U.S. patent number RE45,048 [Application Number 13/751,021] was granted by the patent office on 2014-07-22 for methods for weed control using plants having dicamba-degrading enzymatic activity.
This patent grant is currently assigned to Monsanto Technology LLC. The grantee listed for this patent is Monsanto Technology LLC. Invention is credited to Ronald J. Brinker, Paul C. C. Feng.
United States Patent |
RE45,048 |
Feng , et al. |
July 22, 2014 |
**Please see images for:
( Certificate of Correction ) ** |
Methods for weed control using plants having dicamba-degrading
enzymatic activity
Abstract
The invention provides methods for weed control with dicamba and
related herbicides. It was found that pre-emergent applications of
dicamba at or near planting could be made without significant crop
damage or yield loss. The techniques can be combined with the
herbicide glyphosate to improve the degree of weed control and
permit control of herbicide tolerant weeds.
Inventors: |
Feng; Paul C. C. (Wildwood,
MO), Brinker; Ronald J. (Ellisville, MO) |
Applicant: |
Name |
City |
State |
Country |
Type |
Monsanto Technology LLC |
St. Louis |
MO |
US |
|
|
Assignee: |
Monsanto Technology LLC (St.
Louis, MO)
|
Family
ID: |
38650176 |
Appl.
No.: |
13/751,021 |
Filed: |
January 25, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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60811276 |
Jun 6, 2006 |
|
|
|
Reissue of: |
11758653 |
Jun 5, 2007 |
7855326 |
Dec 21, 2010 |
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Current U.S.
Class: |
800/300; 504/144;
504/129 |
Current CPC
Class: |
A01N
57/20 (20130101); A01N 25/00 (20130101); A01N
25/00 (20130101); A01N 37/40 (20130101); A01N
39/02 (20130101); A01N 39/04 (20130101); A01N
57/20 (20130101); A01N 57/20 (20130101); A01N
2300/00 (20130101) |
Current International
Class: |
A01H
5/00 (20060101); A01N 37/10 (20060101) |
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Sep 2008 |
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Primary Examiner: O Hara; Eileen B
Attorney, Agent or Firm: Dentons US LLP Sisson; Pamela
Parent Case Text
This application claims the priority of U.S. Provisional Patent
Application 60/811,276, filed Jun. 6, 2006, the disclosure of which
is incorporated herein by reference in its entirety.
Claims
What is claimed is:
1. A method for controlling weed growth in a crop-growing
environment comprising: a) applying a herbicidally effective amount
of .[.an auxin-like.]. .Iadd.dicamba .Iaddend.herbicide to a
crop-growing environment; b) planting a transgenic seed of a
dicotyledonous plant comprising a nucleic acid encoding a
polypeptide having dicamba-degrading enzymatic activity in soil of
the crop-growing environment within 21 days of applying the
herbicide; and c) allowing the seed to germinate into a plant.
2. The method of claim 1, wherein the herbicide is applied prior
to, concurrently with, or after the planting of the seed.
3. The method of claim 1, wherein the transgenic seed is planted in
the soil within about 12, 10, 7, or 3 days before or after the
herbicide is applied.
4. The method of claim 1, wherein the transgenic seed germinates
from between about 18 days and 0 days after treating the soil.
5. The method of claim 1, wherein the transgenic seed germinates
from between about 14 days and 0 .[.day.]. .Iadd.days
.Iaddend.after treating the soil.
6. The method of claim 1, wherein the transgenic seed germinates
from between about 7 days and 0 days after treating the soil.
.[.7. The method of claim 1, wherein the auxin-like herbicide is
selected from the group consisting of a phenoxy carboxylic acid
compound, a benzoic acid compound, a pyridine carboxylic acid
compound, a quinoline carboxylic acid compound, and a
benazolinethyl compound..].
.[.8. The method of claim 7, wherein the phenoxy carboxylic acid
compound is selected from the group consisting of:
2,4-dichlorophenoxyacetic acid, (4-chloro-2-methylphenoxy) acetic
acid (MCPA), and 4-(2,4-dichlorophenoxy) butyric acid
(2,4-DB)..].
.[.9. The method of claim 8, wherein the herbicidally effective
amount of 2,4-dichlorophenoxyacetic, (4-chloro-2-methylphenoxy)
acetic acid (MCPA), or 4-(2,4-dichlorophenoxy) butyric acid
(2,4-DB) is lower than about 1120 g/ha..].
.[.10. The method of claim 7, wherein the benzoic acid compound is
dicamba..].
11. The method of claim .[.10.]. .Iadd.1.Iaddend., wherein the
herbicidally effective amount of dicamba is from about 2.5 g/ha to
about 10,080 g/ha.
12. The method of claim 1, wherein the nucleic acid is selected
from the group consisting of (1) a nucleic acid sequence encoding
the polypeptide of SEQ ID NO: 6, (2) a nucleic acid sequence
comprising the sequence of SEQ ID NO: 5, (3) a nucleic acid
sequence that hybridizes to a complement of the nucleic acid
sequence of SEQ ID NO: 5 under conditions of 5.times.SSC, 50%
formamide and 42.degree. C., (4) a nucleic acid sequence having at
least 70% sequence identity to the nucleic acid sequence of SEQ ID
NO: 5, and (5) a nucleic acid sequence encoding a polypeptide
having at least 70% sequence identity to the polypeptide sequence
of SEQ ID NO:6.
13. The method of claim 1, wherein the dicotyledonous plant is
selected from the group consisting of alfalfa, beans, broccoli,
cabbage, carrot, cauliflower, celery, cotton, cucumber, eggplant,
lettuce, melon, pea, pepper, pumpkin, radish, rapeseed, spinach,
soybean, squash, tomato, and watermelon.
14. The method of claim 13, wherein the dicotyledonous plant is a
soybean, cotton or rapeseed plant.
15. The method of claim 1, further comprising applying a second
treatment of .[.an auxin-like.]. .Iadd.dicamba .Iaddend.herbicide
after the seed germinates.
16. The method of claim 15, wherein the second treatment is carried
out at a time selected from the group consisting of between about
the V1 to V2 and V3 to V4 stages, before flowering, at flowering,
after flowering, and at seed formation.
17. The method of claim 1, .Iadd.further .Iaddend.comprising
allowing a spray drift from an application of .[.auxin-like.].
.Iadd.dicamba .Iaddend.herbicide to a second crop-growing
environment to contact said plant, wherein the plant is tolerant to
the spray drift.
18. A method for controlling a glyphosate tolerant weed in a field
comprising: a) planting a transgenic seed in a field comprising a
glyphosate tolerant weed or a seed thereof, wherein the seed
comprises a transgene conferring glyphosate tolerance and a
transgene encoding dicamba monooxygenase, the transgene encoding
dicamba monooxygenase which displays dicamba-degrading enzymatic
activity comprising a nucleic acid sequence selected from the group
consisting of (1) a nucleic acid sequence encoding the polypeptide
of SEQ ID NO:6, (2) a nucleic acid sequence comprising the sequence
of SEQ ID NO:5, and (3) a nucleic acid sequence encoding a
polypeptide with at least 90% sequence identity to the polypeptide
of SEQ ID NO:6, wherein the polypeptide has dicamba monooxygenase
activity; b) growing the seed into a plant; and c) treating the
field with an amount of .[.an auxin-like.]. .Iadd.dicamba
.Iaddend.herbicide and glyphosate effective to control weed growth
of the glyphosate tolerant weed.
19. The method of claim 18, wherein the transgene conferring
glyphosate tolerance encodes a protein selected from the group
consisting of glyphosate resistant
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), glyphosate
oxidoreductase (GOX), glyphosate-N-acetyl transferase (GAT) and
glyphosate decarboxylase.
20. The method of claim 19, wherein the transgene encoding GAT
comprises the nucleic acid sequence of SEQ ID NO:18, or encodes the
polypeptide of SEQ ID NO:19.
21. The method of claim 18, wherein the seed is from a
dicotyledonous plant selected from the group consisting of alfalfa,
beans, broccoli, cabbage, carrot, cauliflower, celery, cotton,
cucumber, eggplant, lettuce, melon, pea, pepper, pumpkin, radish,
rapeseed, spinach, soybean, squash, tomato, and watermelon.
22. The method of claim 21, wherein the dicotyledonous plant is a
soybean, cotton or rapeseed plant.
23. The method of claim 18, wherein treating the field is carried
out at a time selected from the group consisting of between about
the V1 to V2 and V3 to V4 leaf stages, before flowering, at
flowering, after flowering, and at seed formation.
24. The method of claim 18, wherein treating the field is carried
out after the seed germinates.
25. The method of claim 18, wherein treating the field is carried
out about four weeks, three weeks, two weeks, 1 week, or 0 weeks
before step a).
26. The method of claim 18, wherein treating the field is carried
out concurrently with the planting of the seed.
27. The method of claim 18, wherein the transgenic seed is planted
in the soil within about 15, 12, 10, 7 or about 3 days before or
after applying the herbicide.
28. The method of claim 18, wherein the transgenic seed germinates
from between about 0 and about 18, 14, 7, or 1 days after treating
the soil.
.[.29. The method of claim 18, wherein the auxin-like herbicide is
selected from the group consisting of a phenoxy carboxylic acid
compound, benzoic acid compound, pyridine carboxylic acid compound,
quinoline carboxylic acid compound, and benazolinethyl
compound..].
.[.30. The method of claim 29, wherein the phenoxy carboxylic acid
compound is selected from the group consisting of
2,4-dichlorophenoxyacetic acid and (4-chloro-2-methylphenoxy)
acetic acid..].
.[.31. The method of claim 30, wherein the amount of
2,4-dichlorophenoxyacetic compound is lower than about 1120
g/ha..].
.[.32. The method of claim 30, wherein the amount of
2,4-dichlorophenoxyacetic compound is lower than about 280
g/ha..].
.[.33. The method of claim 30, wherein the amount of
(4-chloro-2-methylphenoxy) acetic acid compound is lower than about
1120 g/ha..].
.[.34. The method of claim 30, wherein the amount of
(4-chloro-2-methylphenoxy) acetic acid compound is lower than about
280 g/ha..].
.[.35. The method of claim 29, wherein the benzoic acid is
dicamba..].
36. The method of claim .[.35.]. .Iadd.18.Iaddend., wherein the
amount of dicamba is from about 2.5 g/ha to about 10,080 g/ha.
37. The method of claim 23, wherein the amount of glyphosate is
from about 200 g/ha to about 1,600 g/ha.
38. The method of claim 18, wherein .[.the auxin-like.].
.Iadd.dicamba .Iaddend.herbicide and glyphosate are applied
substantially simultaneously.
.[.39. A method for controlling weed growth in a field comprising:
a) applying a herbicidally effective amount of an auxin-like
herbicide other than dicamba to a field, wherein the field
comprises a transgenic dicotyledonous plant comprising a nucleic
acid encoding a polypeptide having dicamba degrading enzymatic
activity or is planted with a seed that germinates into said
transgenic dicotyledonous plant within 21 days of applying the
herbicide, wherein the herbicidally effective amount is an amount
that does not damage the transgenic dicotyledonous plant but will
damage a plant of the same genotype that lacks the nucleic acid
encoding a polypeptide having dicamba degrading enzymatic activity,
wherein the nucleic acid is selected from the group consisting of
(1) a nucleic acid sequence encoding the polypeptide of SEQ ID
NO:6, (2) a nucleic acid sequence comprising the sequence of SEQ ID
NO:5, (3) a nucleic acid sequence encoding a polypeptide with at
least 90% sequence identity to the polypeptide of SEQ ID NO:6,
wherein the polypeptide has dicamba monooxygenase activity; and b)
allowing the transgenic dicotyledonous plant to grow..].
.[.40. The method of claim 39, wherein step a) comprises applying
the herbicidally effective amount of an auxin-like herbicide to a
growing environment adjacent to a growing environment comprising
the transgenic dicotyledonous plant and allowing the herbicide to
drift onto the plant or soil in which the plant grows..].
.[.41. The method of claim 39, wherein the auxin-like herbicide is
selected from the group consisting of a phenoxy carboxylic acid
compound, benzoic acid compound, pyridine carboxylic acid compound,
quinoline carboxylic acid compound, and benazolinethyl
compound..].
.[.42. The method of claim 41, wherein the phenoxy carboxylic acid
compound is selected from the group consisting of
2,4-dichlorophenoxyacetic acid, (4-chloro-2-methylphenoxy) acetic
acid and 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB)..].
.[.43. The method of claim 39, wherein step b) comprises allowing
the transgenic dicotyledonous plant to grow to maturity..].
.[.44. The method of claim 39, wherein the transgenic
dicotyledonous plant is a selected from the group consisting of
alfalfa, beans, broccoli, cabbage, carrot, cauliflower, celery,
cotton, cucumber, eggplant, lettuce, melon, pea, pepper, pumpkin,
radish, rapeseed, spinach, soybean, squash, tomato, and
watermelon..].
.[.45. The method of claim 39, wherein the transgenic
dicotyledonous plant is a soybean, cotton or rapeseed plant..].
46. A method for increasing the efficiency of use of a herbicide
delivery device comprising: a) obtaining a device that has been
used to deliver a first composition comprising .[.an auxin-like.].
.Iadd.dicamba .Iaddend.herbicide; b) delivering a second
composition to the field using the device without first completely
washing the device so that a herbicide residue comprising .[.the
auxin-like.]. .Iadd.dicamba .Iaddend.herbicide remains in the
device and is delivered with the second composition to the field,
wherein the field comprises a transgenic dicotyledonous plant
expressing a nucleic acid encoding dicamba monooxygenase which
displays dicamba-degrading enzymatic activity or is planted with a
seed that germinates into said transgenic dicotyledonous plant, and
wherein the herbicide residue is present in an amount that does not
damage the transgenic dicotyledonous plant but will damage a plant
of the same genotype that lacks the nucleic acid encoding dicamba
monooxygenase which displays dicamba-degrading enzymatic
activity.
47. The method of claim 46, wherein the nucleic acid is selected
from the group consisting of (1) a nucleic acid sequence encoding
the polypeptide of SEQ ID NO:6, (2) a nucleic acid sequence
comprising the sequence of SEQ ID NO:5, (3) a nucleic acid sequence
that hybridizes to a complement of the nucleic acid sequence of SEQ
ID NO:5 under conditions of 5.times. SSC, 50% formamide and
42.degree. C., (4) a nucleic acid sequence having at least 70%
sequence identity to the nucleic acid sequence of SEQ ID NO:5, and
(5) a nucleic acid sequence encoding a polypeptide having at least
70% sequence identity to the polypeptide sequence of SEQ ID
NO:6.
.[.48. The method of claim 46, wherein the auxin-like herbicide is
selected from the group consisting of a phenoxy carboxylic acid
compound, benzoic acid compound, pyridine carboxylic acid compound,
quinoline carboxylic acid compound, and benazolinethyl
compound..].
.[.49. The method of claim 48, wherein the phenoxy carboxylic acid
compound is 2,4-dichlorophenoxyacetic acid,
(4-chloro-2-methylphenoxy) acetic acid (MCPA), or
4-(2,4-dichlorophenoxy) butyric acid (2,4-DB)..].
.[.50. The method of claim 48, wherein the benzoic acid compound is
dicamba..].
51. The method of claim .[.48.]. .Iadd.46.Iaddend., wherein the
dicotyledonous plant is selected from the group consisting of
alfalfa, beans, broccoli, cabbage, carrot, cauliflower, celery,
cotton, cucumber, eggplant, lettuce, melon, pea, pepper, pumpkin,
radish, rapeseed, spinach, soybean, squash, tomato, and
watermelon.
52. The method of claim 51, wherein the dicotyledonous plant is a
soybean, cotton or rapeseed plant.
53. A method for controlling weed growth in a crop-growing
environment comprising: a) planting a transgenic seed in a field
comprising a weed or a seed thereof, wherein the transgenic seed
comprises a transgene conferring glyphosate tolerance and a
transgene conferring dicamba tolerance; b) treating the field with
a herbicidally effective amount of dicamba, glyphosate, or a
mixture thereof, wherein the planting and the treating is done in a
single pass through the field; and c) growing the transgenic seed
into a plant.
54. The method of claim 53, wherein the transgene conferring
glyphosate tolerance encodes a protein selected from the group
consisting of glyphosate resistant
5-enolpyruvylshikimate-3-phosphate synthase, glyphosate
oxidoreductase, and glyphosate-N-acetyl transferase, and glyphosate
decarboxylase.
55. The method of claim 54, wherein the transgene encoding GAT
comprises the nucleic acid sequence of SEQ ID NO:18, or encodes the
polypeptide of SEQ ID NO:19.
56. The method of claim 53, wherein the transgene conferring
dicamba tolerance encodes a dicamba monooxygenase comprising a
nucleic acid sequence selected from the group consisting of (a) a
nucleic acid sequence encoding the polypeptide of SEQ ID NO:6, (b)
a nucleic acid sequence comprising the sequence of SEQ ID NO:5, (c)
a nucleic acid sequence that hybridizes to a complement of the
nucleic acid sequence of SEQ ID NO:5 under conditions of 5.times.
SSC, 50% formamide and 42.degree. C., (d) a nucleic acid sequence
having at least 70% sequence identity to the nucleic acid sequence
of SEQ ID NO:5, and e) a nucleic acid sequence encoding a
polypeptide having at least 70% sequence identity to the
polypeptide sequence of SEQ ID NO:6.
57. The method of claim 53, wherein the transgenic seed if from a
dicotyledonous plant is selected from the group consisting of
alfalfa, beans, broccoli, cabbage, carrot, cauliflower, celery,
cotton, cucumber, eggplant, lettuce, melon, pea, pepper, pumpkin,
radish, rapeseed, spinach, soybean, squash, tomato, and watermelon
seed.
58. The method of claim 57, wherein the dicotyledonous plant is a
soybean, cotton or rapeseed plant.
59. The method of claim 53, wherein the amount of dicamba is from
about 2.5 g/ha to about 10,080 g/ha.
60. The method of claim 53, wherein the amount of glyphosate is
from about 200 g/ha to about 1,600 g/ha.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates generally to the field of weed management.
More specifically, the invention relates to methods for using
auxin-like herbicides such as dicamba for controlling weeds.
2. Description of the Related Art
Weeds cost farmers billions of dollars annually in crop losses and
the expense of efforts to keep weeds under control. Weeds also
serve as hosts for crop diseases and insect pests. The losses
caused by weeds in agricultural production environments include
decreases in crop yield, reduced crop quality, increased irrigation
costs, increased harvesting costs, decreased land value, injury to
livestock, and crop damage from insects and diseases harbored by
the weeds. The principal means by which weeds cause these effects
are: 1) competing with crop plants for the essentials of growth and
development, 2) production of toxic or irritant chemicals that
cause human or animal health problem, 3) production of immense
quantities of seed or vegetative reproductive parts or both that
contaminate agricultural products and perpetuate the species in
agricultural lands, and 4) production on agricultural and
nonagricultural lands of vast amounts of vegetation that must be
disposed of. The damage caused can be significant. For example, it
is estimated that between 1972 and 1976 corn yields were reduced by
about 10% due to weeds (Chandler, 1981).
Among weeds that serve as hosts for crop pests, for example,
pepperweed and tansymustard (Descurainia sp.) maintain large
populations of diamondback moths during the late fall, winter, and
spring. They are also hosts to the turnip aphid and green peach
aphid. Several weed species of the nightshade family (Solanaceae)
are hosts to insects that commonly attack eggplant, pepper, potato,
and tomato. For example, horsenettle (Solanum carolinense L.) is a
host of the Colorado potato beetle, and black nightshade (S. nigrum
L.) is a host of the cabbage looper. Morning-glory is an important
host of insects attacking sweet potato, especially the highly
destructive sweet potato weevil. Ragweed serves as a host for
Mansonia mosquitoes, an insect vector for the human diseases
encephalitis and rural filariasis.
Some weeds are undesirable in hay, pastures, and range-lands
because of the mechanical injury that they inflict on livestock.
Woody stems, thorns, and stiff seed awns cause injury to the mouth
and digestive tract of livestock; and the hairs and fibers of some
plants tend to ball up and obstruct the intestines, especially in
horses, causing serious problems. Ingested by milk cows, some weeds
such as ragweeds, wild garlic (Allium vineale L.), and mustard,
among others, impart a distinctly distasteful odor or flavor to
milk and butter. Barbed seed dispersal units may become so
entangled in the wool of sheep as to greatly diminish its market
value. Parasitic plants, such as dodder (Cuscuta sp.), broomrape
(Orobanche sp.), and witchweed, rob their host plants of organic
foodstuffs.
Chemical herbicides have provided an effective method of weed
control over the years. Herbicides can generally be applied
pre-emergence and/or post-emergence. Pre-emergence herbicides are
applied in a field before a crop emerges from the soil. Such
applications are typically applied to the soil before, at the same
time, or soon after planting the crop. Such applications may kill
weeds that are growing in the field prior to the emergence of the
crop, and may also prevent or reduce germination of weeds that are
present in the soil. Post-emergence herbicides are typically used
to kill weeds after a crop has emerged in the field. Such
applications may kill weeds in the field and prevent or reduce
future weed germination. In either case, the herbicides may be
applied to the surface of the soil, mixed with the soil, over the
top of the plant, or applied by any other method known to those of
skill in the art.
One weed control strategy is to apply an herbicide such as dicamba
to a field before sowing seeds. However, after applying the
herbicide to a field, a farmer has to wait at least several weeks
before sowing the field with crop seeds such that the herbicide has
killed most of the weeds and has degraded so as not injure the sown
crop. For example, plants are especially sensitive to dicamba and
it has been recommended that dicamba formulations such as
Banvel.TM. or Sterling.TM. be applied 30 days prior to planting for
controlling weeds. A comprehensive list of weeds that are
controlled by dicamba is available (Anonymous, 2007). The herbicide
is particularly useful for control of taller weeds and more
difficult to control weeds such as purslane, sicklepod, morninglory
and wild buckwheat. Dicamba can be used to control weeds not
susceptible to other herbicides. Following the application of
Clarity.TM., another formulation of dicamba, a minimum accumulation
of one inch of rainfall or overhead irrigation followed by a 14 day
waiting period for the 4 to 8 ounce/acre rates or a 28 day waiting
period for the 16 ounce/acre rates has been recommend for
controlling weeds in a soybean field (see Table 22 in VanGessel and
Majek, 2005). Also, the Clarity.RTM. label recommends that it be
applied at least 15 days prior to sorghum planting. Similarly, for
cotton, a waiting period of 21 days is recommended after applying
Clarity.RTM. or Banvel.RTM. to the field, before planting the
cotton seeds (Craig et al., 2005, Crop Profile for Cotton
(Gossypium hirsutum) in Tennessee,
www.ipmcenters.org/cropprofiles/docs/tncotton.html) and no
pre-emergence and post-emergence application are recommended. The
waiting period is also dependent on the crop growing environment at
any give time, such as the type of soil (soil having organic
activity will degrade dicamba faster), moisture content, rainfall,
temperature, as well as type of formulation and rate of
application.
The herbicide 2,4-D has been recommended for controlling certain
weeds in a soybean field such as mustard spp., plantains,
marestail, and 2,4-D susceptible annual broadleaf weeds by applying
it 7 to 30 days prior to planting, depending on rate and
formulation (ester or amine) (see Table 22 in VanGessel and Majek,
2005).
One method that has been successfully used to manage weeds combines
herbicide treatments with crops that are tolerant to the
herbicides. In this manner, herbicides that would normally injure a
crop can be applied before and during growth of the crop without
causing damage. Thus, weeds may be effectively controlled and new
weed control options are made available to the grower. In recent
years, crops tolerant to several herbicides have been developed.
For example, crops tolerant to 2,4-dichlorophenoxyacetic acid
(Streber and Willmitzer, 1989), bromoxynil (Stalker et al., 1988),
glyphosate (Comai et al., 1985) and phosphinothricin (De Block et
al., 1987) have been developed.
Recently, a gene for dicamba monooxygenase (DMO) was isolated from
Pseudomonas maltophilia (US Patent Application No: 20030135879)
which is involved in the conversion of a herbicidal form of the
herbicide dicamba (3,6-dichloro-o-anisic acid) to a non-toxic
3,6-dichlorosalicylic acid. The inventors reported the
transformation of the DMO gene into tobacco and Arabidopsis. The
transformed plant tissue was selected on kanamycin and regenerated
into a plant. However, herbicide tolerance was not demonstrated or
suggested in immature tissues or seedlings or in other plants.
Pre-emergence herbicide tolerance to dicamba was not described.
Transgenic soybean plants and other plants tolerant to application
of dicamba are described in Behrens et al. (2007).
Dicamba is one member of a class of herbicides commonly referred to
as "auxin-like" herbicides or "synthetic auxins." These herbicides
mimic or act like the natural plant growth regulators called
auxins. Auxin-like herbicides appear to affect cell wall plasticity
and nucleic acid metabolism, which can lead to uncontrolled cell
division and growth. The injury symptoms caused by auxin-like
herbicides include epinastic bending and twisting of stems and
petioles, leaf cupping and curling, and abnormal leaf shape and
venation.
Dicamba is one of the many auxin-like herbicides that is a
low-cost, environmentally-friendly herbicide that has been used as
a pre-emergence herbicide (i.e., 30 days prior to planting) in
dicots and as a pre- and/or post-emergence herbicide in corn,
sorghum, small grains, pasture, hay, rangeland, sugarcane,
asparagus, turf, and grass seed crops to effectively control annual
and perennial broadleaf weeds and several grassy weeds (Crop
Protection Chemicals Reference, 1995). Unfortunately, dicamba can
injure many commercial crops including beans, soybeans, cotton,
peas, potatoes, sunflowers, tomatoes, tobacco, and fruit trees,
ornamental plants and trees, and other broadleaf plants when it
comes into contact with them. Soybean and cotton are particularly
sensitive to dicamba. Thus, applications of dicamba must generally
occur several weeks before planting of sensitive crops to ensure
that residual dicamba is sufficiently cleared from the crop
environment before crops emerge. For post-emergent weed control in
corn, dicamba is the 5th most widely used herbicide for broad leaf
weeds. However, although the optimal rate for broad leaf weed
control is between 280 to 560 g/h (grams/hectare), the average use
rate in corn is 168 g/h as at higher use rates and under certain
environmental conditions, dicamba can injure corn.
As noted above, current manufacturer's guidelines typically require
at least a 30 day delay between the application of dicamba and the
planting of sensitive crops. This inability to apply dicamba close
to the time that crops are planted delays sowing time and shortens
the growing season, thereby increasing the risk of exposing crops
to frost in the fall. The delay also means that the farmers have to
go through the field twice; once for planting and once for
spraying, thereby increasing fuel and wear-tear costs to the
farmers. Improvements over the state of the art that would
eliminate the delay would positively impact the quality and
quantity of the crop which could result and reduce economic losses
to farmers. More effective weed control would also reduce the risk
of weeds developing resistance to existing herbicides.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a method for controlling weed
growth in a field comprising: a) applying a herbicidally effective
amount of an auxin-like herbicide to a crop-growing environment;
and planting a transgenic seed of a dicotyledonous plant expressing
a nucleic acid encoding dicamba monooxygenase in soil of the
crop-growing environment, wherein the seed germinates within 30
days or less of applying the herbicide and wherein the dicamba
monooxygenase comprises at least 70% sequence identity to the
polypeptide sequence of SEQ ID NO:2; and c) allowing the seed to
germinate into a plant. In certain embodiments, the seed germinates
within four weeks, three weeks, two weeks, or less than one week
after treating the growing environment with the auxin-like
herbicide. The treated growing environment may be, for example, a
field in which a crop is planted. A population of seeds of a plant
tolerant to the auxin-like herbicide may be planted in the field.
Treating the environment can be carried out according to known
techniques in the art using, for example, commercially available
formulations of auxin-like herbicides such as dicamba. The
environment includes an area for which control of weeds is desired
and in which the seed of a plant tolerant to the auxin-like
herbicide can be planted. A weed can be directly contacted with
herbicide in the environment and soil in the environment can be
contacted with the herbicide, preventing or reducing weed growth in
the soil. The step of treating the environment with a herbicide may
be carried out before, after, or concurrently with the step of
planting the soil with the transgenic seed. The transgenic seed may
be planted into soil in the environment, for example, within three
weeks before or after treatment, including from between about two
weeks, one week and 0 weeks before or after treatment, further
including from between about 1, 2, 3, 4, 5, or 6 days before or
after treatment, including concurrently with treatment. In the
method, the seed may germinate, for example, from between about 30
days and 0 days after treating the environment, including between
about 21, 18, 16, 14, 12, 10, 8, 6, 5, 4, 3, 2, 1 and about 0 days
after treating the environment. The method may further comprise
applying one or more additional treatments of an auxin-like
herbicide after the seed germinates and/or the plant is growing. In
certain embodiments, a second treatment is carried out at a time
selected from the group consisting of between about the 1 to 2 leaf
and 3 to 4 leaf stages, before flowering, at flowering, after
flowering, and at seed formation. In one embodiment, the second
treatment comprises applying dicamba and/or a
2,4-dichlorophenoxyacetic compound (2,4-D).
In a method of the invention, the auxin-like herbicide may be
selected from the group consisting of a phenoxy carboxylic acid
compound, benzoic acid compound, pyridine carboxylic acid compound,
quinoline carboxylic acid compound, and benazolinethyl compound.
Examples of a phenoxy carboxylic acid compound include
2,4-dichlorophenoxyacetic acid and (4-chloro-2-methylphenoxy)acetic
acid. In certain embodiments, a herbicidally effective amount of
2,4-D and/or (4-chloro-2-methylphenoxy)acetic acid used is between
about 2 g/ha (grams/hectare) to about 5000 g/ha, including about 50
g/ha to about 2500 g/ha, about 60 g/ha to about 2000 g/ha, about
100 g/ha to about 2000 g/ha, about 75 g/ha to about 1000 g/ha,
about 100 g/ha to about 500 g/ha, and from about 100 g/ha to about
280 g/ha. In one embodiment found to function particularly well
with the invention, dicamba is used as the herbicide. In certain
embodiments, a herbicidally effective amount of dicamba used may be
from about 2.5 g/ha to about 10,080 g/ha, including about 2.5 g/ha
to about 5,040 g/ha, about 5 g/ha to about 2,020 g/ha, about 10 g/a
to about 820 g/h and about 50 g/ha to about 1,000 g/ha, about 100
g/ha to about 800 g/ha and about 250 g/ha to about 800 g/ha.
In a method of the invention a plant may be used exhibiting
tolerance to auxin-like herbicides including dicamba. Such a plant
may comprise a nucleic acid encoding a dicamba monooxygenase. In
one embodiment, the plant is defined as comprising a nucleic acid
encoding a dicamba monooxygenase that has at least 70% identity to
a polypeptide sequence of any one or more of SEQ ID NOs:2, 4, 6, 8,
10 or 12, including at least about 75%, 80%, 85%, 90%, 95%, 97%,
98%, 99% and greater sequence identity to these sequences.
Polypeptide or polynucleotide comparisons may be carried out and
identity determined as is known in the art, for example, using
MEGAlign (DNAStar, Inc., 1228 S. Park St., Madison, Wis. 53715)
with default parameters. Such software matches similar sequences by
assigning degrees of similarity or identity.
The methods of the invention may be used in connection with plants
that exhibit susceptibility to auxin-like herbicides such as
dicotyledonous (dicot) plants. In certain embodiments, a
dicotyledonous plant is used selected from the group consisting of
alfalfa, beans, broccoli, cabbage, carrot, cauliflower, celery,
cotton, cucumber, eggplant, lettuce, melon, pea, pepper, pumpkin,
radish, rapeseed, spinach, soybean, squash, tomato, and watermelon.
In some embodiments, the dicot is soybean, cotton, or canola.
In another aspect, the invention provides a method for controlling
a weed in a field comprising: a) planting a transgenic seed in a
field, wherein the seed comprises transgenes conferring tolerance
to an auxin-like herbicide and a second herbicide; b) growing the
seed into a plant; and c) treating the field with an amount of the
auxin-like herbicide and the second herbicide in amounts effective
to control weed growth. In some embodiments, the second herbicide
may be glufosinate (De Block et al., 1987), a sulfonylurea
(Sathasiivan et al., 1990), an imidazolinone (U.S. Pat. Nos.
5,633,437; 6,613,963), bromoxynil (Stalker et al., 1988), dalapon
or 2,2-Dichloropropionic acid (Buchanan-Wollaston et al., 1989),
cyclohexanedione (U.S. Pat. No. 6,414,222), a protoporphyrinogen
oxidase inhibitor (U.S. Pat. No. 5,939,602), norflurazon (Misawa et
al., 1993 and Misawa et al., 1994), or isoxaflutole (WO 96/38567)
herbicide, among others. The auxin-like herbicide and the second
herbicide may be applied simultaneously or separately. In a
particular embodiment, the second herbicide is glyphosate and the
auxin-like herbicide is dicamba. In one embodiment, the plant
comprises a nucleic acid that has at least 70% sequence identity to
a nucleic acid sequence of any one or more of SEQ ID NOs: 1, 3, 5,
7, 9, or 11, including at least about 75%, 80%, 85%, 90%, 95%, 97%,
98%, 99% and greater sequence identity to these sequence.
In further embodiments, a plant such as the foregoing is defined as
comprising a transgene conferring glyphosate tolerance. Glyphosate
resistant 5-enolpyruvylshikimate-3-phosphate synthases (EPSPS) are
well known in the art and disclosed, for example, in U.S. Pat. Nos.
5,627,061, 5,633,435, 6,040,497, 5,094,945, WO04074443, and
WO04009761. Nucleic acids encoding glyphosate degrading enzymes,
for example, glyphosate oxidoreductase (GOX, U.S. Pat. No.
5,463,175), and nucleic acids encoding glyphosate inactivating
enzymes, such as glyphosate-N-acetyl transferase (GAT, U.S. Patent
publication 20030083480; U.S. Patent Publication 20070079393) and
glyphosate decarboxylase (WO05003362 and U.S. Patent Application
No. 20040177399) are also known. In certain embodiments, the GAT
enzyme comprises the sequence of GAT4601 (SEQ ID NO:19), or is
encoded by a transgene comprising the nucleic acid sequence of SEQ
ID NO:18. In a particular embodiment, the GAT polypeptide is
expressed using the SCP1 promoter.
In the method, treating the field may be carried out at a time
selected from the group consisting of between about the 1 to 2 leaf
and 3 to 4 leaf stages, before flowering, at flowering, after
flowering, and at seed formation. Treating the field may further be
defined as carried out at a time proximate to step a) such that the
seed germinates while the auxin-like herbicide remains in the soil
in an amount effective to control growth of the weed. In the
method, treating the field may be carried out about three weeks,
two weeks 1 week or 0 weeks before step a). The auxin-like
herbicide may be selected from the group consisting of a phenoxy
carboxylic acid compound, benzoic acid compound, pyridine
carboxylic acid compound, quinoline carboxylic acid compound, and
benazolinethyl compound.
The phenoxy carboxylic acid compound may be selected from the group
consisting of 2,4-dichlorophenoxyacetic acid,
(4-chloro-2-methylphenoxy)acetic acid, and
4-(2,4-dichlorophenoxy)butyric acid (2,4-DB). The amount of
2,4-dichlorophenoxyacetic compound used may be lower than about 280
g/ha. The amount of 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB)
used may be lower than about 1120 g/ha. The amount of
(4-chloro-2-methylphenoxy) acetic acid compound used may be lower
than about 280 g/ha. In one embodiment, the auxin like herbicide is
dicamba. The amount of dicamba used may be, for example, from about
2.5 g/ha to about 10,080 g/ha, including about 2.5 g/ha to about
1040 g/ha, about 5 g/ha to about 2040 g/ha, about 10 g/a to about
820 g/h, and about 50 g/ha to about 1000 g/ha. The amount of
glyphosate may be from about 200 g/ha to about 1,600 g/h, including
from about 200 g/ha to about 1,000 g/h, from about 200 g/ha to
about 800 g/h, from about 200 g/ha to about 400 g/h, and from about
400 g/ha to about 800 g/h.
In yet another aspect, the invention provides a method for
controlling weed growth in a crop-growing environment comprising:
a) applying a herbicidally effective amount of an auxin-like
herbicide to a crop-growing environment; b) planting a transgenic
seed of a monocotyledonous plant comprising a nucleic acid encoding
a dicamba degrading enzymatic activity, such as dicamba
monooxygenase, in soil of the crop-growing environment within 21
days of applying the auxin-like herbicide, wherein the herbicidally
effective amount is an amount that does not damage the transgenic
seed or a plant that germinates therefrom but will damage a seed or
a plant that germinates therefrom of the same genotype that lacks
the nucleic acid and is planted under the same conditions as the
transgenic seed, wherein the nucleic acid is selected from the
group consisting of (1) a nucleic acid sequence encoding the
polypeptide of SEQ ID NO:8, (2) a nucleic acid sequence comprising
the sequence of SEQ ID NO:7, (3) a nucleic acid sequence that
hybridizes to a complement of the nucleic acid sequence of SEQ ID
NO:7 under conditions of 5.times.SSC, 50% formamide and 42.degree.
C., (4) a nucleic acid sequence having at least 70% sequence
identity to the nucleic acid sequence of SEQ ID NO:7, and (5) a
nucleic acid sequence encoding a polypeptide having at least 70%
sequence identity to the polypeptide sequence of SEQ ID NO:8; and
c) allowing the seed to germinate into a plant. The nucleic acid
sequence having at least 70% sequence identity to the nucleic acid
sequence of SEQ ID NO:7 may encode a polypeptide comprising a
cysteine residue at position 112. This embodiment may combined with
any of the methods and compositions provided above.
In particular embodiments of the invention, herbicide treatments to
monocot plants may be made at higher rates and/or in closer
proximity to emergence of crops than previously could be made
without damaging crops. In specific embodiments, a herbicidally
effective amount of 2,4-D and/or MCPA, such as, for example, at
least about 200, 300, 300, 500, 590, 650, 700, 800 or more g/ha of
either or both herbicides, including from about 300 to about 1200
g/ha, from about 500 to about 1200 g/ha, from about 600 to about
1200 g/ha, from about 590 to about 1400 g/ha, and from about 700 to
about 1100 g/ha of either or both herbicides. The herbicide may
also be dicamba and the herbicidally effective amount may be, for
example, at least about 168, 175, 190, 200, 225, 250, 280, 300,
400, 500, 560 or more g/ha of dicamba, including from about 200
g/ha to about 600 g/ha, from about 250 g/ha to about 600 g/ha, from
about 250 g/ha to about 800 g/ha, from about 225 g/ha to about 1120
g/ha, and from about 250 g/ha to about 1200 g/ha, from about 280
g/ha to about 1120 g/ha and from about 560 g/ha to about 1120 g/ha.
In particular embodiment, the monocotyledonous plant is selected
from the group consisting of corn, rice, sorghum, wheat, rye,
millet, sugarcane, oat, triticale, switchgrass, and turfgrass.
Expressing the transgenic dicamba-degrading enzymatic activity such
as a monooxygenase, in a monocotyledonous crop plant, such as corn,
allows application of a higher level of dicamba to the crop for the
purpose of weed control at any stage of plant growth, as compared
to the level of dicamba that may be applied to a monocotyledonous
crop plant that does not comprise a transgene that encodes such a
dicamba-degrading enzymatic activity.
In yet another aspect, the invention provides a method for
controlling weed growth in a field comprising: a) applying a
herbicidally effective amount of an auxin-like herbicide other than
dicamba to a field, wherein the field comprises a transgenic
dicotyledonous plant comprising a nucleic acid encoding dicamba
monooxygenase or is planted with a seed that germinates into said
transgenic dicotyledonous plant within 21 days of applying the
herbicide, wherein the herbicidally effective amount is an amount
that does not damage the transgenic dicotyledonous plant but will
damage a plant of the same genotype that lacks the nucleic acid
encoding dicamba monooxygenase, wherein the nucleic acid is
selected from the group consisting of (1) a nucleic acid sequence
encoding the polypeptide of SEQ ID NO:8, (2) a nucleic acid
sequence comprising the sequence of SEQ ID NO:7, (3) a nucleic acid
sequence that hybridizes to a complement of the nucleic acid
sequence of SEQ ID NO:7 under conditions of 5.times.SSC, 50%
formamide and 42.degree. C., (4) a nucleic acid sequence having at
least 70% sequence identity to the nucleic acid sequence of SEQ ID
NO:7, and (5) a nucleic acid sequence encoding a polypeptide having
at least 70% sequence identity to the polypeptide sequence of SEQ
ID NO:8; and b) allowing the transgenic dicotyledonous plant to
grow. In the method, step a) may comprise applying the herbicidally
effective amount of an auxin-like herbicide to a growing
environment adjacent to a growing environment comprising the
transgenic dicotyledonous plant and allowing the herbicide to drift
onto the plant or soil in which the plant grows. The auxin-like
herbicide may be any herbicide as described herein. In the method,
step b) may comprise allowing the transgenic dicotyledonous plant
to grow to maturity. In specific embodiments, the herbicidally
effective amount may be defined as an amount that does not damage
the transgenic plant.
In yet another aspect, the invention provides a method for
increasing the efficiency of use of a herbicide delivery device
comprising: a) obtaining a device that has been used to deliver a
first composition comprising an auxin-like herbicide; and b)
delivering a second composition to the field using the device
without first completely washing the device so that a herbicide
residue comprising the auxin-like herbicide remains in the device
and is delivered with the second composition to the field, wherein
the field comprises a transgenic dicotyledonous plant expressing a
nucleic acid encoding dicamba monooxygenase or is planted with a
seed that germinates into said transgenic dicotyledonous plant
within 21 days of delivering the second composition, wherein the
herbicide residue is present in an amount that does not damage the
transgenic dicotyledonous plant but will damage a plant of the same
genotype that lacks the nucleic acid encoding dicamba
monooxygenase, wherein the nucleic acid is selected from the group
consisting of (1) a nucleic acid sequence encoding the polypeptide
of SEQ ID NO:8, (2) a nucleic acid sequence comprising the sequence
of SEQ ID NO:7, (3) a nucleic acid sequence that hybridizes to a
complement of the nucleic acid sequence of SEQ ID NO:7 under
conditions of 5.times.SSC, 50% formamide and 42.degree. C., (4) a
nucleic acid sequence having at least 70% sequence identity to the
nucleic acid sequence of SEQ ID NO:7, and (5) a nucleic acid
sequence encoding a polypeptide having at least 70% sequence
identity to the polypeptide sequence of SEQ ID NO:8.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates, in one aspect, to the unexpected discovery
that pre-emergent applications of auxin-like herbicides such as
dicamba may be made close to, or even concurrently with the
planting of crops. The invention provides superior weed control
options, including reduction and/or prevention of herbicide
tolerance in weeds. Pre-emergent applications of auxin-like
herbicides such as dicamba have previously required herbicide
applications well in advance of planting and germination of plants
susceptible to auxin-like herbicides to allow breakdown of the
herbicide in the environment and avoid significant crop damage or
death. Most crop plants, and particularly dicotyledonous plants
such as soybeans and cotton are extremely sensitive to dicamba.
Thus, the recommended post-application delays in planting by
manufacturers must be closely followed.
Young plantlets and seeds are particularly sensitive to herbicides.
Even in transgenic seeds and plants, immature tissues can
insufficiently express the gene needed to render them tolerant to
the herbicide, or may not have accumulated sufficient levels of the
protein to confer tolerance. For example, mature plants have been
found exhibiting high levels of tolerance to the herbicides
Harness.TM. (acetochlor), Lasso.TM. (alachlor), Treflan.TM.
(Trifluralin), Eptam.TM. (EPTC), and/or Far-Go.TM. (triallate) but
susceptibility to the herbicides at germination. As a result of
this variability in young tissues, crop response to post-emergence
applications (e.g., in more mature vegetative tissues) of dicamba
herbicides can significantly differ from the crop response to
pre-emergent applications of herbicides in which younger more
sensitive tissues are exposed. The former does not necessarily
predict the latter. This is underscored in the case of plants
highly sensitive to a given herbicide, such as dicots and the
herbicide dicamba. Thus, the present invention unexpectedly shows
that higher than predicted levels of crop safety can be achieved
from pre-emergence applications of dicamba.
The present invention employs auxin-like herbicides, which are also
called auxinic or growth regulator herbicides, or Group 4
herbicides (based on their mode of action). These types of
herbicides mimic or act like the natural plant growth regulators
called auxins. The action of auxinic herbicides appears to affect
cell wall plasticity and nucleic acid metabolism, which can lead to
uncontrolled cell division and growth.
Auxin-like herbicides include four chemical families: phenoxy,
carboxylic acid (or pyridine), benzoic acid, and quinaline
carboxylic acid. Phenoxy herbicides are most common and have been
used as herbicides since the 1940s when (2,4-dichlorophenoxy)acetic
acid (2,4-D) was discovered. Other examples include
4-(2,4-dichlorophenoxy)butyric acid (2,4-DB),
2-(2,4-dichlorophenoxy)propanoic acid (2,4-DP),
(2,4,5-trichlorophenoxy)acetic acid (2,4,5-T),
2-(2,4,5-Trichlorophenoxy)Propionic Acid (2,4,5-TP),
2-(2,4-dichloro-3-methylphenoxy)-N-phenylpropanamide (clomeprop),
(4-chloro-2-methylphenoxy)acetic acid (MCPA),
4-(4-chloro-o-tolyloxy)butyric acid (MCPB), and
2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP).
The next largest chemical family is the carboxylic acid herbicides,
also called pyridine herbicides. Examples include
3,6-dichloro-2-pyridinecarboxylic acid (Clopyralid),
4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (picloram),
(2,4,5-trichlorophenoxy) acetic acid (triclopyr), and
4-amino-3,5-dichloro-6-fluoro-2-pyridyloxyacetic acid
(fluoroxypyr). Examples of benzoic acids include
3,6-dichloro-o-anisic acid (dicamba) and
3-amino-2,5-dichlorobenzoic acid (choramben). Dicamba is a
particularly useful herbicide for use in the present invention. A
fourth chemical family of auxinic herbicides is the quinaline
carboxylic acid family. Example includes
3,7-dichloro-8-quinolinecarboxylic acid (quinclorac). This
herbicide is unique in that it also will control some grass weeds,
unlike the other auxin-like herbicides which essentially control
only broadleaf or dicotyledonous plants. The other herbicide in
this category is 7-chloro-3-methyl-8-quinolinecarboxylic acid
(quinmerac).
It was found, for example, that soybean plants transformed with
dicamba monooxygenase (DMO)-encoding polynucleotide constructs were
tolerant to even early pre-emergence application of dicamba, with
less than 10% injury rates at even 9.times. the labeled application
rate (5,040 g/ha, 4.5 lb/acre; Table 1). The inventors found that,
even using an 18.times. application rate of 10,080 g/ha (9
lb/acre), injury to transgenic dicamba tolerant plants was less
than 20% (Table 4). At an approximately 2.times. rate of
application of 1122 g/ha, less than 2% injury was observed. It was
therefore indicated the improved weed control associated with pre-
and post-emergence applications of herbicides may be used without
any significant decreases in productivity due to herbicide damage.
Pre-emergent applications of dicamba according to the invention may
therefore be combined with one or more herbicide applications
post-emergence to dicamba-tolerant plants, while maintaining crop
yield and obtaining improved weed control. For example, one such
herbicide application regime involved a late pre-emergence
application of dicamba in conjunction with a post-emergence
application of dicamba at the V2 stage of development. In certain
embodiments, the post-emergence application may be carried out at
any point from emergence to harvest. Particularly beneficial will
be post-emergence application at any V stage until the soybean
canopy closes, for example, at about the V1, V2, V3, V4, V5, V6
and/or later stages.
In accordance with the invention, methods and compositions for the
control of weeds are provided comprising the use of plants
exhibiting tolerance to glyphosate and auxin-like herbicides such
as dicamba. As shown in the working examples, dicamba and
glyphosate allow use of decreased amounts of herbicide to achieve
the same level of control of glyphosate-tolerant weeds and thus
this embodiment provides a significant advance for the control of
herbicide tolerance in commercial production fields. In one
embodiment, a tank mix of glyphosate and dicamba is applied pre-
and/or post-emergence to plants. Glyphosate and dicamba may
additionally be applied separately. In order to obtain the ability
to use decreased amount of herbicide, the glyphosate and dicamba
are preferably applied within a sufficient interval that both
herbicides remain active and able to control weed growth.
This embodiment therefore allows use of lower amounts of either
herbicide to achieve the same degree of weed control as an
application of only one of the herbicides. For example, the
invention provides methods of weed control comprising applying in a
field planted with transgenic plants having tolerance to dicamba
and glyphosate a herbicide composition comprising less than a
1.times. rate of glyphosate and/or dicamba, relative to the
standard manufacturer labeled rate. Examples of respective
glyphosate and dicamba application rates include from about a
0.5.times.-0.95.times. of either herbicide, specifically including
about 0.5.times., 0.6.times., 0.7.times., 0.8.times., 0.85.times.,
0.9.times., and 0.95.times. of either herbicide and all derivable
combinations thereof, as well as higher rates such as 0.97.times.
and 0.99.times.. Alternatively, in the case of more difficult to
control weeds or where a greater degree of weed control is desired,
1.times. and higher application rates may be made in view of the
finding herein that even high application rates of dicamba did not
significantly damage plants. The 1.times. application rates are set
by the manufacturer of a commercially available herbicide
formulation and are known to those of skill in the art. For
example, the label for Fallow Master.TM., a glyphosate and dicamba
mixture having a ratio of glyphosate:dicamba of about 2:1
recommends application rates of about 451 g/ha (311 ae g/ha
glyphosate:140 ae g/ha dicamba) to 621 ae g/ha (428 ae g/ha
glyphosate: 193 ae g/ha dicamba) depending upon the weed species
and weed height.
"Glyphosate" refers to N-phosphonomethylglycine and salts thereof.
Glyphosate is commercially available in numerous formulations.
Examples of these formulations of glyphosate include, without
limitation, those sold by Monsanto Company as ROUNDUP.RTM.,
ROUNDUP.RTM. ULTRA, ROUNDUP.RTM. ULTRAMAX, ROUNDUP.RTM. CT,
ROUNDUP.RTM. EXTRA, ROUNDUP.RTM. BIACTIVE, ROUNDUP.RTM. BIOFORCE,
RODEO.RTM., POLARIS.RTM., SPARK.RTM. and ACCORD.RTM. herbicides,
all of which contain glyphosate as its isopropylammonium salt,
ROUNDUP.RTM. WEATHERMAX containing glyphosate as its potassium
salt; ROUNDUP.RTM. DRY and RIVAL.RTM. herbicides, which contain
glyphosate as its ammonium salt; ROUNDUP.RTM. GEOFORCE, which
contains glyphosate as its sodium salt; and TOUCHDOWN.RTM.
herbicide, which contains glyphosate as its trimethylsulfonium
salt. "Dicamba" refers to 3,6-dichloro-o-anisic acid or
3,6-dichloro-2-methoxy benzoic acid and its acids and salts. Its
salts include isopropylamine, diglycoamine, dimethylamine,
potassium and sodium. Examples of commercial formulations of
dicamba include, without limitation, Banvel.TM. (as DMA salt),
Clarity.TM. (as DGA salt), VEL-58-CS-11.TM. and Vanquish.TM. (as
DGA salt, BASF).
Non-limiting examples of weeds that can be effectively controlled
using dicamba are the following: cheese weed, chick weed, while
clover, cocklebur, Asiatic dayflower, dead-nettle, red stem
filaree, Carolina geranium, hemp sesbania, henbit, field horsetail
(marestail), knotweed, kochia, lambs-quarter, morninglory, indian
mustard, wild mustard, redroot pigweed, smooth pigweed, prickly
sida, cutleaf evening primrose, common purslane, common ragweed,
gaint ragweed, russian thistle, shepardspurse, pennsylvania
smartweed, spurge, velvetleaf, field violet, wild buckwheat, wild
radish, soybeanpurslane, sicklepod, morninglory, wild buckwheat,
common ragweed, horseweed (marestail), hairy fleabane, buckhorn
plantain, and palmer pigweed. Non-limiting examples of weeds that
can be controlled using dicamba and glyphosate are the following:
barnyardgrass, downy brome, volunteer cereals, Persian darnel,
field sandbur, green foxtail, wild oats, wild buckwheat, volunteer
canola, cowcockle, flix-weed, kochia, ladysthumb, lambsquarters,
wild mustard, prickly lettuce, redroot pigweed, smartweed,
stinkgrass, stinkweed, russian thistle, foxtail, and witchgrass.
Combining glyphosate and dicamba achieves the same level of weed
control with reduced herbicide amounts and thus the spectrum of
weeds that may be controlled at any given herbicide application
rate may be increased when the herbicides are combined.
Transgenic plants having herbicide tolerance may be made as
described in the art. Dicamba tolerance may be conferred, for
example, by a gene for dicamba monooxygenase (DMO) from Pseudomonas
maltophilia (US Patent Application No: 20030135879). Examples of
sequences that may be used in this regard are nucleic acid encoding
the polypeptides of SEQ ID Nos: 2, 4, 6, 8, 10, and 12. Examples of
sequences encoding these polypeptides are given as SEQ ID NOS: 1,
3, 5, 7, 9, and 11. SEQ ID NO: 1 shows DMO from Pseudomonas
maltophilia optimized for expression in dicots using Arabidopsis
thaliana codon usage. The polypeptide, predicted to have an Ala,
Thr, Cys at positions 2, 3, 112, respectively, is given in SEQ ID
NO:2. SEQ ID NO:3 shows another Pseudomonas maltophilia DMO
optimized for expression in dicots and encoding the polypeptide of
SEQ ID NO:4, predicted to have an Leu, Thr, Cys at positions 2, 3,
112, respectively. SEQ ID NO:5 shows the coding sequence and SEQ ID
NO:6 the polypeptide for dicot optimized DMO predicted to have a
Leu, Thr, Trp at positions 2, 3, 112, respectively. SEQ ID NOS:7
and 8 show the coding and polypeptide sequences for DMO predicted
to have an Ala, Thr, Cys at position 2, 3, 112, respectively. SEQ
ID NOS:9 and 10 show the dicot-optimized coding sequence and
polypeptide sequences for DMO predicted to have an Ala, Thr, Trp at
positions 2, 3, 112, respectively. SEQ ID NOS:11 and 12 show coding
sequence and polypeptide sequences for DMO from Pseudomonas
maltophilia (US Patent Application No: 20030135879). Another
exemplary DMO sequence may be a DMO predicted to have a Leu, Thr,
Cys at position 2, 3, 112, respectively with codon usage of
Pseudomonas maltophilia (US Patent Application No:
20030135879).
Sequences conferring glyphosate tolerance are also known, including
glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthases
(EPSPS) as described in U.S. Pat. Nos. 5,627,061, 5,633,435,
6,040,497, 5,094,945, WO04074443, WO04009761, all of which are
hereby incorporated by reference; by expression of nucleic acids
encoding glyphosate degrading enzymes, for example, glyphosate
oxidoreductase (GOX, U.S. Pat. No. 5,463,175, herein incorporated
by reference), glyphosate decarboxylase (WO05003362; US Patent
Application 20040177399, herein incorporated by reference); and by
expression of nucleic acids encoding glyphosate inactivating
enzymes, such as glyphosate-N-acetyl transferase (GAT, e.g. U.S.
Patent publications 20030083480 and 20070079393, herein
incorporated by reference).
Variants of proteins having a capability to degrade auxin-like
herbicides, glyphosate or other herbicides can readily be prepared
and assayed for activity according to standard methods. Such
sequences can also be identified by techniques know in the art, for
example, from suitable organisms including bacteria that degrade
auxin-like herbicides such as dicamba or other herbicides (U.S.
Pat. No. 5,445,962; Cork and Krueger, 1991; Cork and Khalil, 1995).
One means of isolating a DMO or other sequence is by nucleic acid
hybridization, for example, to a library constructed from the
source organism, or by RT-PCR using mRNA from the source organism
and primers based on the disclosed desaturases. The invention
therefore encompasses use of nucleic acids hybridizing under
stringent conditions to a DMO encoding sequence described herein.
One of skill in the art understands that conditions may be rendered
less stringent by increasing salt concentration and decreasing
temperature. Thus, hybridization conditions can be readily
manipulated, and thus will generally be a method of choice
depending on the desired results. An example of high stringency
conditions is 5.times.SSC, 50% formamide and 42.degree. C. By
conducting a wash under such conditions, for example, for 10
minutes, those sequences not hybridizing to a particular target
sequence under these conditions can be removed.
Variants can also be chemically synthesized, for example, using the
known DMO polynucleotide sequences according to techniques well
known in the art. For instance, DNA sequences may be synthesized by
phosphoamidite chemistry in an automated DNA synthesizer. Chemical
synthesis has a number of advantages. In particular, chemical
synthesis is desirable because codons preferred by the host in
which the DNA sequence will be expressed may be used to optimize
expression. Not all of the codons need to be altered to obtain
improved expression, but preferably at least the codons rarely used
in the host are changed to host-preferred codons. High levels of
expression can be obtained by changing greater than about 50%, most
preferably at least about 80%, of the codons to host-preferred
codons. The codon preferences of many host cells are known (PCT WO
97/31115; PCT WO 97/11086; EP 646643; EP 553494; and U.S. Patent
Nos: 5,689,052; 5,567,862; 5,567,600; 5,552,299 and 5,017,692). The
codon preferences of other host cells can be deduced by methods
known in the art. Also, using chemical synthesis, the sequence of
the DNA molecule or its encoded protein can be readily changed to,
for example, optimize expression (for example, eliminate mRNA
secondary structures that interfere with transcription or
translation), add unique restriction sites at convenient points,
and delete protease cleavage sites.
Modification and changes may be made to the polypeptide sequence of
a protein such as the DMO sequences provided herein while retaining
enzymatic activity. The following is a discussion based upon
changing the amino acids of a protein to create an equivalent, or
even an improved, modified polypeptide and corresponding coding
sequences. It is known, for example, that certain amino acids may
be substituted for other amino acids in a protein structure without
appreciable loss of interactive binding capacity with structures
such as binding sites on substrate molecules. Since it is the
interactive capacity and nature of a protein that defines that
protein's biological functional activity, certain amino acid
sequence substitutions can be made in a protein sequence, and, of
course, its underlying DNA coding sequence, and nevertheless obtain
a protein with like properties. It is thus contemplated that
various changes may be made in the DMO peptide sequences described
herein or other herbicide tolerance polypeptides and corresponding
DNA coding sequences without appreciable loss of their biological
utility or activity.
In making such changes, the hydropathic index of amino acids may be
considered. The importance of the hydropathic amino acid index in
conferring interactive biologic function on a protein is generally
understood in the art (Kyte et al., 1982). It is accepted that the
relative hydropathic character of the amino acid contributes to the
secondary structure of the resultant protein, which in turn defines
the interaction of the protein with other molecules, for example,
enzymes, substrates, receptors, DNA, antibodies, antigens, and the
like. Each amino acid has been assigned a hydropathic index on the
basis of their hydrophobicity and charge characteristics (Kyte et
al., 1982), these are: isoleucine (+4.5); valine (+4.2); leucine
(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine
(+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine
(-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6);
histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate
(-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
It is known in the art that amino acids may be substituted by other
amino acids having a similar hydropathic index or score and still
result in a protein with similar biological activity, i.e., still
obtain a biological functionally equivalent protein. In making such
changes, the substitution of amino acids whose hydropathic indices
are within .+-.2 is preferred, those which are within .+-.1 are
particularly preferred, and those within .+-.0.5 are even more
particularly preferred.
It is also understood in the art that the substitution of like
amino acids can be made effectively on the basis of hydrophilicity.
U.S. Pat. No. 4,554,101 states that the greatest local average
hydrophilicity of a protein, as governed by the hydrophilicity of
its adjacent amino acids, correlates with a biological property of
the protein. As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent
protein. In such changes, the substitution of amino acids whose
hydrophilicity values are within .+-.2 is preferred, those which
are within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred. Exemplary
substitutions which take these and various of the foregoing
characteristics into consideration are well known to those of skill
in the art and include: arginine and lysine; glutamate and
aspartate; serine and threonine; glutamine and asparagine; and
valine, leucine and isoleucine.
A gene conferring herbicide tolerance will typically be linked to a
plant promoter driving expression of the gene in an amount
sufficient to confer the herbicide tolerance. Promoters suitable
for this and other uses are well known in the art. Examples
describing such promoters include U.S. Pat. No. 6,437,217 (maize
RS81 promoter), U.S. Pat. No. 5,641,876 (rice actin promoter), U.S.
Pat. No. 6,426,446 (maize RS324 promoter), U.S. Pat. No. 6,429,362
(maize PR-1 promoter), U.S. Pat. No. 6,232,526 (maize A3 promoter),
U.S. Pat. No. 6,177,611 (constitutive maize promoters), U.S. Pat.
Nos. 5,322,938, 5,352,605, 5,359,142 and 5,530,196 (35S promoter),
U.S. Pat. No. 6,433,252 (maize L3 oleosin promoter), U.S. Pat. No.
6,429,357 (rice actin 2 promoter as well as a rice actin 2 intron),
U.S. Pat. No. 5,837,848 (root specific promoter), U.S. Pat. No.
6,294,714 (light inducible promoters), U.S. Pat. No. 6,140,078
(salt inducible promoters), U.S. Pat. No. 6,252,138 (pathogen
inducible promoters), U.S. Pat. No. 6,175,060 (phosphorus
deficiency inducible promoters), U.S. Pat. No. 6,388,170 (PC1SV
promoter), U.S. Pat. No. 6,635,806 (gamma-coixin promoter), and
U.S. patent application Ser. No. 09/757,089 (maize chloroplast
aldolase promoter). Additional promoters that may find use are a
nopaline synthase (NOS) promoter (Ebert et al., 1987), the octopine
synthase (OCS) promoter (which is carried on tumor-inducing
plasmids of Agrobacterium tumefaciens), the caulimovirus promoters
such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et
al., 1987), the CaMV 35S promoter (Odell et al., 1985), the figwort
mosaic virus 35S-promoter (Walker et al., 1987), the sucrose
synthase promoter (Yang et al., 1990), the R gene complex promoter
(Chandler et al., 1989), the chlorophyll a/b binding protein gene
promoter, CaMV35S (U.S. Pat. Nos. 5,322,938; 5,352,605; 5,359,142;
and 5,530,196), FMV35S (U.S. Pat. Nos. 6,051,753; 5,378,619), a
PC1SV promoter (U.S. Pat. No. 5,850,019; or SEQ ID NO:20), the SCP
promoter (U.S. Pat. No. 6,677,503), and AGRtu.nos (GenBank
Accession V00087; Depicker et al, 1982; Bevan et al., 1983)
promoters, and the like (see also see Table 1).
Benefit may be obtained for the expression of herbicide tolerance
genes by use of a sequence coding for a transit peptide. For
example, incorporation of a suitable chloroplast transit peptide,
such as, the Arabidopsis thaliana EPSPS CTP (Klee et al., 1987),
and the Petunia hybrida EPSPS CTP (della-Cioppa et al., 1986) has
been shown to target heterologous EPSPS protein sequences to
chloroplasts in transgenic plants. Chloroplast transit peptides
(CTPs) are engineered to be fused to the N-terminus of a protein to
direct the protein into the plant chloroplast. Such sequences may
find use in connection with a nucleic acid conferring dicamba
tolerance in particular. Many chloroplast-localized proteins are
expressed from nuclear genes as precursors and are targeted to the
chloroplast by a chloroplast transit peptide that is removed during
the import process. Examples of chloroplast proteins include the
small subunit (RbcS2) of ribulose-1,5,-bisphosphate carboxylase,
ferredoxin, ferredoxin oxidoreductase, the light-harvesting complex
protein I and protein II, and thioredoxin F. Other exemplary
chloroplast targeting sequences include the maize cab-m7 signal
sequence (Becker et al., 1992; PCT WO 97/41228), the pea
glutathione reductase signal sequence (Creissen et al., 1995; PCT
WO 97/41228), and the CTP of the Nicotiana tabacum ribulose
1,5-bisphosphate carboxylase small subunit chloroplast transit
peptide (SSU-CTP) (Mazur, et al., 1985). Use of AtRbcS4 (CTP1; U.S.
Pat. No. 5,728,925), AtShkG (CTP2; Klee et al., 1987), AtShkGZm
(CTP2synthetic; see SEQ ID NO:14 of WO04009761), and PsRbcS
(Coruzzi et al., 1984), as well as others disclosed, for instance,
in U.S. Provisional Patent Application 60/891,675, peptide and
nucleic acid sequences for which are listed herein at SEQ ID
NOs:21-32, may be of benefit for use with the invention.
A 5' UTR that functions as a translation leader sequence is a DNA
genetic element located between the promoter sequence of a gene and
the coding sequence. The translation leader sequence is present in
the fully processed mRNA upstream of the translation start
sequence. The translation leader sequence may affect processing of
the primary transcript to mRNA, mRNA stability or translation
efficiency. Examples of translation leader sequences include maize
and petunia heat shock protein leaders (U.S. Pat. No. 5,362,865),
plant virus coat protein leaders, plant rubisco leaders, among
others (Turner and Foster, 1995). Non-limiting examples of 5' UTRs
that may in particular be of benefit for use GmHsp (U.S. Pat. No.
5,659,122), PhDnaK (U.S. Pat. No. 5,362,865), AtAnt1, TEV
(Carrington and Freed, 1990), and AGRtunos (GenBank Accession
V00087; Bevan et al., 1983).
The 3' non-translated sequence, 3' transcription termination
region, or poly adenylation region means a DNA molecule linked to
and located downstream of a structural polynucleotide molecule and
includes polynucleotides that provide polyadenylation signal and
other regulatory signals capable of affecting transcription, 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 sequence can be
derived from the natural gene, from a variety of plant genes, or
from T-DNA genes. An example of a 3' transcription termination
region is the nopaline synthase 3' region (nos 3'; Fraley et al.,
1983). The use of different 3' nontranslated regions is exemplified
(Ingelbrecht et al., 1989). Polyadenylation molecules from a Pisum
sativum RbcS2 gene (Ps.R-bcS2-E9; Coruzzi et al., 1984) and
AGRtu.nos (Rojiyaa et al., 1987, Genbank Accession E01312) in
particular may be of benefit for use with the invention.
Intron sequences are known in the art to aid in the expression of
transgenes in monocot plant cells. Examples of introns include the
corn actin intron (U.S. Pat. No. 5,641,876), the corn HSP70 intron
(ZmHSP70; U.S. Pat. No. 5,859,347; U.S. Pat. No. 5,424,412), and
rice TPI intron (OsTPI; U.S. Pat. No. 7,132,528), and are of
benefit in practicing this invention.
Any of the techniques known in the art for introduction of
transgenes into plants may be used to prepare a herbicide tolerant
plant in accordance with the invention (see, for example, Miki et
al., 1993). Suitable methods for transformation of plants are
believed to include virtually any method by which DNA can be
introduced into a cell, such as by electroporation as illustrated
in U.S. Pat. No. 5,384,253; micro-projectile bombardment as
illustrated in U.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880;
6,160,208; 6,399,861; and 6,403,865; Agrobacterium-mediated
transformation as illustrated in U.S. Pat. Nos. 5,635,055;
5,824,877; 5,591,616; 5,981,840; and 6,384,301; and protoplast
transformation as illustrated in U.S. Pat. No. 5,508,184, etc.
Through the application of techniques such as these, the cells of
virtually any plant species may be stably transformed, and these
cells developed into transgenic plants. Techniques that may be
particularly useful in the context of cotton transformation are
disclosed in U.S. Pat. Nos. 5,846,797, 5,159,135, 5,004,863, and
6,624,344; and techniques for transforming Brassica plants in
particular are disclosed, for example, in U.S. Pat. No. 5,750,871;
and techniques for transforming soybean are disclosed in for
example in Zhang et al., 1999 and U.S. Pat. No. 6,384,301). Corn
can be transformed using methods described in WO9506722 and US
patent application 20040244075.
After effecting delivery of exogenous DNA to recipient cells, the
next steps generally concern identifying the transformed cells for
further culturing and plant regeneration. In order to improve the
ability to identify transformants, one may desire to employ a
selectable or screenable marker gene with a transformation vector
prepared in accordance with the invention. In this case, one would
then generally assay the potentially transformed cell population by
exposing the cells to a selective agent or agents, or one would
screen the cells for the desired marker gene trait.
Cells that survive the exposure to the selective agent, or cells
that have been scored positive in a screening assay, may be
cultured in media that supports regeneration of plants. In an
exemplary embodiment, any suitable plant tissue culture media, for
example, MS and N6 media may be modified by including further
substances such as growth regulators. Tissue may be maintained on a
basic media with growth regulators until sufficient tissue is
available to begin plant regeneration efforts, or following
repeated rounds of manual selection, until the morphology of the
tissue is suitable for regeneration, typically at least 2 weeks,
then transferred to media conducive to shoot formation. Cultures
are transferred periodically until sufficient shoot formation has
occurred. Once shoot are formed, they are transferred to media
conducive to root formation. Once sufficient roots are formed,
plants can be transferred to soil for further growth and
maturity.
To confirm the presence of the exogenous DNA or "transgene(s)" in
the regenerating plants, a variety of assays may be performed. Such
assays include, for example, "molecular biological" assays, such as
Southern and Northern blotting and PCR.TM.; "biochemical" assays,
such as detecting the presence of a protein product, e.g., by
immunological means (ELISAs and Western blots) or by enzymatic
function; plant part assays, such as leaf or root assays; and also,
by analyzing the phenotype of the whole regenerated plant.
Once a transgene has been introduced into a plant, that gene can be
introduced into any plant sexually compatible with the first plant
by crossing, without the need for ever directly transforming the
second plant. Therefore, as used herein the term "progeny" denotes
the offspring of any generation of a parent plant prepared in
accordance with the instant invention, wherein the progeny
comprises a selected DNA construct prepared in accordance with the
invention. A "transgenic plant" may thus be of any generation.
"Crossing" a plant to provide a plant line having one or more added
transgenes or alleles relative to a starting plant line, as
disclosed herein, is defined as the techniques that result in a
particular sequence being introduced into a plant line by crossing
a starting line with a donor plant line that comprises a transgene
or allele of the invention. To achieve this one could, for example,
perform the following steps: (a) plant seeds of the first (starting
line) and second (donor plant line that comprises a desired
transgene or allele) parent plants; (b) grow the seeds of the first
and second parent plants into plants that bear flowers; (c)
pollinate a flower from the first parent plant with pollen from the
second parent plant; and (d) harvest seeds produced on the parent
plant bearing the fertilized flower.
The preparation of herbicide compositions for use in connection
with the current invention will be apparent to those of skill in
the art in view of the disclosure. Such compositions, which are
commercially available, will typically include, in addition to the
active ingredient, components such as surfactants, solid or liquid
carriers, solvents and binders. Examples of surfactants that may be
used for application to plants include the alkali metal, alkaline
earth metal or ammonium salts of aromatic sulfonic acids, e.g.,
ligno-, phenol-, naphthalene- and dibutylnaphthalenesulfonic acid,
and of fatty acids of arylsulfonates, of alkyl ethers, of lauryl
ethers, of fatty alcohol sulfates and of fatty alcohol glycol ether
sulfates, condensates of sulfonated naphthalene and its derivatives
with formaldehyde, condensates of naphthalene or of the
naphthalenesulfonic acids with phenol and formaldehyde, condensates
of phenol or phenolsulfonic acid with formaldehyde, condensates of
phenol with formaldehyde and sodium sulfite, polyoxyethylene
octylphenyl ether, ethoxylated isooctyl-, octyl- or nonylphenol,
tributylphenyl polyglycol ether, alkylaryl polyether alcohols,
isotridecyl alcohol, ethoxylated castor oil, ethoxylated
triarylphenols, salts of phosphated triarylphenolethoxylates,
lauryl alcohol polyglycol ether acetate, sorbitol esters,
lignin-sulfite waste liquors or methylcellulose, or mixtures of
these. Common practice in the case of surfactant use is about 0.25%
to 1.0% by weight, and more commonly about 0.25% to 0.5% by
weight.
Compositions for application to plants may be solid or liquid.
Where solid compositions are used, it may be desired to include one
or more carrier materials with the active compound. Examples of
carriers include mineral earths such as silicas, silica gels,
silicates, talc, kaolin, attaclay, limestone, chalk, loess, clay,
dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate,
magnesium oxide, ground synthetic materials, fertilizers such as
ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea
and urea, products of vegetable origin such as cereal meals, tree
bark meal, wood meal and nutshell meal, cellulose powders,
attapulgites, montmorillonites, mica, vermiculites, synthetic
silicas and synthetic calcium silicates, or mixtures of these.
For liquid solutions, water-soluble compounds or salts may be
included, such as sodium sulfate, potassium sulfate, sodium
chloride, potassium chloride, sodium acetate, ammonium hydrogen
sulfate, ammonium chloride, ammonium acetate, ammonium formate,
ammonium oxalate, ammonium carbonate, ammonium hydrogen carbonate,
ammonium thiosulfate, ammonium hydrogen diphosphate, ammonium
dihydrogen monophosphate, ammonium sodium hydrogen phosphate,
ammonium thiocyanate, ammonium sulfamate or ammonium carbamate.
Other exemplary components in herbicidal compositions include
binders such as polyvinylpyrrolidone, polyvinyl alcohol, partially
hydrolyzed polyvinyl acetate, carboxymethylcellulose, starch,
vinylpyrrolidone/vinyl acetate copolymers and polyvinyl acetate, or
mixtures of these; lubricants such as magnesium stearate, sodium
stearate, talc or polyethylene glycol, or mixtures of these;
antifoams such as silicone emulsions, long-chain alcohols,
phosphoric esters, acetylene diols, fatty acids or organofluorine
compounds, and complexing agents such as: salts of
ethylenediaminetetraacetic acid (EDTA), salts of
trinitrilotriacetic acid or salts of polyphosphoric acids, or
mixtures of these.
Equipment and methods known in the art are used to apply various
herbicide treatments as disclosed herein. The application rates of
herbicides maybe varied, for instance as described above, depending
upon the soil texture, pH, organic matter content, tillage systems,
and the size of the weed, and can be determined by consulting the
herbicide label for the proper herbicide rate.
EXAMPLES
The following examples are included to illustrate embodiments of
the invention. It should be appreciated by those of skill in the
art that the techniques disclosed in the examples that follow
represent techniques discovered by the inventor to function well in
the practice of the invention. However, those of skill in the art
should, in light of the present disclosure, appreciate that many
changes can be made in the specific embodiments which are disclosed
and still obtain a like or similar result without departing from
the concept, spirit and scope of the invention. More specifically,
it will be apparent that certain agents which are both chemically
and physiologically related may be substituted for the agents
described herein while the same or similar results would be
achieved. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the invention as defined by the appended
claims.
Example 1
Tolerance of Soybean Plants Containing DMO-Encoding Polynucleotide
Construct to Early Pre-Emergence Application of Dicamba
Transgenic soybean plants were obtained by Agrobacterium
transformation of soybean cotyledonary nodes using standard
procedures and a binary vector containing the DMO-encoding
polynucleotide given as SEQ ID NO:7, which encodes the polypeptide
of SEQ ID NO:8. Four transgenic soybean events were prepared and
designated Events 1-4. Transgenic soybean plants containing the
events were tested for their tolerance to dicamba herbicide
relative to controls, confirming herbicide tolerance.
Non-transgenic soybean plants were used as controls.
Transgenic and control soybean seeds were planted into 3.5-inch
square plastic pots containing Redi-earth.TM. (Scotts-Sierra
Horticultural Products Co., Marysville, Ohio). The soil surface was
treated with various amounts (561 to 5040 g/ha, 0.5 to 4.5 lb/acre,
or 1.times. to 9.times. labeled rates) of dicamba formulations
(Clarity.TM. or Banvel.TM., BASF, Raleigh, N.C.). The pots were
placed on capillary matting in 35 inch.times.60 inch fiberglass
watering trays for overhead and/or sub-irrigation for the duration
of the test period so as to maintain optimum soil moisture for
plant growth and were fertilized with Osmocote (14-14-14 slow
release; Scotts-Sierra Horticultural Products Co., Marysville,
Ohio) at the rate of 100 gm/cu.ft. to sustain plant growth for the
duration of greenhouse trials.
The plants were grown in greenhouses at 27.degree./21.degree. C.
day/night temperature with relative humidity between 25%-75% to
simulate warm season growing conditions of late spring. A 14 h
minimum photoperiod was provided with supplemental light at about
600 .mu.E as needed. Trials were established in a randomized block
design randomized by rate with 4 to 6 replications of each
treatment depending on plant quality, availability, and to account
for any environmental variability that may have occurred within the
confines of each greenhouse.
Treated plants in greenhouse trials were visually assessed at a
particular day after treatment (DAT) for injury on a scale of 0 to
100 percent relative to untreated control plants, with zero
representing "no" injury and 100% representing "complete" injury or
death. Data were collected and analyzed using suitable statistical
methods.
The results of the study surprisingly showed that soybean plants
transformed with the DMO-encoding polynucleotide construct were
tolerant to even early pre-emergence application of dicamba. As
indicated in Table 1 below, injury to the transgenic plants was
less than 10% even at the highest application rate i.e., 5040 g/ha,
4.5 lb/acre, or 9.times. labeled rates of dicamba.
TABLE-US-00001 TABLE 1 Percentage injury to non-transgenic or
transgenic soybean plants from early pre-emergence application of
dicamba at sowing. The % injury was represented as ANOVA mean
comparisons. Similar letters represent no statistical difference at
the p = 0.05 level. % injury at shown Formulation rates (g ae/ha*)
at 14 DAT Clarity .TM. ID 561 840 2244 4485 5040 Control 67.0 a
73.0 b 96.6 a 98.2 a 99.5 a Control 61.0 a 86.0 a 98.1 a 98.3 a
99.8 a Event 1 0.0 b 0.0 c 1.7 bc 0.7 b 3.1 b Event 2 0.0 b 0.0 c
1.1 c 1.0 b 2.2 b Event 3 0.0 b 0.0 c 1.1 c 0.6 b 3.5 b Event 4 0.0
b 0.0 c 4.4 b 0.8 b 7.2 b LSD 9.9 7.2 3.2 2.2 5.1
Example 2
Tolerance of Soybean Plants Containing a DMO-Encoding
Polynucleotide Construct to Early Pre-Emergence Application of
Dicamba at Sowing Followed by Post-Emergence Application of
Dicamba
In addition to the method described in Example 2 for early
pre-emergence (at sowing) application of dicamba, post-emergence
(V2 stage of soybean development) application of dicamba was made
with a track sprayer using the Teejet 9501E flat fan nozzle
(Spraying Systems Co, Wheaton, Ill.) with the air pressure set at a
minimum of 24 psi (165 kpa). The spray nozzle was kept at a height
of about 16 inches above the top of the plant material for
spraying. The spray volume was 10 gallons per acre or 93 liters per
hectare.
As shown in Table 2, soybean plants transformed with the
DMO-encoding polynucleotide construct were tolerant to early
pre-emergence applications of dicamba at sowing followed by
post-emergence application of dicamba. Surprisingly, injury to
transgenic plants was less than 20% at the overall dicamba rate of
10080 g/ha, 9 lb/acre or 18.times. labeled rate.
TABLE-US-00002 TABLE 2 Percentage injury to non-transgenic or
transgenic soybean plants from application of dicamba at sowing
followed by post-emergence application at V2 stage.* Formulation %
injury at shown rates (g ae/ha*) at 28 DAT Clarity .TM. Plants 1122
1680 4488 8970 10080 Control 97.5 a 98.8 a 99.8 a 100.0 a 100.0 a
Control 95.6 a 98.1 a 99.4 a 100.0 a 100.0 a Event 1 0.0 c 1.8 b
4.5 d 11.9 c 16.9 b Event 2 2.6 bc 3.9 b 8.1 bc 13.8 b 16.9 b Event
3 3.1 b 2.9 b 8.8 b 11.9 c 17.5 b Event 4 2.3 bc 2.0 b 6.9 c 11.9 c
15.6 b LSD 3.1 2.2 1.4 1.6 1.9 *The % injury was represented as
ANOVA mean comparisons. Similar letters represent no statistical
difference at the p = 0.05 level.
Example 3
Tolerance of Soybean Plants Containing DMO-Encoding Polynucleotide
Construct to Late Pre-Emergence Application of Dicamba
An analysis was carried out of the effect of late pre-emergence
applications of dicamba at soil cracking due to emergence of
soybean seedling hypocotyls. Dicamba applications were made using a
track sprayer as described in the previous examples. As shown in
Table 3, soybean plants transformed with the DMO-encoding
polynucleotide construct were found to be tolerant to late
pre-emergence application of dicamba at soil cracking.
Significantly, injury in the transgenic events was less than 5%
even at the highest rate i.e., 5040 g/ha, 4.5 lb/acre, or 9.times.
labeled rates of dicamba.
TABLE-US-00003 TABLE 3 Percentage injury to non-transgenic or
transgenic soybean plants from late pre-emergence application of
dicamba at soil cracking.* % injury at shown Formulation rates (g
ae/ha*) at 14 DAT Clarity .TM. Plants 561 840 2244 4485 5040
Control 86.9 a 96.8 a 98.4 A 98.5 a 99.2 a Control 89.6 a 91.9 a
98.4 A 99.0 a 99.4 a Event 1 0.0 b 0.0 b 0.5 C 2.5 bc 2.0 b Event 2
0.0 b 0.0 b 2.9 bc 0.0 c 1.5 b Event 3 0.0 b 0.0 b 1.5 bc 4.4 b 1.3
b Event 4 0.0 b 0.5 b 3.3 B 3.0 bc 1.3 b LSD 8.1 5.4 2.4 3.9 2.3
*The % injury was represented as ANOVA mean comparisons. Similar
letters represent no statistical difference at the p = 0.05
level.
Example 4
Tolerance of Soybean Plants Containing DMO-Encoding Polynucleotide
Construct to Late Pre-Emergence Applications of Dicamba Followed by
Post-Emergence Applications of Dicamba
In addition to the studies above, an analysis was carried out of
the effect of late pre-emergence applications of dicamba at soil
cracking followed by post-emergence application of dicamba at the
V2 stage of development. As shown in Table 4, soybean plants
transformed with the DMO-encoding polynucleotide construct were
tolerant to late pre-emergence application of dicamba at soil
cracking and post-emergence application of dicamba. Injury to
transgenic events was less than 20% even at the overall dicamba
rate of 10080 g/ha, 9 lb/acre, or 18.times. labeled rate.
TABLE-US-00004 TABLE 4 Percentage injury to non-transgenic or
transgenic soybean plants from late pre-emergence application of
dicamba at soil cracking followed by post-emergence application at
V2 stage.* % injury at shown Formulation rates (g ae/ha*) at 28 DAT
Clarity .TM. Plants 1122 1680 4488 8970 10080 Control 95.6 a 98.1 a
100.0 a 100.0 a 100.0 a Control 95.0 a 98.1 a 99.4 a 99.8 a 100.0 a
Event 1 0.3 b 0.9 b 6.3 b 13.1 b 16.3 bc Event 2 0.8 b 1.6 b 6.0 b
11.3 c 15.0 c Event 3 1.0 b 1.4 b 7.5 b 11.3 c 17.5 b Event 4 1.8 b
1.8 b 7.5 b 13.1 b 16.3 bc LSD 4.5 2.7 1.6 1.6 1.9 *The % injury
was represented as ANOVA mean comparisons. Similar letters
represent no statistical difference at the p = 0.05 level.
Example 5
Tolerance of Soybean Plants Containing DMO-Encoding Polynucleotide
Construct to Pre- and Post-Emergence Application of Dicamba in the
Field
Non-transgenic and transgenic soybean seeds were planted around the
beginning of the growing season at the time of optimum growth
conditions depending on soil moisture, temperature, and seeding
depth. Across all locations seeds were planted under split-plot
design with dicamba treatments as whole-plot effects and events as
split-plot effects. The design details were as follows: 6
locations, 2 replications/location, 2 rows/plot, row length 12 feet
(+3 ft alley), 9 seeds/foot, 108 seeds/row, 5 events (Events 1-4
and a fifth event that was segregating); and 4 treatments as shown
below in Table 5. In all 240 plots were planted at 6 locations (40
per location).
TABLE-US-00005 TABLE 5 Details of 4 treatments applied to show the
tolerance of transgenic soybean to dicamba. 1st Application 2nd
Application Treatment Rate Plant Stage Rate Plant Stage 1 NO
Dicamba NO Dicamba NO Dicamba NO Dicamba 2 1.5 lb ae/acre At
Planting N/A N/A 3 N/A N/A 1.5 lb ae/acre V3-4 4 1.5 lb ae/acre At
Planting 1.5 lb ae/acre V3-4
Four non-transgenic border rows were planted all around the trial
using a known commercial line such as A3525. Optimum production and
management practices known in the art were followed. Maximum pest
control and disease control was practiced as needed to prevent
confounding effects of dicamba applications. The field was
irrigated as needed according to standard practices.
All plants in the field were treated with pre-emergence and
post-emergence applications of dicamba and visually assessed at a
particular day after planting for injury on a scale of 0 to 100
percent relative to untreated control plants, with zero
representing "no" injury and 100% representing "complete" injury or
death. Seed planting and pre-emergence treatment were carried out
approximately one-month apart in late spring in Monmouth, Ill. As
shown in Table 6, it was found that all transgenic soybean plants
had no or very little injury. A fifth transgenic event used
appeared to be segregating, so a certain percentage of plants died
after the treatments.
TABLE-US-00006 TABLE 6 Tolerance of soybean plants containing DMO-
encoding polynucleotide construct to pre- and post-emergence
application of dicamba in field.* % % % % % % % Event Inj Inj Inj
Inj GR Inj GR Dead or # Trmt 6/7 6/13 6/20 6/27 6/27 7/5 7/5
Stunted 1 No spray 0 0 0 0 0 2 0 0 1 No spray 0 0 0 0 0 0 0 0 2 No
spray 0 0 0 1 0 3 3 0 2 No spray 0 0 0 0 0 0 0 0 3 No spray 0 0 0 1
0 3 0 0 3 No spray 0 0 0 0 0 3 0 0 4 No spray 0 0 0 2 0 0 0 0 4 No
spray 0 0 0 0 0 5 0 0 5 No spray 0 0 0 7 0 5 2 0 5 No spray 0 0 0 7
3 7 3 0 1 Pre at 0 0 0 1 0 0 0 0 sowing 1 Pre at 0 2 0 2 0 0 0 0
sowing 2 Pre at 0 0 0 5 0 0 0 0 sowing 2 Pre at 0 0 0 2 0 0 0 0
sowing 3 Pre at 0 4 0 1 0 0 0 0 sowing 3 Pre at 0 2 0 5 0 3 0 0
sowing 4 Pre at 0 3 0 5 0 0 0 0 sowing 4 Pre at 0 4 0 2 0 0 0 0
sowing 5 Pre at 0 15 15 5 0 0 0 24 sowing 5 Pre at 0 8 10 2 0 0 0
14 sowing 1 Post at V3 0 0 0 5 0 0 0 0 1 Post at V3 0 0 0 7 0 0 0 2
2 Post at V3 0 0 0 3 0 0 0 0 2 Post at V3 0 0 0 3 0 2 0 1 3 Post at
V3 0 0 0 3 3 0 0 0 3 Post at V3 0 0 0 5 0 3 0 0 4 Post at V3 0 0 0
3 0 0 0 0 4 Post at V3 0 0 0 3 0 0 0 0 5 Post at V3 0 0 0 7 0 2 0
15 5 Post at V3 0 0 0 5 5 2 0 15 1 Pre & Post 0 0 0 5 0 0 0 3 1
Pre & Post 0 2 2 5 3 0 0 0 2 Pre & Post 0 0 0 1 0 2 0 0 2
Pre & Post 0 0 0 2 0 0 0 0 3 Pre & Post 0 0 0 3 0 2 0 0 3
Pre & Post 0 2 0 3 0 0 0 0 4 Pre & Post 0 0 0 3 8 2 2 0 4
Pre & Post 0 1 0 3 3 0 0 0 5 Pre & Post 0 15 10 3 5 0 0 23
5 Pre & Post 0 10 10 1 0 0 0 20 *No spray means no dicamba was
applied to the plants. Pre at sowing means 1.5 lb/acre of dicamba
was applied at planting. Post at V3 means 1.5 lb/acre of dicamba
was applied 4 weeks after planting. Pre and post means 1.5 lb/acre
of dicamba was applied at planting and 1.5 lb/acre of dicamba was
applied 4 weeks after planting. % inj means percentage injury on
given date. % GR means percentage growth reduction.
Example 6
Controlling Glyphosate Tolerant Weeds by Dicamba
Marestail is one of the major weeds in a crop field. Marestail is
effectively controlled by glyphosate, but the development of
methods for controlling this common weed with other herbicides is
important to minimize opportunities for herbicide tolerance to
develop. An analysis was carried out to determine the extent to
which this glyphosate tolerant weed could be controlled by
applications of dicamba. Marestail (Conyza canadensis) plants of
two biotypes, each from a different geographic region, California
(CA) and Kentucky (KY), were grown, and treated at 4-6 inch
diameter rosette leaf stage with dicamba as described in Example 2
and 3. The results of the study, as shown in Table 7, demonstrated
that dicamba was equally effective in controlling both susceptible
and tolerant biotypes of marestail from CA and KY. Dicamba was more
effective in controlling resistant biotypes at lower application
rates than glyphosate. For example, 2100 g/ha of glyphosate was
required to obtain about 77% and 91% inhibition of CA and KY
resistant biotypes, whereas only 280 g/ha of dicamba was required
to obtain about 83% and about 91% control of CA and KY resistant
biotypes.
TABLE-US-00007 TABLE 7 Control of glyphosate tolerant weeds by
dicamba. % Injury (21 DAT) MARESTAIL MARESTAIL MARESTAIL MARESTAIL
Rate (CA) (CA) (KY) (KY) Formulation g/ha Susceptible Resistant
Susceptible Resistant Roundup 840 97.2 55.0 76.7 58.3 WeatherMAX
.TM. 1680 100.0 64.2 97.5 79.2 2100 100.0 76.7 100.0 90.8 Clarity
.TM. 50 68.3 61.7 78.3 78.3 140 82.5 80.8 90.0 88.3 280 85.0 82.5
91.7 90.8
Example 7
Development of a Method for Controlling Glyphosate Tolerant Weeds
in a Field
Transgenic seeds having dicamba tolerance are planted in a field
that has been treated with glyphosate before planting the
transgenic seeds. The field is then treated with a herbicidally
effective amount of dicamba before or after planting the seeds to
control glyphosate resistant weeds. The herbicidally effective
amount of dicamba is such that the growth of glyphosate resistant
weeds is controlled, but is not injurious to the planted crop as
shown in the examples described herein. Thus, transgenic seeds
having dicamba tolerance in combination with an effective amount of
dicamba are useful for control of glyphosate resistant weeds. The
method may be implemented without delaying planting of the dicamba
tolerant crop plants, thus providing a significant advance over the
prior art, in which dicamba must be applied sufficiently prior to
planting such that the dicamba degrades in the environment
sufficiently to avoid injury to crop plants.
Example 8
Combination of Dicamba and Glyphosate for Controlling Glyphosate
Resistant Weeds to Allow Reduced Herbicide Application Rates
As shown in Table 8, dicamba alone was more effective in
controlling resistant biotypes at lower application rates than
glyphosate. Further, it has unexpectedly been found that dicamba in
combination with glyphosate allows control of glyphosate tolerant
and susceptible weeds at lower application rates. For example,
whereas 200 g/ha of glyphosate was able to control only 6% of
marestail (KY resistant biotype) at 18 DAT and 40 g/ha of dicamba
was able to control about 52% of the KY biotype at 18 DAT, a 200
g/ha glyphosate and 40 g/ha dicamba mixture was able to control
about 79% of the KY biotype at 18 DAT.
In general, any formulation containing dicamba appeared to be more
efficacious than glyphosate alone on the resistant biotype. Also,
in general, the following trend in effectiveness of glyphosate to
dicamba ratio on resistant biotype was found to be true at:
4:1>10:1>20:1>40:1>80:1. The results show that a
glyphosate to dicamba mixture ratio of 4:1 containing 200 g/h
glyphosate and 50 g/h dicamba provided superior control than either
glyphosate or dicamba alone.
TABLE-US-00008 TABLE 8 Effect of dicamba and glyphosate for
controlling glyphosate resistant weeds. % Injury % Injury (18 DAT)
(30 DAT) Marestail Marestail Marestail Marestail CHEMICAL Rate
Susceptible Resistant Susceptible Resistant FORMULATION g/ha RATIO
(KY) (KY) (KY) (KY) Roundup 200 86.0 5.8 96.3 0.0 WeatherMAX .TM.
400 99.7 25.0 100.0 18.3 800 100.0 46.7 100.0 44.2 1600 100.0 59.2
100.0 62.5 Clarity .TM. 2.5 6.7 10.8 15.8 7.5 5 18.3 25.0 20.8 35.0
10 34.2 35.8 29.2 39.2 20 40.8 45.8 40.0 45.0 40 50.0 52.5 51.7
68.3 80 68.3 69.2 71.7 84.3 100 83.3 75.8 86.3 87.5 200 89.2 83.3
99.3 94.3 Roundup 200 + 2.5 80:1 50.8 20.8 55.8 31.7 WeatherMAX
.TM. + 400 + 5 80:1 85.8 39.2 97.7 40.8 Clarity .TM. 800 + 10 80:1
99.7 47.5 100.0 45.0 1600 + 20 80:1 100.0 50.8 100.0 63.3 Roundup
200 + 5 40:1 56.7 28.3 64.2 35.0 WeatherMAX .TM. + 400 + 10 40:1
82.5 40.0 94.2 43.3 Clarity .TM. 800 + 20 40:1 99.3 53.3 100.0 60.8
1600 + 40 40:1 100.0 70.8 100.0 80.8 Roundup 200 + 10 20:1 58.3
38.3 66.7 40.0 WeatherMAX .TM. + 400 + 20 20:1 81.7 56.7 93.3 50.0
Clarity .TM. 800 + 40 20:1 99.0 62.5 100.0 73.3 1600 + 80 20:1 99.7
77.5 100.0 88.3 Roundup 200 + 20 10:1 56.7 52.5 70.8 60.0
WeatherMAX .TM. + 400 + 40 10:1 84.2 79.2 93.3 86.3 Clarity .TM.
800 + 80 10:1 98.7 83.3 100.0 96.8 1600 + 160 10:1 99.7 89.2 100.0
99.3 Roundup 200 + 50 4:1 61.7 79.2 83.5 87.2 WeatherMAX .TM. + 400
+ 100 4:1 89.2 88.3 99.7 98.7 Clarity .TM. 800 + 200 4:1 99.7 88.3
100.0 99.3 1600 + 400 4:1 100.0 89.7 100.0 100.0
Example 9
Production of Transgenic Seeds Having Dicamba and Glyphosate
Tolerance
Methods for producing transgenic seeds having glyphosate tolerance
are known in the art and such seeds can be produced by persons of
skill in the art by using a polynucleotide encoding glyphosate
resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) as
described in U.S. Pat. Nos. 5,627,061, 5,633,435, 6,040,497 and in
U.S. Pat. No. 5,094,945, WO04074443 and WO04009761, all of which
are hereby incorporated by reference. Soybean breeding lines
containing the Roundup Ready.RTM. trait event 40-3-2 (Padgette et
al., 1995) have been produced. Seeds from soybean plant designated
as MON19788 have been deposited under ATCC Accession No.
PTA-6708.
Glyphosate tolerant plants can also be produced by incorporating
polynucleotides encoding glyphosate degrading enzymes such as
glyphosate oxidoreductase (GOX, U.S. Pat. No. 5,463,175, herein
incorporated by reference), a glyphosate-N-acetyl transferase (GAT,
U.S. Patent publication 20030083480, herein incorporated by
reference), and a glyphosate decarboxylase (WO05003362; US Patent
Application 20040177399, herein incorporated by reference).
Dicamba tolerant plants are disclosed herein. A suitable line from
each is crossed and progeny seeds screened with herbicide
applications of glyphosate and dicamba to obtain progeny expressing
both genes and exhibiting tolerance to both dicamba and glyphosate.
Alternatively, coding sequences conferring tolerance to one or both
of the herbicides are directly introduced into a given line. Seeds
from these plants are used for developing a method for controlling
weed resistance development in a field as described below.
Transgenic seeds having dicamba and glyphosate tolerances were
tested for their tolerance to dicamba, glyphosate, or both
herbicides. Table 9 shows tolerance of transgenic soybeans carrying
glyphosate and dicamba tolerance transgenes to glyphosate, dicamba,
and glyphosate and dicamba at various stages of plant growth.
Injury was not seen on plants when either or both herbicides were
applied at pre-emergence stage. Post-emergence treatments of either
or both herbicides at V3, R1, and R3-4 showed only little
injury.
TABLE-US-00009 TABLE 9 Tolerance of transgenic soybeans carrying
glyphosate and dicamba tolerance transgenes to glyphosate, dicamba,
and glyphosate and dicamba. Post-emergence Pre-emergence treatment
treatment V3 R1 R3-4 20 8 7 18 Rate DAT DAT DAT DAT gm % injury
Plant Line Herbicide Applied ae/ha (Average of 4 replications)
Non-transgenic Control CLARITY 561 99.0 83.8 71.3 85.0 RWMax 841
0.0 81.3 66.3 67.5 CLARITY + RWMax 561 + 841 99.5 93.8 81.3 99.0
RR1+ DMO Line 1 CLARITY 561 0.0 7.0 6.3 4.5 RWMax 841 0.0 3.5 3.5
11.3 CLARITY + RWMax 561 + 841 0.0 3.0 4.0 10.0 RR1+ DMO Line 2
CLARITY 561 0.0 5.3 6.3 5.3 RWMax 841 0.0 4.5 4.5 11.7 CLARITY +
RWMax 561 + 841 0.0 5.0 4.0 8.8 RR1+ DMO Line 3 CLARITY 561 0.0 9.0
8.8 7.5 RWMax 841 0.0 3.5 4.0 11.3 CLARITY + RWMax 561 + 841 0.0
4.5 3.5 10.0 RR1+ DMO Line 4 CLARITY 561 0.0 8.5 8.8 3.5 RWMax 841
0.0 3.5 3.5 11.3 CLARITY + RWMax 561 + 841 0.0 4.5 4.5 8.8 RR2+ DMO
Line 1 CLARITY 561 0.0 8.5 6.3 5.3 RWMax 841 0.0 3.5 3.5 3.0
CLARITY + RWMax 561 + 841 0.0 5.0 4.5 5.0 RR2+ DMO Line 2 CLARITY
561 0.0 9.0 6.3 3.0 RWMax 841 0.0 3.5 6.3 3.0 CLARITY + RWMax 561 +
841 0.0 9.5 7.0 3.0 RR2+ DMO Line 3 CLARITY 561 0.0 9.5 7.5 3.5
RWMax 841 0.0 3.5 6.3 4.5 CLARITY + RWMax 561 + 841 0.0 8.5 3.5 3.3
RR2+ DMO Line 4 CLARITY 561 0.0 5.3 5.8 3.0 RWMax 841 0.0 16.5 17.0
4.0 CLARITY + RWMax 561 + 841 0.0 11.0 3.5 5.3
Example 10
Development of a Method for Controlling Weed Resistance Development
in a Field
Transgenic seeds having dicamba and glyphosate tolerance prepared
as described above are planted in a field. The field is treated
with dicamba and glyphosate before or after planting the seeds
using a mixture of dicamba and glyphosate in an effective amount to
control weed growth. Typically about a 1.times. application rate of
either herbicide will be effective in controlling weed growth, but
the rate may be varied depending upon environmental conditions and
the type of weeds being controlled, as is known in the art. The
rate of application may also be increased or decreased depending
upon the rate of control desired. Generally speaking, increasing
the rate of one herbicide will allow a decrease in the rate of the
second herbicide in order to obtain the same level of seed control.
In specific embodiments, an application of from about 200 to about
1600 g/ha of glyphosate is combined with from about 20 to about 400
g/ha of dicamba.
A desired application rate may be optimized in any particular
environment or in the context of a particular weed can be
determined using the experimental layout of Example 9 with the
different formulation rates described therein. In addition to
desired level of weed control, the herbicide level is selected to
avoid using more herbicide than is needed on the one hand, and to
avoid poor weed control that could lead to herbicide tolerant
plants. Over application of herbicides could also damage herbicide
tolerant crop. As shown in Example 9 above however, combining
optimized applications of these herbicides provides significant
levels of control of even herbicide tolerant weeds, and thus
represents a major advance in the art.
Example 11
Development of a Method for Controlling Weeds in a Single Pass in a
Field
The procedures in Examples 9 and 10 are applied to develop a method
for controlling weed growth in a crop-growing environment involving
planting a transgenic seed in a field containing a weed or a seed
thereof and treating the field in a single pass though the field.
The treatment comprises a herbicidally effective amount of dicamba,
glyphosate, or a mixture thereof, administered contemporaneously
with the planting of the seed. The planting, treating, and growing
of the transgenic seed are achieved by standard agricultural
methods.
Such a method of planting the transgenic seed and treating the
transgenic seed in one pass eliminates the need for a farmer to
make multiple passes through the field, including once for planting
and once for spraying. The technique therefore reduces fuel and
wear-tear costs to farmers.
Example 12
Tolerance of Plants Containing DMO-Encoding Polynucleotide Molecule
to Other Auxin-Like Herbicides
Herbicide drift and contamination of herbicide delivery equipment
is a serious concern in agriculture and can injure non-target crops
resulting in losses to farmers. However, some level of drift is
often inevitable due to changing environmental conditions such as
wind and the proximity of growing fields. Further, it is often
difficult and expensive to eliminate all residual levels of a
herbicide in a tank following herbicide application and residual
herbicides often result in inadvertent injury to crops. Often
several rinses of herbicide delivery equipment are required before
it can be used for another herbicide, which wastes water and
cleaning chemicals.
As herbicides such as 2,4-D and MCPA are post-emergent herbicides
for some crops, but can cause serious damage to non-target crops,
residual contamination with these herbicides is of particular
concern. A transgenic crop tolerant to at least low levels of these
herbicides would therefore be of significant value in managing
injuries due to spray drift and contamination of herbicide
equipment. This could also reduce the extent of equipment washing
needed for herbicide delivery equipment.
An analysis was therefore carried out to determine whether soybean
plants having DMO-encoding polynucleotide could deactivate other
auxin-like herbicides in addition to dicamba, including 2,4-D and
MCPA. This was carried out by applying various concentrations of
commercially available formulations of other auxin-like herbicides
such as 2,4-D (Helena, Collierville, Tenn.), MCPA (Agriliance, St.
Paul, Minn), triclopyr (GARLON 3A; Dow Elanco, Indianapolis, Ind.),
clopyralid (STINGER; Dow Elanco, Indianapolis, Ind.), picloram
(TORDON 22K; Dow Elanco, Indianapolis, Ind.), or Banvel or CLARITY
(BASF, Raleigh, N.C.) to DMO containing plant tissues or
plants.
Transgenic soybean plants were obtained by Agrobacterium-mediated
transformation of soybean explants with a DMO-encoding
polynucleotide as described above for the events designated Events
1-4. A non-transgenic line was used as a control. Non-transgenic
and transgenic soybean seeds were planted into 3.5-inch square
plastic pots containing Redi-earth.TM. (Scotts-Sierra Horticultural
Products Co., Marysville, Ohio). The pots were placed on capillary
matting in 35 inch.times.60 inch fiberglass watering trays for
overhead and/or sub-irrigation for the duration of the test period
so as to maintain optimum soil moisture for plant growth. The pots
were fertilized with Osmocote (14-14-14 slow release; Scotts-Sierra
Horticultural Products Co., Marysville, Ohio) at the rate of 100
gm/cu.ft. to sustain plant growth for the duration of greenhouse
trials, and grown in greenhouses at 27.degree./21.degree. C.
day/night temperature, with relative humidity between 25%-75% to
simulate warm season growing conditions of late spring. A 14 h
minimum photoperiod was provided with supplemental light at about
600 .mu.E as needed.
All herbicide applications were made with the track sprayer using a
Teejet 9501E flat fan nozzle (Spraying Systems Co, Wheaton, Ill.)
with air pressure set at a minimum of 24 psi (165 kpa). The spray
nozzle was kept at a height of about 16 inches above the top of
plant material for spraying. The spray volume was 10 gallons per
acre or 93 liters per hectare. Applications were made when plants
had reached V-3 stage. All trials were established in a randomized
block design (randomized by rate) with 4 to 6 replications of each
treatment depending on plant quality, availability and to account
for any environmental variability that may have occurred within the
confines of each greenhouse.
All treated plants in greenhouse trials were visually assessed at
about 4, 14, 18, and 21 days after treatment (DAT) for injury on a
scale of 0 to 100 percent relative to untreated control plants,
with zero representing "no" injury and 100% representing "complete"
injury or death. Data were collected using a palm top computer and
analyzed using standard statistical methods. The results shown in
Table 10 clearly indicate tolerance of transgenic soybean to other
auxin-like herbicides such as 2,4-D and MCPA relative to the
non-transgenic line.
TABLE-US-00010 TABLE 10 Percentage injury relative to un-treated
controls at 25 DAT post-V3 applications of different auxin-like
herbicides to non-transgenic or transgenic soybean plants.*
Herbicide Plant/trial 280 561 1120 % injury at shown rates (g
ae/ha**) at 21 DAT Dicamba (Clarity) Non-transgenic 100 100 Event 1
0.0 1.2 Event 2 0.0 1.7 Event 3 0.0 0.7 Event 4 0.0 1.5 Dicamba
(Banvel) Non-transgenic 100.0 100.0 Event 1 0.0 1.5 Event 2 0.0 0.7
Event 3 0.0 0.5 Event 4 0.0 1.3 2,4-D Non-transgenic 86.8 100.0
100.0 Event 1 58.3 75.0 100.0 Event 2 64.2 94.7 100.0 Event 3 40.0
85.0 100.0 Event 4 45.8 84.2 100.0 MCPA Non-transgenic 93.0 98.3
100.0 Event 1 72.5 99.3 100.0 Event 2 55.0 95.0 99.7 Event 3 55.0
95.8 100.0 Event 4 88.3 98.8 100.0 LSD 16.3 10.6 3.7 % injury shown
rates (g ae/ha**) at 14 DAT Triclopyr Non-transgenic 86.7 97.3 98.7
Event 1 88.3 95.7 99.3 Event 2 86.7 98.7 99.3 Event 3 86.7 94.0
96.3 Event 4 90.8 98.0 99.2 Clopyralid Non-transgenic 99.3 100.0
100.0 Event 1 99.2 100.0 100.0 Event 2 98.2 99.7 100.0 Event 3 99.3
100.0 100.0 Event 4 99.7 100.0 100.0 Picloram Non-transgenic 99.3
100.0 100.0 Event 1 99.7 100.0 100.0 Event 2 99.3 100.0 100.0 Event
3 99.3 99.7 100.0 Event 4 99.3 100.0 100.0 % injury at shown rates
(g ae/ha**) at 21 DAT LSD 2.9 1.8 1.4 *The % injury was represented
as ANOVA mean comparisons. **grams of active acid
equivalent/hectare
Another auxin-like herbicide Butyrac 200 (2,4-DB; Albaugh) was also
tested on transgenic soybean plants carrying a DMO gene for testing
the plants tolerance to it. The herbicide was applied as a
post-emergence treatment at three application rates on two
transgenic soybean events and compared with a non-transgenic line
for total crop injury across all three application rates: 280 g/ha
(0.25 lb/a), 561 g/ha (0.5 lb/a) and 841 g/ha (0.75 lb/a) (see
Table 11). Both transgenic soybean lines showed low level of
tolerance to 2,4-DB. This example shows that dicamba tolerant
soybean is also tolerant to low levels of 2,4-DB and should be
useful in managing damage from spray drift from the same or
neighboring fields to prevent crop loses, and would exhibit
tolerance to residual levels of 2,4-DB following incomplete washing
of herbicide delivery equipment.
TABLE-US-00011 TABLE 11 Percentage injury relative to the untreated
control at 16 DAT by the application of 2,4-DB to non-transgenic or
transgenic soybean plants. % injury at shown rates (g ae/ha) at 16
DAT Herbicide Plant 280 561 1120 2,4-DB (Butyrac 200)
Non-transgenic 59.2 70.0 79.2 NE3001 462-1-21 25.0 43.3 75.8
469-13-19 18.3 37.5 70.0
This example shows that transgenic soybean plants exhibit tolerance
to other auxin-like herbicides, indicating a likely common
deactivation mechanism for dicamba and other auxin-like herbicides
such as 2,4-D and MCPA. In case of triclopyr, clopyralid, and
picloram, the application rate of 280 g ae/ha appeared too
stringent in this study and thus lower concentrations may be
desired in most settings to reduce plant damage. Thus, a DMO
polynucleotide containing soybean that is tolerant to dicamba is
also tolerant to low levels of 2,4-D and MCPA and should prevent or
minimize damage from spray drift from same or neighboring fields to
prevent crop loses, and would exhibit tolerance to residual levels
of these herbicides following incomplete washing of herbicide
delivery equipment. The herbicide delivery equipment could include
a tank, container, hose, strainer, boom, sprayer, nozzle, pump, and
accessories such as coupling, elbows, shanks, and valves. The
delivery equipment is operable manually or mechanically for example
on a farm vehicle, airplane, and helicopter, among others.
Example 13
Production of Dicamba Tolerant Transgenic Corn Plants
To test the use of a DMO gene in providing dicamba tolerance to
monocots, transgenic corn plants were produced that comprise a DMO
gene as disclosed above with or without a transit peptide (e.g. Ta
Waxy, CTP1, CTP2synthetic, CTP4) under the control of plant gene
expression elements such as a promoter (e.g. PC1SV, e35S, OsAct1,
OsTPI, OsAct15), and an intron (e.g. OsAct1, OsAct15, OsTPI,
ZmHSP70). This expression element contains first intron and
flanking UTR exon sequences from the rice actin 1 gene and includes
12 nt of exon 1 at the 5' end and 7 nt of exon 2 at the 3' end),
and a 3'UTR (e.g. TaHsp17). Nucleotide sequences/and or patent
references for various expression elements are disclosed in
co-pending application U.S. Ser. No. 60/891,675.
Transgenic corn plants were produced by the methods known in the
art such as WO9506722 and US patent application 20040244075.
Transgenic corn events having single copy were evaluated for
dicamba tolerance at a single location replicated trial. Six events
from each of the six constructs were used. The experimental design
was as follows: rows/entry: 1; treatment: 0.5 lb/a of dicamba at V3
stage followed by 1 lb/a of dicamba at V8 stage (Clarity.RTM.,
BASF, Raleigh, N.C.); replications: 2; row spacing: 30 inches; plot
length: minimum 20 feet; plant density: about 30 plants/17.5 ft.;
alleys: 2.5 feet. The entire plot was fertilized uniformly to
obtain an agronomically acceptable crop. A soil insecticide such as
Force.RTM. 3G (Syngenta Crop Protection, Greensboro, N.C., USA) at
5 oz. per 1000 ft. of row for control of corn rootworm was applied
at planting time. If black cutworm infestation was observed,
POUNCE.RTM. 3.2EC at 4 to 8 oz. per acre rate (FMC Corporation,
Philadelphia, Pa.) was used. In addition, an insecticide spray
program was used to control all above ground lepidopteran pests
including European corn borer, corn earworm, and fall armyworm.
POUNCE.RTM. 3.2EC at 4 to 8 oz. per acre was applied every 3 weeks
to control lepidopteran pests; about 4 applications were made. The
plot was kept weed free with a pre-emergence application of a
herbicide such as Harness.RTM. Xtra 5.6 L (Monsanto, St. Louis,
Mo.) and Degree Xtra.RTM. (Monsanto, St. Louis, Mo.). If weed
escapes were observed in the untreated check, they were controlled
by hand weeding or a post-emergence application of PERMIT
(Monsanto, St. Louis, Mo.) or BUCTRIL.RTM. (Bayer, Research
Triangle Park, NC) over the entire trial.
Corn inbred lines transformed with DNA constructs comprising a DMO
transgene were tested for dicamba tolerance by measuring brace root
injury when treated with 0.5 lb/a of dicamba at V3 stage followed
by 1 lb/a of dicamba at V8 stage. Brace root injury was evaluated
visually by counting the number of plants in a row showing an
"atypical" morphology of having the brace roots fused as compared
to a typical morphology of "finger-like" structure. As shown in
Table 12, corn plants transformed with DNA constructs coding for a
DMO without linking it to a CTP (pMON73699, pMON73704) showed
higher level of brace root injury, i.e. lower level of protection
upon dicamba treatment. The constructs coding for a DMO linked to a
CTP (pMON73716, pMON73700, pMON73715, pMON73703) showed lower level
of brace root injury, i.e. higher level of protection upon dicamba
treatment.
TABLE-US-00012 TABLE 12 Percentage brace root injury as a measure
of dicamba tolerance exhibited by transgenic corn plants
transformed with DNA constructs carrying DMO. Brace Inbreds/ root
Constructs Details injury 01CSI6 Susceptible inbred to dicamba 95.4
LH244 Resistant inbred to dicamba 93.8 pMON73699
PC1SV/I-OsAct1/DMO-Wmc/TaHsp17 93.2 pMON73704
e35S/I-OsAct1/DMO-Wmc/TaHsp17 91.3 pMON73716
PC1SV/I-OsAct1/TaWaxy/DMO-Wmc/TaHsp17 78.8 pMON73700
PC1SV/I-OsAct1/CTP1/DMO-Wmc/TaHsp17 74.4 pMON73715
PC1SV/I-OsAct1/CTP2syn/DMO-Wmc/TaHsp17 68.2 pMON73703
e35S/I-OsAct1/CTP1/DMO-Wmc/TaHsp17 68.8
Example 14
Production of Dicamba Tolerant Transgenic Cotton Plants
To test the use of DMO gene in providing dicamba tolerance to
cotton, transgenic cotton plants were produced. Several DNA
constructs carrying a DMO coding region as disclosed herein with a
transit peptide (e.g., PsRbcS CTP, CTP1, CTP2) under the control of
plant gene expression elements such as a promoter (e.g. PC1SV, FMV,
or e35S), and a 3'UTR (e.g. E6; Accession # U30508) were produced
and transformed into cotton (Gossypium hirsutum) as follows.
Nucleotide sequences/and or patent references for various
expression elements are disclosed in co-pending application U.S.
Ser. No. 60/891,675. Media used are noted in Table 13.
Cotton transformation was performed, for instance as described
according to U.S. Patent Application Publication 20040087030, via
an embryogenic approach. Explants of cotton cv Coker 130 were grown
in vitro and with a liquid suspension of Agrobacterium tumefaciens
carrying a DNA construct of interest, using selection on kanamycin
containing media. Putative transgenic plantlets were then
transferred to soil to obtain mature cotton plants. The transgenic
nature of transformants was confirmed by DNA testing.
TABLE-US-00013 TABLE 13 Composition of various media used for
cotton transformation. Amount/L Components Glucose Sucrose UMO TRP+
SHSU MS basal salts 4.33 g 4.33 g 4.33 g 4.33 g -- (Phytotech.)
Gamborg's B5 vitamins 2 ml 2 ml 2 ml 2 ml -- (Phytotech) (500X)
2,4-D (1 mg/ml) 0.1 ml 0.1 ml -- -- Stewart and Hsu -- -- -- -- 100
ml majors (10X) Stewart and Hsu -- -- -- -- 10 ml minors (100X)
Steward and Hsu -- -- -- -- 10 ml organic (100X) Kinetin (0.5
mg/ml) 1 ml 1 ml -- -- -- Chelated iron (100X) -- -- -- -- 1.5 ml
Glucose 30 g 30 g 30 g 30 g 5 g Potassium nitrate -- -- -- 1.9 g --
Casein hydrolysate -- -- -- 0.1 g -- pH 5.8 5.8 5.8 5.8 6.8
Phytagel (Sigma) 2.5 g 2.5 g -- -- -- Gelrite (Kelco) -- -- 3.5 g
3.5 g 2.2 g Carbenicillin 1.7 ml 1.7 ml 1.7 ml 1.7 ml -- (250
mg/ml) Cefotaxime (100 mg/ml) 1 ml 1 ml 1 ml 1 ml -- Benlate (50
mg/ml) -- -- -- 1 ml 1 ml Kanamycin (50 mg/ml) 0.8-1.0 ml 0.8-1.0
ml 1 ml -- -- Sucrose -- 0.1 g -- -- -- Ascorbic acid -- -- 100
mg
Transformed cotton plants that comprise a DNA construct, i.e, each
comprising a different combination of a DMO coding region with a
transit peptide, a promoter, and a 3'UTR, were treated with dicamba
(Clarity.RTM., BASF, Raleigh, N.C.) as a post-emergent treatment at
V4-5 growth stage at the rate of 561 g ae/ha (0.5 lb/a) and found
to be tolerant whereas untransformed cotton plants showed an injury
rate of 79% to 86%. Transgenic plants showing more than 95%
tolerance (equal to less than 5% injury) were selected for further
studies. Transgenic plants were also tolerant to a subsequent
post-emergent treatment of dicamba. For example, the plants that
were treated with 0.5 lb/acre of dicamba at V3-4 stage followed by
either 1 or 2 lb/acre of dicamba at V5 or later stages were still
tolerant to dicamba. R1 transgenic seeds and plants were also
subjected to pre-emergence or pre-emergence and post-emergence
dicamba treatment and found to be tolerant. This example shows that
a DMO gene can provide dicamba tolerance to cotton at various
stages of growth thus enabling application of dicamba at various
stages to obtain effective weed control.
All of the compositions and/or methods disclosed and claimed herein
can be made and executed without undue experimentation in light of
the present disclosure. While the compositions and methods of this
invention have been described in terms of preferred embodiments, it
will be apparent to those of skill in the art that variations may
be applied to the compositions and/or methods and in the steps or
in the sequence of steps of the method described herein without
departing from the concept, spirit and scope of the invention. More
specifically, it will be apparent that certain agents that are both
chemically and physiologically related may be substituted for the
agents described herein while the same or similar results would be
achieved. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the invention as defined by the appended
claims.
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The references listed below are incorporated herein by reference to
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SEQUENCE LISTINGS
1
3211023DNAArtificialBased on dicamba monooxygenase gene from
Pseudomonas maltophilia 1atggccactt tcgttagaaa cgcttggtac
gttgctgcac ttcctgagga gttgagcgag 60aagcctctag gaagaactat cctcgatact
ccactagctc tctatcgtca acctgacgga 120gttgtcgctg ccctgcttga
tatttgtccg catcgcttcg ctccgttgag tgacggtatt 180ctagtcaacg
gacatctcca gtgtccatat cacggtctgg aatttgacgg aggtggccag
240tgtgtccaca acccgcacgg caacggagcc cgccctgctt ctctgaacgt
gcgatcattc 300cctgtcgtgg aaagagacgc attgatctgg atctgccctg
gagatccagc actcgcagat 360cccggtgcta tccctgactt tgggtgtcgt
gttgatccag cttaccgtac tgtcggaggt 420tacggtcacg tggactgcaa
ctacaagctc cttgtggata acctcatgga tcttggacac 480gctcagtacg
tgcaccgcgc taacgcccaa acagacgcct tcgatagact tgagcgtgag
540gtgatcgttg gcgacggcga gatccaggcg ctcatgaaga tccctggtgg
cacaccctca 600gttctcatgg ctaagttctt gcgtggtgct aacacaccag
ttgacgcctg gaacgacatc 660cggtggaata aggtgtcggc tatgctgaac
ttcatcgcgg tcgcgccgga agggacgccg 720aaggagcagt caatccactc
ccgaggaacc catatcctta ctcctgagac cgaggcaagc 780tgccattact
tcttcggtag ttcccgcaac ttcggtatag acgatccaga gatggacggt
840gttctcagga gctggcaagc tcaagccctg gtgaaggagg acaaagtggt
cgttgaagct 900atcgaaaggc ggagggctta cgtcgaagcg aacgggatca
gacccgccat gttgtcctgc 960gacgaggcag ccgtcagggt atccagggag
attgagaagc tcgaacaact agaagcggcg 1020tga 10232340PRTArtificialBased
on dicamba monooxygenase gene from Pseudomonas maltophilia 2Met Ala
Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu 1 5 10 15
Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu 20
25 30 Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp
Ile 35 40 45 Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu
Val Asn Gly 50 55 60 His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe
Asp Gly Gly Gly Gln 65 70 75 80 Cys Val His Asn Pro His Gly Asn Gly
Ala Arg Pro Ala Ser Leu Asn 85 90 95 Val Arg Ser Phe Pro Val Val
Glu Arg Asp Ala Leu Ile Trp Ile Cys 100 105 110 Pro Gly Asp Pro Ala
Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly 115 120 125 Cys Arg Val
Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val 130 135 140 Asp
Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His 145 150
155 160 Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp
Arg 165 170 175 Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln
Ala Leu Met 180 185 190 Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met
Ala Lys Phe Leu Arg 195 200 205 Gly Ala Asn Thr Pro Val Asp Ala Trp
Asn Asp Ile Arg Trp Asn Lys 210 215 220 Val Ser Ala Met Leu Asn Phe
Ile Ala Val Ala Pro Glu Gly Thr Pro 225 230 235 240 Lys Glu Gln Ser
Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu 245 250 255 Thr Glu
Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly 260 265 270
Ile Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln 275
280 285 Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg
Arg 290 295 300 Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met
Leu Ser Cys 305 310 315 320 Asp Glu Ala Ala Val Arg Val Ser Arg Glu
Ile Glu Lys Leu Glu Gln 325 330 335 Leu Glu Ala Ala 340
31023DNAArtificialBased on dicamba monooxygenase gene from
Pseudomonas maltophilia 3atgctcactt tcgttagaaa cgcttggtac
gttgctgcac ttcctgagga gttgagcgag 60aagcctctag gaagaactat cctcgatact
ccactagctc tctatcgtca acctgacgga 120gttgtcgctg ccctgcttga
tatttgtccg catcgcttcg ctccgttgag tgacggtatt 180ctagtcaacg
gacatctcca gtgtccatat cacggtctgg aatttgacgg aggtggccag
240tgtgtccaca acccgcacgg caacggagcc cgccctgctt ctctgaacgt
gcgatcattc 300cctgtcgtgg aaagagacgc attgatctgg atctgccctg
gagatccagc actcgcagat 360cccggtgcta tccctgactt tgggtgtcgt
gttgatccag cttaccgtac tgtcggaggt 420tacggtcacg tggactgcaa
ctacaagctc cttgtggata acctcatgga tcttggacac 480gctcagtacg
tgcaccgcgc taacgcccaa acagacgcct tcgatagact tgagcgtgag
540gtgatcgttg gcgacggcga gatccaggcg ctcatgaaga tccctggtgg
cacaccctca 600gttctcatgg ctaagttctt gcgtggtgct aacacaccag
ttgacgcctg gaacgacatc 660cggtggaata aggtgtcggc tatgctgaac
ttcatcgcgg tcgcgccgga agggacgccg 720aaggagcagt caatccactc
ccgaggaacc catatcctta ctcctgagac cgaggcaagc 780tgccattact
tcttcggtag ttcccgcaac ttcggtatag acgatccaga gatggacggt
840gttctcagga gctggcaagc tcaagccctg gtgaaggagg acaaagtggt
cgttgaagct 900atcgaaaggc ggagggctta cgtcgaagcg aacgggatca
gacccgccat gttgtcctgc 960gacgaggcag ccgtcagggt atccagggag
attgagaagc tcgaacaact agaagcggcg 1020tga 10234340PRTArtificialBased
on dicamba monooxygenase gene from Pseudomonas maltophilia 4Met Leu
Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu 1 5 10 15
Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu 20
25 30 Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp
Ile 35 40 45 Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu
Val Asn Gly 50 55 60 His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe
Asp Gly Gly Gly Gln 65 70 75 80 Cys Val His Asn Pro His Gly Asn Gly
Ala Arg Pro Ala Ser Leu Asn 85 90 95 Val Arg Ser Phe Pro Val Val
Glu Arg Asp Ala Leu Ile Trp Ile Cys 100 105 110 Pro Gly Asp Pro Ala
Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly 115 120 125 Cys Arg Val
Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val 130 135 140 Asp
Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His 145 150
155 160 Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp
Arg 165 170 175 Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln
Ala Leu Met 180 185 190 Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met
Ala Lys Phe Leu Arg 195 200 205 Gly Ala Asn Thr Pro Val Asp Ala Trp
Asn Asp Ile Arg Trp Asn Lys 210 215 220 Val Ser Ala Met Leu Asn Phe
Ile Ala Val Ala Pro Glu Gly Thr Pro 225 230 235 240 Lys Glu Gln Ser
Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu 245 250 255 Thr Glu
Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly 260 265 270
Ile Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln 275
280 285 Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg
Arg 290 295 300 Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met
Leu Ser Cys 305 310 315 320 Asp Glu Ala Ala Val Arg Val Ser Arg Glu
Ile Glu Lys Leu Glu Gln 325 330 335 Leu Glu Ala Ala 340
51023DNAArtificialBased on dicamba monooxygenase gene from
Pseudomonas maltophilia 5atgctcactt tcgttagaaa cgcttggtac
gttgctgcac ttcctgagga gttgagcgag 60aagcctctag gaagaactat cctcgatact
ccactagctc tctatcgtca acctgacgga 120gttgtcgctg ccctgcttga
tatttgtccg catcgcttcg ctccgttgag tgacggtatt 180ctagtcaacg
gacatctcca gtgtccatat cacggtctgg aatttgacgg aggtggccag
240tgtgtccaca acccgcacgg caacggagcc cgccctgctt ctctgaacgt
gcgatcattc 300cctgtcgtgg aaagagacgc attgatctgg atctggcctg
gagatccagc actcgcagat 360cccggtgcta tccctgactt tgggtgtcgt
gttgatccag cttaccgtac tgtcggaggt 420tacggtcacg tggactgcaa
ctacaagctc cttgtggata acctcatgga tcttggacac 480gctcagtacg
tgcaccgcgc taacgcccaa acagacgcct tcgatagact tgagcgtgag
540gtgatcgttg gcgacggcga gatccaggcg ctcatgaaga tccctggtgg
cacaccctca 600gttctcatgg ctaagttctt gcgtggtgct aacacaccag
ttgacgcctg gaacgacatc 660cggtggaata aggtgtcggc tatgctgaac
ttcatcgcgg tcgcgccgga agggacgccg 720aaggagcagt caatccactc
ccgaggaacc catatcctta ctcctgagac cgaggcaagc 780tgccattact
tcttcggtag ttcccgcaac ttcggtatag acgatccaga gatggacggt
840gttctcagga gctggcaagc tcaagccctg gtgaaggagg acaaagtggt
cgttgaagct 900atcgaaaggc ggagggctta cgtcgaagcg aacgggatca
gacccgccat gttgtcctgc 960gacgaggcag ccgtcagggt atccagggag
attgagaagc tcgaacaact agaagcggcg 1020tga 10236340PRTArtificialBased
on dicamba monooxygenase gene from Pseudomonas maltophilia 6Met Leu
Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu 1 5 10 15
Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu 20
25 30 Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp
Ile 35 40 45 Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu
Val Asn Gly 50 55 60 His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe
Asp Gly Gly Gly Gln 65 70 75 80 Cys Val His Asn Pro His Gly Asn Gly
Ala Arg Pro Ala Ser Leu Asn 85 90 95 Val Arg Ser Phe Pro Val Val
Glu Arg Asp Ala Leu Ile Trp Ile Trp 100 105 110 Pro Gly Asp Pro Ala
Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly 115 120 125 Cys Arg Val
Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val 130 135 140 Asp
Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His 145 150
155 160 Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp
Arg 165 170 175 Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln
Ala Leu Met 180 185 190 Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met
Ala Lys Phe Leu Arg 195 200 205 Gly Ala Asn Thr Pro Val Asp Ala Trp
Asn Asp Ile Arg Trp Asn Lys 210 215 220 Val Ser Ala Met Leu Asn Phe
Ile Ala Val Ala Pro Glu Gly Thr Pro 225 230 235 240 Lys Glu Gln Ser
Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu 245 250 255 Thr Glu
Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly 260 265 270
Ile Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln 275
280 285 Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg
Arg 290 295 300 Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met
Leu Ser Cys 305 310 315 320 Asp Glu Ala Ala Val Arg Val Ser Arg Glu
Ile Glu Lys Leu Glu Gln 325 330 335 Leu Glu Ala Ala 340
71023DNAArtificialBased on dicamba monooxygenase gene from
Pseudomonas maltophilia 7atggccacct tcgtccgcaa tgcctggtat
gtggcggcgc tgcccgagga actgtccgaa 60aagccgctcg gccggacgat tctcgacaca
ccgctcgcgc tctaccgcca gcccgacggt 120gtggtcgcgg cgctgctcga
catctgtccg caccgcttcg cgccgctgag cgacggcatc 180ctcgtcaacg
gccatctcca atgcccctat cacgggctgg aattcgatgg cggcgggcag
240tgcgtccata acccgcacgg caatggcgcc cgcccggctt cgctcaacgt
ccgctccttc 300ccggtggtgg agcgcgacgc gctgatctgg atctgtcccg
gcgatccggc gctggccgat 360cctggggcga tccccgactt cggctgccgc
gtcgatcccg cctatcggac cgtcggcggc 420tatgggcatg tcgactgcaa
ctacaagctg ctggtcgaca acctgatgga cctcggccac 480gcccaatatg
tccatcgcgc caacgcccag accgacgcct tcgaccggct ggagcgcgag
540gtgatcgtcg gcgacggtga gatacaggcg ctgatgaaga ttcccggcgg
cacgccgagc 600gtgctgatgg ccaagttcct gcgcggcgcc aatacccccg
tcgacgcttg gaacgacatc 660cgctggaaca aggtgagcgc gatgctcaac
ttcatcgcgg tggcgccgga aggcaccccg 720aaggagcaga gcatccactc
gcgcggtacc catatcctga cccccgagac ggaggcgagc 780tgccattatt
tcttcggctc ctcgcgcaat ttcggcatcg acgatccgga gatggacggc
840gtgctgcgca gctggcaggc tcaggcgctg gtcaaggagg acaaggtcgt
cgtcgaggcg 900atcgagcgcc gccgcgccta tgtcgaggcg aatggcatcc
gcccggcgat gctgtcgtgc 960gacgaagccg cagtccgtgt cagccgcgag
atcgagaagc ttgagcagct cgaagccgcc 1020tga 10238340PRTArtificialBased
on dicamba monooxygenase gene from Pseudomonas maltophilia 8Met Ala
Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu 1 5 10 15
Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu 20
25 30 Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp
Ile 35 40 45 Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu
Val Asn Gly 50 55 60 His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe
Asp Gly Gly Gly Gln 65 70 75 80 Cys Val His Asn Pro His Gly Asn Gly
Ala Arg Pro Ala Ser Leu Asn 85 90 95 Val Arg Ser Phe Pro Val Val
Glu Arg Asp Ala Leu Ile Trp Ile Cys 100 105 110 Pro Gly Asp Pro Ala
Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly 115 120 125 Cys Arg Val
Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val 130 135 140 Asp
Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His 145 150
155 160 Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp
Arg 165 170 175 Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln
Ala Leu Met 180 185 190 Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met
Ala Lys Phe Leu Arg 195 200 205 Gly Ala Asn Thr Pro Val Asp Ala Trp
Asn Asp Ile Arg Trp Asn Lys 210 215 220 Val Ser Ala Met Leu Asn Phe
Ile Ala Val Ala Pro Glu Gly Thr Pro 225 230 235 240 Lys Glu Gln Ser
Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu 245 250 255 Thr Glu
Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly 260 265 270
Ile Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln 275
280 285 Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg
Arg 290 295 300 Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met
Leu Ser Cys 305 310 315 320 Asp Glu Ala Ala Val Arg Val Ser Arg Glu
Ile Glu Lys Leu Glu Gln 325 330 335 Leu Glu Ala Ala 340
91023DNAArtificialBased on dicamba monooxygenase gene from
Pseudomonas maltophilia 9atggccactt tcgttagaaa cgcttggtac
gttgctgcac ttcctgagga gttgagcgag 60aagcctctag gaagaactat cctcgatact
ccactagctc tctatcgtca acctgacgga 120gttgtcgctg ccctgcttga
tatttgtccg catcgcttcg ctccgttgag tgacggtatt 180ctagtcaacg
gacatctcca gtgtccatat cacggtctgg aatttgacgg aggtggccag
240tgtgtccaca acccgcacgg caacggagcc cgccctgctt ctctgaacgt
gcgatcattc 300cctgtcgtgg aaagagacgc attgatctgg atctggcctg
gagatccagc actcgcagat 360cccggtgcta tccctgactt tgggtgtcgt
gttgatccag cttaccgtac tgtcggaggt 420tacggtcacg tggactgcaa
ctacaagctc cttgtggata acctcatgga tcttggacac 480gctcagtacg
tgcaccgcgc taacgcccaa acagacgcct tcgatagact tgagcgtgag
540gtgatcgttg gcgacggcga gatccaggcg ctcatgaaga tccctggtgg
cacaccctca 600gttctcatgg ctaagttctt gcgtggtgct aacacaccag
ttgacgcctg gaacgacatc 660cggtggaata aggtgtcggc tatgctgaac
ttcatcgcgg tcgcgccgga agggacgccg 720aaggagcagt caatccactc
ccgaggaacc catatcctta ctcctgagac cgaggcaagc 780tgccattact
tcttcggtag ttcccgcaac ttcggtatag acgatccaga gatggacggt
840gttctcagga gctggcaagc tcaagccctg gtgaaggagg acaaagtggt
cgttgaagct 900atcgaaaggc ggagggctta cgtcgaagcg aacgggatca
gacccgccat gttgtcctgc 960gacgaggcag ccgtcagggt atccagggag
attgagaagc tcgaacaact agaagcggcg 1020tga
102310340PRTArtificialBased on dicamba monooxygenase gene from
Pseudomonas maltophilia 10Met Ala Thr Phe
Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu 1 5 10 15 Glu Leu
Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu 20 25 30
Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp Ile 35
40 45 Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu Val Asn
Gly 50 55 60 His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe Asp Gly
Gly Gly Gln 65 70 75 80 Cys Val His Asn Pro His Gly Asn Gly Ala Arg
Pro Ala Ser Leu Asn 85 90 95 Val Arg Ser Phe Pro Val Val Glu Arg
Asp Ala Leu Ile Trp Ile Trp 100 105 110 Pro Gly Asp Pro Ala Leu Ala
Asp Pro Gly Ala Ile Pro Asp Phe Gly 115 120 125 Cys Arg Val Asp Pro
Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val 130 135 140 Asp Cys Asn
Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His 145 150 155 160
Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp Arg 165
170 175 Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln Ala Leu
Met 180 185 190 Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met Ala Lys
Phe Leu Arg 195 200 205 Gly Ala Asn Thr Pro Val Asp Ala Trp Asn Asp
Ile Arg Trp Asn Lys 210 215 220 Val Ser Ala Met Leu Asn Phe Ile Ala
Val Ala Pro Glu Gly Thr Pro 225 230 235 240 Lys Glu Gln Ser Ile His
Ser Arg Gly Thr His Ile Leu Thr Pro Glu 245 250 255 Thr Glu Ala Ser
Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly 260 265 270 Ile Asp
Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln 275 280 285
Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg Arg 290
295 300 Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met Leu Ser
Cys 305 310 315 320 Asp Glu Ala Ala Val Arg Val Ser Arg Glu Ile Glu
Lys Leu Glu Gln 325 330 335 Leu Glu Ala Ala 340
111020DNAPseudomonas maltophilia 11atgaccttcg tccgcaatgc ctggtatgtg
gcggcgctgc ccgaggaact gtccgaaaag 60ccgctcggcc ggacgattct cgacacaccg
ctcgcgctct accgccagcc cgacggtgtg 120gtcgcggcgc tgctcgacat
ctgtccgcac cgcttcgcgc cgctgagcga cggcatcctc 180gtcaacggcc
atctccaatg cccctatcac gggctggaat tcgatggcgg cgggcagtgc
240gtccataacc cgcacggcaa tggcgcccgc ccggcttcgc tcaacgtccg
ctccttcccg 300gtggtggagc gcgacgcgct gatctggatc tggcccggcg
atccggcgct ggccgatcct 360ggggcgatcc ccgacttcgg ctgccgcgtc
gatcccgcct atcggaccgt cggcggctat 420gggcatgtcg actgcaacta
caagctgctg gtcgacaacc tgatggacct cggccacgcc 480caatatgtcc
atcgcgccaa cgcccagacc gacgccttcg accggctgga gcgcgaggtg
540atcgtcggcg acggtgagat acaggcgctg atgaagattc ccggcggcac
gccgagcgtg 600ctgatggcca agttcctgcg cggcgccaat acccccgtcg
acgcttggaa cgacatccgc 660tggaacaagg tgagcgcgat gctcaacttc
atcgcggtgg cgccggaagg caccccgaag 720gagcagagca tccactcgcg
cggtacccat atcctgaccc ccgagacgga ggcgagctgc 780cattatttct
tcggctcctc gcgcaatttc ggcatcgacg atccggagat ggacggcgtg
840ctgcgcagct ggcaggctca ggcgctggtc aaggaggaca aggtcgtcgt
cgaggcgatc 900gagcgccgcc gcgcctatgt cgaggcgaat ggcatccgcc
cggcgatgct gtcgtgcgac 960gaagccgcag tccgtgtcag ccgcgagatc
gagaagcttg agcagctcga agccgcctga 102012339PRTPseudomonas
maltophilia 12Met Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu
Pro Glu Glu 1 5 10 15 Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu
Asp Thr Pro Leu Ala 20 25 30 Leu Tyr Arg Gln Pro Asp Gly Val Val
Ala Ala Leu Leu Asp Ile Cys 35 40 45 Pro His Arg Phe Ala Pro Leu
Ser Asp Gly Ile Leu Val Asn Gly His 50 55 60 Leu Gln Cys Pro Tyr
His Gly Leu Glu Phe Asp Gly Gly Gly Gln Cys 65 70 75 80 Val His Asn
Pro His Gly Asn Gly Ala Arg Pro Ala Ser Leu Asn Val 85 90 95 Arg
Ser Phe Pro Val Val Glu Arg Asp Ala Leu Ile Trp Ile Trp Pro 100 105
110 Gly Asp Pro Ala Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly Cys
115 120 125 Arg Val Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His
Val Asp 130 135 140 Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp
Leu Gly His Ala 145 150 155 160 Gln Tyr Val His Arg Ala Asn Ala Gln
Thr Asp Ala Phe Asp Arg Leu 165 170 175 Glu Arg Glu Val Ile Val Gly
Asp Gly Glu Ile Gln Ala Leu Met Lys 180 185 190 Ile Pro Gly Gly Thr
Pro Ser Val Leu Met Ala Lys Phe Leu Arg Gly 195 200 205 Ala Asn Thr
Pro Val Asp Ala Trp Asn Asp Ile Arg Trp Asn Lys Val 210 215 220 Ser
Ala Met Leu Asn Phe Ile Ala Val Ala Pro Glu Gly Thr Pro Lys 225 230
235 240 Glu Gln Ser Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu
Thr 245 250 255 Glu Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn
Phe Gly Ile 260 265 270 Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser
Trp Gln Ala Gln Ala 275 280 285 Leu Val Lys Glu Asp Lys Val Val Val
Glu Ala Ile Glu Arg Arg Arg 290 295 300 Ala Tyr Val Glu Ala Asn Gly
Ile Arg Pro Ala Met Leu Ser Cys Asp 305 310 315 320 Glu Ala Ala Val
Arg Val Ser Arg Glu Ile Glu Lys Leu Glu Gln Leu 325 330 335 Glu Ala
Ala 13455PRTAgrobacterium tumefaciens 13Met Leu His Gly Ala Ser Ser
Arg Pro Ala Thr Ala Arg Lys Ser Ser 1 5 10 15 Gly Leu Ser Gly Thr
Val Arg Ile Pro Gly Asp Lys Ser Ile Ser His 20 25 30 Arg Ser Phe
Met Phe Gly Gly Leu Ala Ser Gly Glu Thr Arg Ile Thr 35 40 45 Gly
Leu Leu Glu Gly Glu Asp Val Ile Asn Thr Gly Lys Ala Met Gln 50 55
60 Ala Met Gly Ala Arg Ile Arg Lys Glu Gly Asp Thr Trp Ile Ile Asp
65 70 75 80 Gly Val Gly Asn Gly Gly Leu Leu Ala Pro Glu Ala Pro Leu
Asp Phe 85 90 95 Gly Asn Ala Ala Thr Gly Cys Arg Leu Thr Met Gly
Leu Val Gly Val 100 105 110 Tyr Asp Phe Asp Ser Thr Phe Ile Gly Asp
Ala Ser Leu Thr Lys Arg 115 120 125 Pro Met Gly Arg Val Leu Asn Pro
Leu Arg Glu Met Gly Val Gln Val 130 135 140 Lys Ser Glu Asp Gly Asp
Arg Leu Pro Val Thr Leu Arg Gly Pro Lys 145 150 155 160 Thr Pro Thr
Pro Ile Thr Tyr Arg Val Pro Met Ala Ser Ala Gln Val 165 170 175 Lys
Ser Ala Val Leu Leu Ala Gly Leu Asn Thr Pro Gly Ile Thr Thr 180 185
190 Val Ile Glu Pro Ile Met Thr Arg Asp His Thr Glu Lys Met Leu Gln
195 200 205 Gly Phe Gly Ala Asn Leu Thr Val Glu Thr Asp Ala Asp Gly
Val Arg 210 215 220 Thr Ile Arg Leu Glu Gly Arg Gly Lys Leu Thr Gly
Gln Val Ile Asp 225 230 235 240 Val Pro Gly Asp Pro Ser Ser Thr Ala
Phe Pro Leu Val Ala Ala Leu 245 250 255 Leu Val Pro Gly Ser Asp Val
Thr Ile Leu Asn Val Leu Met Asn Pro 260 265 270 Thr Arg Thr Gly Leu
Ile Leu Thr Leu Gln Glu Met Gly Ala Asp Ile 275 280 285 Glu Val Ile
Asn Pro Arg Leu Ala Gly Gly Glu Asp Val Ala Asp Leu 290 295 300 Arg
Val Arg Ser Ser Thr Leu Lys Gly Val Thr Val Pro Glu Asp Arg 305 310
315 320 Ala Pro Ser Met Ile Asp Glu Tyr Pro Ile Leu Ala Val Ala Ala
Ala 325 330 335 Phe Ala Glu Gly Ala Thr Val Met Asn Gly Leu Glu Glu
Leu Arg Val 340 345 350 Lys Glu Ser Asp Arg Leu Ser Ala Val Ala Asn
Gly Leu Lys Leu Asn 355 360 365 Gly Val Asp Cys Asp Glu Gly Glu Thr
Ser Leu Val Val Arg Gly Arg 370 375 380 Pro Asp Gly Lys Gly Leu Gly
Asn Ala Ser Gly Ala Ala Val Ala Thr 385 390 395 400 His Leu Asp His
Arg Ile Ala Met Ser Phe Leu Val Met Gly Leu Val 405 410 415 Ser Glu
Asn Pro Val Thr Val Asp Asp Ala Thr Met Ile Ala Thr Ser 420 425 430
Phe Pro Glu Phe Met Asp Leu Met Ala Gly Leu Gly Ala Lys Ile Glu 435
440 445 Leu Ser Asp Thr Lys Ala Ala 450 455
14448PRTArtificialVariant TIPA EPSPS derived from lettuce 14Lys Pro
Ser Thr Ala Pro Glu Glu Ile Val Leu Gln Pro Ile Lys Glu 1 5 10 15
Ile Ser Gly Thr Val Asn Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg 20
25 30 Ile Leu Leu Leu Ala Ala Leu Ser Glu Gly Thr Thr Val Val Asp
Asn 35 40 45 Leu Leu Asn Ser Asp Asp Val His Tyr Met Leu Gly Ala
Leu Arg Ala 50 55 60 Leu Gly Leu His Val Glu Glu Asn Gly Ala Leu
Lys Arg Ala Ile Val 65 70 75 80 Glu Gly Cys Gly Gly Val Phe Pro Val
Gly Arg Glu Ser Lys Asp Glu 85 90 95 Ile Gln Leu Phe Leu Gly Asn
Ala Gly Ile Ala Met Arg Ala Leu Thr 100 105 110 Ala Ala Val Thr Ala
Ala Gly Gly Ser Ser Ser Tyr Ile Leu Asp Gly 115 120 125 Val Pro Arg
Met Arg Glu Arg Pro Ile Gly Asp Leu Val Thr Gly Leu 130 135 140 Lys
Gln Leu Gly Ala Asp Val Asp Cys Phe Leu Gly Thr Asp Cys Pro 145 150
155 160 Pro Val Arg Val Val Gly Ser Gly Gly Leu Pro Gly Gly Lys Val
Lys 165 170 175 Leu Ser Gly Ser Ile Ser Ser Gln Tyr Leu Thr Ala Leu
Leu Met Ala 180 185 190 Ala Pro Leu Ala Leu Gly Asp Val Glu Ile Glu
Ile Ile Asp Lys Leu 195 200 205 Ile Ser Ile Pro Tyr Val Glu Met Thr
Leu Lys Leu Met Glu Arg Phe 210 215 220 Gly Val Ser Val Gln His Ser
Asp Thr Trp Asp Arg Phe His Val Gln 225 230 235 240 Gly Gly Gln Lys
Tyr Lys Ser Pro Gly Asn Ala Tyr Val Glu Gly Asp 245 250 255 Ala Ser
Ser Ala Ser Tyr Phe Leu Ala Gly Ala Ala Ile Thr Gly Gly 260 265 270
Thr Ile Thr Val Glu Gly Cys Gly Thr Ser Ser Leu Gln Gly Asp Val 275
280 285 Lys Phe Ala Glu Val Leu Gly Gln Met Gly Ala Gln Val Thr Trp
Thr 290 295 300 Glu Asn Ser Val Thr Val Lys Gly Pro Pro Arg Asp Pro
Ser Gly Arg 305 310 315 320 Lys His Leu Arg Pro Val Asp Val Asn Met
Asn Lys Met Pro Asp Val 325 330 335 Ala Met Thr Leu Ala Val Val Ala
Leu Tyr Ala Asp Gly Pro Thr Ala 340 345 350 Ile Arg Asp Val Ala Ser
Trp Arg Val Lys Glu Thr Glu Arg Met Ile 355 360 365 Ala Ile Cys Thr
Glu Leu Arg Lys Leu Gly Ala Thr Val Glu Glu Gly 370 375 380 Pro Asp
Tyr Cys Ile Ile Thr Pro Pro Glu Lys Leu Asn Val Thr Ala 385 390 395
400 Ile Asp Thr Tyr Asp Asp His Arg Met Ala Met Ala Phe Ser Leu Ala
405 410 415 Ala Cys Ala Asp Val Ala Val Thr Ile Lys Asp Pro Gly Cys
Thr Arg 420 425 430 Lys Thr Phe Pro Asp Tyr Phe Glu Val Leu Gln Arg
Phe Ala Lys His 435 440 445 15434PRTArtificialVariant TIPA EPSPS
derived from Zea mays 15Ile Lys Glu Ile Ser Gly Thr Val Lys Leu Pro
Gly Ser Lys Ser Leu 1 5 10 15 Ser Asn Arg Ile Leu Leu Leu Ala Ala
Leu Ser Glu Gly Thr Thr Val 20 25 30 Val Asp Asn Leu Leu Asn Ser
Glu Asp Val His Tyr Met Leu Gly Ala 35 40 45 Leu Arg Thr Leu Gly
Leu Ser Val Glu Ala Asp Lys Ala Ala Lys Arg 50 55 60 Ala Val Val
Val Gly Cys Gly Gly Lys Phe Pro Val Glu Asp Ala Lys 65 70 75 80 Glu
Glu Val Gln Leu Phe Leu Gly Asn Ala Gly Ile Ala Met Arg Ala 85 90
95 Leu Thr Ala Ala Val Thr Ala Ala Gly Gly Asn Ala Thr Tyr Val Leu
100 105 110 Asp Gly Val Pro Arg Met Arg Glu Arg Pro Ile Gly Asp Leu
Val Val 115 120 125 Gly Leu Lys Gln Leu Gly Ala Asp Val Asp Cys Phe
Leu Gly Thr Asp 130 135 140 Cys Pro Pro Val Arg Val Asn Gly Ile Gly
Gly Leu Pro Gly Gly Lys 145 150 155 160 Val Lys Leu Ser Gly Ser Ile
Ser Ser Gln Tyr Leu Ser Ala Leu Leu 165 170 175 Met Ala Ala Pro Leu
Ala Leu Gly Asp Val Glu Ile Glu Ile Ile Asp 180 185 190 Lys Leu Ile
Ser Ile Pro Tyr Val Glu Met Thr Leu Arg Leu Met Glu 195 200 205 Arg
Phe Gly Val Lys Ala Glu His Ser Asp Ser Trp Asp Arg Phe Tyr 210 215
220 Ile Lys Gly Gly Gln Lys Tyr Lys Ser Pro Lys Asn Ala Tyr Val Glu
225 230 235 240 Gly Asp Ala Ser Ser Ala Ser Tyr Phe Leu Ala Gly Ala
Ala Ile Thr 245 250 255 Gly Gly Thr Val Thr Val Glu Gly Cys Gly Thr
Thr Ser Leu Gln Gly 260 265 270 Asp Val Lys Phe Ala Glu Val Leu Glu
Met Met Gly Ala Lys Val Thr 275 280 285 Trp Thr Glu Thr Ser Val Thr
Val Thr Gly Pro Pro Arg Glu Pro Phe 290 295 300 Gly Arg Lys His Leu
Lys Ala Ile Asp Val Asn Met Asn Lys Met Pro 305 310 315 320 Asp Val
Ala Met Thr Leu Ala Val Val Ala Leu Phe Ala Asp Gly Pro 325 330 335
Thr Ala Ile Arg Asp Val Ala Ser Trp Arg Val Lys Glu Thr Glu Arg 340
345 350 Met Val Ala Ile Arg Thr Glu Leu Thr Lys Leu Gly Ala Ser Val
Glu 355 360 365 Glu Gly Pro Asp Tyr Cys Ile Ile Thr Pro Pro Glu Lys
Leu Asn Val 370 375 380 Thr Ala Ile Asp Thr Tyr Asp Asp His Arg Met
Ala Met Ala Phe Ser 385 390 395 400 Leu Ala Ala Cys Ala Glu Val Pro
Val Thr Ile Arg Asp Pro Gly Cys 405 410 415 Thr Arg Lys Thr Phe Pro
Asp Tyr Phe Asp Val Leu Ser Thr Phe Val 420 425 430 Lys Asn
16428PRTXanthomonas campestris 16Met Lys Ile Tyr Lys Leu Gln Thr
Pro Val Asn Ala Ile Leu Glu Asn 1 5 10 15 Ile Ala Ala Asp Lys Ser
Ile Ser His Arg Phe Ala Ile Phe Ser Leu 20 25 30 Leu Thr Gln Glu
Glu Asn Lys Ala Gln Asn Tyr Leu Leu Ala Gln Asp 35 40 45 Thr Leu
Asn Thr Leu Glu Ile Ile Lys Asn Leu Gly Ala Lys Ile Glu 50 55 60
Gln Lys Asp Ser Cys Val Lys Ile Ile Pro Pro Lys Glu Ile Leu Ser 65
70 75 80 Pro Asn Cys Ile Leu Asp Cys Gly Asn Ser Gly Thr Ala Met
Arg Leu 85 90 95 Met Ile Gly Phe Leu Ala Gly Ile Ser Gly Phe Phe
Val Leu Ser Gly 100
105 110 Asp Lys Tyr Leu Asn Asn Arg Pro Met Arg Arg Ile Ser Lys Pro
Leu 115 120 125 Thr Gln Ile Gly Ala Arg Ile Tyr Gly Arg Asn Glu Ala
Asn Leu Ala 130 135 140 Pro Leu Cys Ile Glu Gly Gln Lys Leu Lys Ala
Phe Asn Phe Lys Ser 145 150 155 160 Glu Ile Ser Ser Ala Gln Val Lys
Thr Ala Met Ile Leu Ser Ala Phe 165 170 175 Arg Ala Asp Asn Val Cys
Thr Phe Ser Glu Ile Ser Leu Ser Arg Asn 180 185 190 His Ser Glu Asn
Met Leu Lys Ala Met Lys Ala Pro Ile Arg Val Ser 195 200 205 Asn Asp
Gly Leu Ser Leu Glu Ile Asn Pro Leu Lys Lys Pro Leu Lys 210 215 220
Ala Gln Asn Ile Ile Ile Pro Asn Asp Pro Ser Ser Ala Phe Tyr Phe 225
230 235 240 Val Leu Ala Ala Ile Ile Leu Pro Lys Ser Gln Ile Ile Leu
Lys Asn 245 250 255 Ile Leu Leu Asn Pro Thr Arg Ile Glu Ala Tyr Lys
Ile Leu Gln Lys 260 265 270 Met Gly Ala Lys Leu Glu Met Thr Ile Thr
Gln Asn Asp Phe Glu Thr 275 280 285 Ile Gly Glu Ile Arg Val Glu Ser
Ser Lys Leu Asn Gly Ile Glu Val 290 295 300 Lys Asp Asn Ile Ala Trp
Leu Ile Asp Glu Ala Pro Ala Leu Ala Ile 305 310 315 320 Ala Phe Ala
Leu Ala Lys Gly Lys Ser Ser Leu Ile Asn Ala Lys Glu 325 330 335 Leu
Arg Val Lys Glu Ser Asp Arg Ile Ala Val Met Val Glu Asn Leu 340 345
350 Lys Leu Cys Gly Val Glu Ala Arg Glu Leu Asp Asp Gly Phe Glu Ile
355 360 365 Glu Gly Gly Cys Glu Leu Lys Ser Ser Lys Ile Lys Ser Tyr
Gly Asp 370 375 380 His Arg Ile Ala Met Ser Phe Ala Ile Leu Gly Leu
Leu Cys Gly Ile 385 390 395 400 Glu Ile Asp Asp Ser Asp Cys Ile Lys
Thr Ser Phe Pro Asn Phe Ile 405 410 415 Glu Ile Leu Ser Asn Leu Gly
Ala Arg Ile Asp Tyr 420 425 17443PRTCaulobacter crescentus 17Met
Ser Leu Ala Gly Leu Lys Ser Ala Pro Gly Gly Ala Leu Arg Gly 1 5 10
15 Ile Val Arg Ala Pro Gly Asp Lys Ser Ile Ser His Arg Ser Met Ile
20 25 30 Leu Gly Ala Leu Ala Thr Gly Thr Thr Thr Val Glu Gly Leu
Leu Glu 35 40 45 Gly Asp Asp Val Leu Ala Thr Ala Arg Ala Met Gln
Ala Phe Gly Ala 50 55 60 Arg Ile Glu Arg Glu Gly Val Gly Arg Trp
Arg Ile Glu Gly Lys Gly 65 70 75 80 Gly Phe Glu Glu Pro Val Asp Val
Ile Asp Cys Gly Asn Ala Gly Thr 85 90 95 Gly Val Arg Leu Ile Met
Gly Ala Ala Ala Gly Phe Ala Met Cys Ala 100 105 110 Thr Phe Thr Gly
Asp Gln Ser Leu Arg Gly Arg Pro Met Gly Arg Val 115 120 125 Leu Asp
Pro Leu Ala Arg Met Gly Ala Thr Trp Leu Gly Arg Asp Lys 130 135 140
Gly Arg Leu Pro Leu Thr Leu Lys Gly Gly Asn Leu Arg Gly Leu Asn 145
150 155 160 Tyr Thr Leu Pro Met Ala Ser Ala Gln Val Lys Ser Ala Val
Leu Leu 165 170 175 Ala Gly Leu His Ala Glu Gly Gly Val Glu Val Ile
Glu Pro Glu Ala 180 185 190 Thr Arg Asp His Thr Glu Arg Met Leu Arg
Ala Phe Gly Ala Glu Val 195 200 205 Ile Val Glu Asp Arg Lys Ala Gly
Asp Lys Thr Phe Arg His Val Arg 210 215 220 Leu Pro Glu Gly Gln Lys
Leu Thr Gly Thr His Val Ala Val Pro Gly 225 230 235 240 Asp Pro Ser
Ser Ala Ala Phe Pro Leu Val Ala Ala Leu Ile Val Pro 245 250 255 Gly
Ser Glu Val Thr Val Glu Gly Val Met Leu Asn Glu Leu Arg Thr 260 265
270 Gly Leu Phe Thr Thr Leu Gln Glu Met Gly Ala Asp Leu Val Ile Ser
275 280 285 Asn Val Arg Val Ala Ser Gly Glu Glu Val Gly Asp Ile Thr
Ala Arg 290 295 300 Tyr Ser Gln Leu Lys Gly Val Val Val Pro Pro Glu
Arg Ala Pro Ser 305 310 315 320 Met Ile Asp Glu Tyr Pro Ile Leu Ala
Val Ala Ala Ala Phe Ala Ser 325 330 335 Gly Glu Thr Val Met Arg Gly
Val Gly Glu Met Arg Val Lys Glu Ser 340 345 350 Asp Arg Ile Ser Leu
Thr Ala Asn Gly Leu Lys Ala Cys Gly Val Gln 355 360 365 Val Val Glu
Glu Pro Glu Gly Phe Ile Val Thr Gly Thr Gly Gln Pro 370 375 380 Pro
Lys Gly Gly Ala Thr Val Val Thr His Gly Asp His Arg Ile Ala 385 390
395 400 Met Ser His Leu Ile Leu Gly Met Ala Ala Gln Ala Glu Val Ala
Val 405 410 415 Asp Glu Pro Gly Met Ile Ala Thr Ser Phe Pro Gly Phe
Ala Asp Leu 420 425 430 Met Arg Gly Leu Gly Ala Thr Leu Ala Glu Ala
435 440 18441DNAArtificialArtificial primer 18atg ata gag gtg aaa
ccg att aac gca gag gat acc tat gaa cta agg 48Met Ile Glu Val Lys
Pro Ile Asn Ala Glu Asp Thr Tyr Glu Leu Arg 1 5 10 15 cat aga ata
ctc aga cca aac cag ccg ata gaa gcg tgt atg ttt gaa 96His Arg Ile
Leu Arg Pro Asn Gln Pro Ile Glu Ala Cys Met Phe Glu 20 25 30 agc
gat tta ctt cgt ggt gca ttt cac tta ggc ggc ttt tac agg ggc 144Ser
Asp Leu Leu Arg Gly Ala Phe His Leu Gly Gly Phe Tyr Arg Gly 35 40
45 aaa ctg att tcc ata gct tca ttc cac cag gcc gag cac tcg gaa ctc
192Lys Leu Ile Ser Ile Ala Ser Phe His Gln Ala Glu His Ser Glu Leu
50 55 60 caa ggc cag aaa cag tac cag ctc cga ggt atg gct acc ttg
gaa ggt 240Gln Gly Gln Lys Gln Tyr Gln Leu Arg Gly Met Ala Thr Leu
Glu Gly 65 70 75 80 tat cgt gag cag aaa gcg gga tca act cta gtt aaa
cac gct gaa gaa 288Tyr Arg Glu Gln Lys Ala Gly Ser Thr Leu Val Lys
His Ala Glu Glu 85 90 95 atc ctt cgt aag agg ggg gcg gac atg ctt
tgg tgt aat gcg agg aca 336Ile Leu Arg Lys Arg Gly Ala Asp Met Leu
Trp Cys Asn Ala Arg Thr 100 105 110 tcc gcc tca ggc tac tac aaa aag
tta ggc ttc agc gag cag gga gag 384Ser Ala Ser Gly Tyr Tyr Lys Lys
Leu Gly Phe Ser Glu Gln Gly Glu 115 120 125 ata ttt gac acg ccg cca
gta gga cct cac atc ctg atg tat aaa agg 432Ile Phe Asp Thr Pro Pro
Val Gly Pro His Ile Leu Met Tyr Lys Arg 130 135 140 atc aca taa
441Ile Thr 145 19146PRTArtificialSynthetic Construct 19Met Ile Glu
Val Lys Pro Ile Asn Ala Glu Asp Thr Tyr Glu Leu Arg 1 5 10 15 His
Arg Ile Leu Arg Pro Asn Gln Pro Ile Glu Ala Cys Met Phe Glu 20 25
30 Ser Asp Leu Leu Arg Gly Ala Phe His Leu Gly Gly Phe Tyr Arg Gly
35 40 45 Lys Leu Ile Ser Ile Ala Ser Phe His Gln Ala Glu His Ser
Glu Leu 50 55 60 Gln Gly Gln Lys Gln Tyr Gln Leu Arg Gly Met Ala
Thr Leu Glu Gly 65 70 75 80 Tyr Arg Glu Gln Lys Ala Gly Ser Thr Leu
Val Lys His Ala Glu Glu 85 90 95 Ile Leu Arg Lys Arg Gly Ala Asp
Met Leu Trp Cys Asn Ala Arg Thr 100 105 110 Ser Ala Ser Gly Tyr Tyr
Lys Lys Leu Gly Phe Ser Glu Gln Gly Glu 115 120 125 Ile Phe Asp Thr
Pro Pro Val Gly Pro His Ile Leu Met Tyr Lys Arg 130 135 140 Ile Thr
145 20433DNAArtificial SequenceBased on PClSV promoter sequence
20agatcttgag ccaatcaaag aggagtgatg tagacctaaa gcaataatgg agccatgacg
60taagggctta cgcccatacg aaataattaa aggctgatgt gacctgtcgg tctctcagaa
120cctttacttt ttatgtttgg cgtgtatttt taaatttcca cggcaatgac
gatgtgaccc 180aacgagatct tgagccaatc aaagaggagt gatgtagacc
taaagcaata atggagccat 240gacgtaaggg cttacgccca tacgaaataa
ttaaaggctg atgtgacctg tcggtctctc 300agaaccttta ctttttatat
ttggcgtgta tttttaaatt tccacggcaa tgacgatgtg 360acctgtgcat
ccgctttgcc tataaataag ttttagtttg tattgatcga cacggtcgag
420aagacacggc cat 4332157PRTPisum sativum 21Met Ala Ser Met Ile Ser
Ser Ser Ala Val Thr Thr Val Ser Arg Ala 1 5 10 15 Ser Arg Gly Gln
Ser Ala Ala Met Ala Pro Phe Gly Gly Leu Lys Ser 20 25 30 Met Thr
Gly Phe Pro Val Arg Lys Val Asn Thr Asp Ile Thr Ser Ile 35 40 45
Thr Ser Asn Gly Gly Arg Val Lys Cys 50 55 2285PRTArabidopsis
thaliana 22Met Ala Ser Ser Met Leu Ser Ser Ala Thr Met Val Ala Ser
Pro Ala 1 5 10 15 Gln Ala Thr Met Val Ala Pro Phe Asn Gly Leu Lys
Ser Ser Ala Ala 20 25 30 Phe Pro Ala Thr Arg Lys Ala Asn Asn Asp
Ile Thr Ser Ile Thr Ser 35 40 45 Asn Gly Gly Arg Val Asn Cys Met
Gln Val Trp Pro Pro Ile Glu Lys 50 55 60 Lys Lys Phe Glu Thr Leu
Ser Tyr Leu Pro Asp Leu Thr Asp Ser Gly 65 70 75 80 Gly Arg Val Asn
Cys 85 2376PRTArabidopsis thaliana 23Met Ala Gln Val Ser Arg Ile
Cys Asn Gly Val Gln Asn Pro Ser Leu 1 5 10 15 Ile Ser Asn Leu Ser
Lys Ser Ser Gln Arg Lys Ser Pro Leu Ser Val 20 25 30 Ser Leu Lys
Thr Gln Gln His Pro Arg Ala Tyr Pro Ile Ser Ser Ser 35 40 45 Trp
Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu Leu Arg 50 55
60 Pro Leu Lys Val Met Ser Ser Val Ser Thr Ala Cys 65 70 75
2476PRTArabidopsis thaliana 24Met Ala Gln Val Ser Arg Ile Cys Asn
Gly Val Gln Asn Pro Ser Leu 1 5 10 15 Ile Ser Asn Leu Ser Lys Ser
Ser Gln Arg Lys Ser Pro Leu Ser Val 20 25 30 Ser Leu Lys Thr Gln
Gln His Pro Arg Ala Tyr Pro Ile Ser Ser Ser 35 40 45 Trp Gly Leu
Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu Leu Arg 50 55 60 Pro
Leu Lys Val Met Ser Ser Val Ser Thr Ala Cys 65 70 75 2572PRTPetunia
hybrida 25Met Ala Gln Ile Asn Asn Met Ala Gln Gly Ile Gln Thr Leu
Asn Pro 1 5 10 15 Asn Ser Asn Phe His Lys Pro Gln Val Pro Lys Ser
Ser Ser Phe Leu 20 25 30 Val Phe Gly Ser Lys Lys Leu Lys Asn Ser
Ala Asn Ser Met Leu Val 35 40 45 Leu Lys Lys Asp Ser Ile Phe Met
Gln Lys Phe Cys Ser Phe Arg Ile 50 55 60 Ser Ala Ser Val Ala Thr
Ala Cys 65 70 2669PRTTriticum aestivum 26Met Ala Ala Leu Val Thr
Ser Gln Leu Ala Thr Ser Gly Thr Val Leu 1 5 10 15 Ser Val Thr Asp
Arg Phe Arg Arg Pro Gly Phe Gln Gly Leu Arg Pro 20 25 30 Arg Asn
Pro Ala Asp Ala Ala Leu Gly Met Arg Thr Val Gly Ala Ser 35 40 45
Ala Ala Pro Lys Gln Ser Arg Lys Pro His Arg Phe Asp Arg Arg Cys 50
55 60 Leu Ser Met Val Val 65 27171DNAPisum sativum 27atggcttcta
tgatatcctc ttccgctgtg acaacagtca gccgtgcctc tagggggcaa 60tccgccgcaa
tggctccatt cggcggcctc aaatccatga ctggattccc agtgaggaag
120gtcaacactg acattacttc cattacaagc aatggtggaa gagtaaagtg c
17128255DNAArabidopsis thaliana 28atggcttcct ctatgctctc ttccgctact
atggttgcct ctccggctca ggccactatg 60gtcgctcctt tcaacggact taagtcctcc
gctgccttcc cagccacccg caaggctaac 120aacgacatta cttccatcac
aagcaacggc ggaagagtta actgtatgca ggtgtggcct 180ccgattgaaa
agaagaagtt tgagactctc tcttaccttc ctgaccttac cgattccggt
240ggtcgcgtca actgc 25529228DNAArabidopsis thaliana 29atggcgcaag
ttagcagaat ctgcaatggt gtgcagaacc catctcttat ctccaatctc 60tcgaaatcca
gtcaacgcaa atctccctta tcggtttctc tgaagacgca gcagcatcca
120cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga gtgggatgac
gttaattggc 180tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca cggcgtgc
22830228DNAArtificial sequenceArtificial primer 30atggcgcaag
ttagcagaat ctgcaatggt gtgcagaacc catctcttat ctccaatctc 60tcgaaatcca
gtcaacgcaa atctccctta tcggtttctc tgaagacgca gcagcatcca
120cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga gtgggatgac
gttaattggc 180tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca cggcgtgc
22831216DNAArtificial sequenceArtificial primer 31atggcccaga
tcaacaacat ggcccagggc atccagaccc tgaaccctaa ctctaacttc 60cacaagccgc
aagtgcccaa gtctagctcc ttcctcgtgt tcggctccaa gaagctcaag
120aatagcgcca attccatgct ggtcctgaag aaagactcga tcttcatgca
gaagttctgc 180tcctttcgca tcagtgcttc ggttgcgact gcctgc
21632207DNAArtificial sequenceArtificial primer 32atggcggcac
tggtgacctc ccagctcgcg acaagcggca ccgtcctgtc ggtgacggac 60cgcttccggc
gtcccggctt ccagggactg aggccacgga acccagccga tgccgctctc
120gggatgagga cggtgggcgc gtccgcggct cccaagcaga gcaggaagcc
acaccgtttc 180gaccgccggt gcttgagcat ggtcgtc 207
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References