13, further comprising selecting the plant
comprising said recombinant DNA molecule with at least one enhanced
trait selected from a group consisting of increased yield,
increased nitrogen use efficiency, and increased water use
efficiency as compared to a control plant.
16. The method of claim 14, further comprising selecting the plant
comprising said recombinant DNA molecule with at least one enhanced
trait selected from a group consisting of increased yield,
increased nitrogen use efficiency, and increased water use
efficiency as compared to a control plant.
17. A method for increasing yield, increasing nitrogen use
efficiency, or increasing water use efficiency in a plant
comprising: a) crossing the plant of claim 1 with itself, a second
plant from the same plant line, a wild type plant, or a second
plant from a different line of plants to produce a seed; b) growing
said seed to produce a plurality of progeny plants; and c)
selecting a progeny plant with increased yield, increased nitrogen
use efficiency, or increased water use efficiency relative to a
plant not having said recombinant DNA molecule.
18. A method for increasing yield, increasing nitrogen use
efficiency, or increasing water use efficiency in a plant
comprising: a) crossing the plant of claim 2 with itself, a second
plant from the same plant line, a wild type plant, or a second
plant from a different line of plants to produce a seed; b) growing
said seed to produce a plurality of progeny plants; and c)
selecting a progeny plant with increased yield, increased nitrogen
use efficiency, or increased water use efficiency relative to a
plant not having said recombinant DNA molecule.
19. A plant comprising a recombinant DNA molecule comprising a
polynucleotide encoding a polypeptide, wherein the nucleotide
sequence of the polynucleotide is selected from the group
consisting of: a) a nucleotide sequence encoding a protein having
the amino acid sequence of SEQ ID NO: 12, or 16; and b) a
nucleotide sequence encoding a protein with 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%, at least 99% identity to SEQ
ID NO: 12, or 16; and wherein said plant has at least one phenotype
selected from the group consisting of anthocyanin score, biomass,
canopy area, chlorophyll score, plant height, water applied, water
content score and water use efficiency that is altered for said
plant as compared to a control plant.
20. A plant comprising a recombinant DNA molecule comprising a
nucleotide sequence, wherein said nucleotide sequence suppresses at
least one target gene encoding at least one target protein, and
wherein said nucleotide sequence is selected from the group
consisting of: a) a nucleotide sequence set forth as SEQ ID NO: 20;
b) a nucleotide sequence that suppresses at least one target gene
set forth as SEQ ID NO: 21; c) a nucleotide sequence that
suppresses at least one target gene encoding a target protein set
forth as SEQ ID NO: 22; d) a nucleotide sequence with 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%, at least 99%
identity to SEQ ID NO: 20; e) a nucleotide sequence that suppresses
at least one target gene with 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%, at least 99% identity to SEQ ID NO: 21;
and f) a nucleotide sequence that suppresses at least one target
gene encoding a target protein with 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%, at least 99% identity to SEQ ID NO: 22;
wherein said plant has at least one phenotype selected from the
group consisting of anthocyanin score, biomass, canopy area,
chlorophyll score, plant height, water applied, water content score
and water use efficiency that is altered for said plant as compared
to a control plant.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35USC .sctn.119(e)
of U.S. provisional application Ser. No. 61/730,765, filed on Nov.
28, 2012, and is herein incorporated by reference in its
entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] The sequence listing file named "38-21.sub.--59496_B.txt",
which is 50,544 bytes (measured in MS-WINDOWS) and was created on
Mar. 5, 2013, is filed herewith and incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0003] Disclosed herein are plants having enhanced traits such as
increased yield, increased nitrogen use efficiency and increased
water use efficiency; propagules, progenies and field crops of such
plants; and methods of making and using such plants. Also disclosed
are methods of producing seed from such plants, growing such seed
and/or selecting progeny plants with enhanced traits.
SUMMARY OF THE INVENTION
[0004] An aspect of this disclosure provides a plant comprising a
recombinant DNA molecule comprising a polynucleotide encoding a
polypeptide, wherein the nucleotide sequence of the polynucleotide
is selected from the group consisting of: a nucleotide sequence
encoding a protein having the amino acid sequence of SEQ ID NO: 2,
4, 6, 8, 10, 12, 14, 16; 23, 24, 25, 26, 27, 28, or 29 and b) a
nucleotide sequence encoding a protein with 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%, at least 99% identity to SEQ
ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 23, 24, 25, 26, 27, 28 or 29;
and wherein the plant has at least one enhanced trait as compared
to a control plant.
[0005] Another aspect of this disclosure also provides a plant
comprising a recombinant DNA molecule comprising a polynucleotide,
wherein the nucleotide sequence of the polynucleotide is selected
from the group consisting of: a) a nucleotide sequence as set forth
as SEQ ID NO: 17 or 20; b) a nucleotide sequence that suppresses at
least one target gene set forth as SEQ ID NO: 18 or 21; c) a
nucleotide sequences that expresses an RNA that suppresses the
expression of a protein having the amino acid sequence of SEQ ID
NO: 19 or 22; d) a nucleotide sequence with 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%, at least 99% identity for
SEQ ID NO: 17 or 20; e) a nucleotide sequence with 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%, at least 99% identity to
SEQ ID NO: 18 or 21; and f) a nucleotide sequence encoding a
protein with 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%, at least 99% identity to SEQ ID NO: 19 or 22; and
wherein the plant has at least one enhanced trait as compared to a
control plant.
[0006] Another aspect this invention also provides a plant, wherein
the plant has at least one enhanced trait as compared to a control
plant, and wherein said enhanced trait is selected from the group
consisting of increased yield, increased nitrogen use efficiency,
and increased water use efficiency.
[0007] Yet another aspect of this disclosure provides a plant
comprising a recombinant DNA molecule of the disclosure, wherein
said plant is a monocot plant or is a member of the family Poaceae,
wheat plant, maize plant, sweet corn plant, rice plant, wild rice
plant, barley plant, rye, millet plant, sorghum plant, sugar cane
plant, turfgrass plant, bamboo plant, oat plant, brome-grass plant,
Miscanthus plant, pampas grass plant, switchgrass (Panicum) plant,
and/or teosinte plant, or is a member of the family Alliaceae,
onion plant, leek plant, garlic plant, or a member of the family
Musaceae, banana plant; or wherein the plant is a dicot plant or is
a member of the family Amaranthaceae, spinach plant, quinoa plant,
a member of the family Anacardiaceae, mango plant, a member of the
family Asteraceae, sunflower plant, endive plant, lettuce plant,
artichoke plant, a member of the family Brassicaceae, Arabidopsis
thaliana plant, rape plant, oilseed rape plant, broccoli plant,
Brussels sprouts plant, cabbage plant, canola plant, cauliflower
plant, kohlrabi plant, turnip plant, radish plant, a member of the
family Bromeliaceae, pineapple plant, a member of the family
Caricaceae, papaya plant, a member of the family Chenopodiaceae,
beet plant, a member of the family Curcurbitaceae, melon plant,
cantaloupe plant, squash plant, watermelon plant, honeydew plant,
cucumber plant, pumpkin plant, a member of the family
Dioscoreaceae, yam plant, a member of the family Ericaceae,
blueberry plant, a member of the family Euphorbiaceae, cassava
plant, a member of the family Fabaceae, alfalfa plant, clover
plant, peanut plant, a member of the family Grossulariaceae,
currant plant, a member of the family Juglandaceae, walnut plant, a
member of the family Lamiaceae, mint plant, a member of the family
Lauraceae, avocado plant, a member of the family Leguminosae,
soybean plant, bean plant, pea plant, a member of the family
Malvaceae, cotton plant, a member of the family Marantaceae,
arrowroot plant, a member of the family Myrtaceae, guava plant,
eucalyptus plant, a member of the family Rosaceae, peach plant,
apple plant, cherry plant, plum plant, pear plant, prune plant,
blackberry plant, raspberry plant, strawberry plant, a member of
the family Rubiaceae, coffee plant, a member of the family
Rutaceae, citrus plant, orange plant, lemon plant, grapefruit
plant, tangerine plant, a member of the family Salicaceae, poplar
plant, willow plant, a member of the family Solanaceae, potato
plant, sweet potato plant, tomato plant, Capsicum plant, tobacco
plant, tomatillo plant, eggplant plant, Atropa belladona plant,
Datura stramonium plant, a member of the family Vitaceae, grape
plant, a member of the family Umbelliferae, carrot plant; or
wherein the plant is a member of the family Pinaceae, cedar plant,
fir plant, hemlock plant, larch plant, pine plant, or spruce
plant.
[0008] Another aspect of this disclosure provides a plant
comprising a recombinant DNA molecule of the disclosure, wherein
the recombinant DNA molecule further comprises a promoter that is
operably linked to the polynucleotide encoding a polypeptide,
wherein said promoter is selected from the group consisting of a
constitutive, inducible, tissue specific, diurnally regulated,
tissue enhanced, and cell specific promoter.
[0009] In yet another aspect of this disclosure provides a plant
comprising a recombinant DNA molecule of the disclosure, wherein
said plant is a progeny, propagule, or field crop. Such field crop
is selected from the group consisting of corn, soybean, cotton,
canola, rice, barley, oat, wheat, turf grass, alfalfa, sugar beet,
sunflower, quinoa and sugar cane.
[0010] Yet in another aspect, this disclosure provides a plant
comprising a recombinant DNA molecule of the disclosure, wherein
said plant is a progeny, propagule, or field crop. Such propagule
is selected from the group consisting of a cell, pollen, ovule,
flower, embryo, leaf, root, stem, shoot, meristem, grain and
seed.
[0011] Another aspect of this disclosure provides a method for
producing a plant comprising: introducing into a plant cell a
recombinant DNA comprising a polynucleotide encoding a polypeptide,
wherein the nucleotide sequence of the polynucleotide is selected
from the group consisting of: a) a nucleotide sequence encoding a
protein having the amino acid sequence of SEQ ID NO: 2, 4, 6, 8,
10, 12, 14, 16, 23, 24, 25, 26, 27, 28 or 29; and b) a nucleotide
sequence encoding a protein with 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%, at least 99% identity to SEQ ID NO: 2,
4, 6, 8, 10, 12, 14, 16, 23, 24, 25, 26, 27, 28, or 29; and growing
a plant from the plant cell.
[0012] Another aspect of this disclosure provides a method for
producing a plant comprising: introducing into a plant cell a
recombinant DNA comprising a polynucleotide, wherein the nucleotide
sequence of the polynucleotide suppresses at least one target gene
encoding at least one target protein, and wherein the nucleotide
sequence is selected from the group consisting of: a) a nucleotide
sequence set forth as SEQ ID NO: 17 or 20; b) a nucleotide sequence
that suppresses at least one target gene set forth as SEQ ID NO: 18
or 21; c) a nucleotide sequence that suppresses at least one target
gene encoding a target protein set forth as SEQ ID NO: 19 or 22;
and growing a plant from the plant cell.
[0013] Another aspect of this disclosure provides a method of
producing a plant comprising: introducing into a plant cell a
recombinant DNA molecule of the disclosure; growing a plant from
the plant cell. Still another aspect of this disclose further
comprises selecting a plant comprising a recombinant DNA molecule
of this disclosure, or with at least one enhanced trait selected
from increased yield, increased nitrogen use efficiency, and
increased water use efficiency as compared to a control plant.
[0014] Another aspect of this disclosure provides a method of
increasing yield, increasing nitrogen use efficiency, or increasing
water use efficiency in a plant comprising: producing a plant
comprising a recombinant DNA of the disclosure wherein the plant
has an enhanced trait selected from the group consisting of
increased yield, increased nitrogen use efficiency, and increased
water use efficiency as compared to a control plant; crossing the
plant with itself, a second plant from the same plant line, a wild
type plant, or a second plant from a different line of plants to
produce a seed; growing the seed to produce a plurality of progeny
plants, and selecting a progeny plant with increased yield,
increased nitrogen use efficiency, or increased water use
efficiency.
[0015] Yet another aspect of this disclosure provides a plant
comprising a recombinant DNA molecule comprising a polynucleotide
encoding a polypeptide, wherein the nucleotide sequence of the
polynucleotide is selected from the group consisting of: a) a
nucleotide sequence encoding a protein having the amino acid
sequence of SEQ ID NO: 12, or 16; and b) a nucleotide sequence
encoding a protein with 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%, at least 99% identity to SEQ ID NO: 12, or 16,
wherein the plant has at least one phenotype selected from the
group consisting of anthocyanin score, biomass, canopy area,
chlorophyll score, plant height, water applied, water content score
and water use efficiency that is altered for said plant as compared
to a control plant.
[0016] Another aspect of this disclosure provides a plant
comprising a recombinant DNA molecule comprising a nucleotide
sequence, wherein the nucleotide sequence suppresses at least one
target gene encoding at least one target protein, and wherein the
nucleotide sequence is selected from the group consisting of: a) a
nucleotide sequence set forth as SEQ ID NO: 20; b) a nucleotide
sequence that suppresses at least one target gene set forth as SEQ
ID NO: 21; c) a nucleotide sequence that suppresses at least one
target protein set forth as SEQ ID NO: 22; d) a nucleotide sequence
with 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%,
at least 99% identity to SEQ ID NO: 20; e) a nucleotide sequence
that suppresses at least one target gene with 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%, at least 99% identity to
SEQ ID NO: 21; and f) a nucleotide sequence that suppresses at
least one target gene encoding a target protein with 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%, at least 99%
identity to SEQ ID NO: 22, wherein the plant has at least one
phenotype selected from the group consisting of anthocyanin score,
biomass, canopy area, chlorophyll score, plant height, water
applied, water content score and water use efficiency that is
altered for said plant as compared to a control plant.
DETAILED DESCRIPTION OF THE INVENTION
[0017] In the attached sequence listing:
[0018] SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15 are nucleotide
sequences of the coding strand of the DNA molecules used in the
recombinant DNA constructs imparting an enhanced trait in plants,
each representing a coding sequence for a protein.
[0019] SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16 are amino acid
sequences of the cognate proteins of the DNA molecules with
nucleotide sequences 1, 3, 5, 7, 9, 11, 13, and 15.
[0020] SEQ ID NOs: 17 and 20 are the nucleotide sequences of the
suppression elements used to suppress at least one target gene, SEQ
ID NOs: 18 and 21, which encode protein SEQ ID NOs: 19 and 22, used
in the recombinant DNA constructs to impart an enhanced trait in
plants.
[0021] SEQ ID NOs: 23-29 are amino acid sequences of homologous
proteins.
[0022] As used herein, the term "expression" refers to the activity
level of a gene in a plant, plant cell or plant tissue in producing
a protein. Expression is the process by which information from a
gene is used in the synthesis of a functional gene product. Gene
expression can give rise to the phenotype. Such phenotypes are
often expressed by the synthesis of proteins that control the
organism's shape, or that acts as enzymes catalyzing specific
metabolic pathways. "Expression or altered expression" in reference
to a polynucleotide indicates that the pattern of expression in,
for example, a transgenic plant or plant tissue, is different from
the expression pattern in a wild-type plant or a non-transgenic
plant of the same species. The pattern of expression may also be
compared with a reference expression pattern in a wild-type plant
of the same species. For example, the polynucleotide or polypeptide
is expressed in a cell or tissue type other than a cell or tissue
type in which the sequence is expressed in the wild-type plant, or
by expression at a time other than at the time the sequence is
expressed in the wild-type plant, or by a response to different
inducible agents, such as hormones or environmental signals, or at
different expression levels (either higher or lower) compared with
those found in a wild-type plant. The term also refers to altered
expression patterns that are produced by lowering the levels of
expression to below the detection level or completely abolishing
expression. The resulting expression pattern can be transient or
stable, constitutive or inducible. In reference to a polypeptide,
the term "ectopic expression or altered expression" can relate to
altered activity levels resulting from the interactions of the
polypeptides with exogenous or endogenous modulators or from
interactions with factors or as a result of the chemical
modification of the polypeptides. Variation in expression can occur
when, for example, the genes encoding one or more polypeptides are
under the control of a constitutive promoter (for example, the
cauliflower mosaic virus 35S transcription initiation region).
Expression can also be altered by having the gene under the control
of an endogenous or a heterologous promoter, or an inducible or
tissue specific promoter. Expression can occur throughout a plant,
in specific tissues of the plant, or in the presence or absence of
particular environmental signals, depending on the promoter used.
Expression can also occur in plant cells where endogenous
expression of the present polypeptides or functionally equivalent
molecules normally occurs, but such normal expression is at a lower
level.
[0023] The term "overexpression" as used herein refers to a greater
expression level of a gene in a plant, plant cell or plant tissue,
compared to expression in a wild-type plant, cell or tissue, at any
developmental or temporal stage for the gene. Overexpression can
occur when, for example, the genes encoding one or more
polypeptides are under the control of a promoter (for example, the
cauliflower mosaic virus 35S transcription initiation region).
Overexpression can also be under the control of a heterologous
promoter, or an inducible or tissue specific promoter. Thus,
overexpression can occur throughout a plant, in specific tissues of
the plant, or in the presence or absence of particular
environmental signals, depending on the promoter used.
Overexpression can take place in plant cells normally lacking
expression of polypeptides functionally equivalent or identical to
the present polypeptides. Overexpression can also occur in plant
cells where endogenous expression of the present polypeptides or
functionally equivalent molecules normally occurs, but such normal
expression is at a lower level. Overexpression thus results in a
greater than normal production, or "overproduction" of the
polypeptide in the plant, cell or tissue.
[0024] The term "suppression", as used herein, refers to a lower
expression level of a gene in a plant, plant cell or plant tissue,
compared to the expression in a wild-type or control plant, cell or
tissue, at any developmental or temporal stage for the gene.
Suppression can be applied using numerous approaches. Non limiting
examples include: to suppress an endogenous gene(s) or a subset of
genes in a pathway, to suppress a mutation that has resulted in
decreased activity of a protein, to suppress the production of an
inhibitory agent, to elevate, reduce or eliminate the level of
substrate that an enzyme requires for activity, to produce a new
protein; to activate a normally silent gene; or to accumulate a
product that does not normally increase under natural
conditions.
[0025] In one embodiment. RNAi-mediated gene suppression can be
used to suppress the expression of targeted genes within plants. In
another embodiment, a recombinant DNA construct having a promoter
that is functional in a plant cell is operably-linked to a
polynucleotide. Thus, when the DNA construct is expressed in a
plant cell, the DNA is transcribed into an RNA molecule that
suppresses the level of an endogenous protein in the plant cell
relative to a control, thereby modulating the regulation of gene
expression. In another embodiment, the recombinant DNA construct
comprising a polynucleotide sequence is transcribed into an RNA
molecule, such an RNA molecule can be a dsRNA processed into
siRNAs, a ta-siRNA, which is processed into siRNAs, or a miRNA, all
of which target a messenger RNA encoding the protein; and result in
the suppression of protein expression relative to a control. The
basic mechanisms of RNA silencing are known (See Baulcombe, 2004,
Nature 431: 356-363). The main contributors for RNA silencing
include, but are not limited to, RNA dependent RNA dsRNA, siRNA,
miRNAs, or Dicer and Argonaute nucleases.
[0026] Other methods to suppress a gene include, for example, the
use of antisense, co-suppression, and RNA interference described in
detail in PCT Application Publication No. WO2006073727, which is
incorporated herein by reference. Anti-sense gene suppression in
plants is described in U.S. Pat. No. 5,107,065, U.S. Pat. No.
5,453,566, and U.S. Pat. No. 5,759,829, and are incorporated herein
by reference. US Patent Application Publication Nos. US
2004/0106566 and US 2004/0253604, which are incorporated by
reference in their entirety herein, disclose methods for inducing
gene silencing using nucleic acid constructs containing a gene
silencing molecule (sense or anti-sense or both) within an intron
flanked by multiple protein-coding exons, wherein, upon splicing
and removal of the intron, the protein-coding exons are linked to
form a mature mRNA encoding a protein with desired function and the
gene silencing molecule is released. Methods of inducing gene
silencing using short RNA molecules or DNA constructs encoding
short RNA molecules (commonly referred to as "RNAi") as described
in U.S. Pat. No. 8,097,710, and methods disclosed for screening for
the occurrence of and detecting gene silencing in plants, for
example post transcriptional gene silencing as described in U.S.
Pat. No. 6,753,139, are incorporated herein by reference. The
phased small RNA ("phased sRNA") pathway (see PCT patent
application serial No. PCT/US2007/019283, published as WO
2008/027592) is based on an endogenous locus termed a "phased small
RNA locus", which transcribes to an RNA transcript forming a single
foldback structure that is cleaved in phase in vivo into multiple
small double-stranded RNAs (termed "phased small RNAs") capable of
suppressing a target gene.
[0027] In addition, molecular constructs and methods for use
thereof, including constructs including heterologous miRNA
recognition sites, constructs for gene suppression including a gene
suppression element embedded within an intron flanked on one or on
both sides by non-protein-coding sequence, constructs containing
engineered miRNA or miRNA precursors, construct for use of inverted
repeats for suppression and constructs for suppression of
production of mature microRNA in a cell are described in detail in
U.S. Pat. No. 8,217,227 and are incorporated herein by reference.
The various utilities of miRNAs, their precursors, their
recognition sites are described in detail in US Patent Application
Publication US 2006/0200878 A1. specifically incorporated by
reference herein. Non-limiting examples of these utilities include:
(1) the expression of a native miRNA or miRNA precursor sequence to
suppress a target gene; (2) the expression of an engineered
(non-native) miRNA or miRNA precursor sequence to suppress a target
gene; (3) the expression of a transgene with a miRNA recognition
site, wherein the transgene is suppressed when the corresponding
mature miRNA is expressed, either endogenously or transgenically;
(4) the expression of a transgene driven by a miRNA promoter; and
(5) the expression of a transgene with an RNA molecule, wherein a
RNA molecule is a cleavage blocker of a miRNA or is a miRNA decoy
of a miRNA (Examples of such RNAi-mediated gene suppression
approaches are disclosed in U.S. Patent Application Publication No.
2009/61288019 and incorporated herein by reference). Additionally
MIR genes and mature miRNAs are also described in US Patent
Application Publication Nos. US 2005/0120415 and US 2005/0144669
A1, which is incorporated by reference herein. MIR genes have been
reported to occur in inter-genic regions, both isolated and in
clusters in the genome, but can also be located entirely or
partially within introns of other genes (both protein-coding and
non-protein-coding).
[0028] A method described in US Patent Application Publication No.
US 2011/0296556 A1, herein incorporated by reference, discloses how
to modulate gene expression in plants by using external application
of polynucleotide molecules. The method provides for an RNA or DNA
containing composition for the regulation of plant gene expression
when the composition is applied to a plant surface.
[0029] As used herein "microRNAs" (miRNAs) are non-protein coding
RNAs, generally of between about 19 to about 25 nucleotides
(generally of between about 19 to about 25 nucleotides but commonly
about 20-24 nucleotides in plants), that guide cleavage in trans of
target transcripts, negatively regulating the expression of genes
involved in various regulation and development pathways (See Bartel
(2004) Cell, 116:281-297). In some cases, miRNAs serve to guide
in-phase processing of siRNA primary transcripts (see Allen et al.
(2005) Cell, 121:207-221, which is incorporated herein by
reference). Micro RNAs are regulatory miRNAs that can also control
gene expression at the level of translation and maintain mRNA
stability in the cytoplasm. Recombinant DNA constructs can be used
to modify the activity of native miRNAs by a variety of means. By
increasing the expression of a miRNA, for example, temporally or
spatially, the modulation of expression of a native target gene can
be enhanced. An alternative gene suppression approach for
suppressing the expression of a target protein can include the use
of a recombinant DNA construct that produces a synthetic miRNA that
is designed to bind to a native or synthetic miRNA recognition site
on messenger RNA for the target protein. Alternatively, by reducing
the expression of a miRNA, the modulation of a native target gene
can be diminished resulting in enhanced expression of the target
protein. More specifically, the expression of a target protein can
be enhanced by suppression of the activity of the miRNA that binds
to a recognition site in the messenger RNA that is transcribed from
the native gene for the target protein. Several types of
recombinant DNA constructs can be designed to suppress the activity
of a miRNA. Recombinant DNA encoding an RNA encoding a miRNA, or a
miRNA-sensitive RNA are designed using methods disclosed in US
Patent Application Publication No. US 2009/0070898 A1. The
construction and description of such recombinant DNA constructs is
disclosed in US Patent Application Publication No. US 2009/0070898
A1, and US application publication No. US 2011/0296555 A1, all of
which are incorporated herein by reference.
[0030] As used herein, "double-stranded RNA" ("dsRNA") is RNA
capable of being processed through an RNAi pathway (for example, to
produce small interfering RNAs or microRNAs, see, for example, Xie
et al. (2004) PLoS Biol., 2:642-652; Bartel (2004) Cell,
116:281-297; Murchison and Hannon (2004) Curr. Opin. Cell Biol.,
16:223-229; and Dugas and Bartel (2004) Curr. Opin. Plant Biol.,
7:512-520, all of which are incorporated by reference. The
transcribable DNA that is processed into dsRNA can be flanked on
one or both sides by DNA that transcribes to RNA capable of forming
dsRNA (for example, by forming an inverted repeat where the
transcribable DNA is located in the middle "spacer" region, or by
forming separate dsRNA regions on one or both sides of the
transcribable DNA, which may be processed to small interfering
RNAs, to microRNA precursors such as pre-miRNAs, or to mature
microRNAs).
[0031] As used herein, "siRNA" refers to the siRNA pathway involves
the non-phased cleavage of a longer double-stranded RNA
intermediate to small interfering RNAs ("siRNAs"). The size of
siRNAs can be in a range from about 19 to about 25 base pairs, but
common classes of siRNAs include those containing 21 base pairs or
24 base pairs. See, for example. Hamilton et al. (2002) EMBO J.,
21:4671-4679. siRNAs are typically associated with
posttranscriptional gene silencing triggered by transgenes and
viruses in plants.
[0032] As used herein "trans-acting RNAs" ("ta-siRNA") refer to
miRNAs that serve to guide in-phase processing of siRNA primary
transcripts in a process that requires an RNA-dependent RNA
polymerase for production of a double-stranded RNA precursor;
trans-acting siRNAs are defined by lack of secondary structure, a
miRNA target site that initiates production of double-stranded RNA,
requirements of DCL4 and an RNA-dependent RNA polymerase (RDR6),
and production of multiple phased .about.21-nt small RNAs with
matched duplexes with 2-nucleotide 3' overhangs (see Allen et al.
(2005) Cell, 121:207-221; Vazquez et al. (2004) Mol. Cell,
16:69-79).
[0033] As used herein, "noncoding RNAs" ("ncRNAs") are another
class of RNAs that have functional roles in regulating gene
expression in higher cells. Small RNAs have chain lengths varying
from approximately 60-300 nucleotides in length. Small nuclear RNAs
(snRNAs) can exist as a complex tightly bound to one or more
proteins in particles termed small nuclear ribonucleoproteins
(snRNPs). Some occupy the nucleoplasm, which contains the DNA and
devotes to the production of mRNAs for export to the cytoplasm.
Others occupy the nucleolus, the location where ribosomes are
assembled prior to being directed to the cytoplasm for protein
synthesis.
[0034] As used herein, "natural anti-sense transcript small
interfering RNA" ("nat-siRNA) refers to gene suppression mediated
by small RNAs processed from natural antisense transcripts are
involved in at least two pathways. In the natural antisense
transcript small interfering RNA ("nat-siRNA") pathway (Borsani et
al. (2005) Cell, 123:1279-1291), siRNAs are generated by DCL1
cleavage of a double-stranded RNA formed between the antisense
transcripts of a pair of genes (cis-antisense gene pairs). A
similar natural anti-sense transcript microRNA ("nat-miRNA")
pathway (Lu et al. (2008) Proc. Natl. Acad. Sci. USA, 105:
4951-4956) has also been reported. In metazoan animals, small RNAs
termed Piwi-interacting RNAs ("piRNAs") also have gene-silencing
activity (See Lau et al. (2006) Science, 313:363-367; O'Donnell
& Boeke (2007) Cell, 129:37-44).
[0035] Small RNAs that regulate protein expression can include
miRNAs and ta-siRNAs. A miRNA is a small (typically about 21
nucleotide) RNA that has the ability to modulate the expression of
a target gene by binding to messenger RNA for the target protein
leading to destabilization of the target protein messenger RNA or
translational inhibition of the target protein messenger RNA,
resulting in reduction of the target protein. The design and
construction of ta-siRNA constructs and their use in the modulation
of protein in transgenic plant cells was disclosed by Allen and
Carrington in US Patent Application Publication No. US 2006/0174380
A1 which is incorporated herein by reference. The expression or
suppression of such small RNAs are aspects of the invention
illustrated by reference the use of miRNAs.
[0036] As used herein, "inverted repeat" ("IR") is a sequence of
nucleotides that is the reversed complement of another sequence
further downstream. For example, 5' - - - GACTGC . . . GCAGTC - - -
3'. When no nucleotides intervene between the sequence and its
downstream complement, it is called a palindrome. Inverted repeats
define the boundaries in transposons. Inverted repeats also
indicate regions capable of self-complementary base pairing
(regions within a single sequence which can base pair with each
other).
[0037] As used herein, "miRNA decoy" refers to a sequence that can
be recognized and bound by an endogenous mature miRNA resulting in
base-pairing between the miRNA decoy sequence and the endogenous
mature miRNA, thereby forming a cleavage-resistant RNA duplex that
is not cleaved because of the presence of mismatches between the
miRNA decoy sequence and the mature miRNA. Prediction or designing
of a miRNA decoy sequence have been described in US Patent
Application Publication No. US 2009/0070898 A1.
[0038] As used herein, "RNA cleavage blocker" is the RNA including
single-stranded RNA that binds to the transcript of at least one
target gene, and more specifically refers to the portion(s) of the
single-stranded RNA that forms a hybridized segment of at least
partially double-stranded RNA with the transcript. Cleavage
blockers inhibit double-stranded RNA-mediated suppression of the at
least one target gene, thereby increasing expression of the target
gene (relative to expression in the absence of the cleavage
blocker). The RNA includes single-stranded RNA that binds to the
transcript of at least one target gene to form a hybridized segment
of at least partially double-stranded RNA that imparts to the
transcript resistance to cleavage by an RNase III ribonuclease
within or in the vicinity of the hybridized segment, wherein the
binding of the single-stranded RNA to the transcript (and the
resultant formation of the hybridized segment) inhibits
double-stranded RNA-mediated suppression of the at least one target
gene.
[0039] As used herein, "target gene" include any gene for which
expression is intended to be modified, either in a cell containing
the recombinant DNA construct or in other cells or organisms that
come into contact with the recombinant DNA construct. The target
gene can be native (endogenous) to the cell (for example, a cell of
a plant or animal) in which the recombinant DNA construct is
transcribed, or can be native to a pest or pathogen (or a symbiont
of the pest or pathogen) of the plant or animal in which the
recombinant DNA construct is transcribed. The target gene can also
be an exogenous gene, such as a transgene in a plant. A target gene
can be a native gene targeted for suppression, with or without
concurrent expression of an exogenous transgene. For example, by
including a gene expression element in the recombinant DNA
construct, or in a separate recombinant DNA construct. The
recombinant DNA construct can be designed to be more specifically
modulate the expression of the target gene. For example, by
designing the recombinant DNA construct to include DNA that is
processed to an RNA including single-stranded RNA that binds to the
target gene transcript, wherein the single-stranded RNA includes a
nucleotide sequence substantially non-identical (or
non-complementary) to a non-target gene sequence (and is thus less
likely to bind to a non-target gene transcript). Alternatively,
non-target genes can include any gene for which expression is not
intended to be modified, either in a cell containing the
recombinant DNA construct or in other cells or organisms that come
into contact with the recombinant DNA construct.
[0040] As used herein, "target sequence" is the sequence suppress
the expression of a protein encoded by a target gene endogenous or
exogenous to a plant. The target sequence can include nucleotide
sequence to target for suppression gene of interest (for example an
mRNA encoding a protein), or a sequence that is targeted by an RNA
that is designed and processed to an siRNA or miRNA. The target
sequence can be translatable (coding) sequence, or can be
non-coding sequence (such as non-coding regulatory sequence), or
both. The target sequence can include at least one eukaryotic
target sequence, at least one non-eukaryotic target sequence, or
both. A target sequence can include any sequence from any species
(including, but not limited to, non-eukaryotes such as bacteria,
and viruses; fungi; plants, including monocots and dicots, such as
crop plants). The recombinant DNA construct can be designed to more
specifically modulate the expression of the target gene, for
example, by designing the recombinant DNA construct to include DNA
that is processed to an RNA including single-stranded RNA that
binds to the target gene transcript, wherein the single-stranded
RNA includes a nucleotide sequence substantially non-identical (or
non-complementary) to a non-target gene sequence (and is thus less
likely to bind to a non-target gene transcript).
[0041] In one embodiment, the modulation of protein in transgenic
plant cells can be achieved by a variety of approaches involving
the use of recombinant DNA constructs. None limiting examples of
such recombinant DNA constructs include recombinant DNA constructs
that produce messenger RNA for the target protein where native
miRNA recognition sites in the mRNA for the target protein are
modified or deleted, recombinant DNA constructs that produce an RNA
gene suppression element such as a miRNA or a dsRNA comprising
sense and anti-sense sequences from the gene encoding the target
protein, recombinant DNA constructs that produce a transacting
short interfering RNA (ta-siRNA) and recombinant DNA constructs
that produce a miRNA element such as a decoy miRNA that is a target
for native miRNA or RNA that sequesters target messenger RNA away
from native miRNA.
[0042] As used herein, "gene suppression elements" refer to a
genetic element(s) that can be transcribable DNA of any suitable
length, and will generally include at least about 19 to about 27
nucleotides (for example 19, 20, 21, 22, 23, or 24 nucleotides) for
every target gene that the recombinant DNA construct is intended to
suppress. In one embodiment, the gene suppression element includes
more than 23 nucleotides (for example, more than about 30, about
50, about 100, about 200, about 300, about 500, about 1000, about
1500, about 2000, about 3000, about 4000, or about 5000
nucleotides) for every target gene that the recombinant DNA
construct is intended to suppress.
[0043] In another embodiment, gene suppression elements refer to,
but are not limited to, elements that include transcribable
exogenous DNAs: DNA that includes at least one anti-sense DNA
segment to at least one segment of the at least one target gene, or
DNA that includes multiple copies of at least one anti-sense DNA
segment that is anti-sense to at least one segment of the at least
one target gene; DNA that includes at least one sense DNA segment
that is at least one segment of the at least one target gene, or
DNA that includes multiple copies of at least one sense DNA segment
that is at least one segment of the at least one target gene; DNA
that transcribes to RNA for suppressing at least one target gene by
forming double-stranded RNA and includes at least one anti-sense
DNA segment that is anti-sense to at least one segment of the at
least one target gene and at least one sense DNA segment that is at
least one segment of the at least one target gene; DNA that
transcribes to RNA for suppressing the at least one target gene by
forming a single double-stranded RNA and includes multiple serial
anti-sense DNA segments that are anti-sense to at least one segment
of the at least one target gene and multiple serial sense DNA
segments that are at least one segment of the at least one target
gene; DNA that transcribes to RNA for suppressing the at least one
target gene by forming multiple double strands of RNA and includes
multiple anti-sense DNA segments that are anti-sense to at least
one segment of the at least one target gene and multiple sense DNA
segments that are at least one segment of the at least one target
gene, and wherein said multiple anti-sense DNA segments and the
multiple sense DNA segments are arranged in a series of inverted
repeats; and DNA that includes nucleotides derived from a miRNA, or
DNA that includes nucleotides of a siRNA. Various arrangements of
double-stranded RNA (dsRNA) that can be transcribed from
embodiments of the gene suppression elements and transcribable
exogenous DNAs and can suppress one or more target genes, and can
form a single double-stranded RNA or multiple double strands of
RNA, or a single dsRNA "stem" or multiple "stems". In some
embodiments, an intron is used to deliver a gene suppression
element in the absence of any protein-coding exons (coding
sequence). In a non-limiting example, an intron, such as an
expression-enhancing intron, is interrupted by embedding within the
intron a gene suppression element, wherein, upon transcription, the
gene suppression element is excised from the intron. Additional
gene suppression elements are described in detail in US Patent
Application Publication No. US 2006/0200878 A1, which disclosure is
specifically incorporated herein by reference, and include one or
more of: (a) DNA that includes at least one anti-sense DNA segment
that is anti-sense to at least one segment of the gene to be
suppressed; (b) DNA that includes multiple copies of at least one
anti-sense DNA segment that is anti-sense to at least one segment
of the gene to be suppressed; (c) DNA that includes at least one
sense DNA segment that is at least one segment of the gene to be
suppressed; (d) DNA that includes multiple copies of at least one
sense DNA segment that is at least one segment of the gene to be
suppressed; (e) DNA that transcribes to RNA for suppressing the
gene to be suppressed by forming double-stranded RNA and includes
at least one anti-sense DNA segment that is anti-sense to at least
one segment of the gene to be suppressed and at least one sense DNA
segment that is at least one segment of the gene to be suppressed;
(f) DNA that transcribes to RNA for suppressing the gene to be
suppressed by forming a single double-stranded RNA and includes
multiple serial anti-sense DNA segments that are anti-sense to at
least one segment of the gene to be suppressed and multiple serial
sense DNA segments that are at least one segment of the gene to be
suppressed; (g) DNA that transcribes to RNA for suppressing the
gene to be suppressed by forming multiple double strands of RNA and
includes multiple anti-sense DNA segments that are anti-sense to at
least one segment of the gene to be suppressed and multiple sense
DNA segments that are at least one segment of the gene to be
suppressed, and wherein the multiple anti-sense DNA segments and
the multiple sense DNA segments are arranged in a series of
inverted repeats; (h) DNA that includes nucleotides derived from a
plant miRNA; (i) DNA that includes nucleotides of a siRNA; any of
these gene suppression elements, whether transcribing to a single
double-stranded RNA or to multiple double-stranded RNAs, can be
designed to suppress at least one target gene, including, for
example, more than one allele of a target gene, multiple target
genes (or multiple segments of at least one target gene) from a
single species, or target genes from different species.
[0044] As used herein a "plant" includes whole plant, transgenic
plant, meristem, shoot organ/structure (for example, leaf, stem and
tuber), root, flower and floral organ/structure (for example,
bract, sepal, petal, stamen, carpel, anther and ovule), seed
(including embryo, endosperm, and seed coat) and fruit (the mature
ovary), plant tissue (for example, vascular tissue, ground tissue,
and the like) and cell (for example, guard cell, egg cell, pollen,
mesophyll cell, and the like), and progeny of same. The classes of
plants that can be used in the disclosed methods are generally as
broad as the classes of higher and lower plants amenable to
transformation and breeding techniques, including angiosperms
(monocotyledonous and dicotyledonous plants), gymnosperms, ferns,
horsetails, psilophytes, lycophytes, bryophytes, and algae.
[0045] As used herein, a "transgenic plant" refers to a plant whose
genome has been altered by the stable integration of recombinant
DNA. A transgenic plant includes a plant regenerated from an
originally-transformed plant cell and progeny transgenic plants
from later generations or crosses of a transgenic plant.
[0046] As used herein, a "control plant" refers to a plant that
does not contain the recombinant DNA that imparts an enhanced
trait. A control plant is used to identify and select a transgenic
plant that has an enhanced trait. A suitable control plant can be a
non-transgenic plant of the parental line used to generate a
transgenic plant, for example, a wild type plant devoid of a
recombinant DNA. A suitable control plant can also be a transgenic
plant that contains the recombinant DNA that imparts other traits,
for example, a transgenic plant having enhanced herbicide
tolerance. A suitable control plant can in some cases be a progeny
of a hemizygous transgenic plant line that does not contain the
recombinant DNA, known as a negative segregant, or a negative
isoline.
[0047] As used herein, a "transgenic plant cell" refers to a plant
cell that is transformed with stably-integrated, recombinant DNA,
for example, by Agrobacterium-mediated transformation or by
bombardment using microparticles coated with recombinant DNA or by
other means. A plant cell of this disclosure can be an
originally-transformed plant cell that exists as a microorganism or
as a progeny plant cell that is regenerated into differentiated
tissue, for example, into a transgenic plant with
stably-integrated, recombinant DNA, or seed or pollen derived from
a progeny transgenic plant.
[0048] As used herein, a "propagule" includes all products of
meiosis and mitosis, including but not limited to, plant, seed and
part of a plant able to propagate a new plant. Propagules include
whole plants, cells, pollen, ovules, flowers, embryos, leaves,
roots, stems, shoots, meristems, grains or seeds, or any plant part
that is capable of growing into an entire plant. Propagule also
includes graft where one portion of a plant is grafted to another
portion of a different plant (even one of a different species) to
create a living organism. Propagule also includes all plants and
seeds produced by cloning or by bringing together meiotic products,
or allowing meiotic products to come together to form an embryo or
a fertilized egg (naturally or with human intervention).
[0049] As used herein, a "progeny" includes any plant, seed, plant
cell, and/or regenerable plant part comprising a recombinant DNA of
the present disclosure derived from an ancestor plant. A progeny
can be homozygous or heterozygous for the transgene. Progeny can be
grown from seeds produced by a transgenic plant comprising a
recombinant DNA of the present disclosure, and/or from seeds
produced by a plant fertilized with pollen or ovule from a
transgenic plant comprising a recombinant DNA of the present
disclosure.
[0050] As used herein, a "trait" is a physiological, morphological,
biochemical, or physical characteristic of a plant or particular
plant material or cell. In some instances, this characteristic is
visible to the human eye, such as seed or plant size, or can be
measured by biochemical techniques, such as detecting the protein,
starch, certain metabolites, or oil content of seed or leaves, or
by observation of a metabolic or physiological process, for
example, by measuring tolerance to water deprivation or particular
salt or sugar concentrations, or by the measurement of the
expression level of a gene or genes, for example, by employing
Northern analysis, RT-PCR, microarray gene expression assays, or
reporter gene expression systems, or by agricultural observations
such as hyperosmotic stress tolerance or yield. Any technique can
be used to measure the amount of, comparative level of, or
difference in any selected chemical compound or macromolecule in
the transgenic plants.
[0051] As used herein, an "enhanced trait" means a characteristic
of a transgenic plant as a result of stable integration and
expression of a recombinant DNA in the transgenic plant. Such
traits include, but are not limited to, an enhanced agronomic trait
characterized by enhanced plant morphology, physiology, growth and
development, yield, nutritional enhancement, disease or pest
resistance, or environmental or chemical tolerance. An enhanced
trait can also be increased drought tolerance, increased water use
efficiency, cold tolerance, increased nitrogen use efficiency,
increased yield, and altered phenotypes as shown in Tables 4-6
(corn, altered phenotypes), Tables 7-12 (corn), Table 13 (soybean)
and Table 14 (canola). In another aspect, the trait is increased
yield under non-stress conditions or increased yield under
environmental stress conditions. Stress conditions can include, for
example, drought, shade, fungal disease, viral disease, bacterial
disease, insect infestation, nematode infestation, cold temperature
exposure, heat exposure, osmotic stress, reduced nitrogen nutrient
availability, reduced phosphorus nutrient availability and high
plant density. "Yield" can be affected by many properties including
without limitation, plant height, plant biomass, pod or silique
number, pod position on the plant, number of internodes, incidence
of pod shatter, grain size, efficiency of nodulation and nitrogen
fixation, efficiency of nutrient assimilation, resistance to biotic
and abiotic stress, carbon assimilation, plant architecture,
resistance to lodging, percent seed germination, seedling vigor,
and juvenile traits. Yield can also be affected by efficiency of
germination (including germination in stressed conditions), growth
rate (including growth rate in stressed conditions), ear number,
seed number per ear, seed size, seed weight, composition of seed
(starch, oil, protein) and characteristics of seed fill.
[0052] Also used herein, the term "trait modification" encompasses
altering the naturally occurring trait by producing a detectable
difference in a characteristic in a plant comprising a recombinant
DNA of the present disclosure relative to a plant not comprising
the recombinant DNA, such as a wild-type plant, or a negative
segregant. In some cases, the trait modification can be evaluated
quantitatively. For example, the trait modification can entail an
increase or decrease, in an observed trait as compared to a control
plant. It is known that there can be natural variations in the
modified trait. Therefore, the trait modification observed entails
a change of the normal distribution and magnitude of the trait in
the plants as compared to a control plant.
[0053] Increased yield of a plant of the present disclosure can be
measured in a number of ways, including test weight, seed number
per plant, seed weight, seed number per unit area (for example,
seeds, or weight of seeds, per acre), bushels per acre, tons per
acre, or kilo per hectare. For example, corn yield can be measured
as production of shelled corn kernels per unit of production area,
for example in bushels per acre or metric tons per hectare.
Increased yield can result from improved utilization of key
biochemical compounds, such as nitrogen, phosphorous and
carbohydrate, or from improved responses to environmental stresses,
such as cold, heat, drought, salt, shade, high plant density, and
attack by pests or pathogens. This disclosure can also be used to
provide plants with improved growth and development, and ultimately
increased yield, as the result of modified expression of plant
growth regulators or modification of cell cycle or photosynthesis
pathways. Also of interest is the generation of plants that
demonstrate increased yield with respect to a seed component that
may or may not correspond to an increase in overall plant
yield.
[0054] In an embodiment, the present disclosure relates to a plant
with improved economically important characteristics, more
specifically increased yield. More specifically the present
disclosure relates to a plant comprising a polynucleotide of this
disclosure that encodes a polypeptide, wherein the plant has
increased yield as compared to a control plant. Many plants of this
disclosure exhibited increased yield as compared to a control
plant. In an embodiment, a plant of the present disclosure
exhibited an improved trait that is a component of yield.
[0055] Yield can be defined as the measurable produce of economic
value from a crop. Yield can be defined in the scope of quantity
and/or quality. Yield can be directly dependent on several factors,
for example, the number and size of organs, plant architecture
(such as the number of branches, plant biomass, etc.), seed
production and more. Root development, photosynthetic efficiency,
nutrient uptake, stress tolerance, early vigor, delayed senescence
and functional stay green phenotypes can be important factors in
determining yield. Optimizing the above mentioned factors can
therefore contribute to increasing crop yield.
[0056] Reference herein to an increase in yield-related traits can
also be taken to refer to an increase in biomass (weight) of one or
more parts of a plant, which can include above ground and/or below
ground (harvestable) plant parts. In particular, such harvestable
parts are seeds, and performance of the methods of the disclosure
results in plants with increased yield and in particular increased
seed yield relative to the seed yield of suitable control plants.
The term "yield" of a plant can relate to vegetative biomass (root
and/or shoot biomass), to reproductive biomass (such as corn ear
biomass (unit) or corn ear biomass per plot (unit), and/or to
propagules (such as seeds) of that plant.
[0057] In an embodiment, "alfalfa yield" can be measured in forage
yield, the amount of above ground biomass at harvest. Factors
contributing to increased biomass include increased vegetative
growth, branches, nodes and internodes, leaf area, and leaf area
index.
[0058] In another embodiment, "canola yield" can also be measured
in silique number, number of siliques per plant, number of siliques
per node, number of internodes, incidence of silique shatter, seeds
per silique, seed weight per silique, improved seed, oil, or
protein composition.
[0059] Additionally, "corn or maize yield" can also be measured as
production of shelled corn kernels per unit of production area,
ears per acre, number of kernel rows per ear, kernels per ear,
weight per kernel, ear number, ear biomass and ear biomass per
plot.
[0060] In yet another embodiment, "cotton yield" can be measured as
bolls per plant, size of bolls, fiber quality, seed cotton yield in
grams (g)/plant, seed cotton yield in pounds (lbs)/acre, lint yield
in lb/acre, and number of bales.
[0061] Specific embodiment for "rice yield" can also include
panicles per hill, grain per hill, and filled grains per
panicle.
[0062] Still further embodiment for "soybean yield" can also
include pods per plant, pods per acre, seeds per plant, seeds per
pod, weight per seed, weight per pod, pods per node, number of
nodes, and the number of internodes per plant.
[0063] In still further embodiment, "sugarcane yield" can be
measured as cane yield (tons per acre; kilograms (kg)/hectare),
total recoverable sugar (pounds per ton), and sugar yield
(tons/acre).
[0064] In yet still further embodiment, "wheat yield" can include:
cereal per unit area, grain number, grain weight, grain size,
grains per head, seeds per head, seeds per plant, heads per acre,
number of viable tillers per plant, composition of seed (for
example, carbohydrates, starch, oil, and protein) and
characteristics of seed fill.
[0065] The terms "yield", "seed yield" are defined above for a
number of core crops. The terms "increased", "improved", "enhanced"
are interchangeable and are defined herein.
[0066] In another embodiment, the present disclosure also provides
a method for the production of plants having increased yield.
Performance of the method gives plants increased yield. "Increased
yield" can manifest as one or more of the following: (i) increased
plant biomass (weight) of one or more parts of a plant,
particularly aboveground (harvestable) parts, of a plant, increased
root biomass (increased number of roots, increased root thickness,
increased root length) or increased biomass of any other
harvestable part; (ii) increased early vigor, defined herein as an
improved seedling aboveground area approximately three weeks
post-germination. "Early vigor" refers to active healthy plant
growth especially during early stages of plant growth, and can
result from increased plant fitness due to, for example, the plants
being better adapted to their environment (for example, optimizing
the use of energy resources, uptake of nutrients and partitioning
carbon allocation between shoot and root). Early vigor in corn, for
example, is a combination of the ability of corn seeds to germinate
and emerge after planting and the ability of the young corn plants
to grow and develop after emergence. Plants having early vigor also
show increased seedling survival and better establishment of the
crop, which often results in highly uniform fields with the
majority of the plants reaching the various stages of development
at substantially the same time, which often results in increased
yield. Therefore early vigor can be determined by measuring various
factors, such as kernel weight, percentage germination, percentage
emergence, seedling growth, seedling height, root length, root and
shoot biomass, canopy size and color and others; (iii) increased
total seed yield, which includes an increase in seed biomass (seed
weight) and which can be an increase in the seed weight per plant
or on an individual seed basis; increased number of panicles per
plant; increased pods, increased number of nodes, increased number
of flowers ("florets") per panicle/plant; increased seed fill rate;
increased number of filled seeds; increased seed size (length,
width, area, perimeter), which can also influence the composition
of seeds; increased seed volume, which can also influence the
composition of seeds. Increased yield can also result in modified
architecture, or can occur because of modified plant architecture;
(iv) increased harvest index, which is expressed as a ratio of the
yield of harvestable parts, such as seeds, over the total biomass;
(v) increased kernel weight, which is extrapolated from the number
of filled seeds counted and their total weight. An increased kernel
weight can result from an increased seed size and/or seed weight,
an increase in embryo size, endosperm size, aleurone and/or
scutellum, or other parts of the seed; and vi) increased ear
biomass, which is the weight of the ear and can be represented on a
per ear, per plant or per plot basis.
[0067] In one embodiment, increased yield can be increased seed
yield, and is selected from one of the following: (i) increased
seed weight; (ii) increased number of filled seeds; and (iii)
increased harvest index.
[0068] The disclosure also extends to harvestable parts of a plant
such as, but not limited to seeds, leaves, fruits, flowers, bolls,
stems, rhizomes, tubers and bulbs. The disclosure furthermore
relates to products derived from a harvestable part of such a
plant, such as dry pellets, powders, oil, fat and fatty acids,
starch or proteins.
[0069] The present disclosure provides a method for increasing
"yield" of a plant or "broad acre yield" of a plant or plant part
defined as the harvestable plant parts per unit area, for example
seeds, or weight of seeds, per acre, pounds per acre, bushels per
acre, tones per acre, tons per acre, kilo per hectare.
[0070] This disclosure further provides a method of increasing
yield in a plant by crossing a plant comprising a recombinant DNA
molecule of the present disclosure with itself, a second plant from
the same plant line, a wild type plant, or a plant from a different
line of plants to produce a seed. The seed of the resultant plant
can be harvested from fertile plants and be used to grow progeny
generations of plant(s) of this disclosure. In addition to direct
transformation of a plant with a recombinant DNA, transgenic plants
can be prepared by crossing a first plant having a recombinant DNA
with a second plant lacking the DNA. For example, recombinant DNA
can be introduced into a first plant line that is amenable to
transformation to produce a transgenic plant which can be crossed
with a second plant line to introgress the recombinant DNA into the
second plant line. A transgenic plant with a recombinant DNA having
the polynucleotide of this disclosure provides at least one
enhanced trait of increased yield, increased nitrogen use
efficiency or increased water use efficiency compared to a control
plant. Genetic markers associated with recombinant DNA can be used
to identify transgenic progeny that is homozygous for the desired
recombinant DNA. Progeny plants carrying the recombinant DNA can be
back crossed into a parent line or other transgenic line multiple
times, for example usually 6 to 8 generations, to produce a progeny
plant with substantially the same genotype as the original
transgenic parental line. The term "progeny" denotes the offspring
of any generation of a parent plant prepared by the methods of this
disclosure comprising the recombinant polynucleotides as described
herein.
[0071] As used herein, "nitrogen use efficiency" refers to the
processes which lead to an increase in the plant's yield, biomass,
vigor, and growth rate per nitrogen unit applied. The processes can
include the uptake, assimilation, accumulation, signaling, sensing,
retranslocation (within the plant) and use of nitrogen by the
plant.
[0072] As used herein, "nitrogen limiting conditions" refers to
growth conditions or environments that provide less than optimal
amounts of nitrogen needed for adequate or successful plant
metabolism, growth, reproductive success and/or viability.
[0073] As used herein, "increased nitrogen stress tolerance" refers
to the ability of plants to grow, develop, or yield normally, or
grow, develop, or yield faster or better when subjected to less
than optimal amounts of available/applied nitrogen, or under
nitrogen limiting conditions.
[0074] As used herein, "increased nitrogen use efficiency" refers
to the ability of plants to grow, develop, or yield faster or
better than normal when subjected to the same amount of
available/applied nitrogen as under normal or standard conditions;
ability of plants to grow, develop, or yield normally, or grow,
develop, or yield faster or better when subjected to less than
optimal amounts of available/applied nitrogen, or under nitrogen
limiting conditions.
[0075] Increased plant nitrogen use efficiency can be translated in
the field into either harvesting similar quantities of yield, while
supplying less nitrogen, or increased yield gained by supplying
optimal/sufficient amounts of nitrogen. The increased nitrogen use
efficiency can improve plant nitrogen stress tolerance, and can
also improve crop quality and biochemical constituents of the seed
such as protein yield and oil yield. The terms "increased nitrogen
use efficiency", "enhanced nitrogen use efficiency", and "nitrogen
stress tolerance" are used inter-changeably in the present
disclosure to refer to plants with improved productivity under
nitrogen limiting conditions.
[0076] As used herein, "water use efficiency" refers to the amount
of carbon dioxide assimilated by leaves per unit of water vapor
transpired. "Drought tolerance" refers to the degree to which a
plant is adapted to arid or drought conditions. The physiological
responses of plants to a deficit of water include leaf wilting, a
reduction in leaf area, leaf abscission, and the stimulation of
root growth by directing nutrients to the underground parts of the
plants. Plants are more susceptible to drought during flowering and
seed development (the reproductive stages), as plant's resources
are deviated to support root growth. In addition, abscisic acid
(ABA), a plant stress hormone, induces the closure of leaf stomata
(microscopic pores involved in gas exchange), thereby reducing
water loss through transpiration, and decreasing the rate of
photosynthesis. These responses improve the water-use efficiency of
the plant on the short term. The terms "increased water use
efficiency", "enhanced water use efficiency", and "increased
drought tolerance" are used inter-changeably in the present
disclosure to refer to plants with improved productivity under
water-limiting conditions.
[0077] As used herein, "increased water use efficiency" refers to
the ability of plants to grow, develop, or yield faster or better
than normal when subjected to the same amount of available/applied
water as under normal or standard conditions; ability of plants to
grow, develop, or yield normally, or grow, develop, or yield faster
or better when subjected to reduced amounts of available/applied
water (water input) or under conditions of water stress or water
deficit stress.
[0078] As used herein, "increased drought tolerance" refers to the
ability of plants to grow, develop, or yield normally, or grow,
develop, or yield faster or better than normal when subjected to
reduced amounts of available/applied water and/or under conditions
of acute or chronic drought.
[0079] As used herein, "drought stress" refers to a period of
dryness (acute or chronic/prolonged) that results in water deficit
and subjects plants to stress and/or damage to plant tissues and/or
negatively affects grain/crop yield; a period of dryness (acute or
chronic/prolonged) that results in water deficit and/or higher
temperatures and subjects plants to stress and/or damage to plant
tissues and/or negatively affects grain/crop yield.
[0080] As used herein, "water deficit" refers to the conditions or
environments that provide less than optimal amounts of water needed
for adequate/successful growth and development of plants.
[0081] As used herein, "water stress" refers to the conditions or
environments that provide improper (either less/insufficient or
more/excessive) amounts of water than that needed for
adequate/successful growth and development of plants/crops thereby
subjecting the plants to stress and/or damage to plant tissues
and/or negatively affecting grain/crop yield.
[0082] As used herein, "water deficit stress" refers to the
conditions or environments that provide less/insufficient amounts
of water than that needed for adequate/successful growth and
development of plants/crops thereby subjecting the plants to stress
and/or damage to plant tissues and/or negatively affecting grain
yield.
[0083] As used herein, a "polynucleotide" is a nucleic acid
molecule comprising a plurality of polymerized nucleotides. A
polynucleotide may be referred to as a nucleic acid,
oligonucleotide, nucleotide, or any fragment thereof. In many
instances, a polynucleotide encodes a polypeptide (or protein) or a
domain or fragment thereof. Additionally, a polynucleotide can
comprise a promoter, an intron, an enhancer region, a
polyadenylation site, a translation initiation site, 5' or 3'
untranslated regions, a reporter gene, a selectable marker, a
scorable marker, or the like. A polynucleotide can be
single-stranded or double-stranded DNA or RNA. A polynucleotide can
also comprise modified bases or a modified backbone. A
polynucleotide can be, for example, genomic DNA or RNA, a
transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA,
a synthetic DNA or RNA, or the like. A polynucleotide can be
combined with carbohydrate(s), lipid(s), protein(s), or other
materials to perform a particular activity such as transformation
or form a composition such as a peptide nucleic acid (PNA). A
polynucleotide can comprise a sequence in either sense or antisense
orientations. "Oligonucleotide" is substantially equivalent to the
terms amplimer, primer, oligomer, element, target, and probe and is
typically single-stranded.
[0084] As used herein, a "recombinant polynucleotide" or
"recombinant DNA" is a polynucleotide that is not in its native
state, for example, a polynucleotide comprises a series of
nucleotides (represented as a nucleotide sequence) not found in
nature, or a polynucleotide is in a context other than that in
which it is naturally found; for example, separated from
polynucleotides with which it typically is in proximity in nature,
or adjacent (or contiguous with) polynucleotides with which it
typically is not in proximity. The "recombinant polynucleotide" or
"recombinant DNA" refers to polynucleotide or DNA which has been
genetically engineered and constructed outside of a cell including
DNA containing naturally occurring DNA or cDNA or synthetic DNA.
For example, the polynucleotide at issue can be cloned into a
vector, or otherwise recombined with one or more additional nucleic
acids.
[0085] As used herein, a "polypeptide" comprises a plurality of
consecutive polymerized amino acid residues for example, at least
about 15 consecutive polymerized amino acid residues. In many
instances, a polypeptide comprises a series of polymerized amino
acid residues that is a transcriptional regulator or a domain or
portion or fragment thereof. Additionally, the polypeptide can
comprise: (i) a localization domain; (ii) an activation domain;
(iii) a repression domain; (iv) an oligomerization domain; (v) a
protein-protein interaction domain; (vi) a DNA-binding domain; or
the like. The polypeptide optionally comprises modified amino acid
residues, naturally occurring amino acid residues not encoded by a
codon, non-naturally occurring amino acid residues.
[0086] As used herein, "protein" refers to a series of amino acids,
oligopeptide, peptide, polypeptide or portions thereof whether
naturally occurring or synthetic.
[0087] As used herein a "recombinant polypeptide" is a polypeptide
produced by translation of a recombinant polynucleotide.
[0088] A "synthetic polypeptide" is a polypeptide created by
consecutive polymerization of isolated amino acid residues using
methods well known in the art.
[0089] Recombinant DNA constructs are assembled using methods known
to persons of ordinary skill in the art and typically comprise a
promoter operably linked to DNA, the expression of which provides
an enhanced agronomic trait. Other construct components can include
additional regulatory elements, such as 5' leaders and introns for
enhancing transcription, 3' untranslated regions (such as
polyadenylation signals and sites), and DNA for transit or
targeting or signal peptides. A "DNA construct" as used in the
present disclosure comprises at least one expression cassette
having a promoter operable in plant cells and a polynucleotide of
the present disclosure encoding a protein or variant of a protein
or fragment of a protein that is functionally defined to maintain
activity in host cells including plant cells, plant parts, explants
and plants. DNA constructs are made that contain various genetic
elements necessary for the expression of noncoding and coding
polynucleotides in plants. Promoters, leaders, enhancers, introns,
transit or targeting or signal peptide sequences, 3'
transcriptional termination regions are genetic elements that can
be operably linked in a DNA construct.
[0090] Percent identity describes the extent to which
polynucleotides or protein segments are invariant in an alignment
of sequences, for example, nucleotide sequences or amino acid
sequences. An alignment of sequences is created by manually
aligning two sequences, for example, a stated sequence, as provided
herein, as a reference, and another sequence, to produce the
highest number of matching elements, for example, individual
nucleotides or amino acids, while allowing for the introduction of
gaps into either sequence. An "identity fraction" for a sequence
aligned with a reference sequence is the number of matching
elements, divided by the full length of the reference sequence, not
including gaps introduced by the alignment process into the
reference sequence. "Percent identity" ("% identity") as used
herein is the identity fraction times 100.
[0091] As used herein, a "functional fragment" refers to a portion
of a polypeptide provided herein which retains full or partial
molecular, physiological or biochemical function of the full length
polypeptide. A functional fragment often contains the domain(s),
such as Pfam domains, identified in the polypeptide provided in the
sequence fisting.
[0092] As used herein, a "homolog" or "homologues" means a protein
in a group of proteins that perform the same biological function,
for example, proteins that belong to the same Pfam protein family
and that provide a common enhanced trait in transgenic plants of
this disclosure. Homologs are expressed by homologous genes. With
reference to homologous genes, homologs include orthologs, for
example, genes expressed in different species that evolved from a
common ancestral genes by speciation and encode proteins retain the
same function, but do not include paralogs, for example, genes that
are related by duplication but have evolved to encode proteins with
different functions. Homologous genes include naturally occurring
alleles and artificially-created variants. Degeneracy of the
genetic code provides the possibility to substitute at least one
base of the protein encoding sequence of a gene with a different
base without causing the amino acid sequence of the polypeptide
produced from the gene to be changed. When optimally aligned,
homolog proteins, or their respective nucleotides, have typically
at least about 60% identity, in some instances at least about 70%,
at least about 75%, at least about 80%, at least about 85%, at
least about 90%, at least about 92%, at least about 94%, at least
about 95%, at least about 96%, at least about 97%, at least about
98%, at least about 99%, and even at least about 99.5% identity
over the full length of a protein identified as being associated
with imparting an enhanced trait when expressed in plant cells. In
one aspect of the disclosure homolog proteins have amino acid
sequences or corresponding nucleotide sequences that have at least
about 80%, at least about 85%, at least about 90%, at least about
92%, at least about 94%, at least about 95%, at least about 96%, at
least about 97%, at least about 98%, at least about 99%, and at
least about 99.5% identity to a consensus amino acid sequence of
proteins and homologs that can be built from sequences disclosed
herein.
[0093] Homologs are inferred from sequence similarity, by
comparison of protein sequences, for example, manually or by use of
a computer-based tool using well-known sequence comparison
algorithms such as BLAST and FASTA. A sequence search and local
alignment program, for example, BLAST, can be used to search query
protein sequences of a base organism against a database of protein
sequences of various organisms, to find similar sequences, and the
summary Expectation value (E-value) can be used to measure the
level of sequence similarity. Because a protein hit with the lowest
E-value for a particular organism may not necessarily be an
ortholog or be the only ortholog, a reciprocal query is used to
filter hit sequences with significant E-values for ortholog
identification. The reciprocal query entails search of the
significant hits against a database of protein sequences of the
base organism. A hit can be identified as an ortholog, when the
reciprocal query's best hit is the query protein itself or a
paralog of the query protein. With the reciprocal query process
orthologs are further differentiated from paralogs among all the
homologs, which allows for the inference of functional equivalence
of genes. A further aspect of the homologs encoded by DNA useful in
the transgenic plants of the invention are those proteins that
differ from a disclosed protein as the result of deletion or
insertion of one or more amino acids in a native sequence.
[0094] Other functional homolog proteins differ in one or more
amino acids from those of a trait-improving protein disclosed
herein as the result of one or more of the well-known conservative
amino acid substitutions, for example, valine is a conservative
substitute for alanine and threonine is a conservative substitute
for serine. Conservative substitutions for an amino acid within the
native sequence can be selected from other members of a class to
which the naturally occurring amino acid belongs. Representative
amino acids within these various classes include, but are not
limited to: (1) acidic (negatively charged) amino acids such as
aspartic acid and glutamic acid; (2) basic (positively charged)
amino acids such as arginine, histidine, and lysine; (3) neutral
polar amino acids such as glycine, serine, threonine, cysteine,
tyrosine, asparagine, and glutamine; and (4) neutral nonpolar
(hydrophobic) amino acids such as alanine, leucine, isoleucine,
valine, proline, phenylalanine, tryptophan, and methionine.
Conserved substitutes for an amino acid within a native protein or
polypeptide can be selected from other members of the group to
which the naturally occurring amino acid belongs. For example, a
group of amino acids having aliphatic side chains is glycine,
alanine, valine, leucine, and isoleucine; a group of amino acids
having aliphatic-hydroxyl side chains is serine and threonine; a
group of amino acids having amide-containing side chains is
asparagine and glutamine; a group of amino acids having aromatic
side chains is phenylalanine, tyrosine, and tryptophan; a group of
amino acids having basic side chains is lysine, arginine, and
histidine; and a group of amino acids having sulfur-containing side
30 chains is cysteine and methionine. Naturally conservative amino
acids substitution groups are: valine-leucine, valine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alaninevaline, aspartic
acid-glutamic acid, and asparagine-glutamine. A further aspect of
the disclosure includes proteins that differ in one or more amino
acids from those of a described protein sequence as the result of
deletion or insertion of one or more amino acids in a native
sequence.
[0095] Homologs can be identified for the polypeptide sequences
provided in Table 1, using the reciprocal search process as
described in paragraph [0091]. The NCBI "blastp" program can be
used for the sequence search, with E-value cutoff of Ie-4 to
identify the initial significant hits. NCBI non-redundant
amino-acid dataset can be used as the database of protein sequences
of various organisms. Homologs with at least 95% identity over 95%
of the length of the polypeptide sequences provided in Table 1
would be kept. From the sequences of the proteins identified in SEQ
ID NOs: 6, 8, 12, and 16, the corresponding homologous protein
sequences set forth as SEQ ID NOs: 23 (homolog of SEQ ID NO: 6), 24
(homolog of SEQ ID NO: 8), SEQ ID NOs: 25 and 26 (homologs of SEQ
ID NO: 12), SEQ ID NO: 27 (homolog of SEQ ID NO: 14), and SEQ ID
NOs: 28 and 29 (homologs of SEQ ID NO: 16) were identified for
preparing additional transgenic seeds and plants with enhanced
agronomic traits.
[0096] In general, the term "variant" refers to molecules with some
differences, generated synthetically or naturally, in their
nucleotide or amino acid sequences as compared to a reference
(native) polynucleotides or polypeptides, respectively. These
differences include substitutions, insertions, deletions or any
desired combinations of such changes in a native polynucleotide or
amino acid sequence.
[0097] With regard to polynucleotide variants, differences between
presently disclosed polynucleotides and polynucleotide variants are
limited so that the nucleotide sequences of the former and the
latter are similar overall and, in many regions, identical. Due to
the degeneracy of the genetic code, differences between the former
and the latter nucleotide sequences can be silent (for example, the
amino acids encoded by the polynucleotide are the same, and the
variant polynucleotide sequence encodes the same amino acid
sequence as the presently disclosed polynucleotide). Variant
nucleotide sequences can encode different amino acid sequences, in
which case such nucleotide differences will result in amino acid
substitutions, additions, deletions, insertions, truncations or
fusions with respect to the similarly disclosed polynucleotide
sequences. These variations can result in polynucleotide variants
encoding polypeptides that share at least one functional
characteristic. The degeneracy of the genetic code also dictates
that many different variant polynucleotides can encode identical
and/or substantially similar polypeptides.
[0098] As used herein "gene" or "gene sequence" refers to the
partial or complete coding sequence of a gene, its complement, and
its 5' and/or 3' untranslated regions. A gene is also a functional
unit of inheritance, and in physical terms is a particular segment
or sequence of nucleotides along a molecule of DNA (or RNA, in the
case of RNA viruses) involved in producing a polypeptide chain. The
latter can be subjected to subsequent processing such as chemical
modification or folding to obtain a functional protein or
polypeptide. By way of example, a transcriptional regulator gene
encodes a transcriptional regulator polypeptide, which can be
functional or require processing to function as an initiator of
transcription.
[0099] As used herein, the term "promoter" refers generally to a
DNA molecule that is involved in recognition and binding of RNA
polymerase II and other proteins (trans-acting transcription
factors) to initiate transcription. A promoter can be initially
isolated from the 5' untranslated region (5' UTR) of a genomic copy
of a gene. Alternately, promoters can be synthetically produced or
manipulated DNA molecules. Promoters can also be chimeric, that is
a promoter produced through the fusion of two or more heterologous
DNA molecules. Plant promoters include promoter DNA obtained from
plants, plant viruses, fungi and bacteria such as Agrobacterium and
Bradyrhizobium bacteria.
[0100] Promoters which initiate transcription in all or most
tissues of the plant are referred to as "constitutive" promoters.
Promoters which 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. Promoters which express
within a specific tissue 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, for example a microspore mother cell, is referred to as a
"cell type specific" promoter. An "inducible" promoter is a
promoter in which transcription is initiated in response to an
environmental stimulus such as cold, drought or light; or other
stimuli such as wounding or chemical application. Many
physiological and biochemical processes in plants exhibit
endogenous rhythms with a period of about 24 hours. A "diurnal
promoter" is a promoter which exhibits altered expression profiles
under the control of a circadian oscillator. Diurnal regulation is
subject to environmental inputs such as light and temperature and
coordination by the circadian clock.
[0101] As used herein, the term "leader" refers to a DNA molecule
isolated from the untranslated 5' region (5' UTR) of a genomic copy
of a gene and is defined generally as a nucleotide segment between
the transcription start site (TSS) and the protein coding sequence
start site. Alternately, leaders can be synthetically produced or
manipulated DNA elements. A leader can be used as a 5' regulatory
element for modulating expression of an operably linked
transcribable polynucleotide molecule.
[0102] As used herein, the term "intron" refers to a DNA molecule
that can be isolated or identified from the genomic copy of a gene
and can be defined generally as a region spliced out during mRNA
processing prior to translation. Alternately, an intron can be a
synthetically produced or manipulated DNA element. An intron can
contain enhancer elements that effect the transcription of operably
linked genes. An intron can be used as a regulatory element for
modulating expression of an operably linked transcribable
polynucleotide molecule. A DNA construct can comprise an intron,
and the intron may or may not be heterologous with respect to the
transcribable polynucleotide molecule.
[0103] As used herein, the term "enhancer" or "enhancer element"
refers to a cis-acting transcriptional regulatory element, a.k.a.
cis-element, which confers an aspect of the overall expression
pattern, but is usually insufficient alone to drive transcription,
of an operably linked polynucleotide. Unlike promoters, enhancer
elements do not usually include a transcription start site (TSS) or
TATA box or equivalent sequence. A promoter can naturally comprise
one or more enhancer elements that affect the transcription of an
operably linked polynucleotide. An isolated enhancer element can
also be fused to a promoter to produce a chimeric promoter
cis-element, which confers an aspect of the overall modulation of
gene expression. A promoter or promoter fragment can comprise one
or more enhancer elements that effect the transcription of operably
linked genes. Many promoter enhancer elements are believed to bind
DNA-binding proteins and/or affect DNA topology, producing local
conformations that selectively allow or restrict access of RNA
polymerase to the DNA template or that facilitate selective opening
of the double helix at the site of transcriptional initiation. An
enhancer element can function to bind transcription factors that
regulate transcription. Some enhancer elements bind more than one
transcription factor, and transcription factors can interact with
different affinities with more than one enhancer domain.
[0104] Expression cassettes of this disclosure can include a
"transit peptide" or "targeting peptide" or "signal peptide"
molecule located either 5' or 3' to or within the gene(s). These
terms generally refer to peptide molecules that when linked to a
protein of interest directs the protein to a particular tissue,
cell, subcellular location, or cell organelle. Examples include,
but are not limited to, chloroplast transit peptides (CTPs),
chloroplast targeting peptides, mitochondrial targeting peptides,
nuclear targeting signals, nuclear exporting signals, vacuolar
targeting peptides, vacuolar sorting peptides. For description of
the use of chloroplast transit peptides see U.S. Pat. No. 5,188,642
and U.S. Pat. No. 5,728,925. For description of the transit peptide
region of an Arabidopsis EPSPS gene see Klee, H. J. et al (MGG
(1987) 210:437-442. Expression cassettes of this disclosure can
also include an intron or introns. Expression cassettes of this
disclosure can contain a DNA near the 3' end of the cassette that
acts as a signal to terminate transcription from a heterologous
nucleic acid and that directs polyadenylation of the resultant
mRNA. These are commonly referred to as "3'-untranslated regions"
or "3'-non-coding sequences" or "3'-UTRs". The "3' non-translated
sequences" means DNA sequences located downstream of a structural
nucleotide sequence and include sequences encoding polyadenylation
and other regulatory signals capable of affecting mRNA processing
or gene expression. The polyadenylation signal functions in plants
to cause the addition of polyadenylate nucleotides to the 3' end of
the mRNA precursor. The polyadenylation signal can be derived from
a natural gene, from a variety of plant genes, or from T-DNA. An
example of a polyadenylation sequence is the nopaline synthase 3'
sequence (nos 3'; Fraley et al., Proc. Natl. Acad. Sci. USA 80:
4803-4807, 1983). The use of different 3' non-translated sequences
is exemplified by Ingelbrecht et al., Plant Cell 1:671-680, 1989.
Recombinant DNA constructs in this disclosure generally include a
3' element that typically contains a polyadenylation signal and
site. Well-known 3' elements include those from Agrobacterium
tumefaciens genes such as nos 3', tml 3', tmr 3', tms 3', ocs 3',
tr7 3', for example disclosed in U.S. Pat. No. 6,090,627; 3'
elements from plant genes such as wheat (Triticum aesevitum) heat
shock protein 17 (Hsp17 3'), a wheat ubiquitin gene, a wheat
fructose-1,6-biphosphatase gene, a rice glutelin gene, a rice
lactate dehydrogenase gene and a rice beta-tubulin gene, all of
which are disclosed in US Patent Application Publication No. US
2002/0192813 A1; and the pea (Pisum sativum) ribulose biphosphate
carboxylase gene (rbs 3'), and 3' elements from the genes within
the host plant.
[0105] Expression cassettes of this disclosure can also contain one
or more genes that encode selectable markers and confer resistance
to a selective agent such as an antibiotic or a herbicide. A number
of selectable marker genes are known in the art and can be used in
the present disclosure: selectable marker genes conferring
tolerance to antibiotics like kanamycin and paromomycin (nptll),
hygromycin B (aph IV), spectinomycin (aadA), US Patent Publication
No. US 2009/0138985 A1 and gentamycin (aac3 and aacC4) or tolerance
to herbicides like glyphosate (for example,
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), U.S. Pat. No.
5,627,061; U.S. Pat. No. 5,633,435; U.S. Pat. No. 6,040,497; U.S.
Pat. No. 5,094,945), sulfonyl herbicides (for example,
acetohydroxyacid synthase or acetolactate synthase conferring
tolerance to acetolactate synthase inhibitors such as sulfonylurea,
imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates and
phthalide (U.S. Pat. No. 6,225,105; U.S. Pat. No. 5,767,366; U.S.
Pat. No. 4,761,373; U.S. Pat. No. 5,633,437; U.S. Pat. No.
6,613,963; U.S. Pat. No. 5,013,659; U.S. Pat. No. 5,141,870; U.S.
Pat. No. 5,378,824; and U.S. Pat. No. 5,605,011)), bialaphos or
phosphinothricin or derivatives (for example, phosphinothricin
acetyltransferase (bar) tolerance to phosphinothricin or
glufosinate (U.S. Pat. No. 5,646,024; U.S. Pat. No. 5,561,236; U.S.
Pat. No. 5,276,268; U.S. Pat. No. 5,637,489; U.S. Pat. No.
5,273,894); dicamba (dicamba monooxygenase, US Patent Application
Publications No. US 2003/0115626 A1), or sethoxydim (modified
acetyl-coenzyme A carboxylase for conferring tolerance to
cyclohexanedione (sethoxydim)), and aryloxyphenoxypropionate
(haloxyfop, U.S. Pat. No. 6,414,222).
[0106] Transformation vectors of this disclosure can contain one or
more "expression cassettes", each comprising a native or non-native
plant promoter operably linked to a polynucleotide sequence of
interest, which is operably linked to a 3' UTR termination signal,
for expression in an appropriate host cell. It also typically
comprises sequences required for proper translation of the
polynucleotide or transgene. As used herein, the term "transgene"
refers to a polynucleotide molecule artificially incorporated into
a host cell's genome. Such a transgene can be heterologous to the
host cell. The expression cassette comprising the nucleotide
sequence of interest can be chimeric, meaning that at least one of
its components is heterologous with respect to at least one of its
other components. As used herein the term "chimeric" refers to a
DNA molecule that is created from two or more genetically diverse
sources, for example, a first molecule from one gene or organism
and a second molecule from another gene or organism.
[0107] As used herein "operably linked" means the association of
two or more DNA fragments in a recombinant DNA construct so that
the function of one, for example, protein-encoding DNA, is
controlled by the other, for example, a promoter.
[0108] As used herein "expressed" means produced, for example, the
information from a gene is used in the synthesis of a functional
gene product. These products are often proteins. For example, a
protein is expressed in a plant cell when its cognate DNA is
transcribed to mRNA that is translated to the protein. In the case
of non-protein coding gene/sequence, the product is a functional
RNA. An "expressed" protein can also include its truncated version
(for example, N-terminal truncated, C-terminal truncated or
internal truncated) as long as the truncated version maintains the
same or similar functionality as the full length version.
[0109] Transgenic plants can comprise a stack of one or more
polynucleotides disclosed herein resulting in the production of
multiple polypeptide sequences. Transgenic plants comprising stacks
of polynucleotides can be obtained by either or both of traditional
breeding methods or through genetic engineering methods. These
methods include, but are not limited to, crossing individual
transgenic lines each comprising a polynucleotide of interest,
transforming a transgenic plant comprising a first gene disclosed
herein with a second gene, and co-transformation of genes into a
single plant cell. Co-transformation of genes can be carried out
using single transformation vectors comprising multiple genes or
multiples vectors each comprising one or more genes.
[0110] Transgenic plants comprising or derived from plant cells of
this disclosure transformed with recombinant DNA can be further
enhanced with stacked traits, for example, a crop plant having an
enhanced trait resulting from expression of DNA disclosed herein in
combination with herbicide and/or pest resistance traits. For
example, genes of the current disclosure can be stacked with other
traits of agronomic interest, such as a trait providing herbicide
resistance, or insect resistance, such as using a gene from
Bacillus thuringensis to provide resistance against lepidopteran,
coliopteran, homopteran, hemiopteran, and other insects, or
improved quality traits such as improved nutritional value.
Herbicides to which transgenic plant tolerance has been
demonstrated and the method of the present disclosure can be
applied include, but are not limited to, glyphosate, dicamba,
glufosinate, sulfonylurea, bromoxynil and norflurazon herbicides.
Polynucleotide molecules encoding proteins involved in herbicide
tolerance are well-known in the art and include, but are not
limited to, a polynucleotide molecule encoding
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) disclosed in
U.S. Pat. No. 5,094,945; U.S. Pat. No. 5,627,061; U.S. Pat. No.
5,633,435 and U.S. Pat. No. 6,040,497 for imparting glyphosate
tolerance; polynucleotide molecules encoding a glyphosate
oxidoreductase (GOX) disclosed in U.S. Pat. No. 5,463,175 and a
glyphosate-N-acetyl transferase (GAT) disclosed in US Patent
Application Publication No. US 2003/0083480 A1 also for imparting
glyphosate tolerance; dicamba monooxygenase disclosed in US Patent
Application Publication No. US 2003/0135879 A1 for imparting
dicamba tolerance; a polynucleotide molecule encoding bromoxynil
nitrilase (Bxn) disclosed in U.S. Pat. No. 4,810,648 for imparting
bromoxynil tolerance; a polynucleotide molecule encoding phytoene
desaturase (crtl) described in Misawa et al, (1993) Plant J.
4:833-840 and in Misawa et al, (1994) Plant J. 6:481-489 for
norflurazon tolerance; a polynucleotide molecule encoding
acetohydroxyacid synthase (AHAS, aka ALS) described in Sathasiivan
et al. (1990) Nucl. Acids Res. 18:2188-2193 for imparting tolerance
to sulfonylurea herbicides; polynucleotide molecules known as bar
genes disclosed in DeBlock, et al. (1987) EMBO J. 6:2513-2519 for
imparting glufosinate and bialaphos tolerance as disclosed in U.S.
Pat. No. 7,112,665; polynucleotide molecules disclosed in U.S. Pat.
No. 6,107,549 for imparting pyridine herbicide resistance;
molecules and methods for imparting tolerance to multiple
herbicides such as glyphosate, atrazine, ALS inhibitors,
isoxoflutole and glufosinate herbicides are disclosed in U.S. Pat.
No. 6,376,754 and US Patent Application Publication No. US
2002/0112260. Molecules and methods for imparting
insect/nematode/virus resistance are disclosed in U.S. Pat. No.
5,250,515; U.S. Pat. No. 5,880,275; U.S. Pat. No. 6,506,599; U.S.
Pat. No. 5,986,175 and US Patent Application Publication No. US
2003/0150017 A1.
Plant Cell Transformation Methods
[0111] Numerous methods for transforming chromosomes in a plant
cell with recombinant DNA are known in the art and are used in
methods of producing a transgenic plant cell and plant. Two
effective methods for such transformation are
Agrobacterium-mediated transformation and microprojectile
bombardment-mediated transformation. Microprojectile bombardment
methods are illustrated in U.S. Pat. No. 5,015,580 (soybean); U.S.
Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880 (corn); U.S.
Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208 (corn); U.S.
Pat. No. 6,399,861 (corn); U.S. Pat. No. 6,153,812 (wheat) and U.S.
Pat. No. 6,365,807 (rice). Agrobacterium-mediated transformation
methods are described in U.S. Pat. No. 5,159,135 (cotton); U.S.
Pat. No. 5,824,877 (soybean); U.S. Pat. No. 5,463,174 (canola);
U.S. Pat. No. 5,591,616 (corn); U.S. Pat. No. 5,846,797 (cotton);
U.S. Pat. No. 6,384,301 (soybean), U.S. Pat. No. 7,026,528 (wheat)
and U.S. Pat. No. 6,329,571 (rice), US Patent Application
Publication No. US 2004/0087030 A1 (cotton), and US Patent
Application Publication No. US 2001/0042257 A1 (sugar beet), all of
which are incorporated herein by reference for enabling the
production of transgenic plants. Transformation of plant material
is carried out in tissue culture on nutrient media, for example, a
mixture of nutrients that allow cells to grow in vitro. Recipient
cell targets include, but are not limited to, meristem cells, shoot
tips, hypocotyls, calli, immature or mature embryos, and gametic
cells such as microspores, pollen, sperm and egg cells. Callus can
be initiated from tissue sources including, but not limited to,
immature or mature embryos, hypocotyls, seedling apical meristems,
microspores and the like. Cells containing a transgenic nucleus are
grown into transgenic plants.
[0112] In addition to direct transformation of a plant material
with a recombinant DNA, a transgenic plant can be prepared by
crossing a first plant comprising a recombinant DNA with a second
plant lacking the recombinant DNA. For example, recombinant DNA can
be introduced into a first plant line that is amenable to
transformation, which can be crossed with a second plant line to
introgress the recombinant DNA into the second plant line. A
transgenic plant with recombinant DNA providing an enhanced trait,
for example, enhanced yield, can be crossed with a transgenic plant
line having another recombinant DNA that confers another trait, for
example herbicide resistance, pest resistance or enhanced water use
efficiency to produce progeny plants having recombinant DNA that
confers both traits. Typically, in such breeding for combining
traits the transgenic plant donating the additional trait is the
male line and the transgenic plant carrying the base traits is the
female line. The progeny of this cross will segregate such that
some of the plants will carry the DNA for both parental traits and
some will carry DNA for one parental trait; such plants can be
identified by markers associated with parental recombinant DNA, for
example, marker identification by analysis for recombinant DNA or,
in the case where a selectable marker is linked to the recombinant
DNA, by application of a selective agent such as a herbicide for
use with a herbicide tolerance marker, or by selection for the
enhanced trait. Progeny plants carrying DNA for both parental
traits can be crossed back into the female parent line multiple
times, for example usually 6 to 8 generations, to produce a progeny
plant with substantially the same genotype as the original
transgenic parental line but for the recombinant DNA of the other
transgenic parental line.
[0113] In transformation, DNA is typically introduced into only a
small percentage of target plant cells in any one transformation
experiment. Marker genes are used to provide an efficient system
for identification of those cells that are stably transformed by
receiving and integrating a recombinant DNA molecule into their
genomes. Preferred marker genes provide selective markers which
confer resistance to a selective agent, such as an antibiotic or a
herbicide. Any of the herbicides to which plants of this disclosure
can be resistant is a agent for selective markers. Potentially
transformed cells are exposed to the selective agent. In the
population of surviving cells are those cells where, generally, the
resistance-conferring gene is integrated and expressed at
sufficient levels to permit cell survival. Cells can be tested
further to confirm stable integration of the exogenous DNA.
Commonly used selective marker genes include those conferring
resistance to antibiotics such as kanamycin and paromomycin
(nptII), hygromycin B (aph IV), spectinomycin (aadA) and gentamycin
(aac3 and aacC4) or resistance to herbicides such as glufosinate
(bar or pat), dicamba (DMO) and glyphosate (aroA or EPSPS).
Examples of such selectable markers are illustrated in U.S. Pat.
No. 5,550,318; U.S. Pat. No. 5,633,435; U.S. Pat. No. 5,780,708 and
U.S. Pat. No. 6,118,047. Markers which provide an ability to
visually screen transformants can also be employed, for example, a
gene expressing a colored or fluorescent protein such as a
luciferase or green fluorescent protein (GFP) or a gene expressing
a beta-glucuronidase or uidA gene (GUS) for which various
chromogenic substrates are known.
[0114] Plant cells that survive exposure to a selective agent, or
plant cells that have been scored positive in a screening assay,
may be cultured in vitro to regenerate plantlets. Developing
plantlets regenerated from transformed plant cells can be
transferred to plant growth mix, and hardened off, for example, in
an environmentally controlled chamber at about 85% relative
humidity, 600 ppm CO.sub.2, and 25-250 micro-einsteins m.sup.-2
s.sup.-1 of light, prior to transfer to a greenhouse or growth
chamber for maturation. Plants are regenerated from about 6 weeks
to 10 months after a transformant is identified, depending on the
initial tissue, and plant species. Plants can be pollinated using
conventional plant breeding methods known to those of skill in the
art to produce seeds, for example self-pollination is commonly used
with transgenic corn. The regenerated transformed plant or its
progeny seed or plants can be tested for expression of the
recombinant DNA and selected for the presence of an enhanced
agronomic trait.
Transgenic Plants and Seeds
[0115] Transgenic plants derived from transgenic plant cells having
a transgenic nucleus of this disclosure are grown to produce
transgenic seed and haploid pollen of this disclosure. Such plants
can be identified either by the presence of the transgene(s) using
molecular techniques known in the art, or by selection of
transformed plants or progeny seed for an enhanced trait. For
efficiency a selection method is designed to evaluate multiple
transgenic plants (events) comprising the recombinant DNA, for
example, multiple plants from 2 to 20 or more transgenic events.
Transgenic plants grown from transgenic seeds provided herein
demonstrate improved agronomic traits that contribute to increased
yield or other traits that provide increased plant value,
including, for example, improved seed quality. Of particular
interest are plants having increased water use efficiency or
drought tolerance, enhanced high temperature or cold tolerance,
increased yield, and increased nitrogen use efficiency.
[0116] Table 1 provides a list of protein-encoding DNA ("genes") as
recombinant DNA for production of transgenic plants with enhanced
traits, the elements of Table 1 are described by reference to:
[0117] "PEP SEQ ID NO" which identifies an amino acid sequence.
[0118] "NUC SEQ ID NO" which identifies a DNA sequence.
[0119] "Gene ID" which refers to an arbitrary identifier.
[0120] "Protein Name" which is a common name for protein encoded by
the recombinant DNA.
TABLE-US-00001 TABLE 1 NUC PEP SEQ SEQ ID ID NO NO Gene ID Protein
Name 1 2 TRDX2-1 Arabidopsis basic helix-loop-helix protein 3 4
TRDX2-2 Corn uridine phosphate glycosyl transferase protein 5 6
TRDX2-3 Arabidopsis At3g60820 proteosome component protein 7 8
TRDX2-4 Arabidopsis actin-like protein 4 9 10 TRDX2-5 Arabidopsis
transcription factor S-II domain- containing protein 11 12 TRDX2-6
Arabidopsis protein homologous to rice OSJNBa0064D20.11 13 14
TRDX2-7 Pyropia petJ_18146963 protein 15 16 TRDX2-8 Corn
A1ZM043652_s_at_Os01g0678600 protein
[0121] Table 2 provides a list of suppression elements as
recombinant DNA for production of transgenic plants with enhanced
traits, the elements of Table 2 are described by reference to:
[0122] "SUP SEQ ID NO" which identifies a suppression element
sequence.
[0123] "Target Gene NUC SEQ ID NO" which identifies a target gene
nucleotide sequence for suppression.
[0124] "Target Gene PEP SEQ ID NO Gene ID" which identifies an
amino acid sequence of a target gene
[0125] "Gene ID", which refers to an identifier.
[0126] "Target Protein Name" which is a common name for protein
encoded by the target gene DNA.
TABLE-US-00002 TABLE 2 SUP (NUC) Target Gene Target Gene SEQ ID
(NUC) (PEP) Target NO SEQ ID NO SEQ ID NO Gene ID Protein Name 17
18 19 TRDX2-9 Corn DWARF4-like protein 20 21 22 TRDX2-10 Corn
ribozyme inactivating protein
Selection Methods for Transgenic Plants with Enhanced Traits
[0127] Within a population of transgenic plants each regenerated
from a plant cell with recombinant DNA, many plants that survive to
fertile transgenic plants that produce seeds and progeny plants
will not exhibit an enhanced agronomic trait. Selection from the
population is necessary to identify one or more transgenic plants
with an enhanced trait. Transgenic plants having enhanced traits
are selected from populations of plants regenerated or derived from
plant cells transformed as described herein by evaluating the
plants in a variety of assays to detect an enhanced trait, for
example, increased water use efficiency or drought tolerance,
enhanced high temperature or cold tolerance, increased yield,
increased nitrogen use efficiency, enhanced seed composition such
as enhanced seed protein and enhanced seed oil. These assays can
take many forms including, but not limited to, direct screening for
the trait in a greenhouse or field trial or by screening for a
surrogate trait. Such analyses can be directed to detecting changes
in the chemical composition, biomass, physiological property, or
morphology of the plant. Changes in chemical compositions such as
nutritional composition of grain can be detected by analysis of the
seed composition and content of protein, free amino acids, oil,
free fatty acids, starch or tocopherols. Changes in chemical
compositions can also be detected by analysis of contents in
leaves, such as chlorophyll or carotenoid contents. Changes in
biomass characteristics can be evaluated on greenhouse or field
grown plants and can include plant height, stem diameter, root and
shoot dry weights, canopy size; and, for corn plants, ear length,
ear diameter, ear biomass and ear biomass per plot. Changes in
physiological properties can be identified by evaluating responses
to stress conditions, for example, assays using imposed stress
conditions such as water deficit, nitrogen deficiency, cold growing
conditions, pathogen or insect attack or light deficiency, or
increased plant density. Changes in morphology can be measured by
visual observation of tendency of a transformed plant to appear to
be a normal plant as compared to changes toward bushy, taller,
thicker, narrower leaves, striped leaves, knotted trait, chlorosis,
albino, anthocyanin production, or altered tassels, ears or roots.
Other selection properties include days to pollen shed, days to
silking, leaf extension rate, chlorophyll content, leaf
temperature, stand, seedling vigor, internode length, plant height,
leaf number, leaf area, tillering, brace roots, stay green or
delayed senescence, stalk lodging, root lodging, plant health,
barreness/prolificacy, green snap, and pest resistance. In
addition, phenotypic characteristics of harvested grain can be
evaluated, including number of kernels per row on the ear, number
of rows of kernels on the ear, kernel abortion, kernel weight,
kernel size, kernel density, ear biomass, and physical grain
quality.
[0128] Assays for screening for a desired trait are readily
designed by those practicing in the art. The following illustrates
screening assays for corn traits using hybrid corn plants. The
assays can be readily adapted for screening other plants such as
canola, wheat, cotton and soybean either as hybrids or inbreds.
[0129] Transgenic corn plants having increased nitrogen use
efficiency can be identified by screening transgenic plants in the
field under the same and sufficient amount of nitrogen supply as
compared to control plants, where such plants provide higher yield
as compared to control plants. Transgenic corn plants having
increased nitrogen use efficiency can also be identified where such
plants provide higher yield as compared to by screening transgenic
plants in the field under reduced amount of nitrogen supply as
control plants under the same nitrogen limiting conditions.
[0130] Transgenic corn plants having increased yield are identified
by screening progenies of the transgenic plants over multiple
locations for several years with plants grown under optimal
production management practices and maximum weed and pest control.
Selection methods can be applied in multiple and diverse geographic
locations, for example up to 16 or more locations, over one or more
planting seasons, for example at least two planting seasons, to
statistically distinguish yield improvement from natural
environmental effects.
[0131] Transgenic corn plants having increased water use efficiency
or drought tolerance are identified by screening plants in an assay
where water is withheld for a period to induce stress followed by
watering to revive the plants. For example, a selection process
imposes 3 drought/re-water cycles on plants over a total period of
15 days after an initial stress free growth period of 11 days. Each
cycle consists of 5 days, with no water being applied for the first
four days and a water quenching on the 5th day of the cycle. The
primary phenotypes analyzed by the selection method are the changes
in plant growth rate as determined by height and biomass during a
vegetative drought treatment.
[0132] Transgenic cotton plants with increased yield and increased
water use efficiency are identified by growing under variable water
conditions. Specific conditions for cotton include growing a first
set of transgenic and control plants under "wet" conditions, i.e.
irrigated in the range of 85 to 100 percent of evapotranspiration
to provide leaf water potential of -14 to -18 bars, and growing a
second set of transgenic and control plants under "dry" conditions,
for example, irrigated in the range of 40 to 60 percent of
evapotranspiration to provide a leaf water potential of -21 to -25
bars. Pest control, such as weed and insect control is applied
equally to both wet and dry treatments as needed. Data gathered
during the trial includes weather records throughout the growing
season including detailed records of rainfall; soil
characterization information; any herbicide or insecticide
applications; any gross agronomic differences observed such as leaf
morphology, branching habit, leaf color, time to flowering, and
fruiting pattern; plant height at various points during the trial;
stand density; node and fruit number including node above white
flower and node above crack boll measurements; and visual wilt
scoring. Cotton boll samples are taken and analyzed for lint
fraction and fiber quality. The cotton is harvested at the normal
harvest timeframe for the trial area. Increased water use
efficiency is indicated by increased yield, improved relative water
content, enhanced leaf water potential, increased biomass, enhanced
leaf extension rates, and improved fiber parameters.
[0133] Although the plant cells and methods of this disclosure can
be applied to any plant cell, plant, seed or pollen, for example,
any fruit, vegetable, grass, tree or ornamental plant, the various
aspects of the disclosure are applied to corn, soybean, cotton,
canola, rice, barley, oat, wheat, turf grass, alfalfa, sugar beet,
sunflower, quinoa and sugar cane plants.
[0134] The following examples are included to demonstrate aspects
of the disclosure. Those of skill in the art should, in light of
the present disclosure, appreciate that many changes can be made in
the specific aspects which are disclosed and still obtain a like or
similar results without departing from the spirit and scope of the
disclosure.
Example 1
Corn Transformation
[0135] This example illustrates transformation methods in producing
a transgenic corn plant cell, plant, and seed having an enhanced
trait, for example, altered phenotypes as shown in Tables 4-6 or
increased water use efficiency or drought tolerance, increased
yield, and increased nitrogen use efficiency as shown in Tables
7-12.
[0136] For Agrobacterium-mediated transformation of corn embryo
cells corn plants were grown in the greenhouse and ears were
harvested when the embryos were 1.5 to 2.0 mm in length. Ears were
surface-sterilized by spraying or soaking the ears in 80% ethanol,
followed by air drying. Immature embryos were isolated from
individual kernels on surface-sterilized ears. Shortly after
excision, immature maize embryos were inoculated with overnight
grown Agrobacterium cells, and incubated at room temperature with
Agrobacterium for 5-20 minutes. Inoculated immature embryos were
then co-cultured with Agrobacterium for 1 to 3 days at 23.degree.
C. in the dark. Co-cultured embryos were transferred to selection
media and cultured for approximately two weeks to allow embryogenic
callus to develop. Embryogenic calli were transferred to culture
medium containing glyphosate and subcultured at about two week
intervals. Transformed plant cells were recovered 6 to 8 weeks
after initiation of selection.
[0137] For Agrobacterium-mediated transformation of maize callus
immature embryos are cultured for approximately 8-21 days after
excision to allow callus to develop. Callus is then incubated for
about 30 minutes at room temperature with the Agrobacterium
suspension, followed by removal of the liquid by aspiration. The
callus and Agrobacterium are co-cultured without selection for 3-6
days followed by selection on paromomycin for approximately 6
weeks, with biweekly transfers to fresh media. Paromomycin
resistant calli are identified about 6-8 weeks after initiation of
selection.
[0138] To regenerate transgenic corn plants individual transgenic
calli resulting from transformation and selection were placed on
media to initiate shoot and root development into plantlets.
Plantlets were transferred to potting soil for initial growth in a
growth chamber at 26.degree. C. followed by a mist bench before
transplanting to 5 inch pots where plants were grown to maturity.
The regenerated plants were self-fertilized and seeds were
harvested for use in one or more methods to select seeds, seedlings
or progeny second generation transgenic plants (R2 plants) or
hybrids, for example, by selecting transgenic plants exhibiting an
enhanced trait as compared to a control plant.
[0139] The above process can be repeated to produce multiple events
of transgenic corn plants from cells that were transformed with
recombinant DNA from the genes identified in Table 1. Progeny
transgenic plants and seeds of the transformed plants were screened
for the presence and single copy of the inserted nucleotide
sequences set forth in Tables 1 and 2, and for increased water use
efficiency, increased yield, increased nitrogen use efficiency, and
altered phenotypes as shown in Tables 4-6 or Tables 7-12. From each
group of multiple events of transgenic plants with a specific
recombinant DNA from Table 1 the event(s) that showed increased
yield, increased water use efficiency, increased nitrogen use
efficiency, and altered phenotypes was (were) identified.
Example 2
Soybean Transformation
[0140] This example illustrates plant transformation in producing a
transgenic soybean plant cell, plant, and seed having an enhanced
trait, for example, increased water use efficiency, increased
yield, increased nitrogen use efficiency, and altered
phenotypes.
[0141] For Agrobacterium mediated transformation, soybean seeds
were imbibed overnight and the meristem explants excised. Soybean
explants were mixed with induced Agrobacterium cells containing
plasmid DNA with the gene of interest cassette and a plant
selectable marker cassette no later than 14 hours from the time of
initiation of seed imbibition, and wounded using sonication.
Following wounding, explants were placed in co-culture for 2-5 days
at which point they were transferred to selection media to allow
selection and growth of transgenic shoots. Resistant shoots were
harvested in approximately 6-8 weeks and placed into selective
rooting media for 2-3 weeks. Shoots producing roots were
transferred to the greenhouse and potted in soil. Shoots that
remained healthy on selection, but did not produce roots were
transferred to non-selective rooting media for an additional two
weeks. Roots from any shoots that produced roots off selection were
tested for expression of the plant selectable marker before they
were transferred to the greenhouse and potted in soil.
[0142] The above process can be repeated to produce multiple events
of transgenic soybean plants from cells that were transformed with
recombinant DNA from the genes identified in Table 1. Progeny
transgenic plants and seed of the transformed plant cells were
screened for the presence and single copy of the inserted gene, and
for increased water use efficiency, increased yield, increased
nitrogen use efficiency, and altered phenotypes.
Example 3
Canola Transformation
[0143] This example illustrates plant transformation in producing
the transgenic canola plants of this disclosure and the production
and identification of transgenic seed for transgenic canola having
increased water use efficiency, increased yield, and increased
nitrogen use efficiency.
[0144] Tissues from in vitro grown canola seedlings were prepared
and inoculated with overnight-grown Agrobacterium cells containing
plasmid DNA with a gene of interest cassette and a plant selectable
marker cassette. Following co-cultivation with Agrobacterium, the
infected tissues were allowed to grow on selection to promote
growth of transgenic shoots, followed by growth of roots from the
transgenic shoots. The selected plantlets were then transferred to
the greenhouse and potted in soil. Molecular characterizations were
performed to confirm the presence of the gene of interest, and its
expression in transgenic plants and progenies. Progeny transgenic
plants were selected from a population of transgenic canola events
under specified growing conditions and were compared with control
canola plants.
[0145] The above process can be repeated to produce multiple events
of transgenic canola plants from cells that were transformed with
recombinant DNA identified in Table 1 and Table 2. Progeny
transgenic plants and seed of the transformed plant cells were
screened for the presence and single copy of the inserted gene or
DNA, and for increased water use efficiency, increased yield, and
increased nitrogen use efficiency. From each group of multiple
events of transgenic plants with a specific recombinant DNA from
Table 1 and Table 2 the event(s) that showed increased yield,
increased water use efficiency, increased nitrogen use efficiency
and altered phenotypes was (were) identified.
Example 4
Identification of Altered Phenotypes in Automated Greenhouse
[0146] This example illustrates screening and identification of
transgenic plants for altered phenotypes in an automated greenhouse
(AGH). The apparatus and the methods for automated phenotypic
screening of plants are disclosed in US Patent Application
Publication No. US 2011/0135161 A1, which is incorporated by
reference herein in its entirety.
[0147] Screening and Identification of Transgenic Corn Plants for
Altered Phenotypes.
[0148] Corn plants were tested in 3 screens in AGH under different
conditions including non-stress, nitrogen deficit and water deficit
stress conditions. All screens began with a non-stress condition
during day 0-5 germination phase, after which the plants were grown
for 22 days under screen specific conditions as shown in Table
3.
[0149] Water deficit is defined as a specific Volumetric Water
Content (VWC) that is lower than the VWC of non-stress plant. For
example, a non-stressed plant might be maintained at 55% VWC and
water-deficit assay might be defined around 30% VWC as shown in
Table 3. Data were collected using visible light and hyperspectral
imaging as well as direct measurement of pot weight and amount of
water and nutrient applied to individual plants on a daily
basis.
[0150] Eight parameters were measured for each screen. The visible
light color imaging based measurements are: biomass, canopy area
and plant height. Biomass (B) is defined as estimated shoot fresh
weight (g) of the plant obtained from images acquired from multiple
angles of view. Canopy Area (Can) is defined as area of leaf as
seen in top-down image (mm.sup.2). Plant Height (H) refers to the
distance from the top of the pot to the highest point of the plant
derived from side image (mm). Anthocyanin score, chlorophyll score
and water content score are hyperspectral imaging based parameters.
Anthocyanin Score (An) is an estimate of anthocyanin content in the
leaf canopy obtained from a top-down hyperspectral image.
Chlorophyll Score (Chl) is a measurement of chlorophyll in the leaf
canopy obtained from a top-down hyperspectral image. Water Content
Score (WC) is a measurement of water in the leaf canopy obtained
from a top-down hyperspectral image. Water Use Efficiency (WUE) is
derived from the grams of plant biomass per liter of water added.
Water Applied (WA) is a direct measurement of water added to a pot
(pot with no hole) during the course of an experiment.
[0151] These physiological screen runs were set up so that tested
transgenic lines were compared to a control line. The collected
data were analyzed against the control using % delta and certain
p-value cutoff. Tables 4-6 are summaries of transgenic corn plants
comprising the disclosed recombinant DNA molecules with altered
phenotypes under non stress, nitrogen deficit, and water deficit
conditions, respectively.
[0152] "+" denotes an increase in the tested parameter at
p.ltoreq.0.1; whereas denotes a decrease in the tested parameter at
p.ltoreq.0.1. The numbers in parenthesis show penetrance of the
altered phenotypes, where the denominators represent total number
of transgenic events tested for a given parameter in a specific
screen, and the numerators represent the number of events showing a
particular altered phenotype. For example, transgenic plants scored
for anthocyanin content in the nitrogen limiting screens for TRDX2
SEQ ID NO: 6 and TRDX2 SEQ ID NO: 8 (Table 5), showed increased
anthocyanin content at p.ltoreq.0.1 under nitrogen deficit
conditions.
TABLE-US-00003 TABLE 3 Description of the 3 AGH screens for corn
plants. Screen specific Germination phase phase Screen Description
(5 days) (22 days) Non-stress well watered 55% VWC 55% VWC
sufficient nitrogen water 8 mM nitrogen Water deficit limited
watered 55% VWC 30% VWC sufficient nitrogen water 8 mM nitrogen
Nitrogen deficit well watered 55% VWC 55% VWC low nitrogen water 2
mM nitrogen
TABLE-US-00004 TABLE 4 Summary of transgenic corn plants with
altered phenotypes in AGH non-stress screens Non-Stress Gene_ID An
B Can Chl H WA WC WUE TRDX2-6 -(3/3) -(1/3) -(3/3) TRDX2-10 -(1/5)
-(4/5) -(3/5) -(1/5) -(5/5) -(5/5) +(1/5) -(4/5)
TABLE-US-00005 TABLE 5 Summary of transgenic corn plants with
altered phenotypes in AGH nitrogen deficit screens Nitrogen Deficit
Gene_ID An B Can Chl H WA WC WUE TRDX2-6 +(1/3) -(2/3) -(3/3)
-(1/3) -(3/3) -(1/3) -(1/3) TRDX2-8 +(1/5) -(2/5) -(3/5) +(1/5)
+(3/5) TRDX2-10 -(1/5) -(3/5) +(3/5) -(3/5) -(5/5) +(1/5)
TABLE-US-00006 TABLE 6 Summary of transgenic corn plants with
altered phenotypes in AGH water deficit screens Water Deficit
Gene_ID An B Can Chl H WA WC WUE TRDX2-6 -(2/3) -(2/3) -(2/3)
-(2/3) -(2/3) -(2/3) -(2/3) TRDX2-8 -(2/5) -(5/5) -(5/5) -(2/5)
-(5/5) -(5/5) -(3/5) -(2/5) TRDX2-10 -(1/5) +(1/5) +(3/5) +(1/5)
+(1/5)
[0153] Screening and Identification of Transgenic Soybean Plants
for Altered Phenotypes.
[0154] Soybean plants were tested in 2 screens in AGH under
non-stress and water deficit stress conditions. For non-stress
screen, the plants were kept under constant VWC of 55% throughout
the screen length of 27 days. For water deficit screen, the VWC was
kept at 55% for the first 12 days after sowing, followed by gradual
dry down at a rate of 0.025 VWC per day, followed by water recovery
to 55% VWC at 25 days after sowing.
[0155] Water deficit is defined as a specific Volumetric Water
Content (VWC) that is lower than the VWC of non-stress plant. For
example, a non-stressed plant might be maintained at 55% VWC and
water-deficit assay might be defined around 30% VWC as shown in
Table 3. Data were collected using visible light and hyperspectral
imaging as well as direct measurement of pot weight and amount of
water and nutrient applied to individual plants on a daily
basis.
[0156] Eight parameters were measured for each screen. The visible
light color imaging based measurements are: biomass, canopy area
and plant height. Biomass (B) is defined as estimated shoot fresh
weight (grams) of the plant obtained from images acquired from
multiple angles of view. Canopy Area (Can) is defined as area of
leaf as seen in top-down image (mm.sup.2). Plant Height (H) refers
to the distance from the top of the pot to the highest point of the
plant derived from side image (mm). Chlorophyll score is a
hyperspectral imaging based parameter. Chlorophyll Score (Chl) is a
measurement of chlorophyll in the leaf canopy obtained from a
top-down hyperspectral image. Water Use Efficiency (WUE) is derived
from the grams of plant biomass per liter of water added. Water
Applied (WA) is a direct measurement of water added to a pot (pot
with no hole) during the course of an experiment.
[0157] These physiological screen runs were set up so that tested
transgenic lines were compared to a control line. The collected
data were analyzed against the control using % delta and/or certain
p-value cutoff.
Example 5
Phenotypic Evaluation of Transgenic Plants for Increased Nitrogen
Use Efficiency
[0158] Corn nitrogen field efficacy trials were conducted to
identify genes that can improve nitrogen use efficiency under
nitrogen limiting conditions leading to increased yield performance
as compared to non transgenic controls. A yield increase in corn
can be manifested as one or more of the following: an increase in
the number of ears per plant, an increase in the number of rows,
number of kernels per row, kernel weight, thousand kernel weight,
dry/wet ear length/diameter/weight, an increase in ear biomass, and
increase in ear biomass per plot, an increase in the seed filling
rate (which is the number of filled seeds divided by the total
number of seeds and multiplied by 100), among others.
[0159] Table 7 provides a list of protein encoding DNA or
polynucleotide sequence ("gene") for producing transgenic corn
plant with increased nitrogen use efficiency as compared to a
control plant. The element of Table 7 is described by reference
to:
[0160] "SEQ ID NO: Polynucleotide" which identifies a nucleotide
sequence.
[0161] "SEQ ID NO: Polypeptide" which identifies an amino acid
sequence.
[0162] "Gene identifier" which refers to an arbitrary
identifier.
[0163] "NUE results" which represents to the result of a nitrogen
field trial for plants comprising a sequence in a construct with at
least one event showing significant yield increase at p.ltoreq.0.2
across locations. The first number refers to the number of events
with significant yield increase, whereas the second number refers
to the total number of events tested for each sequence in the
construct.
TABLE-US-00007 TABLE 7 Recombinant DNA for increased nitrogen use
efficiency in corn SEQ ID NO: SEQ ID NO: Gene NUE Polynucleotide
Polypeptide Identifier Results 15 16 TRDX2-8 1/5
[0164] Table 8 provides a nucleotide sequence for producing
transgenic corn plant with increased nitrogen use efficiency as
compared to a control plant. The suppression element of Table 8 is
described by reference to:
[0165] "SEQ ID NO: Polynucleotide" which identifies a nucleotide
sequence SEQ ID NO: 20 to suppress an endogenous target protein
from corn;
[0166] "SEQ ID NO: Target Gene" which identifies a polynucleotide
coding sequence from SEQ ID NO: 21 targeted for suppression.
[0167] "SEQ ID NO: Target Protein" which identifies an amino acid
sequence from SEQ ID NO: 22.
[0168] "Gene identifier" which refers to an arbitrary
identifier.
[0169] "NUE results" which refers to the sequence in a construct
with at least one event showing significant yield increase at
p.ltoreq.0.2 across locations. The first number refers to the
number of events with significant yield increase, whereas the
second number refers to the total number of events tested for each
sequence in the construct.
TABLE-US-00008 TABLE 8 Table 8. Recombinant DNA for suppression of
a target gene to provide increased nitrogenuse efficiency in corn
SEQ ID NO: SEQ ID NO: SEQ ID NO: Target Target Gene NUE
Polynucleotide Gene Protein Identifier Results 20 21 22 TRDX2-10
1/5
Example 6
Selection of Transgenic Plants with Increased Yield
[0170] This example illustrates selection and identification of
transgenic plants for increased yield in both dicotyledonous and
monocotyledonous plants with primary examples presented for corn,
soybean, and canola in Tables 9-14 respectively.
[0171] Effective selection of increased and/or enhanced yielding
transgenic plants uses hybrid progenies of the transgenic plants
for corn, cotton, and canola, or inbred progenies of transgenic
plants for soybean plants plant such as corn, cotton, canola, or
inbred plant such as soy, canola and cotton over multiple locations
with plants grown under optimal production management practices. An
exemplary target for improved yield is a 2% to 10% increase in
yield as compared to yield produced by plants grown from seed of a
control plant. Selection methods can be applied in multiple and
diverse geographic locations, for example up to 16 or more
locations, over one or more planting seasons, for example at least
two planting seasons, to statistically distinguish yield
improvement from natural environmental effects.
[0172] Increased Yield in Corn
[0173] Table 9 provides a list of protein encoding DNA or
polynucleotide sequence ("gene") in the production of transgenic
corn plants with increased yield as compared to a control plant.
The elements of Table 9 are described by reference to:
[0174] "SEQ ID NO: Polynucleotide" which identifies a nucleotide
sequence.
[0175] "SEQ ID NO: Polypeptide" which identifies an amino acid
sequence.
[0176] "Gene identifier" which refers to an arbitrary
identifier.
[0177] "Broad acre yield results" represent results from broad acre
yield field trial for plants comprising the sequence in constructs
with at least one event showing significant yield increase at
p.ltoreq.0.2 across locations. The first number refers to the
number of events with significant yield increase, whereas the
second number refers to the total number of events tested for each
sequence in a construct. As indicated in Table 9, genes TRDX2-2,
TRDX2-3, TRDX2-4, TRDX2-5 and TRDX2-6 resulted in at least on
significant positive events identified for increased yield.
TRDX2-2, TRDX2-4, TRDX2-5, TRDX2-6 and TRDX2-8 resulted in positive
broad acre yield increases in one broad acre yield trial. For
example, as indicated in Table 9, gene TRDX2-3 was tested in two
broad acre yield trials with 2 of 6 total events in trial 1 and 1
of 12 total events in trial 2 resulted in significantly positive
yield compared to non-transgenic control plants.
TABLE-US-00009 TABLE 9 Recombinant DNA for increased yield in corn
Broad Acre Broad Acre Yield SEQ ID NO: SEQ ID NO: Gene Yield
Results Results Polynucleotide Polypeptide Identifier Trial 1 Trial
2 3 4 TRDX2-2 1/8 -- 5 6 TRDX2-3 2/6 1/12 7 8 TRDX2-4 1/6 -- 9 10
TRDX2-5 1/8 -- 11 12 TRDX2-6 2/8 -- 15 16 TRDX2-8 2/8 --
TABLE-US-00010 TABLE 10 Recombinant DNA for suppression of target
genes for increased yield in corn SEQ ID NO: Broad Acre SEQ ID NO:
SEQ ID NO: Target Gene Yield Polynucleotide Target Gene Protein
Identifier Results 17 18 19 TRDX2-9 2/8 20 21 22 TRDX2-10 1/6
[0178] Transgenic corn plants having increased yield are identified
by screening using progenies of the transgenic plants over multiple
locations for several years with plants grown under optimal
production management practices and maximum weed and pest control.
Selection methods can be applied in multiple and diverse geographic
locations, for example up to 16 or more locations, over one or more
planting seasons, for example at least two planting seasons, to
statistically distinguish yield improvement from natural
environmental effects.
[0179] A yield increase in corn can also be manifested as an
increase in corn ear biomass which can be calculated on a per plant
or per plot basis using a determination for weight per ear for any
number of events in a plot compared to a control. This example
illustrates the selection of a corn plant with increased yield
measured as an increase in ear biomass.
[0180] Corn ear biomass was measured for plants grown under high
planting density (52,000 plants per acre), nitrogen limiting
conditions of 60 pounds (lbs) per acre or water limiting conditions
(chronic drought condition).
[0181] A correction factor was applied to achieve a corrected plot
ear biomass that was used to correct for ear biomass if there was a
discrepancy in the number of plants per plot. To apply a corrected
value to plot ear biomass, an estimate of plot ear biomass was
measured in the full field trials, which was determined on a field
by field location basis for plot ear biomass and stand. This
analysis for ear biomass derived from the full field trials was
used to calculate a correction factor that effectively reduced and
accounted for the projected ear biomass per plot (fresh ear weight
per plot basis). The factor for corrected ear biomass was applied
to the plots and used to provide ear biomass per plot in the
density, NUE and WUE trials.
[0182] The change or delta between transgenic events and
non-transgenic control events for ear biomass in a plot was used to
calculate a percent change for plot ear biomass.
[0183] "Corn ear biomass" was used as a parameter to predict
increased yield for an individual event on a per plot basis. Table
11 presents events positive for corn ear biomass for plants
comprising the sequences in constructs with at least one event
showing significant increase in ear biomass or fresh weight per
plot at a significant p.ltoreq.0.2 across three locations. The ears
were individually collected and ear biomass was measured by taking
a fresh weight on the corn ear, which was the mass (grams) of the
non-shelled whole ear (grain+cob) at measured at a physiological
maturity stage of R6. Corn ear biomass per plot was used as an
estimate of predicted yield increase in the field and was
determined for each transgenic event in a construct as compared to
non-transgenic wild-type control plants. The positive events for
ear biomass are reported with the number of events with significant
increase in ear biomass (first number N/N) compared to the total
number of plants tested for each event (second number N/N). The
field screens for density and NUE resulted in positive events which
met the statistical criteria for significance across locations at
p.ltoreq.0.2 across three locations and are reported in Table
11.
[0184] Table 11 provides a reference to:
[0185] "SEQ ID NO: Polynucleotide" which identifies a nucleotide
sequence.
[0186] "SEQ ID NO: Polypeptide" which identifies an amino acid
sequence.
[0187] "Gene identifier" which refers to an arbitrary
identifier.
[0188] "Event" which refers to an individual event for a given
construct.
[0189] "Density" refers to a spacing of plants to estimate a field
density of 52,000 plants per acre.
[0190] "NUE" refers to nitrogen use efficiency or increased yield
under nitrogen limiting conditions of 60 pounds (lbs) nitrogen
applied per acre.
TABLE-US-00011 TABLE 11 Recombinant DNA for increased corn ear
biomass [NS = non- statistically significant] SEQ ID NO: SEQ ID NO:
Gene Polynucleotide Polypeptide Identifier Event Density NUE 3 4
TRDX2-2 1 NS 2/4 3 4 TRDX2-2 2 NS 2/4 7 8 TRDX2-4 1 1/4 2/4 7 8
TRDX2-4 2 1/4 NS 9 10 TRDX2-5 1 NS 3/4 9 10 TRDX2-5 2 NS 2/4 11 12
TRDX2-6 1 NS 2/4 11 12 TRDX2-6 2 NS 2/4 11 12 TRDX2-6 3 3/4 NS
[0191] Table 12 provides a reference to:
[0192] "SEQ ID NO: Polynucleotide" which identifies a nucleotide
sequence.
[0193] "SEQ ID NO: Target Gene" which identifies a nucleotide acid
sequence.
[0194] "SEQ ID NO: Target Protein: which identifies an amino acid
sequence
[0195] "Gene identifier" which refers to an arbitrary
identifier.
[0196] "Event" which refers to an individual event for a given
construct.
[0197] "Density" refers to a spacing of plants to estimate a field
density of 52,000 plants per acre.
[0198] "NUE" refers to nitrogen use efficiency or increased yield
under nitrogen limiting conditions of 60 pounds (lbs) nitrogen
applied per acre.
[0199] "Corn ear biomass" was used as a parameter to predict
increased yield for an individual event on a per plot basis. Table
12 presents events positive for corn ear biomass taken for plants
comprising the sequences in constructs with at least one event
showing significant increase in ear biomass or fresh weight per
plot at a significant p.ltoreq.0.2 across three locations. The ears
were individually collected and ear biomass was measured by taking
a fresh weight on the corn ear, which was the mass (grams) of the
non-shelled whole ear (grain+cob) at measured at a physiological
maturity stage of R6. Corn ear biomass per plot was used as an
estimate of predicted yield increase in the field and was
determined for each transgenic event in a construct as compared to
non-transgenic wild-type control plants. The positive events for
ear biomass are reported with the number of events with significant
increase in ear biomass (first number N/N) compared to the total
number of plants tested for each event (second number N/N). Only
the high density screens that resulted in at least one positive
event for corn ear biomass and met the statistical criteria at
p.ltoreq.0.2 across three locations and are reported in Table
12.
TABLE-US-00012 TABLE 12 Recombinant DNA for suppression of target
genes for increased corn ear biomass SEQ ID NO: SEQ ID NO: Poly-
SEQ ID NO: Target Gene nucleotide Target Gene Protein Identifier
Event Density 20 21 22 TRDX2-10 1 3/4
[0200] Increased Yield in Soybean
[0201] A yield increase in soybean can be manifested as one or more
of the following: an increase in pods per plant, pods per acre,
seeds per plant, seeds per pod, weight per seed, weight per pod,
pods per node, number of nodes, and the number of internodes per
plant.
[0202] Table 13 provides a list of protein encoding DNA or
polynucleotide sequences used ("genes") in the production of
transgenic soybean plants with increased yield as compared to a
control plant. The elements of Table 13 are described by reference
to:
[0203] "SEQ ID NO: Polynucleotide" which identifies a nucleotide
sequence.
[0204] "SEQ ID NO: Polypeptide" which identifies an amino acid
sequence.
[0205] "Gene identifier" which refers to an arbitrary
identifier.
[0206] "Broad acre yield results" which refers to the sequence in a
construct with at least one event showing significant yield
increase at p.ltoreq.0.2 across locations. The first number refers
to the number of events with significant yield increase, whereas
the second number refers to the total number of events tested for
each sequence in a construct.
TABLE-US-00013 TABLE 13 Recombinant DNA for increased yield in
soybean Broad Acre SEQ ID NO: SEQ ID NO: Gene Yield Polynucleotide
Polypeptide Identifier Results 13 14 TRDX2-7 1/7
[0207] Increased Yield in Canola
[0208] A yield increase in canola can be manifested as one or more
of the following: an increase in silique number, number of siliques
per plant, number of siliques per node, number of internodes,
incidence of silique shatter, seeds per silique, seed weight per
silique, improved seed, oil, or protein composition.
[0209] Table 14 provides a list of protein encoding DNA or
polynucleotide sequences used ("genes") in the production of
transgenic canola plants with increased yield as compared to a
control plant. The element of Table 14 is described by reference
to:
[0210] "SEQ ID NO: Polynucleotide" which identifies a nucleotide
sequence.
[0211] "SEQ ID NO: Polypeptide" which identifies an amino acid
sequence.
[0212] "Gene identifier" which refers to an arbitrary
identifier.
[0213] "Broad acre yield results" which refers to the sequence in a
construct with at least one event showing significant yield
increase at p.ltoreq.0.2 across locations. The first number refers
to the number of events with significant yield increase, whereas
the second number refers to the total number of events tested for
each sequence in a construct.
TABLE-US-00014 TABLE 14 Recombinant DNA for increased Yield in
Canola Broad Acre SEQ ID NO: SEQ ID NO: Gene Yield Polynucleotide
Polypeptide Identifier Results 1 2 TRDX2-1 3/8
Example 7
Suppression of Corn DWARF4 & DWARF4-Like Protein
[0214] This example illustrates the use of a suppression construct
for use with RNA interference, for example, inverted repeats to
suppress at least one DWARF4 and DWARF4-like protein from Zea mays
(Zm.DWF4), which encodes a cytochrome P450 that was reported to
mediate multiple 22alpha-hydroxylation steps in brassicosteroid
biosynthesis expressed in actively growing tissues (Choe et al.,
2001, Plant J. 26: 573-582). More specifically, this example
illustrates the usage of a inverted repeats designed to target the
gene from Zm.DWF4-like and suppress the Zm.DWF4-like protein in
corn. Transgenic corn plants were stably transformed with inverted
repeats and were used to suppress at least one Zm.DWF4-like protein
and resulted in plants with an increased yield phenotype compared
to control plants. In addition, this example provides methods for
suppression using inverted repeats and a recombinant DNA construct
with suppression elements of inverted repeats to suppress the
Zm.DWF4-like protein for providing corn plants with increased
yield, increased water use efficiency and increased nitrogen use
efficiency.
[0215] In this embodiment, the Zm.DWF4-like protein in corn was
suppressed using an inverted repeat comprising a sense and an
antisense region. A specific example is provided by using SEQ ID
NO: 17. The polynucleotide of SEQ ID NO: 17 encodes an antisense
RNA molecule to target the complementary sense sequence. The RNA
molecule is complementary, such that the RNA molecule is capable of
forming a hairpin structure comprising a "sense" region and an
"antisense" region. The regulatory RNA molecule provided by SEQ ID
NO: 17 was designed to encode one or both strands of a
double-stranded RNA molecule, such that one or both strands of a
double-stranded RNA molecule can form a hairpin structure having a
double-stranded region. In this example, the DNA molecule of SEQ ID
NO: 17 was designed such that the sense and antisense regions are
each about, but not limited to, 325 nucleotides in length. In
another such embodiment, the loop region un-bound by the inverted
repeats (Inverted Repeat 1 and Inverted Repeat 2) is about but not
limited to 150 nucleotides in length. Following expression of such
a RNA molecule, the sense and antisense regions of the inverted
repeat form a double-stranded structure. The double-stranded region
of the inverted repeat can be formed by two separate RNA strands,
or by self-complementary portions of a single RNA having a hairpin
structure and where one strand of the double-stranded region
targets a region of the nucleic acid sequence of a Zm.DWF4-like
gene and suppresses at least of protein, encoded by Zm.DWF4-like
target gene.
[0216] A DNA molecule such as provided by SEQ ID NO: 17 that
encodes an antisense RNA molecule to target the complementary sense
sequence can also be designed to comprise a double-stranded region,
wherein one strand of the double-stranded region is substantially
identical (typically at least about 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.5%, 100% identical) in sequence to a nucleic
acid sequence of a target gene, such as provided by SEQ ID NO: 18.
The other strand of the double-stranded region is fully or
partially complementary to the nucleic acid target from the target
gene (typically at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 99.5% identical to the complement of a region of
the target nucleic acid). In another embodiment, the
double-stranded region can be formed by two separate RNA strands,
or by self-complementary portions of a single RNA having a hairpin
structure and where one strand of the double-stranded region is
identical to the target nucleic acid sequence over this region. In
a non-limiting embodiment, this example can be used to suppress a
protein if a target nucleic acid sequence is derived from a gene
that is a member of a Zm.DWF4 or Zm.DWF4-like gene family, the
sequence of the double-stranded region of a regulatory RNA molecule
can be chosen with the aid of sequence comparison tools such that
only the desired gene is down regulated. Alternatively, the
inverted repeat sequence of a double-stranded region of a
regulatory RNA molecule in this example can be used to down
regulate a plurality of related genes that encode a Zm.DWF4 or
Zm.DWF4-like protein simultaneously.
[0217] In a non-limiting embodiment, this example provides the
nucleic acid sequence containing two inverted repeats in SEQ ID NO:
17, which was designed to a region of an mRNA derived from a target
gene of SEQ ID NO: 18 encoding a corn Zm.DWF4-like protein SEQ ID
NO: 19 and targeted to be down regulated. The use of nucleotide
sequence of SEQ ID NO: 17 was used to target and suppress at least
one of the Zm.DWF4 target gene in corn. The nucleic acid construct
is provided, wherein the nucleic acid construct comprises (a) a
first transcription unit comprising a polynucleotide operably
linked to an constitutive promoter; (b) a second transcription unit
comprising a selectable marker for example selectable marker genes
conferring tolerance to spectinomycin; and (c) a pair of inverted
repeats, wherein one of the inverted repeats is 5' of (a) and (b),
and the other of the inverted repeats is 3' of (a) and (b). In one
such embodiment, the corn Zm.DWF4 inverted repeats comprises a
polynucleotide sequences presented in the 5' or 3' orientation to
comprise a pair of inverted repeats as set forth as SEQ ID NO: 17,
which may be used to suppress target gene sequence Zm.DWF4-like set
forth as SEQ ID NO: 18. The inverted repeats used to suppress a
corn Zm.DWF4-like target gene, for example, SEQ ID NO: 18,
comprises the nucleic acid sequence of Inverted Repeat
Configuration 1 (nucleotides 1-325 of SEQ ID NO:17) and Inverted
Repeat Configuration 2 (nucleotides 486-800 of SEQ ID NO:17).
[0218] To construct transformation vectors for suppressing a target
gene as identified as SEQ ID NO: 18, the amplified protein coding
nucleotides are assembled in a sense and antisense arrangement and
inserted into the base vector at the insertion site in the gene of
interest expression cassette to provide a transcribed RNA molecule
that will form a double-stranded RNA for RNA interference
suppression of a target protein, such as provided as SEQ ID NO: 19
or a plurality of similar proteins. Inverted Repeat Configuration 1
& 2 are embedded in nucleic acid sequences used for the
suppression of an endogenous corn Zm.DWF4-like target gene in corn
such as set forth as SEQ ID NO: 17 (nucleotides 1-800). The target
gene encoding a Corn DWF4-like protein set forth as SEQ ID NO: 18
(nucleotides 1-1527) was used to design the inverted repeats to
suppress the corn DWF4-like protein set forth as SEQ ID NO: 19.
[0219] In the present example, the endogenous proteins that are
targeted by suppression construct SEQ ID NO: 17 include but are not
limited to Zm.DWF4-like proteins, for example SEQ ID NO: 22.
Suppression of this endogenous Zm.DWF4-like protein resulted in
increased yield relative to control plants lacking the transgene
with at least one event showing significant yield increase at
p.ltoreq.0.2 across locations under standard field conditions
(Table 10).
Example 8
Suppression of Ribozyme Inactivating Protein in Corn Plants
[0220] This example illustrates the use of an miRNAs to suppress
ribozyme inactivating protein in corn. More specifically, it
illustrates the use of a miRNA to suppress the expression of at
least one target gene encoding a ribozyme inactivating protein
(Zm.RIP), which a repressor of translation in corn plants. In this
example, a transgene comprising the synthetic miRNA of SEQ ID NO:
20 was designed to suppress the expression of Zm.RIP in transgenic
corn plants. Various other recombinant DNA constructs are available
for use in suppressing the expression of a Zm.RIP target gene
encoding the Zm.RIP protein in transgenic plants.
[0221] In this embodiment, the suppression approach for suppressing
the expression of a Zm.RIP target protein include the use of a
recombinant DNA construct that produces a synthetic miRNA that is
designed to bind to a native or synthetic miRNA recognition site of
the messenger RNA for the Zm.RIP target protein. Recombinant DNA
constructs were used in transformation of a corn cell to produce
multiple events that are each regenerated into transgenic corn
plants, and further screened to identify the presence of the
recombinant DNA construct containing the miRNA for Zm.RIP. For
example, a recombinant DNA construct was used to deliver the miRNA
set forth as SEQ ID NO: 20 to target the Zm.Z1P gene set forth as
SEQ ID NO: 21 and suppress the expression of an endogenously
expressed Zm.RIP protein, set forth as SEQ ID NO: 22.
[0222] The activity of the miRNA which suppresses an endogenous Zm.
RIP protein was enhanced by enhancing the expression of the miRNA
or by enhancing the ability of the miRNA to bind an RNA encoding a
Zm.RIP target protein. A recombinant DNA encoding an RNA encoding
the miRNA can be designed to enhance miRNA activity resulting in
the enhanced suppression of the target mRNA and cognate protein,
for example, Zm.RIP. Recombinant DNA encoding an RNA encoding a
miRNA were designed using methods disclosed in US Patent
Application Publication No. US 2009/0070898 A1.
[0223] The population of transgenic plants from multiple transgenic
events were screened to identify the transgenic plants for the
recombinant construct with SEQ ID NO: 20 and further screened for
those transgenic events that exhibit enhanced yield. Suppression
approaches using a recombinant DNA construct containing the
suppression element of SEQ ID NO: 20 to suppress endogenously
expressed Zm.RIP proteins, for example SEQ ID NO: 22 resulted in
increased yield relative to control plants lacking the transgene
with at least one event showing significant yield increase at
p.ltoreq.0.2 across locations under standard field conditions
(Table 10). Additionally, suppression approaches using a
recombinant DNA construct containing the suppression element of SEQ
ID NO: 20 to suppress endogenously expressed Zm.RIP proteins in
corn resulted in enhanced phenotypes such as increased chlorophyll
and water content under non-stress, nitrogen deficit and water
deficit conditions contributing to increased water use efficiency
in transgenic corn with the Zm.RIP miRNA compared to non-transgenic
control plants (Tables 4-6).
Sequence CWU 1
1
291504DNAArabidopsis thaliana 1atgatgaaca cgtacaacat ggtgaagcaa
gaatttatca agaaatggat aaatactctc 60cacatgttag attcttctat cgaacatcct
ttgaacgtaa cggaaaggaa aaatgcgatt 120agactatcat cggacttagc
catggcagct gctcgaaatg gctctaccgt atggagccgc 180gctcttattt
ctaggagcgg aaataagaca gcaaacaaac ccatggcacg tcgaatacta
240aaaaaagctc gaaatcggat gaagaaccgt tgtaacattc ttagacgaaa
tggcaatttc 300acggcgaaaa cctgggtgag aaaacgtacg gacttgctta
agagtcttgt accgggaggt 360gagttgatag acgacaaaga ttatttgata
agagagacac ttgactacat tgtctatctc 420cgagcacaag tggacgtcat
gcgaaccgtc gcagccgtcg atttattcac ccgaaactta 480accaacgatc
gtaggaacaa ataa 5042167PRTArabidopsis thaliana 2Met Met Asn Thr Tyr
Asn Met Val Lys Gln Glu Phe Ile Lys Lys Trp 1 5 10 15 Ile Asn Thr
Leu His Met Leu Asp Ser Ser Ile Glu His Pro Leu Asn 20 25 30 Val
Thr Glu Arg Lys Asn Ala Ile Arg Leu Ser Ser Asp Leu Ala Met 35 40
45 Ala Ala Ala Arg Asn Gly Ser Thr Val Trp Ser Arg Ala Leu Ile Ser
50 55 60 Arg Ser Gly Asn Lys Thr Ala Asn Lys Pro Met Ala Arg Arg
Ile Leu 65 70 75 80 Lys Lys Ala Arg Asn Arg Met Lys Asn Arg Cys Asn
Ile Leu Arg Arg 85 90 95 Asn Gly Asn Phe Thr Ala Lys Thr Trp Val
Arg Lys Arg Thr Asp Leu 100 105 110 Leu Lys Ser Leu Val Pro Gly Gly
Glu Leu Ile Asp Asp Lys Asp Tyr 115 120 125 Leu Ile Arg Glu Thr Leu
Asp Tyr Ile Val Tyr Leu Arg Ala Gln Val 130 135 140 Asp Val Met Arg
Thr Val Ala Ala Val Asp Leu Phe Thr Arg Asn Leu 145 150 155 160 Thr
Asn Asp Arg Arg Asn Lys 165 31329DNAArabidopsis thaliana
3atggagccaa cgttccatgc ttttatgttt ccctggtttg cttttggtca tatgattcct
60tttctacatc ttgcaaacaa actagctgag aaaggtcatc aaatcacttt cttgctacct
120aagaaagccc aaaaacagtt ggaacatcac aatctgttcc cagacagtat
tgtctttcac 180cctctcacaa tccctcatgt caatggcctc cctgctggtg
ctgagacaac ctcggatatc 240tcaatctcga tggacaactt actgtcggaa
gccttggatc tcactcgcga tcaggttgaa 300gctgcggttc gtgctctgag
accggacttg atcttttttg attttgctca ttggattcca 360gaaattgcca
aagagcatat gatcaagagt gtgagttaca tgatagtatc tgcaacaaca
420atagcttata catttgcccc tggtggtgta ttaggtgttc ccccaccagg
ttatccttca 480tcaaaggtgt tgtaccgtga aaacgatgct catgccttag
caaccttatc tatcttctat 540aagagacttt atcatcagat cactacaggt
tttaagagct gtgacatcat tgcattgagg 600acatgtaatg aaatcgaagg
taaattctgc gactatatat caagtcaata ccataagaag 660gttctcttga
ctggtccaat gctccctgag caagacacaa gtaaaccact agaagaacag
720ttgagtcatt ttctgagcag gttcccaccg aggtcagtgg tgttttgtgc
acttggtagc 780cagatcgttc ttgaaaagga tcaattccaa gaactctgct
tagggatgga gctgacaggt 840ttaccgtttc ttatagcggt aaagccaccg
agaggatcat cgacggtcga agaagggtta 900ccagaagggt tccaggagcg
ggtgaaaggg cgtggtgtgg tttggggagg atgggtgcaa 960caaccattga
tattggatca tccgtcaata ggctgctttg tgaaccattg tggtccggga
1020acaatatggg agtgtcttat gactgattgt caaatggttt tgcttccatt
tttaggtgat 1080caagttctct tcacaagatt gatgaccgag gaattcaagg
tgtctgtaga agtgtcgaga 1140gaaaaaacag gatggttttc aaaggagagc
ttgagcgatg cgatcaagtc tgtgatggat 1200aaagatagcg acctcggaaa
gctagtgagg agtaaccacg ccaaattgaa ggagactctt 1260ggtagtcatg
gattattaac tggttacgtg gataaatttg tagaggaatt gcaagagtat
1320ttgatttag 13294442PRTArabidopsis thaliana 4Met Glu Pro Thr Phe
His Ala Phe Met Phe Pro Trp Phe Ala Phe Gly 1 5 10 15 His Met Ile
Pro Phe Leu His Leu Ala Asn Lys Leu Ala Glu Lys Gly 20 25 30 His
Gln Ile Thr Phe Leu Leu Pro Lys Lys Ala Gln Lys Gln Leu Glu 35 40
45 His His Asn Leu Phe Pro Asp Ser Ile Val Phe His Pro Leu Thr Ile
50 55 60 Pro His Val Asn Gly Leu Pro Ala Gly Ala Glu Thr Thr Ser
Asp Ile 65 70 75 80 Ser Ile Ser Met Asp Asn Leu Leu Ser Glu Ala Leu
Asp Leu Thr Arg 85 90 95 Asp Gln Val Glu Ala Ala Val Arg Ala Leu
Arg Pro Asp Leu Ile Phe 100 105 110 Phe Asp Phe Ala His Trp Ile Pro
Glu Ile Ala Lys Glu His Met Ile 115 120 125 Lys Ser Val Ser Tyr Met
Ile Val Ser Ala Thr Thr Ile Ala Tyr Thr 130 135 140 Phe Ala Pro Gly
Gly Val Leu Gly Val Pro Pro Pro Gly Tyr Pro Ser 145 150 155 160 Ser
Lys Val Leu Tyr Arg Glu Asn Asp Ala His Ala Leu Ala Thr Leu 165 170
175 Ser Ile Phe Tyr Lys Arg Leu Tyr His Gln Ile Thr Thr Gly Phe Lys
180 185 190 Ser Cys Asp Ile Ile Ala Leu Arg Thr Cys Asn Glu Ile Glu
Gly Lys 195 200 205 Phe Cys Asp Tyr Ile Ser Ser Gln Tyr His Lys Lys
Val Leu Leu Thr 210 215 220 Gly Pro Met Leu Pro Glu Gln Asp Thr Ser
Lys Pro Leu Glu Glu Gln 225 230 235 240 Leu Ser His Phe Leu Ser Arg
Phe Pro Pro Arg Ser Val Val Phe Cys 245 250 255 Ala Leu Gly Ser Gln
Ile Val Leu Glu Lys Asp Gln Phe Gln Glu Leu 260 265 270 Cys Leu Gly
Met Glu Leu Thr Gly Leu Pro Phe Leu Ile Ala Val Lys 275 280 285 Pro
Pro Arg Gly Ser Ser Thr Val Glu Glu Gly Leu Pro Glu Gly Phe 290 295
300 Gln Glu Arg Val Lys Gly Arg Gly Val Val Trp Gly Gly Trp Val Gln
305 310 315 320 Gln Pro Leu Ile Leu Asp His Pro Ser Ile Gly Cys Phe
Val Asn His 325 330 335 Cys Gly Pro Gly Thr Ile Trp Glu Cys Leu Met
Thr Asp Cys Gln Met 340 345 350 Val Leu Leu Pro Phe Leu Gly Asp Gln
Val Leu Phe Thr Arg Leu Met 355 360 365 Thr Glu Glu Phe Lys Val Ser
Val Glu Val Ser Arg Glu Lys Thr Gly 370 375 380 Trp Phe Ser Lys Glu
Ser Leu Ser Asp Ala Ile Lys Ser Val Met Asp 385 390 395 400 Lys Asp
Ser Asp Leu Gly Lys Leu Val Arg Ser Asn His Ala Lys Leu 405 410 415
Lys Glu Thr Leu Gly Ser His Gly Leu Leu Thr Gly Tyr Val Asp Lys 420
425 430 Phe Val Glu Glu Leu Gln Glu Tyr Leu Ile 435 440
5672DNAArabidopsis thaliana 5atgactaaac agcacgcgaa ctggtctcct
tacgataaca atggaggaac atgtgtggcc 60atcgctggat cggattactg tgttatcgcc
gccgatactc ggatgtctac tggttacagt 120attcttagtc gcgattactc
caaaatccat aaactagcgg acagagctgt tttgtcttcc 180tctggcttcc
aggctgatgt gaaagctttg cagaaggttc tcaaatccag acacttgatc
240tatcaacatc agcataacaa gcagatgagc tgtcctgcaa tggcccagct
tctctccaac 300acgctttatt tcaagcggtt tttcccctac tatgccttta
atgttctagg agggcttgac 360gaggaaggaa aagggtgtgt ctttacttac
gacgctgttg gctcatacga gagagttgga 420tacggtgctc aaggttctgg
ttccacactc atcatgcctt tccttgacaa tcagctcaag 480tctccaagtc
ctcttttgct acctaaacag gattcaaaca cgcccctttc cgaagctgaa
540gcagttgact tggttaaaac tgttttcgca tctgccacag agagggatat
ctacactgga 600gacaagcttg agattatgat acttaaggcc gacggtatca
agaccgaact catggacctg 660aggaaagact aa 6726223PRTArabidopsis
thaliana 6Met Thr Lys Gln His Ala Asn Trp Ser Pro Tyr Asp Asn Asn
Gly Gly 1 5 10 15 Thr Cys Val Ala Ile Ala Gly Ser Asp Tyr Cys Val
Ile Ala Ala Asp 20 25 30 Thr Arg Met Ser Thr Gly Tyr Ser Ile Leu
Ser Arg Asp Tyr Ser Lys 35 40 45 Ile His Lys Leu Ala Asp Arg Ala
Val Leu Ser Ser Ser Gly Phe Gln 50 55 60 Ala Asp Val Lys Ala Leu
Gln Lys Val Leu Lys Ser Arg His Leu Ile 65 70 75 80 Tyr Gln His Gln
His Asn Lys Gln Met Ser Cys Pro Ala Met Ala Gln 85 90 95 Leu Leu
Ser Asn Thr Leu Tyr Phe Lys Arg Phe Phe Pro Tyr Tyr Ala 100 105 110
Phe Asn Val Leu Gly Gly Leu Asp Glu Glu Gly Lys Gly Cys Val Phe 115
120 125 Thr Tyr Asp Ala Val Gly Ser Tyr Glu Arg Val Gly Tyr Gly Ala
Gln 130 135 140 Gly Ser Gly Ser Thr Leu Ile Met Pro Phe Leu Asp Asn
Gln Leu Lys 145 150 155 160 Ser Pro Ser Pro Leu Leu Leu Pro Lys Gln
Asp Ser Asn Thr Pro Leu 165 170 175 Ser Glu Ala Glu Ala Val Asp Leu
Val Lys Thr Val Phe Ala Ser Ala 180 185 190 Thr Glu Arg Asp Ile Tyr
Thr Gly Asp Lys Leu Glu Ile Met Ile Leu 195 200 205 Lys Ala Asp Gly
Ile Lys Thr Glu Leu Met Asp Leu Arg Lys Asp 210 215 220
71470DNASaccharomyces cerevisiae 7atgtccaatg ctgctttgca agtttatggc
ggcgacgaag tttccgcagt agtcattgat 60cctggctcat acacaacaaa tattggctat
tcgggttctg acttccctca atcaattctg 120ccctctgttt acggtaaata
cactgcagat gaaggcaata agaaaatatt ttctgaacaa 180tcaattggaa
ttccaagaaa agattatgaa ctgaaaccta ttatcgagaa cggtctagtc
240atagactggg ataccgcaca agaacagtgg caatgggcat tgcagaacga
actctatttg 300aattccaact ccggaatacc agctctgtta actgagcccg
tttggaatag cacagaaaac 360aggaaaaaat ctttagaagt gctcttagaa
ggcatgcaat ttgaagcctg ttacttagca 420cccacatcga catgcgtttc
ttttgcagca ggtagaccca attgtttggt tgttgatatt 480ggacatgata
cttgcagcgt cagtccaata gtggatggta tgacattatc aaagagtaca
540agaagaaatt ttattgctgg gaaatttatc aatcacttga ttaaaaaggc
attggaaccc 600aaagaaatca taccactttt cgcaatcaag caaagaaaac
cagagtttat aaaaaaaaca 660ttcgactatg aggttgataa atcgctgtat
gattacgcca ataaccgagg gttctttcaa 720gagtgcaaag aaacactttg
tcatatatgc ccaacaaaaa ctttggaaga aacgaaaaca 780gaattaagtt
ctacggctaa aagatctatt gaaagtcctt ggaatgagga gattgttttt
840gacaacgaaa ctcgttacgg ctttgctgaa gagcttttcc ttccaaaaga
agatgacatc 900ccagcaaatt ggcctcgctc gaactctgga gttgtgaaaa
cttggcggaa tgattacgtg 960ccgctaaaaa gaaccaagcc aagcggagtg
aacaaatcag acaagaaagt tacaccaact 1020gaagaaaagg aacaggaagc
tgtaagcaaa tctacttctc cggctgcaaa tagtgcagac 1080actccaaatg
aaaccggtaa gagaccgtta gaagaagaaa agcctcctaa agaaaataat
1140gaattgattg gtctagcgga tcttgtttat tcgtctataa tgagcagtga
tgtggatcta 1200agagcgacac tagctcataa tgttgtcctt acaggcggta
catcctctat tcctggatta 1260agtgataggt taatgacaga actaaacaaa
atactaccat cccttaaatt tagaatatta 1320acaacagggc acactatcga
aaggcaatac cagtcatggc ttggcggtag tatacttaca 1380agtctgggaa
catttcacca actgtgggtt gggaaaaagg aatacgaaga ggtgggcgtc
1440gaaagattgc ttaacgatag gtttagatag 14708489PRTSaccharomyces
cerevisiae 8Met Ser Asn Ala Ala Leu Gln Val Tyr Gly Gly Asp Glu Val
Ser Ala 1 5 10 15 Val Val Ile Asp Pro Gly Ser Tyr Thr Thr Asn Ile
Gly Tyr Ser Gly 20 25 30 Ser Asp Phe Pro Gln Ser Ile Leu Pro Ser
Val Tyr Gly Lys Tyr Thr 35 40 45 Ala Asp Glu Gly Asn Lys Lys Ile
Phe Ser Glu Gln Ser Ile Gly Ile 50 55 60 Pro Arg Lys Asp Tyr Glu
Leu Lys Pro Ile Ile Glu Asn Gly Leu Val 65 70 75 80 Ile Asp Trp Asp
Thr Ala Gln Glu Gln Trp Gln Trp Ala Leu Gln Asn 85 90 95 Glu Leu
Tyr Leu Asn Ser Asn Ser Gly Ile Pro Ala Leu Leu Thr Glu 100 105 110
Pro Val Trp Asn Ser Thr Glu Asn Arg Lys Lys Ser Leu Glu Val Leu 115
120 125 Leu Glu Gly Met Gln Phe Glu Ala Cys Tyr Leu Ala Pro Thr Ser
Thr 130 135 140 Cys Val Ser Phe Ala Ala Gly Arg Pro Asn Cys Leu Val
Val Asp Ile 145 150 155 160 Gly His Asp Thr Cys Ser Val Ser Pro Ile
Val Asp Gly Met Thr Leu 165 170 175 Ser Lys Ser Thr Arg Arg Asn Phe
Ile Ala Gly Lys Phe Ile Asn His 180 185 190 Leu Ile Lys Lys Ala Leu
Glu Pro Lys Glu Ile Ile Pro Leu Phe Ala 195 200 205 Ile Lys Gln Arg
Lys Pro Glu Phe Ile Lys Lys Thr Phe Asp Tyr Glu 210 215 220 Val Asp
Lys Ser Leu Tyr Asp Tyr Ala Asn Asn Arg Gly Phe Phe Gln 225 230 235
240 Glu Cys Lys Glu Thr Leu Cys His Ile Cys Pro Thr Lys Thr Leu Glu
245 250 255 Glu Thr Lys Thr Glu Leu Ser Ser Thr Ala Lys Arg Ser Ile
Glu Ser 260 265 270 Pro Trp Asn Glu Glu Ile Val Phe Asp Asn Glu Thr
Arg Tyr Gly Phe 275 280 285 Ala Glu Glu Leu Phe Leu Pro Lys Glu Asp
Asp Ile Pro Ala Asn Trp 290 295 300 Pro Arg Ser Asn Ser Gly Val Val
Lys Thr Trp Arg Asn Asp Tyr Val 305 310 315 320 Pro Leu Lys Arg Thr
Lys Pro Ser Gly Val Asn Lys Ser Asp Lys Lys 325 330 335 Val Thr Pro
Thr Glu Glu Lys Glu Gln Glu Ala Val Ser Lys Ser Thr 340 345 350 Ser
Pro Ala Ala Asn Ser Ala Asp Thr Pro Asn Glu Thr Gly Lys Arg 355 360
365 Pro Leu Glu Glu Glu Lys Pro Pro Lys Glu Asn Asn Glu Leu Ile Gly
370 375 380 Leu Ala Asp Leu Val Tyr Ser Ser Ile Met Ser Ser Asp Val
Asp Leu 385 390 395 400 Arg Ala Thr Leu Ala His Asn Val Val Leu Thr
Gly Gly Thr Ser Ser 405 410 415 Ile Pro Gly Leu Ser Asp Arg Leu Met
Thr Glu Leu Asn Lys Ile Leu 420 425 430 Pro Ser Leu Lys Phe Arg Ile
Leu Thr Thr Gly His Thr Ile Glu Arg 435 440 445 Gln Tyr Gln Ser Trp
Leu Gly Gly Ser Ile Leu Thr Ser Leu Gly Thr 450 455 460 Phe His Gln
Leu Trp Val Gly Lys Lys Glu Tyr Glu Glu Val Gly Val 465 470 475 480
Glu Arg Leu Leu Asn Asp Arg Phe Arg 485 9360DNAArabidopsis thaliana
9atggagaaat ctagagaaag cgagttcttg ttctgtaatt tgtgtgggac tatgcttgtc
60ttgaaatcaa ccaagtatgc agaatgtcca cattgcaaaa caacacggaa tgcaaaagat
120atcatcgaca aggaaatagc ttacacagtt tctgctgagg atatcagaag
agaactagga 180atatcattgt ttggtgaaaa aacgcaggca gaagctgagc
taccaaagat caaaaaggca 240tgcgagaaat gccagcatcc tgagcttgta
tacacaacca gacagacgag atcagctgat 300gaaggacaga caacatatta
cacttgcccc aattgtgctc atagattcac agaaggttag 36010119PRTArabidopsis
thaliana 10Met Glu Lys Ser Arg Glu Ser Glu Phe Leu Phe Cys Asn Leu
Cys Gly 1 5 10 15 Thr Met Leu Val Leu Lys Ser Thr Lys Tyr Ala Glu
Cys Pro His Cys 20 25 30 Lys Thr Thr Arg Asn Ala Lys Asp Ile Ile
Asp Lys Glu Ile Ala Tyr 35 40 45 Thr Val Ser Ala Glu Asp Ile Arg
Arg Glu Leu Gly Ile Ser Leu Phe 50 55 60 Gly Glu Lys Thr Gln Ala
Glu Ala Glu Leu Pro Lys Ile Lys Lys Ala 65 70 75 80 Cys Glu Lys Cys
Gln His Pro Glu Leu Val Tyr Thr Thr Arg Gln Thr 85 90 95 Arg Ser
Ala Asp Glu Gly Gln Thr Thr Tyr Tyr Thr Cys Pro Asn Cys 100 105 110
Ala His Arg Phe Thr Glu Gly 115 11393DNAArabidopsis thaliana
11atggctctcg aatgggttgt gttaggttac gcagcagcag ctgaagcgat catggtgatt
60ctcttgacga tgcctggact tgacgctctc cgcaaaggat tagtcgctgt aactcgtaat
120ctcttgaaac cgtttctctc gataatcccg ttttgtctct tccttcttat
ggatatttac 180tggaagtatg agactcgacc ttcttgcgat ggtgattcgt
gtactccttc tgagcatctt 240cgtcaccaga aatcgatcat gaagtctcag
cgtaacgcgc ttctgattgc gtcggctctt 300gttttctact ggattttgta
ctctgtcacg aatttggttg tgaggattga gcagcttaat 360cagagggttg
agaggctcaa gaacaaggat tag 39312130PRTArabidopsis thaliana 12Met Ala
Leu Glu Trp Val Val Leu Gly Tyr Ala Ala Ala Ala Glu Ala 1 5 10 15
Ile Met Val Ile Leu Leu Thr Met Pro Gly Leu Asp Ala Leu Arg Lys 20
25 30 Gly Leu Val Ala Val Thr Arg Asn Leu Leu Lys Pro Phe Leu Ser
Ile 35 40 45 Ile Pro Phe Cys Leu Phe Leu Leu Met Asp Ile Tyr Trp
Lys Tyr Glu 50 55 60
Thr Arg Pro Ser Cys Asp Gly Asp Ser Cys Thr Pro Ser Glu His Leu 65
70 75 80 Arg His Gln Lys Ser Ile Met Lys Ser Gln Arg Asn Ala Leu
Leu Ile 85 90 95 Ala Ser Ala Leu Val Phe Tyr Trp Ile Leu Tyr Ser
Val Thr Asn Leu 100 105 110 Val Val Arg Ile Glu Gln Leu Asn Gln Arg
Val Glu Arg Leu Lys Asn 115 120 125 Lys Asp 130 13333DNAPyropia
yezoensis 13atgaagaaga agctttcagt tcttttcact gtttttagtt tttttgtaat
aggtttcgca 60caaattgctt ttgctgcaga tctagataat ggagaaaaag ttttttctgc
taattgtgca 120gcatgtcatg ctggcggtaa taacgccatt atgccagata
aaaccttaaa aaaagatgta 180cttgaagcta atagtatgaa tactattgat
gctattactt atcaagtaca aaatggtaaa 240aatgccatgc ctgctttcgg
aggtagactg gttgatgaag atattgaaga tgcagcaaat 300tatgtattat
ctcaatctga aaaaggttgg tag 33314110PRTPyropia yezoensis 14Met Lys
Lys Lys Leu Ser Val Leu Phe Thr Val Phe Ser Phe Phe Val 1 5 10 15
Ile Gly Phe Ala Gln Ile Ala Phe Ala Ala Asp Leu Asp Asn Gly Glu 20
25 30 Lys Val Phe Ser Ala Asn Cys Ala Ala Cys His Ala Gly Gly Asn
Asn 35 40 45 Ala Ile Met Pro Asp Lys Thr Leu Lys Lys Asp Val Leu
Glu Ala Asn 50 55 60 Ser Met Asn Thr Ile Asp Ala Ile Thr Tyr Gln
Val Gln Asn Gly Lys 65 70 75 80 Asn Ala Met Pro Ala Phe Gly Gly Arg
Leu Val Asp Glu Asp Ile Glu 85 90 95 Asp Ala Ala Asn Tyr Val Leu
Ser Gln Ser Glu Lys Gly Trp 100 105 110 15585DNAZea mays
15atggtcaccg ccaccacctc cccgctcttc tccctctcct ccctccgcgc ctccctccct
60tcccccaccc gatttcacac gtctctctcg ctccgagccc tctccccacg tgctcgtctc
120tctgccgccc tccctttcgc ctccccactc gtttcaggcg ggtacgggac
ctgggcggcg 180acttcaatct cgtccgcggg aaggttgaga cggcgggggc
tggaggtggt gtgcgaggcc 240acgaccgggc ggcggccgga ctcggttaag
aagagggagc gccagaacga caagcaccgc 300atccgcaatc acgcgcgcaa
ggccgagatg cgcactagga tgaaaaaggt cttaagagct 360cttgaaaagc
ttaggaagaa acctgacgcg cagcctgaag aaataattga gatagagaag
420ctgatcgctg aggcatacaa agccatcgac aagacggtga aggttggcgc
catgcatagg 480aacacggcga accatcggaa gtctcgactg gcaaggagga
agaaggccat cgagatactc 540cgtggttggt atgtcccaaa cgctgaacct
gtcgctgcca cctag 58516194PRTZea mays 16Met Val Thr Ala Thr Thr Ser
Pro Leu Phe Ser Leu Ser Ser Leu Arg 1 5 10 15 Ala Ser Leu Pro Ser
Pro Thr Arg Phe His Thr Ser Leu Ser Leu Arg 20 25 30 Ala Leu Ser
Pro Arg Ala Arg Leu Ser Ala Ala Leu Pro Phe Ala Ser 35 40 45 Pro
Leu Val Ser Gly Gly Tyr Gly Thr Trp Ala Ala Thr Ser Ile Ser 50 55
60 Ser Ala Gly Arg Leu Arg Arg Arg Gly Leu Glu Val Val Cys Glu Ala
65 70 75 80 Thr Thr Gly Arg Arg Pro Asp Ser Val Lys Lys Arg Glu Arg
Gln Asn 85 90 95 Asp Lys His Arg Ile Arg Asn His Ala Arg Lys Ala
Glu Met Arg Thr 100 105 110 Arg Met Lys Lys Val Leu Arg Ala Leu Glu
Lys Leu Arg Lys Lys Pro 115 120 125 Asp Ala Gln Pro Glu Glu Ile Ile
Glu Ile Glu Lys Leu Ile Ala Glu 130 135 140 Ala Tyr Lys Ala Ile Asp
Lys Thr Val Lys Val Gly Ala Met His Arg 145 150 155 160 Asn Thr Ala
Asn His Arg Lys Ser Arg Leu Ala Arg Arg Lys Lys Ala 165 170 175 Ile
Glu Ile Leu Arg Gly Trp Tyr Val Pro Asn Ala Glu Pro Val Ala 180 185
190 Ala Thr 17800DNAartificial sequencesuppression element to
target gene in Zea mays 17gctcaattta gacgcccctc ttagcctttg
tctcctagca atcaggagat gctcctcccg 60gagttcttgc acggccttag ggcacccttc
gaggaagaag atggcgaggg cgagcgccat 120ggacgaagtc tcgtgccccg
cgaagagcag gctcagcaag aggtccagga tctgttcctt 180ggacaggttg
gattgcttca gggcccatcc aagaaggtcg tcctcctcca cgcttgactt
240ctccctgctc atcttctcaa gcctgccctc catcttcctc tctatcactc
caagtatgga 300cgcgcgtgac ttgagcgcct tccagaagta ctgcgatcgc
gttaacgctt tatcacgata 360ccttctacca catatcacta acaacatcaa
cactcatcac tctcgacgac atccactcga 420tcactactct cacacgaccg
attaactcct catccacgcg gccgcctgca ggagcctgga 480aggcgctcaa
gtcacgcgcg tccatacttg gagtgataga gaggaagatg gagggcaggc
540ttgagaagat gagcagggag aagtcaagcg tggaggagga cgaccttctt
ggatgggccc 600tgaagcaatc caacctgtcc aaggaacaga tcctggacct
cttgctgagc ctgctcttcg 660cggggcacga gacttcgtcc atggcgctcg
ccctcgccat cttcttcctc gaagggtgcc 720ctaaggccgt gcaagaactc
cgggaggagc atctcctgat tgctaggaga caaaggctaa 780gaggggcgtc
taaattgagc 800181527DNAZea mays 18atgggcgcca tgatggcctc cataaccagc
gagctcctct tcttccttcc cttcatcctg 60ctggccctcc tcgccttgta caccaccacc
gtcgccaaat gccacggcac ccacccgtgg 120cgccgtcaga agaagaagcg
gcccaacctg cccccgggcg cccgcggatg gcccttggtc 180ggcgaaactt
tcggctacct ccgcgcccac ccggccacct ccgtgggccg cttcatggag
240cggcatgtcg cacggtacgg gaagatatac cggtcgagcc tgttcgggga
gcggacggtg 300gtgtcggcgg acgcggggct gaaccgctac atcctgcaga
acgaggggcg gctgttcgag 360tgcagctacc cgcgcagcat cggcggcatc
ctgggcaagt ggtccatgct ggtgctcgtg 420ggcgacgcgc accgcgagat
gcgcgctatc tcgctcaact tcctcagctc cgtccgcctc 480cgcgccgtgc
tgctccccga ggtggagcgc cacaccctgc tggtcctccg ctcgtggccg
540ccctccgacg gcaccttctc cgcccagcac gaagccaaga agttcacgtt
taacctgatg 600gcgaagaaca taatgagcat ggaccccggc gaggaggaga
cggagcggct gcggctggag 660tacatcacct tcatgaaggg cgtcgtgtca
gcgccgctca acttcccggg cacggcctac 720tggaaggcgc tcaagtcgcg
cgcgtccata cttggagtga tagagaggaa gatggaggac 780aggcttgaga
agatgagcag ggagaagtca agcgtggagg aggacgacct tcttggatgg
840gccctgaagc aatccaacct gtccaaggaa cagatcctgg acctcttgct
gagcctgctc 900ttcgcggggc acgagacttc gtccatggcg ctcgccctcg
ccatcttctt cctcgaaggg 960tgccctaagg ccgtgcaaga actccgggag
gagcatctcc tgattgctag gagacaaagg 1020ctaagggggg cgtccaaatt
gagctgggaa gactacaagg aaatggtttt cacgcagtgt 1080gttataaacg
agacattgcg gctcggcaac gtggtcaggt tcctgcaccg gaaggtcatc
1140cgagatgtac actacaatgg gtacgacata ccgcgggggt ggaaaatcct
gccggttcta 1200gcggcggtgc acctggactc gtcgctgtac gaggacccca
gccggttcaa cccttggaga 1260tggaagctgc agagcaacaa cgcgccaagc
agcttcatgc cgtacggcgg cgggccgcgg 1320ctgtgcgccg ggtcggagct
ggccaagctg gagatggcca tcttcctgca ccacctggtg 1380ctcaacttcc
ggtgggagct ggcggagccg gaccaggcct tcgtctaccc tttcgtcgac
1440ttccccaagg gcctcccgat cagggtccag cgggtcgccg acgaccaagg
ccatcgtagc 1500gttttgaccg agagcacaag aggctga 152719508PRTZea mays
19Met Gly Ala Met Met Ala Ser Ile Thr Ser Glu Leu Leu Phe Phe Leu 1
5 10 15 Pro Phe Ile Leu Leu Ala Leu Leu Ala Leu Tyr Thr Thr Thr Val
Ala 20 25 30 Lys Cys His Gly Thr His Pro Trp Arg Arg Gln Lys Lys
Lys Arg Pro 35 40 45 Asn Leu Pro Pro Gly Ala Arg Gly Trp Pro Leu
Val Gly Glu Thr Phe 50 55 60 Gly Tyr Leu Arg Ala His Pro Ala Thr
Ser Val Gly Arg Phe Met Glu 65 70 75 80 Arg His Val Ala Arg Tyr Gly
Lys Ile Tyr Arg Ser Ser Leu Phe Gly 85 90 95 Glu Arg Thr Val Val
Ser Ala Asp Ala Gly Leu Asn Arg Tyr Ile Leu 100 105 110 Gln Asn Glu
Gly Arg Leu Phe Glu Cys Ser Tyr Pro Arg Ser Ile Gly 115 120 125 Gly
Ile Leu Gly Lys Trp Ser Met Leu Val Leu Val Gly Asp Ala His 130 135
140 Arg Glu Met Arg Ala Ile Ser Leu Asn Phe Leu Ser Ser Val Arg Leu
145 150 155 160 Arg Ala Val Leu Leu Pro Glu Val Glu Arg His Thr Leu
Leu Val Leu 165 170 175 Arg Ser Trp Pro Pro Ser Asp Gly Thr Phe Ser
Ala Gln His Glu Ala 180 185 190 Lys Lys Phe Thr Phe Asn Leu Met Ala
Lys Asn Ile Met Ser Met Asp 195 200 205 Pro Gly Glu Glu Glu Thr Glu
Arg Leu Arg Leu Glu Tyr Ile Thr Phe 210 215 220 Met Lys Gly Val Val
Ser Ala Pro Leu Asn Phe Pro Gly Thr Ala Tyr 225 230 235 240 Trp Lys
Ala Leu Lys Ser Arg Ala Ser Ile Leu Gly Val Ile Glu Arg 245 250 255
Lys Met Glu Asp Arg Leu Glu Lys Met Ser Arg Glu Lys Ser Ser Val 260
265 270 Glu Glu Asp Asp Leu Leu Gly Trp Ala Leu Lys Gln Ser Asn Leu
Ser 275 280 285 Lys Glu Gln Ile Leu Asp Leu Leu Leu Ser Leu Leu Phe
Ala Gly His 290 295 300 Glu Thr Ser Ser Met Ala Leu Ala Leu Ala Ile
Phe Phe Leu Glu Gly 305 310 315 320 Cys Pro Lys Ala Val Gln Glu Leu
Arg Glu Glu His Leu Leu Ile Ala 325 330 335 Arg Arg Gln Arg Leu Arg
Gly Ala Ser Lys Leu Ser Trp Glu Asp Tyr 340 345 350 Lys Glu Met Val
Phe Thr Gln Cys Val Ile Asn Glu Thr Leu Arg Leu 355 360 365 Gly Asn
Val Val Arg Phe Leu His Arg Lys Val Ile Arg Asp Val His 370 375 380
Tyr Asn Gly Tyr Asp Ile Pro Arg Gly Trp Lys Ile Leu Pro Val Leu 385
390 395 400 Ala Ala Val His Leu Asp Ser Ser Leu Tyr Glu Asp Pro Ser
Arg Phe 405 410 415 Asn Pro Trp Arg Trp Lys Leu Gln Ser Asn Asn Ala
Pro Ser Ser Phe 420 425 430 Met Pro Tyr Gly Gly Gly Pro Arg Leu Cys
Ala Gly Ser Glu Leu Ala 435 440 445 Lys Leu Glu Met Ala Ile Phe Leu
His His Leu Val Leu Asn Phe Arg 450 455 460 Trp Glu Leu Ala Glu Pro
Asp Gln Ala Phe Val Tyr Pro Phe Val Asp 465 470 475 480 Phe Pro Lys
Gly Leu Pro Ile Arg Val Gln Arg Val Ala Asp Asp Gln 485 490 495 Gly
His Arg Ser Val Leu Thr Glu Ser Thr Arg Gly 500 505
201085DNAartificial sequencesuppression element to target gene in
Zea mays 20aggtagttga cggccgtggt catttcggca cggcccatgg tgacggtctc
caggccctta 60ctgccgatga ggtcctggta ccggccgcca aagccgagcc acttggcgtt
gtcgtcgagg 120aggtgggtgt cgccgtcctt gccgaactcc caccacaccc
cgccgggggt cctgaagccg 180accaggtaga ggttgtccat acgtatggcg
agcgtgatgg acctggtctt cgttttgagc 240tcggtgtaga accagagctc
ggggacattc ttctccagcg gcagcacggg ctggacgatg 300cctgtatggt
tggtgcagta tttgatcact tctttccgga cggaggtgat gaaggcgctg
360taagggtagg ccgtgtcctc cacggggaag atttcggtga actttggcac
tatatttttc 420ttctttgttt gagtaataag accactcaac tctgggtttg
gctccgaagt actgcgatcg 480cgttaacgct ttatcacgat accttctacc
acatatcact aacaacatca acactcatca 540ctctcgacga catccactcg
atcactactc tcacacgacc gattaactcc tcatccacgc 600ggccgcctgc
aggagccgga gccaaaccca gagttgagtg gtcttattac tcaaacaaag
660aagaaaaata tagtgccaaa gttcaccgaa atcttccccg tggaggacac
ggcctaccct 720tacagcgcct tcatcacctc cgtccggaaa gaagtgatca
aatactgcac caaccataca 780ggcatcgtcc agcccgtgct gccgctggag
aagaatgtcc ccgagctctg gttctacacc 840gagctcaaaa cgaagaccag
gtccatcacg ctcgccatac gtatggacaa cctctacctg 900gtcggcttca
ggacccccgg cggggtgtgg tgggagttcg gcaaggacgg cgacacccac
960ctcctcgacg acaacgccaa gtggctcggc tttggcggcc ggtaccagga
cctcatcggc 1020agtaagggcc tggagaccgt caccatgggc cgtgccgaaa
tgaccacggc cgtcaactac 1080cttag 108521837DNAZea mays 21atggcggagc
caaacccaga gttgagtggt cttattactc aaacaaagaa gaaaaatata 60gtgccaaagt
tcaccgaaat cttccccgtg gaggacacgg cctaccctta cagcgccttc
120atcacctccg tccggaaaga agtgatcaaa tactgcacca accatacagg
catcgtccag 180cccgtgctgc cgctggagaa gaatgtcccc gagctctggt
tctacaccga gctcaaaacg 240aagaccaggt ccatcacgct cgccatacgt
atggacaacc tctacctggt cggcttcagg 300acccccggcg gggtgtggtg
ggagttcggc aaggacggcg acacccacct cctcgacgac 360aacgccaagt
ggctcggctt tggcggccgg taccaggacc tcatcggcag taagggcctg
420gagaccgtca ccatgggccg tgccgaaatg accacggccg tcaactacct
ggcgaagaag 480acgacgacga cactagcaga ggcggcggag gaggaggagg
agctgctgct gctgcaggca 540gcggctgacc ccaaagccga ggagaagagc
aacctggcga agctagtgat catggtatgc 600gaggggctgc ggttcttcac
cgtgtcccgc aaggtagacg aggggttcaa gaagccgcaa 660gcggtgacca
tatcggcgct ggaggggaag caggtgcaga aatgggacag gatctcgaaa
720gccgtcttca ggtgggccgt cgacccgacc gctgagatcc ccgacatgaa
ggatcttggc 780atcaaagata aaaacgcagc agcgcagatc gttgcgctcg
ttaaggacca aaactag 83722278PRTZea mays 22Met Ala Glu Pro Asn Pro
Glu Leu Ser Gly Leu Ile Thr Gln Thr Lys 1 5 10 15 Lys Lys Asn Ile
Val Pro Lys Phe Thr Glu Ile Phe Pro Val Glu Asp 20 25 30 Thr Ala
Tyr Pro Tyr Ser Ala Phe Ile Thr Ser Val Arg Lys Glu Val 35 40 45
Ile Lys Tyr Cys Thr Asn His Thr Gly Ile Val Gln Pro Val Leu Pro 50
55 60 Leu Glu Lys Asn Val Pro Glu Leu Trp Phe Tyr Thr Glu Leu Lys
Thr 65 70 75 80 Lys Thr Arg Ser Ile Thr Leu Ala Ile Arg Met Asp Asn
Leu Tyr Leu 85 90 95 Val Gly Phe Arg Thr Pro Gly Gly Val Trp Trp
Glu Phe Gly Lys Asp 100 105 110 Gly Asp Thr His Leu Leu Asp Asp Asn
Ala Lys Trp Leu Gly Phe Gly 115 120 125 Gly Arg Tyr Gln Asp Leu Ile
Gly Ser Lys Gly Leu Glu Thr Val Thr 130 135 140 Met Gly Arg Ala Glu
Met Thr Thr Ala Val Asn Tyr Leu Ala Lys Lys 145 150 155 160 Thr Thr
Thr Thr Leu Ala Glu Ala Ala Glu Glu Glu Glu Glu Leu Leu 165 170 175
Leu Leu Gln Ala Ala Ala Asp Pro Lys Ala Glu Glu Lys Ser Asn Leu 180
185 190 Ala Lys Leu Val Ile Met Val Cys Glu Gly Leu Arg Phe Phe Thr
Val 195 200 205 Ser Arg Lys Val Asp Glu Gly Phe Lys Lys Pro Gln Ala
Val Thr Ile 210 215 220 Ser Ala Leu Glu Gly Lys Gln Val Gln Lys Trp
Asp Arg Ile Ser Lys 225 230 235 240 Ala Val Phe Arg Trp Ala Val Asp
Pro Thr Ala Glu Ile Pro Asp Met 245 250 255 Lys Asp Leu Gly Ile Lys
Asp Lys Asn Ala Ala Ala Gln Ile Val Ala 260 265 270 Leu Val Lys Asp
Gln Asn 275 23223PRTArabidopsis thaliana 23Met Thr Lys Gln His Ala
Asn Trp Ser Pro Tyr Asp Asn Asn Gly Gly 1 5 10 15 Thr Cys Val Ala
Ile Ala Gly Ser Asp Tyr Cys Val Ile Ala Ala Asp 20 25 30 Thr Arg
Met Ser Thr Gly Tyr Ser Ile Leu Ser Arg Asp Tyr Ser Lys 35 40 45
Ile His Lys Leu Ala Asp Arg Ala Val Leu Ser Ser Ser Gly Phe Gln 50
55 60 Ala Asp Val Lys Ala Leu Gln Lys Val Leu Lys Ser Arg His Leu
Ile 65 70 75 80 Tyr Gln His Gln His Asn Lys Gln Met Ser Cys Pro Ala
Met Ala Gln 85 90 95 Leu Leu Ser Asn Thr Leu Tyr Phe Lys Arg Phe
Phe Pro Tyr Tyr Ala 100 105 110 Phe Asn Val Leu Gly Gly Leu Asp Glu
Glu Gly Lys Gly Cys Val Phe 115 120 125 Thr Tyr Asp Ala Val Gly Ser
Tyr Glu Arg Val Gly Tyr Gly Ala Gln 130 135 140 Gly Ser Gly Ser Thr
Leu Ile Met Pro Phe Leu Asp Asn Gln Leu Lys 145 150 155 160 Ser Pro
Ser Pro Leu Leu Leu Pro Lys Gln Asp Ser Asn Thr Pro Leu 165 170 175
Ser Glu Ala Glu Ala Val Asp Leu Val Lys Thr Val Phe Ala Ser Ala 180
185 190 Thr Glu Arg Asp Ile Tyr Thr Val Asn Lys Leu Glu Ile Met Ile
Leu 195 200 205 Lys Ala Asp Gly Ile Lys Thr Glu Leu Met Asp Leu Arg
Lys Asp 210 215 220 24489PRTSaccharomyces cerevisiae 24Met Ser Asn
Ala Ala Leu Gln Val Tyr Gly Gly Asp Glu Val Ser Ala 1 5 10 15 Val
Val Ile Asp Pro Gly Ser Tyr Thr Thr Asn Ile Gly Tyr Ser Gly 20 25
30 Ser Asp Phe Pro Gln Ser Ile Leu Pro Ser Val Tyr Gly Lys Tyr Thr
35 40 45 Ala Asp Glu Gly Asn Lys Lys Ile Phe Ser Glu Gln Ser Ile
Gly Ile 50
55 60 Pro Arg Lys Asp Tyr Glu Leu Lys Pro Ile Ile Glu Asn Gly Leu
Val 65 70 75 80 Ile Asp Trp Asp Thr Ala Gln Glu Gln Trp Gln Trp Ala
Leu Gln Asn 85 90 95 Glu Leu Tyr Leu Asn Ser Asn Ser Gly Ile Pro
Ala Leu Leu Thr Glu 100 105 110 Pro Val Trp Asn Ser Thr Glu Asn Arg
Lys Lys Ser Leu Glu Val Leu 115 120 125 Leu Glu Gly Met Gln Phe Glu
Ala Cys Tyr Leu Ala Pro Thr Ser Thr 130 135 140 Cys Val Ser Phe Ala
Ala Gly Arg Pro Asn Cys Leu Val Val Asp Ile 145 150 155 160 Gly His
Asp Thr Cys Ser Val Ser Pro Ile Val Asp Gly Met Thr Leu 165 170 175
Ser Lys Ser Thr Arg Arg Asn Phe Ile Ala Gly Lys Phe Ile Asn His 180
185 190 Leu Ile Lys Lys Ala Leu Glu Pro Lys Glu Ile Ile Pro Leu Phe
Ala 195 200 205 Ile Lys Gln Arg Lys Pro Glu Phe Ile Lys Lys Thr Phe
Asp Tyr Glu 210 215 220 Val Asp Lys Ser Leu Tyr Asp Tyr Ala Asn Asn
Arg Gly Phe Phe Gln 225 230 235 240 Glu Cys Lys Glu Thr Leu Cys His
Ile Cys Pro Thr Lys Thr Leu Glu 245 250 255 Glu Thr Lys Thr Glu Leu
Ser Ser Thr Ala Lys Arg Ser Ile Glu Ser 260 265 270 Pro Trp Asn Glu
Glu Ile Val Phe Asp Asn Glu Thr Arg Tyr Gly Phe 275 280 285 Ala Glu
Glu Leu Phe Leu Pro Lys Glu Asp Asp Ile Pro Ala Asn Trp 290 295 300
Pro Arg Ser Asn Ser Gly Val Val Lys Thr Trp Arg Asn Asp Tyr Val 305
310 315 320 Pro Leu Lys Arg Thr Lys Pro Ser Gly Val Asn Lys Ser Asp
Lys Lys 325 330 335 Val Thr Pro Thr Glu Glu Lys Glu Gln Glu Ala Val
Ser Lys Ser Thr 340 345 350 Ser Pro Ala Ala Asn Ser Ala Asp Thr Pro
Asn Glu Thr Gly Lys Arg 355 360 365 Pro Leu Glu Glu Gly Lys Pro Pro
Lys Glu Asn Asn Glu Leu Ile Gly 370 375 380 Leu Ala Asp Leu Val Tyr
Ser Ser Ile Met Ser Ser Asp Val Asp Leu 385 390 395 400 Arg Ala Thr
Leu Ala His Asn Val Val Leu Thr Gly Gly Thr Ser Ser 405 410 415 Ile
Pro Gly Leu Ser Asp Arg Leu Met Thr Glu Leu Asn Lys Ile Leu 420 425
430 Pro Ser Leu Lys Phe Arg Ile Leu Thr Thr Gly His Thr Ile Glu Arg
435 440 445 Gln Tyr Gln Ser Trp Leu Gly Gly Ser Ile Leu Thr Ser Leu
Gly Thr 450 455 460 Phe His Gln Leu Trp Val Gly Lys Lys Glu Tyr Glu
Glu Val Gly Val 465 470 475 480 Glu Arg Leu Leu Asn Asp Arg Phe Arg
485 25130PRTArabidopsis thaliana 25Met Ala Leu Glu Trp Val Val Leu
Gly Tyr Ala Ala Ala Ala Glu Ala 1 5 10 15 Ile Met Val Ile Leu Leu
Thr Met Pro Gly Leu Asp Ala Leu Arg Lys 20 25 30 Gly Leu Val Ala
Val Thr Arg Asn Leu Leu Lys Pro Phe Leu Ser Ile 35 40 45 Ile Pro
Phe Cys Leu Phe Leu Leu Met Asp Ile Tyr Trp Lys Tyr Glu 50 55 60
Thr Arg Pro Ser Cys Asp Gly Asp Ser Cys Thr Pro Ser Glu His Leu 65
70 75 80 Arg His Gln Lys Ser Ile Met Lys Ser Gln Arg Asn Ala Leu
Leu Ile 85 90 95 Ala Ser Ala Leu Val Phe Tyr Trp Ile Leu Tyr Ser
Val Thr Asn Leu 100 105 110 Val Val Arg Ile Glu Gln Leu Asn Gln Arg
Val Glu Arg Leu Lys Asn 115 120 125 Lys Asn 130 26130PRTArabidopsis
lyrata 26Met Ala Leu Glu Trp Val Val Leu Gly Tyr Ala Ala Ala Ala
Glu Ala 1 5 10 15 Ile Met Val Ile Leu Leu Thr Met Pro Gly Leu Asp
Ala Leu Arg Lys 20 25 30 Gly Leu Val Ala Val Thr Arg Asn Leu Leu
Lys Pro Phe Leu Ser Ile 35 40 45 Ile Pro Phe Cys Leu Phe Leu Leu
Met Asp Ile Tyr Trp Lys Tyr Glu 50 55 60 Thr Arg Pro Ser Cys Asp
Ser Asp Ser Cys Thr Pro Ser Glu His Leu 65 70 75 80 Arg His Gln Lys
Ser Ile Met Lys Ser Gln Arg Asn Ala Leu Leu Ile 85 90 95 Ala Ser
Ala Leu Val Phe Tyr Trp Ile Leu Tyr Ser Val Thr Asn Leu 100 105 110
Val Val Arg Ile Glu Gln Leu Asn Gln Arg Val Glu Arg Leu Lys Asn 115
120 125 Lys Asp 130 27110PRTPorphyra tenera 27Met Lys Lys Lys Phe
Ser Val Leu Phe Thr Val Phe Ser Phe Phe Val 1 5 10 15 Ile Gly Phe
Ala Gln Ile Ala Phe Ala Ala Asp Leu Asp Asn Gly Glu 20 25 30 Lys
Val Phe Ser Ala Asn Cys Ala Ala Cys His Ala Gly Gly Asn Asn 35 40
45 Ala Ile Met Pro Asp Lys Thr Leu Lys Lys Asp Val Leu Glu Ala Asn
50 55 60 Ser Met Asn Thr Ile Asp Ala Ile Thr Tyr Gln Val Gln Asn
Gly Lys 65 70 75 80 Asn Ala Met Pro Ala Phe Gly Gly Arg Leu Val Asp
Glu Asp Ile Glu 85 90 95 Asp Ala Ala Asn Tyr Val Leu Ser Gln Ser
Glu Lys Gly Trp 100 105 110 28194PRTZea mays 28Met Val Thr Ala Thr
Thr Ser Pro Leu Phe Ser Leu Ser Ser Leu Arg 1 5 10 15 Ala Ser Leu
Pro Ser Pro Thr Arg Phe His Thr Ser Leu Ser Leu Arg 20 25 30 Ala
Leu Ser Pro Arg Ala Arg Leu Ser Ala Ala Leu Pro Phe Ala Ser 35 40
45 Pro Leu Val Ser Gly Gly Tyr Gly Thr Trp Ala Ala Thr Ser Ile Ser
50 55 60 Ser Ala Gly Arg Leu Arg Arg Arg Gly Leu Glu Val Val Cys
Glu Ala 65 70 75 80 Thr Thr Gly Arg Arg Pro Asp Ser Val Lys Lys Arg
Glu Arg Gln Asn 85 90 95 Asp Lys His Arg Ile Arg Asn His Ala Arg
Lys Ala Glu Met Arg Thr 100 105 110 Arg Met Lys Lys Val Leu Arg Ala
Leu Glu Lys Leu Arg Lys Lys Pro 115 120 125 Asp Ala Gln Pro Glu Glu
Ile Ile Glu Ile Glu Lys Leu Ile Ala Glu 130 135 140 Ala Tyr Lys Ala
Ile Asp Lys Thr Val Lys Val Gly Ala Met His Arg 145 150 155 160 Asn
Thr Ala Asn His Arg Lys Ser Arg Leu Ala Arg Arg Lys Lys Ala 165 170
175 Ile Glu Ile Leu Arg Gly Trp Tyr Val Pro Asn Ala Glu Pro Val Ala
180 185 190 Ala Thr 29193PRTZea mays 29Met Val Thr Ala Thr Thr Ser
Pro Leu Phe Ser Leu Ser Ser Leu Arg 1 5 10 15 Ala Ser Leu Pro Ser
Pro Thr Arg Phe His Thr Ser Leu Ser Leu Arg 20 25 30 Ala Leu Ser
Pro Arg Ala Arg Leu Ser Ala Ser Leu Pro Phe Ala Ser 35 40 45 Pro
Leu Val Ser Gly Gly Tyr Gly Thr Trp Ala Ala Thr Ser Val Ser 50 55
60 Ser Ala Gly Arg Leu Arg Arg Arg Gly Leu Glu Val Val Cys Glu Ala
65 70 75 80 Thr Thr Gly Arg Arg Pro Asp Ser Val Lys Lys Arg Glu Arg
Gln Asn 85 90 95 Asp Lys His Arg Ile Arg Asn His Ala Arg Lys Ala
Glu Met Arg Thr 100 105 110 Arg Met Lys Lys Val Leu Arg Ala Leu Glu
Lys Leu Arg Lys Lys Pro 115 120 125 Asp Ala Gln Pro Glu Glu Ile Ile
Glu Ile Glu Lys Leu Ile Ala Glu 130 135 140 Ala Tyr Lys Ala Ile Asp
Lys Thr Val Lys Val Gly Ala Met His Arg 145 150 155 160 Asn Thr Ala
Asn His Arg Lys Ser Arg Leu Ala Arg Arg Lys Lys Ala 165 170 175 Ile
Glu Ile Leu Arg Gly Trp Tyr Val Pro Asn Ala Glu Pro Val Ala 180 185
190 Ala
* * * * *