U.S. patent application number 17/403476 was filed with the patent office on 2022-03-31 for novel herbicide resistance genes.
The applicant listed for this patent is DOW AGROSCIENCES LLC. Invention is credited to Nicole ARNOLD, Justin LIRA, Donald MERLO, Terry WRIGHT.
Application Number | 20220098610 17/403476 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-31 |
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United States Patent
Application |
20220098610 |
Kind Code |
A1 |
WRIGHT; Terry ; et
al. |
March 31, 2022 |
NOVEL HERBICIDE RESISTANCE GENES
Abstract
The subject invention provides novel plants that are not only
resistant to 2,4-D and other phenoxy auxin herbicides, but also to
aryloxyphenoxypropionate herbicides. Heretofore, there was no
expectation or suggestion that a plant with both of these
advantageous properties could be produced by the introduction of a
single gene. The subject invention also includes plants that
produce one or more enzymes of the subject invention alone or
"stacked" together with another herbicide resistance gene,
preferably a glyphosate resistance gene, so as to provide broader
and more robust weed control, increased treatment flexibility, and
improved herbicide resistance management options. More
specifically, preferred enzymes and genes for use according to the
subject invention are referred to herein as AAD (aryloxyalkanoate
dioxygenase) genes and proteins. No .alpha.-ketoglutarate-dependent
dioxygenase enzyme has previously been reported to have the ability
to degrade herbicides of different chemical classes and modes of
action. This highly novel discovery is the basis of significant
herbicide tolerant crop trait opportunities as well as development
of selectable marker technology. The subject invention also
includes related methods of controlling weeds. The subject
invention enables novel combinations of herbicides to be used in
new ways. Furthermore, the subject invention provides novel methods
of preventing the formation of, and controlling, weeds that are
resistant (or naturally more tolerant) to one or more herbicides
such as glyphosate.
Inventors: |
WRIGHT; Terry; (Westfield,
IN) ; LIRA; Justin; (Zionsville, IN) ; MERLO;
Donald; (Carmel, IN) ; ARNOLD; Nicole;
(Indianapolis, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DOW AGROSCIENCES LLC |
Indianapolis |
IN |
US |
|
|
Appl. No.: |
17/403476 |
Filed: |
August 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17175966 |
Feb 15, 2021 |
11149283 |
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17403476 |
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17143824 |
Jan 7, 2021 |
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17175966 |
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15288406 |
Oct 7, 2016 |
10947555 |
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17143824 |
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14820893 |
Aug 7, 2015 |
10174337 |
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15288406 |
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12951813 |
Nov 22, 2010 |
9127289 |
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14820893 |
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11587893 |
May 22, 2008 |
7838733 |
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PCT/US2005/014737 |
May 2, 2005 |
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12951813 |
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60567052 |
Apr 30, 2004 |
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International
Class: |
C12N 15/82 20060101
C12N015/82; C12N 9/02 20060101 C12N009/02 |
Claims
1. A transgenic soybean plant cell comprising a recombinant
polynucleotide that encodes an AAD- 1 protein that exhibits
aryloxyalkanoate dioxygenase activity wherein said activity
enzymatically degrades a phenoxy auxin herbicide and an
(R)-aryloxyphenoxypropionate herbicide, which are aryloxyalkanoate
herbicides, further wherein said AAD-1 protein comprises: i) an
amino acid sequence having at least 85% sequence identity with SEQ
ID NO: 9; and ii) an AAD- 1 motif having the general formula of:
HX.sub.112D(X).sub.114-137T(X).sub.139-269H(X).sub.271-280R (SEQ ID
NO: 34), wherein X.sub.112 represents a single amino acid at
position 112, relative to the sequence of SEQ ID NO: 9;
(X).sub.114-137 represents a sequence of 24 amino acids;
(X).sub.139-269 represents a sequence of 131 amino acids; and
(X).sub.271-280 represents a sequence of 10 amino acids, wherein
said AAD- 1 protein when expressed in a soybean plant cell renders
said soybean plant cell tolerant to a phenoxy auxin herbicide and
an (R)-aryloxyphenoxypropionate herbicide, as compared to an
untransformed soybean plant cell.
2. The plant cell of claim 1 wherein said AAD-1 motif has the
general formula of:
HX.sub.112D(X).sub.114-137T(X).sub.139-269H(X).sub.271-280R(X).sub.282-28-
4R (SEQ ID NO: 35), wherein (X).sub.282-284 represents a sequence
of 3 amino acids.
3. A transgenic soybean plant comprising a plurality of the soybean
plant cell of claim 1, wherein expression of said polynucleotide
renders said plant tolerant to said aryloxyalkanoate herbicide.
4. The plant of claim 3 wherein said aryloxyalkanoate herbicide
selected from the group consisting of 2,4-dichlorophenoxyacetic
acid, MCPA, dichlorprop, and mecoprop.
5. The plant of claim 3 wherein said aryloxyalkanoate herbicide is
an (R)-aryloxyphenoxypropionate.
6. The plant of claim 3 wherein said aryloxyalkanoate herbicide is
selected from the group consisting of (R)-fluazifop, (R)-haloxyfop,
(R)-diclofop, (R)-quizalofop, (R)-fenoxaprop, (R)-metamifop,
(R)-cyhalofop, and (R)-clodinofop.
7. The plant of claim 3 wherein said plant further comprises a
second herbicide resistance gene.
8. The plant of claim 7 wherein said second herbicide resistance
gene renders said plant resistant to an herbicide selected from the
group consisting of glyphosate, glufosinate, acetolactate synthase
(ALS) inhibitors, inhibitors of
4-hydroxyphenyl-pyruvate-dioxygenase (HPPD), dicamba, and
inhibitors of protoporphyrinogen oxidase (PPO).
9. A method of controlling at least one weed in a field, wherein
said field contains at least one plant of claim 3 wherein said
method comprises applying to at least a portion of said field a
first herbicide selected from the group consisting of a phenoxy
auxin herbicide and an (R)-aryloxyphenoxypropionate herbicide.
10. The method of claim 9 wherein said phenoxy auxin herbicide is
an R-enantiomer of a chiral phenoxy auxin.
11. The method of claim 9 wherein said phenoxy auxin herbicide is
an achiral phenoxy auxin selected from the group consisting of
2,4-D and MCPA.
12. The method of claim 9 wherein said (R)-aryloxyphenoxypropionate
herbicide is selected from the group consisting of (R)-fluazifop,
(R)-haloxyfop, (R)-diclofop, (R)-quizalofop, (R)-fenoxaprop,
(R)-metamifop, (R)-cyhalofop, and (R)-clodinofop.
13. The method of claim 9 wherein said method comprises applying a
second herbicide.
14. The method of claim 13 wherein said first herbicide and said
second herbicide are applied sequentially.
15. The method of claim 13 wherein said first herbicide and said
second herbicide are applied concurrently.
16. The method of claim 13 wherein said first herbicide is a
phenoxy auxin and said second herbicide is an
(R)-aryloxyphenoxypropionate.
17. The method of claim 13 wherein said second herbicide is
selected from the group consisting of glyphosate, glufosinate,
dicamba, acetolactate synthase (ALS) inhibitors, protoporphyrinogen
oxidase (PPO) inhibitors, and 4-hydroxyphenyl-pyruvate-dioxygenase
(HPPD) inhibitors.
18. The method of claim 13, wherein said first herbicide is 2,4-D
and said second herbicide is glyphosate or glufosinate.
19. The method of claim 13, wherein said first herbicide is an
(R)-aryloxyphenoxypropionate and said second herbicide is
glyphosate or glufosinate.
20. The method of claim 13 wherein said plant further comprises a
second herbicide resistance gene that renders said plant resistant
to said second herbicide.
21. The method of claim 20 wherein said second herbicide resistance
gene is selected from the group consisting of a modified
acetolactate synthase (ALS) gene, a glyphosate resistance gene, a
glufosinate resistance gene, and a dicamba-degrading enzyme
gene.
22. The method of claim 13 wherein said method further comprises
applying a third herbicide.
23. The method of claim 22, wherein said third herbicide is
selected from the group consisting of glyphosate, glufosinate,
HPPD-inhibitors, PPO-inhibitors, ALS inhibitors, and dicamba.
24. The method of claim 23 wherein said first, second and third
herbicides are 2,4-D, quizalofop, and glyphosate.
25. A seed comprising a plurality of the plant cell of claim 1.
26. A method of controlling weeds in a field, wherein said method
comprises applying an aryloxyalkanoate herbicide to said field and
planting a seed of claim 25 in said field within 14 days after
applying said aryloxyalkanoate herbicide.
27. A plant grown from the seed of claim 25, wherein said plant
comprises said polynucleotide.
28. A part, progeny, or asexual propagate of the plant of claim 27,
wherein said part, progeny, or sexual propagate comprises said
polynucleotide.
29. An (R)-aryloxyphenoxypropionate herbicide tolerant transgenic
soybean plant cell comprising a recombinant polynucleotide that
encodes an AAD-1 protein that exhibits aryloxyalkanoate dioxygenase
activity wherein said activity enzymatically degrades a phenoxy
auxin herbicide and an (R)-aryloxyphenoxypropionate herbicide,
further wherein said AAD-1 protein comprises: i) an amino acid
sequence having at least 85% sequence identity with SEQ ID NO: 9;
and ii) an AAD-1 motif having the general formula of:
HX.sub.122D(X).sub.114-137T(X).sub.139-269H(X).sub.271-280R (SEQ DI
NO: 34), wherein X.sub.112 represents a single amino acid at
position 112, relative to the sequence of SEQ ID NO: 9;
(X).sub.114-137 represents a sequence of 24 amino acids;
(X).sub.139-269 represents a sequence of 131 amino acids; and
(X).sub.271-280 represents a sequence of 10 amino acids, wherein
said motif has 90% sequence identity with corresponding amino acids
of position 111 to 281 of SEQ ID NO: 9 wherein said AAD-1 protein
when expressed in a soybean plant cell renders said soybean plant
cell tolerant to a phenoxy auxin herbicide and an
(R)-aryloxyphenoxypropionate herbicide, as compared to an
untransformed soybean plant cell.
30. A nucleic acid encoding a variant AAD-1 protein, wherein said
variant AAD-1 protein enzymatically degrades a phenoxy auxin
herbicide and an (R)-aryloxyphenoxypropionate herbicide, further
wherein said variant AAD-1 protein comprises: i) an amino acid
sequence having at least 85%, but less than 95% sequence identity
with SEQ ID NO: 9; and ii) an AAD-1 motif having the general
formula of:
HX.sub.112D(X).sub.114-137T(X).sub.139-269(X).sub.271-280R, wherein
X.sub.112 represents a single amino acid at position 112, relative
to the sequence of SEQ ID NO: 9; (X).sub.114-137 represents a
sequence of 24 amino acids; (X).sub.139-269 represents a sequence
of 131 amino acids; and (X).sub.271-280 represents a sequence of 10
amino acids.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation application of U.S. Ser.
No. 17/175,966, filed Feb. 15, 2021, which is a continuation
application of U.S. Ser. No. 17/143,824, filed Jan. 7, 2021, which
is a continuation application of LLS, Ser. No. 15/288,406, filed
Oct. 7, 2016, now patented as U.S. Pat. No. 10,947,555, which is a
continuation application of U.S. Ser. No, 14/820,893, filed Aug. 7,
2015, now patented as U.S. Pat. No. 10,174,337, which is a
continuation application of U.S. Ser. No. 12/951,813, filed Nov.
22, 2010, now patented as U.S. Pat. No. 9,127,289, which is a
continuation application of U.S. Ser. No. 11/587,893, filed May 22,
2008. now patented as U.S. Pat. No. 7,838,733, which is a national
stage entry of PCT/US2005/014737, filed May 2, 2005, which claims
the benefit of U.S. Provisional Application Serial No. 60/567,052,
filed Apr. 30, 2004 which are hereby incorporated by reference in
their entirety, including any figures, tables, nucleic acid
sequences, amino acid sequences, or drawings.
INCORPORATION BY REFERENCES OF MATERIAL SUBMITTED
ELECTRONICALLY
[0002] Incorporated by reference in its entirety is a
computer-readable nucleotide/amino acid sequence listing submitted
concurrently herewith and identified as follows: 42 kilobytes ACII
(Text) file named "346146_ST25.txt," created on August 16,
2021.
BACKGROUND OF THE INVENTION
[0003] Weeds can quickly deplete soil of valuable nutrients needed
by crops and other desirable plants. There are many different types
of herbicides presently used for the control of weeds. One
extremely popular herbicide is glyphosate.
[0004] Crops, such as corn, soybeans, canola, cotton, sugar beets,
wheat, turf, and rice, have been developed that are resistant to
glyphosate. Thus, fields with actively growing glyphosate resistant
corn, for example, can be sprayed to control weeds without
significantly damaging the corn plants.
[0005] With the introduction of genetically engineered, glyphosate
tolerant crops (GTCs) in the mid-1990's, growers were enabled with
a simple, convenient, flexible, and inexpensive tool for
controlling a wide spectrum of broadleaf and grass weeds
unparalleled in agriculture. Consequently, producers were quick to
adopt GTCs and in many instances abandon many of the accepted best
agronomic practices such as crop rotation, herbicide mode of action
rotation, tank mixing, incorporation of mechanical with chemical
and cultural weed control. Currently glyphosate tolerant soybean,
cotton, corn, and canola are commercially available in the United
States and elsewhere in the Western Hemisphere. More GTCs (e.g.,
wheat, rice, sugar beets, turf, etc.) are poised for introduction
pending global market acceptance. Many other glyphos ate resistant
species are in experimental to development stages (e.g., alfalfa,
sugar cane, sunflower, beets, peas, carrot, cucumber, lettuce,
onion, strawberry, tomato, and tobacco; forestry species like
poplar and sweetgum; and horticultural species like marigold,
petunia, and begonias; see "isb.vt.edu/cfdocs/fieldtestsl.cfm,
2005" website). Additionally, the cost of glyphosate has dropped
dramatically in recent years to the point that few conventional
weed control programs can effectively compete on price and
performance with glyphosate GTC systems.
[0006] Glyphosate has been used successfully in burndown and other
non-crop areas for total vegetation control for more than 15 years.
In many instances, as with GTCs, glyphosate has been used 1-3 times
per year for 3, 5, 10, up to 15 years in a row. These circumstances
have led to an over-reliance on glyphosate and GTC technology and
have placed a heavy selection pressure on native weed species for
plants that are naturally more tolerant to glyphosate or which have
developed a mechanism to resist glyphosate's herbicidal
activity.
[0007] Extensive use of glyphosate-only weed control programs is
resulting in the selection of glyphosate-resistant weeds, and is
selecting for the propagation of weed species that are inherently
more tolerant to glyphosate than most target species (i.e., weed
shifts). (Ng et al., 2003; Simarmata et al., 2003; Lorraine-Colwill
et al., 2003; Sfiligoj, 2004; Miller et al., 2003; Heap, 2005;
Murphy et al., 2002; Martin et al., 2002.) Although glyphosate has
been widely used globally for more than 15 years, only a handful of
weeds have been reported to have developed resistance to glyphosate
(Heap, 2005); however, most of these have been identified in the
past 3-5 years. Resistant weeds include both grass and broadleaf
species--Lolium rigidum, Lolium multiflorum, Eleusine indica,
Ambrosia artemisiifolia, Conyza canadensis, Conyza bonariensis, and
Plantago lanceolata. Additionally, weeds that had previously not
been an agronomic problem prior to the wide use of GTCs are now
becoming more prevalent and difficult to control in the context of
GTCs, which comprise >80% of U.S. cotton and soybean acres and
>20% of U.S. corn acres (Gianessi, 2005). These weed shifts are
occurring predominantly with (but not exclusively)
difficult-to-control broadleaf weeds. Some examples include
Ipomoea, Amaranthus, Chenopodium, Taraxacum, and Commelina
species.
[0008] In areas where growers are faced with glyphosate resistant
weeds or a shift to more difficult-to-control weed species, growers
can compensate for glyphosate's weaknesses by tank mixing or
alternating with other herbicides that will control the missed
weeds. One popular and efficacious tankmix partner for controlling
broadleaf escapes in many instances has been
2,4-diclorophenoxyacetic acid (2,4-D). 2,4-D has been used
agronomically and in non-crop situations for broad spectrum,
broadleaf weed control for more than 60 years. Individual cases of
more tolerant species have been reported, but 2,4-D remains one of
the most widely used herbicides globally. A limitation to further
use of 2,4-D is that its selectivity in dicot crops like soybean or
cotton is very poor, and hence 2,4-D is not typically used on (and
generally not near) sensitive dicot crops. Additionally, 2,4-D's
use in grass crops is somewhat limited by the nature of crop injury
that can occur. 2,4-D in combination with glyphosate has been used
to provide a more robust burndown treatment prior to planting
no-till soybeans and cotton; however, due to these dicot species'
sensitivity to 2,4-D, these burndown treatments must occur at least
14-30 days prior to planting (Agriliance, 2003).
[0009] 2,4-D is in the phenoxy acid class of herbicides, as are
MCPA, mecoprop, and dichlorprop. 2,4-D has been used in many
monocot crops (such as corn, wheat, and rice) for the selective
control of broadleaf weeds without severely damaging the desired
crop plants. 2,4-D is a synthetic auxin derivative that acts to
deregulate normal cell-hormone homeostasis and impede balanced,
controlled growth; however, the exact mode of action is still not
known.
[0010] 2,4-D has different levels of selectivity on certain plants
(e.g., dicots are more sensitive than grasses). Differential
metabolism of 2,4-D by different plants is one explanation for
varying levels of selectivity. In general, plants metabolize 2,4-D
slowly, so varying plant response to 2,4-D may be more likely
explained by different activity at the target site(s) (WSSA, 2002).
Plant metabolism of 2,4-D typically occurs via a two-phase
mechanism, typically hydroxylation followed by conjugation with
amino acids or glucose (WSSA, 2002).
[0011] Over time, microbial populations have developed an
alternative and efficient pathway for degradation of this
particular xenobiotic, which results in the complete mineralization
of 2,4-D. Successive applications of the herbicide select for
microbes that can utilize the herbicide as a carbon source for
growth, giving them a competitive advantage in the soil. For this
reason, 2,4-D is currently formulated to have a relatively short
soil half-life, and no significant carryover effects to subsequent
crops are encountered. This adds to the herbicidal utility of
2,4-D.
[0012] One organism that has been extensively researched for its
ability to degrade 2,4-D is Ralstonia eutropha (Streber et al.,
1987). The gene that codes for the first enzymatic step in the
mineralization pathway is tfdA. See U.S. Pat. No. 6,153,401 and
GENBANK Acc. No. M16730. TfdA catalyzes the conversion of 2,4-D
acid to dichlorophenol (DCP) via an .alpha.-ketoglutarate-dependent
dioxygenase reaction (Smejkal et al., 2001). DCP has little
herbicidal activity compared to 2,4-D. TfdA has been used in
transgenic plants to impart 2,4-D resistance in dicot plants (e.g.,
cotton and tobacco) normally sensitive to 2,4-D (Streber et al.
(1989), Lyon et al. (1989), Lyon (1993), and U.S. Pat. No.
5,608,147).
[0013] A large number of tfdA-type genes that encode proteins
capable of degrading 2,4-D have been identified from the
environment and deposited into the Genbank database. Many
homologues are similar to tfdA (>85% amino acid identity) and
have similar enzymatic properties to tfdA. However, there are a
number of homologues that have a significantly lower identity to
tfdA (25-50%), yet have the characteristic residues associated with
.alpha.-ketoglutarate dioxygenase Fe.sup.2+ dioxygenases. It is
therefore not obvious what the substrate specificities of these
divergent dioxygenases are.
[0014] One unique example with low homology to tfdA (28% amino acid
identity) is rdpA from Sphingobium herbicidovorans (Kohler et al.,
1999, Westendorf et al., 2002). This enzyme has been shown to
catalyze the first step in (R)-dichlorprop (and other
(R)-phenoxypropionic acids) as well as 2,4-D (a phenoxyacetic acid)
mineralization (Westendorf et al., 2003). Although the organisms
that degrade phenoxypropionic acid were described some time ago,
little progress had been made in characterizing this pathway until
recently (Horvath et al., 1990). An additional complication to
dichlorprop degradation is the stereospecificity (R vs. S) involved
in both the uptake (Kohler, 1999) and initial oxidation of
dichlorprop (Westendorf et al., 2003). Heterologous expression of
rdpA in other microbes, or transformation of this gene into plants,
has not heretofore been reported. Literature has focused primarily
around close homologues of tfdA that primarily degrade achiral
phenoxyacetic acids (e.g., 2,4-D).
[0015] Development of new herbicide-tolerant crop (HTC)
technologies has been limited in success due largely to the
efficacy, low cost, and convenience of GTCs. Consequently, a very
high rate of adoption for GTCs has occurred among producers. This
created little incentive for developing new HTC technologies.
[0016] Aryloxyalkanoate chemical substructures are a common entity
of many commercialized herbicides including the phenoxy auxins
(such as 2,4-D and dichlorprop), pyridyloxy auxins (such as
fluroxypyr and triclopyr), aryloxyphenoxypropionates (AOPP)
acetyl-coenzyme A carboxylase (ACCase) inhibitors (such as
haloxyfop, quizalofop, and diclofop), and 5-substituted
phenoxyacetate protoporphyrinogen oxidase IX inhibitors (such as
pyraflufen and flumiclorac). However, these classes of herbicides
are all quite distinct, and no evidence exists in the current
literature for common degradation pathways among these chemical
classes. Discovery of a multifunctional enzyme for the degradation
of herbicides covering multiple modes would be both unique and
valuable as an HTC trait.
BRIEF SUMMARY OF THE INVENTION
[0017] The subject invention provides novel plants that are not
only resistant to 2,4-D, but also to AOPP herbicides. Heretofore,
there was no expectation or suggestion that a plant with both of
these advantageous properties could be produced by the introduction
of a single gene. The subject invention also includes plants that
produce one or more enzymes of the subject invention "stacked"
together with one or more other herbicide resistance genes,
including, but not limited to, glyphosate-, imidazolinone-, and
glufosinate-resistance genes, so as to provide herbicide-tolerant
plants compatible with broader and more robust weed control and
herbicide resistance management options. The present invention
further includes methods and compositions utilizing homologues of
the genes and proteins exemplified herein.
[0018] In some embodiments, the invention provides monocot and
dicot plants tolerant to 2,4-D, AOPP, and one or more commercially
available herbicides (e.g., glyphosate, imidazolinones,
glufosinate, sulfonylureas, dicamba, bromoxynil, and others).
Vectors comprising nucleic acid sequences responsible for such
herbicide tolerance are also disclosed, as are methods of using
such tolerant plants and combinations of herbicides for weed
control and prevention of weed population shifts. The subject
invention enables novel combinations of herbicides to be used in
new ways. Furthermore, the subject invention provides novel methods
of preventing the development of, and controlling, strains of weeds
that are resistant to one or more herbicides such as glyphosate.
The subject invention enables novel uses of novel combinations of
herbicides and crops, including preplant application to an area to
be planted immediately prior to planting with seed for plants that
would otherwise be sensitive to that herbicide (such as 2,4-D).
[0019] The subject invention relates in part to the identification
of an enzyme that is not only able to degrade 2,4-D, but also
surprisingly possesses novel properties, which distinguish the
enzyme of the subject invention from previously known tfdA
proteins, for example. More specifically, the subject invention
relates to the use of an enzyme that is capable of degrading both
2,4-D and AOPP herbicides, in an enantiospecific manner No
.alpha.-ketoglutarate-dependent dioxygenase enzyme has previously
been reported to have the ability to degrade herbicides of
different chemical classes and modes of action. The preferred
enzyme and gene for use according to the subject invention are
referred to herein as AAD-1 (AryloxyAlkanoate Dioxygenase). This
highly novel discovery is the basis of significant HTC trait and
selectable marker opportunities.
[0020] There was no prior motivation to produce plants comprising
an AAD-1 gene (preferably an AAD-1 polynucleotide that has a
sequence optimized for expression in one or more types of plants,
as exemplified herein), and there was no expectation that such
plants could effectively produce an AAD-1 enzyme to render the
plants resistant to not only phenoxy acid herbicides (such as
2,4-D) but also AOPP herbicides (such as quizalofop, haloxyfop, et
al.). Thus, the subject invention provides many advantages that
were not heretofore thought to be possible in the art.
[0021] This invention also relates in part to the identification
and use of genes encoding aryloxyalkanoate dioxygenase enzymes that
are capable of degrading phenoxy auxin and aryloxyphenoxypropionate
herbicides. Methods of screening proteins for these activities are
within the scope of the subject invention. Thus, the subject
invention includes degradation of 2,4-dichlorophenoxyacetic acid,
other phenoxyalkanoate auxin herbicides, and
aryloxyphenoxypropionate herbicides by a recombinantly expressed
AAD-1 enzyme. The subject invention also includes methods of
controlling weeds wherein said methods comprise applying one or
more AOPP, phenoxy auxin, or other aryloxyalkanoate herbicides to
plants comprising an AAD-1 gene. The subject invention also
provides methods of using an AAD-1 gene as a selectable marker for
identifying plant cells and whole plants transformed with AAD-1,
optionally including one, two, or more exogenous genes
simultaneously inserted into target plant cells. Methods of the
subject invention include selecting transformed cells that are
resistant to appropriate levels of an herbicide. The subject
invention further includes methods of preparing a polypeptide,
having the biological activity of aryloxyalkanoate dioxygenase, by
culturing plants and/or cells of the subject invention.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 shows a general scheme for dioxygenase cleavage of
phenoxy auxin or AOPP herbicides.
[0023] FIG. 2 shows loss of herbicidal activity from a 2,4-D
solution treated with AAD-1.
[0024] FIG. 3 shows loss of herbicidal activity from a haloxyfop
solution treated with AAD-1.
[0025] FIG. 4 shows anticipated phenols produced from
representative herbicides catalyzed by AAD-1.
[0026] FIG. 5 shows 2,4-dichlorophenol production by recombinant
AAD-1.
[0027] FIGS. 6A and 6B show phenol production by recombinant AAD-1
from various herbicide substrates.
[0028] FIG. 7 shows AAD-1 reaction rate to substrate concentration
for four herbicide substrates.
[0029] FIGS. 8A and 8B show that AAD-1 (v3) was expressed equally
in Arabidopsis leaves of different ages but continued to accumulate
throughout the 25 days of experiment. Plants that were not sprayed
with the herbicide 2,4-D (panel A) expressed a little more AAD-1
(v3) than those had been sprayed (panel B). Bars represent the
mean.+-.SEM of 5 leaves from 5 different plants, with percent
expression of AAD-1 (v3) normalized to total soluble protein. Light
bars represent the third young leaves (N-3) collected from the top,
dark bars represent the 5.sup.th oldest leaves from the bottom.
[0030] FIGS. 9A, 9B, and 9C show injury of Arabidopsis plants after
2,4-D treatment.
[0031] Four different lines were each treated with four different
doses of 2,4-D and their injury was graded 4 (panel A) and 14
(panel B) days after treatment. Their expression of AAD-1 (v3) in
leaves was also determined using ELISA (panel C). The results were
mean.+-.SEM of five leaves from five different plants received the
same treatment.
[0032] FIG. 10 illustrates that pDAB3230-transformed Arabidopsis
(AAD-1+EPSPS) shows >14-fold level of glyphosate tolerance 7 DAT
vs. wildtype and transformed control Arabidopsis lines.
[0033] FIG. 11 shows dose response of callused maize suspensions to
R-haloxyfop.
[0034] FIG. 12 shows that at 1 .mu.M cyhalofop phenol, growth is
still 76% as high as the control without cyhalofop phenol.
[0035] FIG. 13 illustrates dose-response data on one transgenic
event, 3404-006, to haloxyfop.
[0036] FIG. 14 shows the responses of several AAD-1
(v3)-transformed and non-transformed event clones to lethal doses
of two AOPP herbicides (haloxyfop and quizalofop) applied as a
postemergence spray 1 week prior.
[0037] FIG. 15 shows three different T2 lineages from 3404
transformations that were pre-screened with Liberty.RTM. to remove
nulls, which were chosen to compare their tolerance to quizalofop
with respect to their AAD-1 expression. Expression was measured at
14 DAT (data not shown) and at 30 DAT.
[0038] FIG. 16 shows AAD-1 (v3)-transformed corn tolerant to 8X
field rates of quizalofop (Assure II) under field conditions.
[0039] FIG. 17 illustrates data from immature maize embryos grown
on cyhalofop-containing media.
[0040] FIG. 18 shows Western Blotting analysis on soybean calli
transformed with AAD-1 (V3) gene indicating that the callus cells
are expressing AAD-1 (v3) protein.
[0041] FIG. 19 shows fitted curves for 2,4-D degradation rates by
AAD-2 (v1) vs. AAD-1 (v1).
[0042] FIG. 20 shows the response of AAD-1 v3 (plant optimized), or
AAD-1 (v2) (native), AAD-2 (v1) (native), or AAD-2 (v2) (plant
optimized)-transformed Ti Arabidopsis to a range of 2,4-D rates
applied postemergence. Each pot represents an individual
transformation event within each gene T.sub.1 family
[0043] FIG. 21 shows western blot analysis of individual native
AAD-2 (v1)-transformed T.sub.1 Arabidopsis plants. This shows that
plants expressing the AAD-2 (v1) protein are suffering severe
injury from 200 g ae (acid equivalent)/ha 2,4-D treatments, which
normally causes little injury to native AAD-1 (v2) or plant
optimized AAD-1 (v3)-transformed Arabidopsis. AAD-2 protein is
identified on the gel. Several background bands were detected in
AAD-2-transformed and Pat/Cry1F-transformed samples.
[0044] FIG. 22 shows that the relative AAD-2 (v1) activity on the
substrates was
2,4-D=dichlorprop>(R,S)-haloxyfop>>(R)-haloxyfop.
BRIEF DESCRIPTION OF THE SEQUENCES
[0045] SEQ ID NO:1 is the sequence of a forward primer used to
amplify the rdpA/AAD-1 (v1) gene.
[0046] SEQ ID NO:2 is the sequence of a reverse primer used to
amplify the rdpA/AAD-1 (v1) gene.
[0047] SEQ ID NO:3 is the nucleotide sequence of AAD-1 (v1) from
Sphingobium herbicidovorans.
[0048] SEQ ID NO:4 is the nucleic acid sequence of the native AAD-1
gene with internal
[0049] Notl restriction site removed. This gene is designated AAD-1
(v2). DNA sequencing confirmed that the correct PCR product was
generated, but an inadvertent change was made at amino acid #212
from arginine to cysteine.
[0050] SEQ ID NO:5 is a "plant-optimized" DNA sequence AAD-1 (v3).
This "gene" encodes SEQ ID NO:11, which is the same as SEQ ID NO:9
except for the addition of an alanine residue at the second
position. The additional alanine codon (GCT) was included to encode
an Nco I site (CCATGG) spanning the ATG start codon, to enable
subsequent cloning operations.
[0051] SEQ ID NO:6 ("rdpA(ncoI)") and SEQ ID NO:7 ("3'saci") were
used to amplify a DNA fragment using the Fail Safe PCR System
(Epicenter).
[0052] SEQ ID NO:8 is another PCR primer ("BstEII/Del Notl") that
was used with the "3' SacI" primer.
[0053] SEQ ID NO:9 is the native amino acid sequence encoded by the
AAD-1 (v1) gene from Sphingobium herbicidovorans.
[0054] SEQ ID NO:10 is the amino acid sequence encoded by the AAD-1
(v2) DNA sequence of SEQ ID NO:4.
[0055] SEQ ID NO:11 is the amino acid sequence encoded by the AAD-1
(v3) plant-optimized DNA sequence of SEQ ID NO:5.
[0056] SEQ ID NO:12 is the DNA sequence of the native AAD-2 (v1)
gene.
[0057] SEQ ID NO:13 is the amino acid sequence of the AAD-2 (v1)
protein.
[0058] SEQ ID NO:14 is a forward primer used to amplify AAD-2 (v1)
DNA for cloning.
[0059] SEQ ID NO:15 is a reverse primer used to amplify AAD-2 (v1)
DNA for cloning.
[0060] SEQ ID NO:16 is the M13 forward primer.
[0061] SEQ ID NO:17 is the M13 reverse primer.
[0062] SEQ ID NO:18 is a forward primer used to amplify AAD-2 (v1)
DNA for cloning.
[0063] SEQ ID NO:19 is a reverse primer used to amplify AAD-2 (v1)
DNA for cloning.
[0064] SEQ ID NO:20 is the native soybean EPSPS protein.
[0065] SEQ ID NO:21 is a doubly mutated soybean EPSPS protein
sequence, containing a mutation at residue 183 (threonine of native
protein replaced with isoleucine), and at residue 187 (proline in
native protein replaced with serine).
[0066] SEQ ID NO:22 is the soybean-biased DNA sequence that encodes
the EPSPS protein of SEQ ID NO:21.
[0067] SEQ ID NO:23 is primer Pat 5-3.
[0068] SEQ ID NO:24 is primer Pat 3-3.
[0069] SEQ ID NO:25 is forward primer AAD-1 PTU.
[0070] SEQ ID NO:26 is reverse primer AAD-1 PTU.
[0071] SEQ ID NO:27 is the forward primer for the Coding Region PCR
AAD-1.
[0072] SEQ ID NO:28 is the reverse primer for the Coding Region PCR
AAD-1.
[0073] SEQ ID NO:29 is the AAD-2 (v2) nucleotide (plant
optimized).
[0074] SEQ ID NO:30 is the translated AAD-2 (v2) protein
sequence.
[0075] SEQ ID NO:31 is the Southern fragment PCR AAD-1 forward
primer.
[0076] SEQ ID NO:32 is the Southern fragment PCR AAD-1 reverse
primer.
DETAILED DESCRIPTION OF THE INVENTION
[0077] The subject development of a 2,4-D resistance gene and
subsequent resistant crops provides excellent options for
controlling broadleaf, glyphosate-resistant (or highly tolerant and
shifted) weed species for in-crop applications. 2,4-D is a
broad-spectrum, relatively inexpensive, and robust broadleaf
herbicide that would provide excellent utility for growers if
greater crop tolerance could be provided in dicot and monocot crops
alike. 2,4-D-tolerant transgenic dicot crops would also have
greater flexibility in the timing and rate of application. An
additional utility of an herbicide tolerance trait for 2,4-D would
be its utility to prevent damage to normally sensitive crops from
2,4-D drift, volatilization, inversion (or other off-site movement
phenomenon), misapplication, vandalism and the like. An additional
benefit of the AAD-1 gene is that unlike all tfdA homologues
characterized to date, AAD-1 is able to degrade the R-enantiomers
(herbicidally active isomers) of the chiral phenoxy auxins (e.g.,
dichlorprop and mecoprop) in addition to achiral phenoxy auxins
(e.g., 2,4-D, MCPA, 4-chlorophenoxyacetic acid). See Table 1.
Multiple mixes of different phenoxy auxin combinations have been
used globally to address specific weed spectra and environmental
conditions in various regions. Use of the AAD-1 gene in plants
would afford protection to a much wider spectrum of phenoxy auxin
herbicides, thereby increasing the flexibility and spectra of weeds
that can be controlled, protecting from drift or other off-site
phenoxy herbicide injury for the full breadth of commercially
available phenoxy auxins.
[0078] Table 1. Commercially available phenoxy auxins. Reference to
phenoxy auxin herbicides is generally made to the active acid but
some are commercially formulated as any of a variety of
corresponding ester formulations and these are likewise considered
as substrates for AAD-1 enzyme in planta as general plant esterases
convert these esters to the active acids in planta. Likewise
reference can also be for the corresponding organic or inorganic
salt of the corresponding acid. When chiral propionic acid, salt,
or ester herbicides are indicated, racemic (R,S) or optically
purified (R or S) enantiomers are considered the same herbicides
for the purpose of naming these herbicides, even though different
CAS numbers may correspond to optically pure compounds. Possible
use rate ranges can be as stand-alone treatments or in combination
with other herbicides in both crop and non-crop uses.
TABLE-US-00001 TABLE 1 Commercially available phenoxy auxins. Pre-
Che- Possible ferred mi- use rate use rate cal ranges ranges name
CAS no (g ae/ha) (g ae/ha) Structure 2,4-D 94-75-7 25-4000 280-
1120 ##STR00001## 2,4,5-T 93-76-5 25-4000 25- 4000 ##STR00002##
4-CPA 122-88-3 25-4000 25- 4000 ##STR00003## 3,4-DA 588-22-7
25-4000 25- 4000 ##STR00004## MCPA 94-74-6 25-4000 125- 1550
##STR00005## Di- chlor- prop 120-36-5 25-12000 100- 2240
##STR00006## Meco- prop 7085- 19-0 25-4000 250- 3360 ##STR00007##
Clo- prop 101-10-0 25-4000 25- 4000 ##STR00008## 4-CPP 3307- 39-9
25-4000 25- 4000 ##STR00009## Feno- prop 93-72-1 25-4000 25- 4000
##STR00010## 3,4-DP 3307- 41-3 25-4000 25- 4000 ##STR00011##
[0079] An additional benefit of the AAD-1 gene is its unprecedented
ability to concomitantly degrade a host of commercial and
non-commercial graminicidal compounds of the general class
aryloxyphenoxypropionates (AOPPs). See Table 2. This attribute may
allow the use of any of a number of AOPP compounds in transgenic
crops containing AAD-1, where tolerance in those crops had not
previously warranted use in those crops. These will most commonly
include grass crops such as corn, rice, wheat, barley, rye, oats,
sorghum, warm and cool-season turf species, grass pasture species,
and many others, but could also include dicot crops where AOPP
tolerance (naturally present in most dicots) is not at commercially
acceptable levels to allow AOPP use in said dicot crop.
[0080] Table 2. AOPP graminicidal compounds listed by accepted
common names Reference to AOPP herbicides is generally made to the
active acid but most are commercially formulated as any of a
variety of corresponding ester formulations and these are likewise
considered as substrates for AAD-1 enzyme in planta as general
plant esterases convert these esters to the active acids in planta.
Likewise reference can also be for the corresponding organic or
inorganic salt of the corresponding acid. When chiral propionic
acid, salt, or ester herbicides are indicated, racemic (R,S) or
optically purified (R or S) enantiomers are considered the same
herbicides for the purpose of naming these herbicides, even though
different CAS numbers may correspond to optically pure compounds.
Possible use rate ranges can be as stand-alone treatments or in
combination with other herbicides in both crop and non-crop
uses.
TABLE-US-00002 TABLE 2 AOPP graminicidal compounds listed by
accepted common names. Possible Preferred use rate use rate
Chemical ranges ranges name CAS no (g ae/ha) (g ae/ha) Structure
Chlorazifop 72492-94-7 10-2000 10-2000 ##STR00012## Clodinafop
105512-06-9 10-2000 20-200 ##STR00013## Clofop 59621-49-7 10-2000
10-2000 ##STR00014## Cyhalofop 122008-85-9 10-2000 105-560
##STR00015## Diclofop 71283-65-3 10-2000 280-2000 ##STR00016##
Fenoxaprop 66441-23-4 10-2000 20-200 ##STR00017## Fenthiaprop
95721-12-3 10-2000 10-2000 ##STR00018## Fluazifop 69335-91-7
10-2000 25-420 ##STR00019## Haloxyfop 69806-40-2 10-2000 20-600
##STR00020## Isoxapyrifop 87757-18-4 10-2000 30-240 ##STR00021##
Metamifop 256412-89-2 10-2000 35-280 ##STR00022## Propa- quizafop
111479-05-1 10-2000 30-240 ##STR00023## Quizalofop 76578-14-8
10-2000 20-240 ##STR00024## Trifop 58597-74-4 10-2000 10-2000
##STR00025##
[0081] A single gene (AAD-1) has now been identified which, when
genetically engineered for expression in plants, has the properties
to allow the use of phenoxy auxin herbicides in plants where
inherent tolerance never existed or was not sufficiently high to
allow use of these herbicides. Additionally, AAD-1 can provide
protection in planta to AOPP herbicides where natural tolerance
also was not sufficient to allow selectivity. Plants containing
AAD-1 alone now may be treated sequentially or tank mixed with one,
two, or a combination of several phenoxy auxin herbicides. The rate
for each phenoxy auxin herbicide may range from 25 to 4000 g ae/ha,
and more typically from 100 to 2000 g ae/ha for the control of a
broad spectrum of dicot weeds. Likewise, one, two, or a mixture of
several AOPP graminicidal compounds may be applied to plants
expressing AAD-1 with reduced risk of injury from said herbicides.
The rate for each AOPP may range from 10 to 2000 g ae/ha, and more
typically from 20-500 g ae/ha for the control of a broad spectrum
of monocot weeds. Combinations of these different chemistry classes
and herbicides with different modes of action and spectra in the
same field (either sequentially or in tank mix combination) shall
provide control of most potential weeds for which herbicidal
control is desired.
[0082] Glyphosate is used extensively because it controls a very
wide spectrum of broadleaf and grass weed species. However,
repeated use of glyphosate in GTCs and in non-crop applications
has, and will continue to, select for weed shifts to naturally more
tolerant species or glyphosate-resistant biotypes. Tankmix
herbicide partners used at efficacious rates that offer control of
the same species but having different modes of action is prescribed
by most herbicide resistance management strategies as a method to
delay the appearance of resistant weeds. Stacking AAD-1 with a
glyphosate tolerance trait (and/or with other herbicide-tolerance
traits) could provide a mechanism to allow for the control of
glyphosate resistant weed species (either grass weed species with
one or more AOPP herbicides, or broadleaf weed species with one or
more phenoxy auxins) in GTCs by enabling the use of glyphosate,
phenoxy auxin(s) (e.g., 2,4-D) and AOPP herbicide(s) (e.g.,
quizalofop) selectively in the same crop. Applications of these
herbicides could be simultaneously in a tank mixture comprising two
or more herbicides of different modes of action; individual
applications of single herbicide composition in sequential
applications as pre-plant, preemergence, or postemergence and split
timing of applications ranging from 2 hours to 3 months; or,
alternatively, any combination of any number of herbicides
representing each chemical class can be applied at any timing
within 7 months of planting the crop up to harvest of the crop (or
the preharvest interval for the individual herbicide, whichever is
shortest).
[0083] It is important to have flexibility in controlling a broad
spectrum of grass and broadleaf weeds in terms of timing of
application, rate of individual herbicides, and the ability to
control difficult or resistant weeds. Glyphosate applications in a
crop with a glyphosate resistance gene/AAD-1 stack could range from
250-2500 g ae/ha; phenoxy auxin herbicide(s) (one or more) could be
applied from 25-4000 g ae/ha; and AOPP herbicide(s) (one or more)
could be applied from 10-2000 g ae/ha. The optimal combination(s)
and timing of these application(s) will depend on the particular
situation, species, and environment, and will be best determined by
a person skilled in the art of weed control and having the benefit
of the subject disclosure.
[0084] Herbicide formulations (e.g., ester, acid, or salt
formulation; or soluble concentrate, emulsifiable concentrate, or
soluble liquid) and tankmix additives (e.g., adjuvants or
compatibility agents) can significantly affect weed control from a
given herbicide or combination of one or more herbicides. Any
combination of these with any of the aforementioned herbicide
chemistries is within the scope of this invention.
[0085] One skilled in the art would also see the benefit of
combining two or more modes of action for increasing the spectrum
of weeds controlled and/or for the control of naturally more
tolerant species or resistant weed species could also extend to
chemistries for which herbicide tolerance was enabled in crops
through human involvement (either transgenically or
non-transgenically) beyond GTCs. Indeed, traits encoding glyphosate
resistance (e.g., resistant plant or bacterial EPSPS, GOX, GAT),
glufosinate resistance (e.g., Pat, bar), acetolactate synthase
(ALS)-inhibiting herbicide resistance (e.g., imidazolinone,
sulfonylurea, triazolopyrimidine sulfonanilide,
pyrmidinylthiobenzoates, and other chemistries=AHAS, Csr1, SurA, et
al.), bromoxynil resistance (e.g., Bxn), resistance to inhibitors
of HPPD (4-hydroxlphenyl-pyruvate-dioxygenase) enzyme, resistance
to inhibitors of phytoene desaturase (PDS), resistance to
photosystem II inhibiting herbicides (e.g., psbA), resistance to
photosystem I inhibiting herbicides, resistance to
protoporphyrinogen oxidase IX (PPO)-inhibiting herbicides (e.g.,
PPO-1), resistance to phenylurea herbicides (e.g., CYP76B1),
dicamba-degrading enzymes (see, e.g., US 20030135879), and others
could be stacked alone or in multiple combinations to provide the
ability to effectively control or prevent weed shifts and/or
resistance to any herbicide of the aforementioned classes.
[0086] Regarding additional herbicides, some additional preferred
ALS inhibitors include the triazolopyrimidine sulfonanilides (such
as cloransulam-methyl, diclosulam, florasulam, flumetsulam,
metosulam, and penoxsulam), pyrimidinylthiobenzoates (such as
bispyribac and pyrithiobac), and flucarbazone. Some preferred HPPD
inhibitors include mesotrione, isoxaflutole, and sulcotrione. Some
preferred PPO inhibitors include flumiclorac, flumioxazin,
flufenpyr, pyraflufen, fluthiacet, butafenacil, carfentrazone,
sulfentrazone, and the diphenylethers (such as acifluorfen,
fomesafen, lactofen, and oxyfluorfen).
[0087] Additionally, AAD-1 alone or stacked with one or more
additional HTC traits can be stacked with one or more additional
input (e.g., insect resistance, fungal resistance, or stress
tolerance, et al.) or output (e.g., increased yield, improved oil
profile, improved fiber quality, et al.) traits. Thus, the subject
invention can be used to provide a complete agronomic package of
improved crop quality with the ability to flexibly and cost
effectively control any number of agronomic pests.
[0088] The subject invention relates in part to the identification
of an enzyme that is not only able to degrade 2,4-D, but also
surprisingly possesses novel properties, which distinguish the
enzyme of the subject invention from previously known tfdA
proteins, for example. Even though this enzyme has very low
homology to tfdA, the genes of the subject invention can still be
generally classified in the same overall family of
.alpha.-ketoglutarate-dependent dioxygenases. This family of
proteins is characterized by three conserved histidine residues in
a "HX(D/E)X.sub.23-26(T/S)X.sub.114-183HX.sub.10-13R" motif which
comprises the active site. The histidines coordinate Fe' ion in the
active site that is essential for catalytic activity (Hogan et al.,
2000). The preliminary in vitro expression experiments discussed
herein were tailored to help select for novel attributes.
[0089] More specifically, the subject invention relates in part to
the use of an enzyme that is not only capable of degrading 2,4-D,
but also AOPP herbicides. No .alpha.-ketoglutarate-dependent
dioxygenase enzyme has previously been reported to have the ability
to degrade herbicides of different chemical classes and modes of
action. Preferred enzymes and genes for use according to the
subject invention are referred to herein as AAD-1 (AryloxyAlkanoate
Dioxygenase) genes and proteins.
[0090] This invention also relates in part to the identification
and use of genes encoding aryloxyalkanoate dioxygenase enzymes that
are capable of degrading phenoxy auxin and aryloxyphenoxypropionate
herbicides. Thus, the subject invention relates in part to the
degradation of 2,4-dichlorophenoxyacetic acid, other
phenoxyalkanoic auxin herbicides, and aryloxyphenoxyalkanoate
herbicides by a recombinantly expressed AAD-1 enzyme.
[0091] The subject proteins tested positive for 2,4-D conversion to
2,4-dichlorophenol ("DCP"; herbicidally inactive) in analytical and
biological assays. Partially purified proteins of the subject
invention can rapidly convert 2,4-D to DCP (ranging from 50-100%
conversion) in vitro. An additional advantage that
AAD-1-transformed plants provide is that parent herbicide(s) are
metabolized to inactive forms, thereby reducing the potential for
harvesting herbicidal residues in grain or stover.
[0092] The subject invention also includes methods of controlling
weeds wherein said methods comprise applying an AOPP herbicide
and/or a phenoxy auxin herbicide to plants comprising an AAD-1
gene.
[0093] In light of these discoveries, novel plants that comprise a
polynucleotide encoding this type of enzyme are now provided.
Heretofore, there was no motivation to produce such plants, and
there was no expectation that such plants could effectively produce
this enzyme to render the plants resistant to not only phenoxy acid
herbicides (such as 2,4-D) but also AOPP herbicides. Thus, the
subject invention provides many advantages that were not heretofore
thought to be possible in the art.
[0094] Publicly available strains (deposited in culture collections
like ATCC or DSMZ) can be acquired and screened, using techniques
disclosed herein, for novel genes. Sequences disclosed herein can
be used to amplify and clone the homologous genes into a
recombinant expression system for further screening and testing
according to the subject invention.
[0095] As discussed above in the Background section, one organism
that has been extensively researched for its ability to degrade
2,4-D is Ralstonia eutropha (Streber et al., 1987). The gene that
codes for the first enzyme in the degradation pathway is tfdA. See
U.S. Pat. No. 6,153,401 and GENBANK Acc. No. M16730. TfdA catalyzes
the conversion of 2,4-D acid to herbicidally inactive DCP via an
a-ketoglutarate-dependent dioxygenase reaction (Smejkal et al.,
2001). TfdA has been used in transgenic plants to impart 2,4-D
resistance in dicot plants (e.g., cotton and tobacco) normally
sensitive to 2,4-D (Streber et al., 1989; Lyon et al., 1989; Lyon
et al., 1993). A large number of tfdA-type genes that encode
proteins capable of degrading 2,4-D have been identified from the
environment and deposited into the Genbank database. Many
homologues are quite similar to tfdA (>85% amino acid identity)
and have similar enzymatic properties to tfdA. However, a small
collection of .alpha.-ketoglutarate-dependent dioxygenase
homologues are presently identified that have a low level of
homology to tfdA.
[0096] RdpA, from Sphingobium herbicidovorans (Westendorf et al.,
2002), is one unique example with low homology (28% amino acid
identity). This enzyme has been shown to catalyze the first step in
(R)-dichlorprop (and other (R)-phenoxypropionic acids) as well as
2,4-D (a phenoxyacetic acid) mineralization (Westendorf et al.,
2003). Although the organism responsible for phenoxypropionic acid
degradation has been known for some time, little progress has been
made in characterizing this pathway until recently (Horvath et al.,
1990). An additional complication to dichlorprop degradation is the
stereospecificity (R vs. S) involved in both the uptake (Kohler,
1999) and initial oxidation of dichlorprop (Westendorf et al.,
2003). Heterologous expression of rdpA in other microbes or
transformation of this gene into plants, heretofore, was not
reported. Literature has focused primarily around close homologues
of tfdA that primarily degrade achiral phenoxyacetic acids. There
was no prior expectation that rdpA or AAD-1 genes could be
successfully expressed in plants to render the plants resistant to
2,4-D (not to mention the completely surprising AOPP
resistance).
[0097] As described in more detail in the Examples below, rdpA was
cloned from Sphingobium herbicidovorans and tested for substrate
promiscuity among various herbicide chemical classes. This
.alpha.-ketoglutarate-dependent dioxygenase enzyme purified in its
native form had previously been shown to degrade 2,4-D and
dichlorprop (Westendorf et al., 2002 and 2003). However, no
a-ketoglutarate-dependent dioxygenase enzyme has previously been
reported to have the ability to degrade herbicides of different
chemical classes and modes of action. RdpA has never been expressed
in plants, nor was there any motivation to do so because
development of new HTC technologies has been limited due largely to
the efficacy, low cost, and convenience of GTCs (Devine, 2005).
[0098] In light of the novel activity, proteins and genes of the
subject invention are referred to herein as AAD-1 proteins and
genes. AAD-1 was presently confirmed to degrade a variety of
phenoxyacetic and phenoxypropionic auxin herbicides in vitro.
However, this enzyme, as reported for the first time herein, was
surprisingly found to also be capable of degrading additional
substrates of the class of aryloxyalkanoate molecules. Substrates
of significant agronomic importance are the
aryloxyphenoxypropionate (AOPP) grass herbicides. This highly novel
discovery is the basis of significant Herbicide Tolerant Crop (HTC)
and selectable marker trait opportunities.
[0099] The broad spectrum grass AOPP herbicides are reported herein
to be excellent substrates for AAD-1 as well as 2,4-D, dichlorprop,
and other phenoxy auxins. This enzyme is unique in its ability to
deliver herbicide degradative activity to a range of broad spectrum
broadleaf herbicides (phenoxy auxins) and a range of broad
spectrum, highly active grass herbicides (AOPPs).
[0100] Thus, the subject invention relates in part to the
degradation of 2,4-dichlorophenoxyacetic acid, other
phenoxyalkanoic auxin herbicides, and aryloxyphenoxy-alkanoate
herbicides by a recombinantly expressed aryloxyalkanoate
dioxygenase enzyme (AAD-1). This invention also relates in part to
identification and uses of genes encoding an aryloxyalkanoate
dioxygenase degrading enzyme (AAD-1) capable of degrading phenoxy
auxin and aryloxyphenoxypropionate herbicides.
[0101] The subject enzyme enables transgenic expression resulting
in tolerance to combinations of herbicides that would control
nearly all broadleaf and grass weeds. AAD-1 can serve as an
excellent herbicide tolerant crop (HTC) trait to stack with other
HTC traits (e.g., glyphosate resistance, glufosinate resistance,
imidazolinone resistance, bromoxynil resistance, et al.), and
insect resistance traits (Cry1F, Cry1Ab, Cry 34/45, et al.) for
example. Additionally, AAD-1 can serve as a selectable marker to
aid in selection of primary transformants of plants genetically
engineered with a second gene or group of genes.
[0102] In addition, the subject microbial gene has been redesigned
such that the protein is encoded by codons having a bias toward
both monocot and dicot plant usage (hemicot). Arabidopsis, corn,
tobacco, cotton, soybean, canola, and rice have been transformed
with AAD-1-containing constructs and have demonstrated high levels
of resistance to both the phenoxy auxin and AOPP herbicides. Thus,
the subject invention also relates to "plant optimized" genes that
encode proteins of the subject invention. As shown below in Example
6, the exemplified rebuilt gene was more efficacious in conveying
herbicide resistance to the plant, as compared to the bacterial
gene.
[0103] Oxyalkanoate groups are useful for introducing a stable acid
functionality into herbicides. The acidic group can impart phloem
mobility by "acid trapping," a desirable attribute for herbicide
action and therefore could be incorporated into new herbicides for
mobility purposes. Aspects of the subject invention also provide a
mechanism of creating HTCs. There exist many potential commercial
and experimental herbicides that can serve as substrates for AAD-1.
Thus, the use of the subject genes can also result in herbicide
tolerance to those other herbicides as well.
[0104] HTC traits of the subject invention can be used in novel
combinations with other HTC traits (including but not limited to
glyphosate tolerance). These combinations of traits give rise to
novel methods of controlling weed (and like) species, due to the
newly acquired resistance or inherent tolerance to herbicides
(e.g., glyphosate). Thus, in addition to the HTC traits, novel
methods for controlling weeds using herbicides, for which herbicide
tolerance was created by said enzyme in transgenic crops, are
within the scope of the invention.
[0105] Additionally, glyphosate tolerant crops grown worldwide are
prevalent. Many times in rotation with other glyphosate tolerant
crops, control of glyphosate-resistant volunteers may be difficult
in rotational crops. Thus, the use of the subject transgenic
traits, stacked or transformed individually into crops, provides a
tool for controlling other HTC volunteer crops.
[0106] This invention can be applied in the context of
commercializing a 2,4-D resistance trait stacked with current
glyphosate resistance traits in soybeans, for example. Thus, this
invention provides a tool to combat broadleaf weed species shifts
and/or selection of herbicide resistant broadleaf weeds, which
culminates from extremely high reliance by growers on glyphosate
for weed control with various crops.
[0107] The transgenic expression of the subject AAD-1 genes is
exemplified in, for example, Arabidopsis, corn (maize), tobacco,
cotton, rice, soybean, and canola. However, the subject invention
can be applied to any other desired types of plants. Soybeans are a
preferred crop for transformation according to the subject
invention. However, this invention can be applied to multiple other
grass and other broadleaf crops. Likewise, 2,4-D can be more
positively utilized in grass crops where tolerance to 2,4-D is
moderate, and increased tolerance via this trait would provide
growers the opportunity to use 2,4-D at more efficacious rates and
over a wider application timing without the risk of crop
injury.
[0108] Still further, the subject invention provides a single gene
that can provide resistance to herbicides that control broadleaf
weed (auxins) and grass weeds (AOPPs). This gene may be utilized in
multiple crops to enable the use of a broad spectrum herbicide
combination. The subject invention can also control weeds resistant
to current chemicals, and aids in the control of shifting weed
spectra resulting from current agronomic practices. The subject
AAD-1 can also be used in efforts to effectively detoxify
additional herbicide substrates to non-herbicidal forms. Thus, the
subject invention provides for the development of additional HTC
traits and/or selectable marker technology.
[0109] Separate from, or in addition to, using the subject genes to
produce HTCs, the subject genes can also be used as selectable
markers for successfully selecting transformants in cell cultures,
greenhouses, and in the field. There is high inherent value for the
subject genes simply as a selectable marker for biotechnology
projects. The promiscuity of AAD-1 for other phenoxyalkanoic
auxinic herbicides provides many opportunities to utilize this gene
for HTC and/or selectable marker purposes.
[0110] One gene of the subject invention, referred to herein as
AAD-1 (aryloxyalkanoate dioxygenase), was cloned from Sphingobium
herbicidovorans (ATCC 700291) by PCR into pET 280-S/S (designated
pDAB 3203) and expressed in BL-21 Star E. coli. When this gene is
overexpressed (by induction of 1mM IPTG and culture lysate combined
with the following reaction mix: 112.5 .mu.g/ml 2,4-D, 1 mM
Ascorbic acid, 1 mM a-ketoglutarate, 50 .mu.M
Fe(NH.sub.4).sub.2(SO.sub.4).sub.2, the recombinantly produced
enzyme degrades 2,4-D into herbicidally inactive DCP (as determined
by HPLC, mass spectrometry, colorimetric assay, and Arabidopsis
plate assay). Additionally, AAD-1 has been demonstrated to convert
the following herbicides into their corresponding inactive phenol:
dichlorprop, mecoprop, haloxyfop, dichlofop, and others (See Tables
3 and 4).
[0111] Table 3: Effect of purified AAD-1 (v1) on various herbicidal
auxins and auxin analogs. Substrates were assayed at 1 mM in 25 mM
MOPS pH 6.8, 200 .mu.M Fe.sup.2+, 200 .mu.M Na ascorbate, 1 mM
.alpha.-ketoglutarate using either 1 .mu.g or 10 .mu.g (10.times.)
purified AAD-1 (v1) per 0.16 ml assay.
TABLE-US-00003 TABLE 3 Effect of purified AAD-1 (v1) on various
herbicidal auxins and auxin analogs AAD1 STRUCTURE Registry ID
Compound AAD1 (10X) ##STR00026## 117613 (R,S)- dichlorprop 0.566
2.594 ##STR00027## 188874 (R,S)-mecoprop 0.341 2.085 ##STR00028##
83293 (R,S)-2-chloro, 4- fluorophenoxy- proprionate 0.304 2.358
##STR00029## 11113675 (R,S)-3- amino- dichlorpop 0.228 2.676
##STR00030## 188476 0.077 0.687 ##STR00031## 192132 0.064 0.204
##STR00032## 195517 2,4-D 0.034 0.383 ##STR00033## 398166 sesone
0.02 0.177 ##STR00034## 190252 0.008 0.211 ##STR00035## 124988
0.007 0.058 ##STR00036## 11263526 0.004 0.069 ##STR00037## 178577
0.003 0.021 ##STR00038## 178587 0.003 0.02 ##STR00039## 188527
0.003 0.036
[0112] Table 4: Effect of purified AAD-1 (v1) on various AOPP
graminicides and analogs, and on cloquintocet. Substrates were
assayed at 1 mM in 25 mM MOPS pH 6.8, 200 .mu.M Fe.sup.2+, 200
.mu.M Na ascorbate, 1 mM .alpha.-ketoglutarate using either 1 .mu.g
or 10 .mu.g (10.times.) purified AAD-1 (v1) per 0.16 ml assay.
TABLE-US-00004 TABLE 4 Effect of purified AAD-1 (v1) on various
AOPP graminicides and analogs, and on cloquintocet. AAD1 STRUCTURE
Registry ID Compound AAD1 (10X) ##STR00040## 18706 (R)-quizalofop
0.43 2.1 ##STR00041## 67131 (R,S)-fluazifop 0.427 2.17 ##STR00042##
11044492 (R)-fenoxaprop 0.408 0.597 ##STR00043## 34697
(R,S)-clodinofop 0.295 1.98 ##STR00044## 14603 (R)-cyhalofop 0.222
1.989 ##STR00045## 14623 (R,S)-cyhalofop 0.215 1.815 ##STR00046##
62942 (R,S)-fenthiaprop 0.199 1.055 ##STR00047## 66905 haloxyfop
0.172 1.63 ##STR00048## 460511 (R,S)-diclofop 0.155 1.663
##STR00049## 25646 0.144 1.69 ##STR00050## 70222 (R,S)-chlorazifop
0.128 1.584 ##STR00051## 199608 Cyhalofop 0.114 1.26 ##STR00052##
43865 haloxyfop-oxyacetate 0.004 0.053 ##STR00053## 7466
(S)-cyhalofop 0.003 0.017 ##STR00054## 204558 Cloquinotocet 0
0.001
[0113] Proteins (and source isolates) of the subject invention. The
present invention provides functional proteins. By "functional
activity" (or "active") it is meant herein that the
proteins/enzymes for use according to the subject invention have
the ability to degrade or diminish the activity of a herbicide
(alone or in combination with other proteins). Plants producing
proteins of the subject invention will preferably produce "an
effective amount" of the protein so that when the plant is treated
with a herbicide, the level of protein expression is sufficient to
render the plant completely or partially resistant or tolerant to
the herbicide (at a typical rate, unless otherwise specified;
typical application rates can be found in the well-known Herbicide
Handbook (Weed Science Society of America, Eighth Edition, 2002),
for example). The herbicide can be applied at rates that would
normally kill the target plant, at normal field use rates and
concentrations. (Because of the subject invention, the level and/or
concentration can optionally be higher than those that were
previously used.) Preferably, plant cells and plants of the subject
invention are protected against growth inhibition or injury caused
by herbicide treatment. Transformed plants and plant cells of the
subject invention are preferably rendered resistant or tolerant to
an herbicide, as discussed herein, meaning that the transformed
plant and plant cells can grow in the presence of effective amounts
of one or more herbicides as discussed herein. Preferred proteins
of the subject invention have catalytic activity to metabolize one
or more aryloxyalkanoate compounds.
[0114] Transfer of the functional activity to plant or bacterial
systems can involve a nucleic acid sequence, encoding the amino
acid sequence for a protein of the subject invention, integrated
into a protein expression vector appropriate to the host in which
the vector will reside. One way to obtain a nucleic acid sequence
encoding a protein with functional activity is to isolate the
native genetic material from the bacterial species which produce
the protein of interest, using information deduced from the
protein's amino acid sequence, as disclosed herein. The native
sequences can be optimized for expression in plants, for example,
as discussed in more detail below. Optimized polynucleotide can
also be designed based on the protein sequence.
[0115] The subject invention provides classes of proteins having
novel activities as identified herein. One way to characterize
these classes of proteins and the polynucleotides that encode them
is by defining a polynucleotide by its ability to hybridize, under
a range of specified conditions, with an exemplified nucleotide
sequence (the complement thereof and/or a probe or probes derived
from either strand) and/or by their ability to be amplified by PCR
using primers derived from the exemplified sequences.
[0116] There are a number of methods for obtaining proteins for use
according to the subject invention. For example, antibodies to the
proteins disclosed herein can be used to identify and isolate other
proteins from a mixture of proteins. Specifically, antibodies may
be raised to the portions of the proteins that are most conserved
or most distinct, as compared to other related proteins. These
antibodies can then be used to specifically identify equivalent
proteins with the characteristic activity by immunoprecipitation,
enzyme linked immunosorbent assay (ELISA), or immuno-blotting.
Antibodies to the proteins disclosed herein, or to equivalent
proteins, or to fragments of these proteins, can be readily
prepared using standard procedures. Such antibodies are an aspect
of the subject invention. Antibodies of the subject invention
include monoclonal and polyclonal antibodies, preferably produced
in response to an exemplified or suggested protein.
[0117] One skilled in the art would readily recognize that proteins
(and genes) of the subject invention can be obtained from a variety
of sources. Since entire herbicide degradation operons are known to
be encoded on transposable elements such as plasmids, as well as
genomically integrated, proteins of the subject invention can be
obtained from a wide variety of microorganisms, for example,
including recombinant and/or wild-type bacteria. Other members of
the orders Firmicutes and Proteobacteria, and specific genera with
known rdpA's, such as Sphingobium, Delftia, Rodoferax, and
Comamonas for example, can be used as source isolates.
[0118] Mutants of bacterial isolates can be made by procedures that
are well known in the art. For example, asporogenous mutants can be
obtained through ethylmethane sulfonate (EMS) mutagenesis of an
isolate. The mutants can be made using ultraviolet light and
nitrosoguanidine by procedures well known in the art.
[0119] A protein "from" or "obtainable from" any of the subject
isolates referred to or suggested herein means that the protein (or
a similar protein) can be obtained from the isolate or some other
source, such as another bacterial strain or a plant. "Derived from"
also has this connotation, and includes proteins obtainable from a
given type of bacterium that are modified for expression in a
plant, for example. One skilled in the art will readily recognize
that, given the disclosure of a bacterial gene and protein, a plant
can be engineered to produce the protein. Antibody preparations,
nucleic acid probes (DNA, RNA, or PNA, for example), and the like
can be prepared using the polynucleotide and/or amino acid
sequences disclosed herein and used to screen and recover other
related genes from other (natural) sources.
[0120] Standard molecular biology techniques may be used to clone
and sequence the proteins and genes described herein. Additional
information may be found in Sambrook et al., 1989, which is
incorporated herein by reference.
[0121] Polynucleotides and probes. The subject invention further
provides nucleotide sequences that encode proteins for use
according to the subject invention. The subject invention further
provides methods of identifying and characterizing genes that
encode proteins having the desired herbicidal activity. In one
embodiment, the subject invention provides unique nucleotide
sequences that are useful as hybridization probes and/or primers
for PCR techniques. The primers produce characteristic gene
fragments that can be used in the identification, characterization,
and/or isolation of specific genes of interest. The nucleotide
sequences of the subject invention encode proteins that are
distinct from previously described proteins.
[0122] The polynucleotides of the subject invention can be used to
form complete "genes" to encode proteins or peptides in a desired
host cell. For example, as the skilled artisan would readily
recognize, the subject polynucleotides can be appropriately placed
under the control of a promoter in a host of interest, as is
readily known in the art. The level of gene expression and
temporal/tissue specific expression can greatly impact the utility
of the invention. Generally, greater levels of protein expression
of a degradative gene will result in faster and more complete
degradation of a substrate (in this case a target herbicide).
Promoters will be desired to express the target gene at high levels
unless the high expression has a consequential negative impact on
the health of the plant. Typically, one would wish to have the
AAD-1 gene constitutively expressed in all tissues for complete
protection of the plant at all growth stages. However, one could
alternatively use a vegetatively expressed resistance gene; this
would allow use of the target herbicide in-crop for weed control
and would subsequently control sexual reproduction of the target
crop by application during the flowering stage.
[0123] As the skilled artisan knows, DNA typically exists in a
double-stranded form. In this arrangement, one strand is
complementary to the other strand and vice versa. As DNA is
replicated in a plant (for example), additional complementary
strands of DNA are produced. The "coding strand" is often used in
the art to refer to the strand that binds with the anti-sense
strand. The mRNA is transcribed from the "anti-sense" strand of
DNA. The "sense" or "coding" strand has a series of codons (a codon
is three nucleotides that can be read as a three-residue unit to
specify a particular amino acid) that can be read as an open
reading frame (ORF) to form a protein or peptide of interest. In
order to produce a protein in vivo, a strand of DNA is typically
transcribed into a complementary strand of mRNA which is used as
the template for the protein. Thus, the subject invention includes
the use of the exemplified polynucleotides shown in the attached
sequence listing and/or equivalents including the complementary
strands. RNA and PNA (peptide nucleic acids) that are functionally
equivalent to the exemplified DNA molecules are included in the
subject invention.
[0124] In one embodiment of the subject invention, bacterial
isolates can be cultivated under conditions resulting in high
multiplication of the microbe. After treating the microbe to
provide single-stranded genomic nucleic acid, the DNA can be
contacted with the primers of the invention and subjected to PCR
amplification. Characteristic fragments of genes of interest will
be amplified by the procedure, thus identifying the presence of the
gene(s) of interest.
[0125] Further aspects of the subject invention include genes and
isolates identified using the methods and nucleotide sequences
disclosed herein. The genes thus identified can encode herbicidal
resistance proteins of the subject invention.
[0126] Proteins and genes for use according to the subject
invention can be identified and obtained by using oligonucleotide
probes, for example. These probes are detectable nucleotide
sequences that can be detectable by virtue of an appropriate label
or may be made inherently fluorescent as described in International
Application No. WO 93/16094. The probes (and the polynucleotides of
the subject invention) may be DNA, RNA, or PNA. In addition to
adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U;
for RNA molecules), synthetic probes (and polynucleotides) of the
subject invention can also have inosine (a neutral base capable of
pairing with all four bases; sometimes used in place of a mixture
of all four bases in synthetic probes) and/or other synthetic
(non-natural) bases. Thus, where a synthetic, degenerate
oligonucleotide is referred to herein, and "N" or "n" is used
generically, "N" or "n" can be G, A, T, C, or inosine. Ambiguity
codes as used herein are in accordance with standard IUPAC naming
conventions as of the filing of the subject application (for
example, R means A or G, Y means C or T, etc.).
[0127] As is well known in the art, if a probe molecule hybridizes
with a nucleic acid sample, it can be reasonably assumed that the
probe and sample have substantial homology/similarity/identity.
Preferably, hybridization of the polynucleotide is first conducted
followed by washes under conditions of low, moderate, or high
stringency by techniques well-known in the art, as described in,
for example, Keller, G. H., M. M. Manak (1987) DNA Probes, Stockton
Press, New York, NY, pp. 169-170. For example, as stated therein,
low stringency conditions can be achieved by first washing with
2.times.SSC (Standard Saline Citrate)/0.1% SDS (Sodium Dodecyl
Sulfate) for 15 minutes at room temperature. Two washes are
typically performed. Higher stringency can then be achieved by
lowering the salt concentration and/or by raising the temperature.
For example, the wash described above can be followed by two
washings with 0.1.times.SSC/0.1% SDS for 15 minutes each at room
temperature followed by subsequent washes with 0.1.times.SSC/0.1%
SDS for 30 minutes each at 55.degree. C. These temperatures can be
used with other hybridization and wash protocols set forth herein
and as would be known to one skilled in the art (SSPE can be used
as the salt instead of SSC, for example). The 2.times.SSC/0.1% SDS
can be prepared by adding 50 ml of 20.times.SSC and 5 ml of 10% SDS
to 445 ml of water. 20.times.SSC can be prepared by combining NaCl
(175.3 g/0.150 M), sodium citrate (88.2 g/0.015 M), and water,
adjusting pH to 7.0 with 10 N NaOH, then adjusting the volume to 1
liter. 10% SDS can be prepared by dissolving 10 g of SDS in 50 ml
of autoclaved water, then diluting to 100 ml.
[0128] Detection of the probe provides a means for determining in a
known manner whether hybridization has been maintained. Such a
probe analysis provides a rapid method for identifying genes of the
subject invention. The nucleotide segments used as probes according
to the invention can be synthesized using a DNA synthesizer and
standard procedures. These nucleotide sequences can also be used as
PCR primers to amplify genes of the subject invention.
[0129] Hybridization characteristics of a molecule can be used to
define polynucleotides of the subject invention. Thus the subject
invention includes polynucleotides (and/or their complements,
preferably their full complements) that hybridize with a
polynucleotide exemplified herein. That is, one way to define a
gene (and the protein it encodes), for example, is by its ability
to hybridize (under any of the conditions specifically disclosed
herein) with a known or specifically exemplified gene.
[0130] As used herein, "stringent" conditions for hybridization
refers to conditions which achieve the same, or about the same,
degree of specificity of hybridization as the conditions employed
by the current applicants. Specifically, hybridization of
immobilized DNA on Southern blots with .sup.32P-labeled
gene-specific probes can be performed by standard methods (see,
e.g., Maniatis et al. 1982). In general, hybridization and
subsequent washes can be carried out under conditions that allow
for detection of target sequences. For double-stranded DNA gene
probes, hybridization can be carried out overnight at 20-25.degree.
C. below the melting temperature (Tm) of the DNA hybrid in
6.times.SSPE, 5.times. Denhardt's solution, 0.1% SDS, 0.1 mg/ml
denatured DNA. The melting temperature is described by the
following formula (Beltz et al. 1983): [0131] Tm=81.5.degree.
C.+16.6 Log[Na+]0.41(%G+C)-0.61(%formamide) -600/length of duplex
in base pairs.
[0132] Washes can typically be carried out as follows: [0133] (1)
Twice at room temperature for 15 minutes in 1.times.SSPE, 0.1% SDS
(low stringency wash). [0134] (2) Once at Tm-20.degree. C. for 15
minutes in 0.2.times.SSPE, 0.1% SDS (moderate stringency wash).
[0135] For oligonucleotide probes, hybridization can be carried out
overnight at 10-20.degree. C. below the melting temperature (Tm) of
the hybrid in 6.times.SSPE, 5.times.Denhardt's solution, 0.1% SDS,
0.1 mg/ml denatured DNA. Tm for oligonucleotide probes can be
determined by the following formula:
[0136] Tm (.degree. C.)=2(number T/A base pairs)+4(number G/C base
pairs) (Suggs et al., 1981).
[0137] Washes can typically be out as follows: [0138] (1) Twice at
room temperature for 15 minutes 1.times.SSPE, 0.1% SDS (low
stringency wash). [0139] (2) Once at the hybridization temperature
for 15 minutes in lx SSPE, 0.1% SDS (moderate stringency wash).
[0140] In general, salt and/or temperature can be altered to change
stringency. With a labeled DNA fragment >70 or so bases in
length, the following conditions can be used: [0141] Low: 1 or
2.times.SSPE, room temperature [0142] Low: 1 or 2.times.SSPE,
42.degree. C. [0143] Moderate: 0.2.times. or 1.times.SSPE,
65.degree. C. [0144] High: 0.1.times.SSPE, 65.degree. C.
[0145] Duplex formation and stability depend on substantial
complementarity between the two strands of a hybrid, and, as noted
above, a certain degree of mismatch can be tolerated. Therefore,
the probe sequences of the subject invention include mutations
(both single and multiple), deletions, insertions of the described
sequences, and combinations thereof, wherein said mutations,
insertions and deletions permit formation of stable hybrids with
the target polynucleotide of interest. Mutations, insertions, and
deletions can be produced in a given polynucleotide sequence in
many ways, and these methods are known to an ordinarily skilled
artisan. Other methods may become known in the future.
[0146] PCR technology. Polymerase Chain Reaction (PCR) is a
repetitive, enzymatic, primed synthesis of a nucleic acid sequence.
This procedure is well known and commonly used by those skilled in
this art (see Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and
4,800,159; Saiki et al., 1985). PCR is based on the enzymatic
amplification of a DNA fragment of interest that is flanked by two
oligonucleotide primers that hybridize to opposite strands of the
target sequence. The primers are preferably oriented with the 3'
ends pointing towards each other. Repeated cycles of heat
denaturation of the template, annealing of the primers to their
complementary sequences, and extension of the annealed primers with
a DNA polymerase result in the amplification of the segment defined
by the 5' ends of the PCR primers. The extension product of each
primer can serve as a template for the other primer, so each cycle
essentially doubles the amount of DNA fragment produced in the
previous cycle. This results in the exponential accumulation of the
specific target fragment, up to several million-fold in a few
hours. By using a thermostable DNA polymerase such as Taq
polymerase, isolated from the thermophilic bacterium Thermus
aquaticus, the amplification process can be completely automated.
Other enzymes which can be used are known to those skilled in the
art.
[0147] Exemplified DNA sequences, or segments thereof, can be used
as primers for PCR amplification. In performing PCR amplification,
a certain degree of mismatch can be tolerated between primer and
template. Therefore, mutations, deletions, and insertions
(especially additions of nucleotides to the 5' end) of the
exemplified primers fall within the scope of the subject invention.
Mutations, insertions, and deletions can be produced in a given
primer by methods known to an ordinarily skilled artisan.
[0148] Modification of genes and proteins. The subject genes and
proteins can be fused to other genes and proteins to produce
chimeric or fusion proteins. The genes and proteins useful
according to the subject invention include not only the
specifically exemplified full-length sequences, but also portions,
segments and/or fragments (including contiguous fragments and
internal and/or terminal deletions compared to the full-length
molecules) of these sequences, variants, mutants, chimerics, and
fusions thereof. Proteins of the subject invention can have
substituted amino acids so long as they retain desired functional
activity. "Variant" genes have nucleotide sequences that encode the
same proteins or equivalent proteins having activity equivalent or
similar to an exemplified protein. The terms "variant proteins" and
"equivalent proteins" refer to proteins having the same or
essentially the same biological/functional activity against the
target pests and equivalent sequences as the exemplified proteins.
As used herein, reference to an "equivalent" sequence refers to
sequences having amino acid substitutions, deletions, additions, or
insertions that improve or do not adversely affect activity to a
significant extent. Fragments retaining activity are also included
in this definition. Fragments and other equivalents that retain the
same or similar function or activity as a corresponding fragment of
an exemplified protein are within the scope of the subject
invention. Changes, such as amino acid substitutions or additions,
can be made for a variety of purposes, such as increasing (or
decreasing) protease stability of the protein (without
materially/substantially decreasing the functional activity of the
protein), removing or adding a restriction site, and the like.
Variations of genes may be readily constructed using standard
techniques for making point mutations, for example.
[0149] In addition, U.S. Pat. No. 5,605,793, for example, describes
methods for generating additional molecular diversity by using DNA
reassembly after random or focused fragmentation. This can be
referred to as gene "shuffling," which typically involves mixing
fragments (of a desired size) of two or more different DNA
molecules, followed by repeated rounds of renaturation. This can
improve the activity of a protein encoded by a starting gene. The
result is a chimeric protein having improved activity, altered
substrate specificity, increased enzyme stability, altered
stereospecificity, or other characteristics.
[0150] "Shuffling" can be designed and targeted after obtaining and
examining the atomic 3D (three dimensional) coordinates and crystal
structure of a protein of interest. Thus, "focused shuffling" can
be directed to certain segments of a protein that are ideal for
modification, such as surface-exposed segments, and preferably not
internal segments that are involved with protein folding and
essential 3D structural integrity.
[0151] Variant genes can be used to produce variant proteins;
recombinant hosts can be used to produce the variant proteins.
Using these "gene shuffling" techniques, equivalent genes and
proteins can be constructed that comprise any 5, 10, or 20
contiguous residues (amino acid or nucleotide) of any sequence
exemplified herein. As one skilled in the art knows, the gene
shuffling techniques, for example, can be adjusted to obtain
equivalents having, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,
112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,
125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,
138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,
151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,
164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,
177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,
190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,
203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,
216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,
229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,
242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,
255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267,
268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,
281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293,
294, 295, 296, or 297 contiguous residues (amino acid or
nucleotide), corresponding to a segment (of the same size) in any
of the exemplified or suggested sequences (or the complements (full
complements) thereof). Similarly sized segments, especially those
for conserved regions, can also be used as probes and/or
primers.
[0152] Fragments of full-length genes can be made using
commercially available exonucleases or endonucleases according to
standard procedures. For example, enzymes such as Bal31 or
site-directed mutagenesis can be used to systematically cut off
nucleotides from the ends of these genes. Also, genes that encode
active fragments may be obtained using a variety of restriction
enzymes. Proteases may be used to directly obtain active fragments
of these proteins.
[0153] It is within the scope of the invention as disclosed herein
that proteins can be truncated and still retain functional
activity. By "truncated protein" it is meant that a portion of a
protein may be cleaved off while the remaining truncated protein
retains and exhibits the desired activity after cleavage. Cleavage
can be achieved by various proteases. Furthermore, effectively
cleaved proteins can be produced using molecular biology techniques
wherein the DNA bases encoding said protein are removed either
through digestion with restriction endonucleases or other
techniques available to the skilled artisan. After truncation, said
proteins can be expressed in heterologous systems such as E. coli,
baculoviruses, plant-based viral systems, yeast, and the like and
then placed in insect assays as disclosed herein to determine
activity. It is well-known in the art that truncated proteins can
be successfully produced so that they retain functional activity
while having less than the entire, full-length sequence. For
example, B.t. proteins can be used in a truncated (core protein)
form (see, e.g., Hate et al. (1989), and Adang et al. (1985)). As
used herein, the term "protein" can include functionally active
truncations.
[0154] In some cases, especially for expression in plants, it can
be advantageous to use truncated genes that express truncated
proteins. Preferred truncated genes will typically encode 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, or 99% of the full-length protein.
[0155] Certain proteins of the subject invention have been
specifically exemplified herein. As these proteins are merely
exemplary of the proteins of the subject invention, it should be
readily apparent that the subject invention comprises variant or
equivalent proteins (and nucleotide sequences coding for
equivalents thereof) having the same or similar activity of the
exemplified proteins. Equivalent proteins will have amino acid
similarity (and/or homology) with an exemplified protein. The amino
acid identity will typically be at least 60%, preferably at least
75%, more preferably at least 80%, even more preferably at least
90%, and can be at least 95%. Preferred proteins of the subject
invention can also be defined in terms of more particular identity
and/or similarity ranges. For example, the identity and/or
similarity can be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,
78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, or 99% as compared to a sequence exemplified or
suggested herein. Any number listed above can be used to define the
upper and lower limits.
[0156] Unless otherwise specified, as used herein, percent sequence
identity and/or similarity of two nucleic acids is determined using
the algorithm of Karlin and Altschul, 1990, modified as in Karlin
and Altschul 1993. Such an algorithm is incorporated into the
NBLAST and XBLAST programs of Altschul et al., 1990. BLAST
nucleotide searches are performed with the NBLAST program,
score=100, wordlength=12. Gapped BLAST can be used as described in
Altschul et al., 1997. When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (NBLAST
and XBLAST) are used. See NCBI/NIH website. To obtain gapped
alignments for comparison purposes, the AlignX function of Vector
NTI Suite 8 (InforMax, Inc., North Bethesda, Md., U.S.A.), was used
employing the default parameters. These were: a Gap opening penalty
of 15, a Gap extension penalty of 6.66, and a Gap separation
penalty range of 8.
[0157] Various properties and three-dimensional features of the
protein can also be changed without adversely affecting the
activity/functionality of the protein. Conservative amino acid
substitutions can be tolerated/made to not adversely affect the
activity and/or three-dimensional configuration of the molecule.
Amino acids can be placed in the following classes: non-polar,
uncharged polar, basic, and acidic. Conservative substitutions
whereby an amino acid of one class is replaced with another amino
acid of the same type fall within the scope of the subject
invention so long as the substitution is not adverse to the
biological activity of the compound. Table 5 provides a listing of
examples of amino acids belonging to each class.
TABLE-US-00005 TABLE 5 Class of Amino Acid Examples of Amino Acids
Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar
Gly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic Asp, Glu Basic Lys, Arg,
His
[0158] In some instances, non-conservative substitutions can also
be made. However, preferred substitutions do not significantly
detract from the functional/biological activity of the protein.
[0159] As used herein, reference to "isolated" polynucleotides
and/or "purified" proteins refers to these molecules when they are
not associated with the other molecules with which they would be
found in nature. Thus, reference to "isolated" and/or "purified"
signifies the involvement of the "hand of man" as described herein.
For example, a bacterial "gene" of the subject invention put into a
plant for expression is an "isolated polynucleotide." Likewise, a
protein derived from a bacterial protein and produced by a plant is
an "isolated protein."
[0160] Because of the degeneracy/redundancy of the genetic code, a
variety of different DNA sequences can encode the amino acid
sequences disclosed herein. It is well within the skill of a person
trained in the art to create alternative DNA sequences that encode
the same, or essentially the same, proteins. These variant DNA
sequences are within the scope of the subject invention. This is
also discussed in more detail below in the section entitled
"Optimization of sequence for expression in plants."
[0161] Optimization of sequence for expression in plants. To obtain
high expression of heterologous genes in plants it is generally
preferred to reengineer the genes so that they are more efficiently
expressed in (the cytoplasm of) plant cells. Maize is one such
plant where it may be preferred to re-design the heterologous
gene(s) prior to transformation to increase the expression level
thereof in said plant. Therefore, an additional step in the design
of genes encoding a bacterial protein is reengineering of a
heterologous gene for optimal expression, using codon bias more
closely aligned with the target plant sequence, whether a dicot or
monocot species. Sequences can also be optimized for expression in
any of the more particular types of plants discussed elsewhere
herein.
[0162] Transgenic hosts. The protein-encoding genes of the subject
invention can be introduced into a wide variety of microbial or
plant hosts. The subject invention includes transgenic plant cells
and transgenic plants. Preferred plants (and plant cells) are corn,
Arabidopsis, tobacco, soybeans, cotton, canola, rice, wheat, turf
and pasture grasses, and the like. Other types of transgenic plants
can also be made according to the subject invention, such as
fruits, vegetables, and trees. More generally, dicots and/or
monocots can be used in various aspects of the subject
invention.
[0163] In preferred embodiments, expression of the gene results,
directly or indirectly, in the intracellular production (and
maintenance) of the protein(s) of interest. Plants can be rendered
herbicide-resistant in this manner Such hosts can be referred to as
transgenic, recombinant, transformed, and/or transfected hosts
and/or cells. In some aspects of this invention (when cloning and
preparing the gene of interest, for example), microbial (preferably
bacterial) cells can be produced and used according to standard
techniques, with the benefit of the subject disclosure.
[0164] Plant cells transfected with a polynucleotide of the subject
invention can be regenerated into whole plants. The subject
invention includes cell cultures including tissue cell cultures,
liquid cultures, and plated cultures. Seeds produced by and/or used
to generate plants of the subject invention are also included
within the scope of the subject invention. Other plant tissues and
parts are also included in the subject invention. The subject
invention likewise includes methods of producing plants or cells
comprising a polynucleotide of the subject invention. One preferred
method of producing such plants is by planting a seed of the
subject invention.
[0165] Insertion of genes to form transgenic hosts. One aspect of
the subject invention is the transformation/transfection of plants,
plant cells, and other host cells with polynucleotides of the
subject invention that express proteins of the subject invention.
Plants transformed in this manner can be rendered resistant to a
variety of herbicides with different modes of action.
[0166] A wide variety of methods are available for introducing a
gene encoding a desired protein into the target host under
conditions that allow for stable maintenance and expression of the
gene. These methods are well known to those skilled in the art and
are described, for example, in U.S. Pat. No. 5,135,867.
[0167] Vectors comprising an AAD-1 polynucleotide are included in
the scope of the subject invention. For example, a large number of
cloning vectors comprising a replication system in E. coli and a
marker that permits selection of the transformed cells are
available for preparation for the insertion of foreign genes into
higher plants. The vectors comprise, for example, pBR322, pUC
series, M13mp series, pACYC184, etc. Accordingly, the sequence
encoding the protein can be inserted into the vector at a suitable
restriction site. The resulting plasmid is used for transformation
into E. coli. The E. coli cells are cultivated in a suitable
nutrient medium, then harvested and lysed. The plasmid is recovered
by purification away from genomic DNA. Sequence analysis,
restriction analysis, electrophoresis, and other
biochemical-molecular biological methods are generally carried out
as methods of analysis. After each manipulation, the DNA sequence
used can be restriction digested and joined to the next DNA
sequence. Each plasmid sequence can be cloned in the same or other
plasmids. Depending on the method of inserting desired genes into
the plant, other DNA sequences may be necessary. If, for example,
the Ti or Ri plasmid is used for the transformation of the plant
cell, then at least the right border, but often the right and the
left border of the Ti or Ri plasmid T-DNA, has to be joined as the
flanking region of the genes to be inserted. The use of T-DNA for
the transformation of plant cells has been intensively researched
and described in EP 120 516; Hoekema (1985); Fraley et al. (1986);
and An et al. (1985).
[0168] A large number of techniques are available for inserting DNA
into a plant host cell. Those techniques include transformation
with T-DNA using Agrobacterium tumefaciens or Agrobacterium
rhizogenes as transformation agent, fusion, injection, biolistics
(microparticle bombardment), silicon carbide whiskers, aerosol
beaming, PEG, or electroporation as well as other possible methods.
If Agrobacteria are used for the transformation, the DNA to be
inserted has to be cloned into special plasmids, namely either into
an intermediate vector or into a binary vector. The intermediate
vectors can be integrated into the Ti or Ri plasmid by homologous
recombination owing to sequences that are homologous to sequences
in the T-DNA. The Ti or Ri plasmid also comprises the vir region
necessary for the transfer of the T-DNA. Intermediate vectors
cannot replicate themselves in Agrobacteria. The intermediate
vector can be transferred into Agrobacterium tumefaciens by means
of a helper plasmid (conjugation). Binary vectors can replicate
themselves both in E. coli and in Agrobacteria. They comprise a
selection marker gene and a linker or polylinker which are framed
by the right and left T-DNA border regions. They can be transformed
directly into Agrobacteria (Holsters, 1978). The Agrobacterium used
as host cell is to comprise a plasmid carrying a vir region. The
vir region is necessary for the transfer of the T-DNA into the
plant cell. Additional T-DNA may be contained. The bacterium so
transformed is used for the transformation of plant cells. Plant
explants can be cultivated advantageously with Agrobacterium
tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA
into the plant cell. Whole plants can then be regenerated from the
infected plant material (for example, pieces of leaf, segments of
stalk, roots, but also protoplasts or suspension-cultivated cells)
in a suitable medium, which may contain antibiotics or biocides for
selection. The plants so obtained can then be tested for the
presence of the inserted DNA. No special demands are made of the
plasmids in the case of injection and electroporation. It is
possible to use ordinary plasmids, such as, for example, pUC
derivatives.
[0169] The transformed cells grow inside the plants in the usual
manner They can form germ cells and transmit the transformed
trait(s) to progeny plants. Such plants can be grown in the normal
manner and crossed with plants that have the same transformed
hereditary factors or other hereditary factors. The resulting
hybrid individuals have the corresponding phenotypic
properties.
[0170] In some preferred embodiments of the invention, genes
encoding the bacterial protein are expressed from transcriptional
units inserted into the plant genome. Preferably, said
transcriptional units are recombinant vectors capable of stable
integration into the plant genome and enable selection of
transformed plant lines expressing mRNA encoding the proteins.
[0171] Once the inserted DNA has been integrated in the genome, it
is relatively stable there (and does not come out again). It
normally contains a selection marker that confers on the
transformed plant cells resistance to a biocide or an antibiotic,
such as kanamycin, G418, bleomycin, hygromycin, or chloramphenicol,
inter alia. Plant selectable markers also typically can provide
resistance to various herbicides such as glufosinate, (PAT),
glyphosate (EPSPS), imazethyapyr (AHAS), and many others. The
individually employed marker should accordingly permit the
selection of transformed cells rather than cells that do not
contain the inserted DNA. The gene(s) of interest are preferably
expressed either by constitutive or inducible promoters in the
plant cell. Once expressed, the mRNA is translated into proteins,
thereby incorporating amino acids of interest into protein. The
genes encoding a protein expressed in the plant cells can be under
the control of a constitutive promoter, a tissue-specific promoter,
or an inducible promoter.
[0172] Several techniques exist for introducing foreign recombinant
vectors into plant cells, and for obtaining plants that stably
maintain and express the introduced gene. Such techniques include
the introduction of genetic material coated onto microparticles
directly into cells (U.S. Pat. No. 4,945,050 to Cornell and U.S.
Pat. No. 5,141,131 to DowElanco, now Dow AgroSciences, LLC). In
addition, plants may be transformed using Agrobacterium technology,
see U.S. Pat. No. 5,177,010 to University of Toledo; U.S. Pat. No.
5,104,310 to Texas A&M; European Patent Application 0131624B1;
European Patent Applications 120516, 159418B1 and 176,112 to
Schilperoot; U.S. Patent Nos. 5,149,645, 5,469,976, 5,464,763 and
4,940,838 and 4,693,976 to Schilperoot; European Patent
Applications 116718, 290799, 320500, all to Max Planck; European
Patent Applications 604662 and 627752, and U.S. Pat. No. 5,591,616,
to Japan Tobacco; European Patent Applications 0267159 and 0292435,
and U.S. Patent No. 5,231,019, all to Ciba Geigy, now Syngenta;
U.S. Pat. Nos. 5,463,174 and 4,762,785, both to Calgene; and U.S.
Pat. Nos. 5,004,863 and 5,159,135, both to Agracetus. Other
transformation technology includes whiskers technology. See U.S.
Patent Nos. 5,302,523 and 5,464,765, both to Zeneca, now Syngenta.
Electroporation technology has also been used to transform plants.
See WO 87/06614 to Boyce Thompson Institute; U.S. Patent Nos.
5,472,869 and 5,384,253, both to Dekalb; and WO 92/09696 and WO
93/21335, both to Plant Genetic Systems. Furthermore, viral vectors
can also be used to produce transgenic plants expressing the
protein of interest. For example, monocotyledonous plants can be
transformed with a viral vector using the methods described in U.S.
Pat. No. 5,569,597 to Mycogen Plant Science and Ciba-Geigy (now
Syngenta), as well as U.S. Pat. Nos. 5,589,367 and 5,316,931, both
to Biosource, now Large Scale Biology.
[0173] As mentioned previously, the manner in which the DNA
construct is introduced into the plant host is not critical to this
invention. Any method that provides for efficient transformation
may be employed. For example, various methods for plant cell
transformation are described herein and include the use of Ti or
Ri-plasmids and the like to perform Agrobacterium mediated
transformation. In many instances, it will be desirable to have the
construct used for transformation bordered on one or both sides by
T-DNA borders, more specifically the right border. This is
particularly useful when the construct uses Agrobacterium
tumefaciens or Agrobacterium rhizogenes as a mode for
transformation, although T-DNA borders may find use with other
modes of transformation. Where Agrobacterium is used for plant cell
transformation, a vector may be used which may be introduced into
the host for homologous recombination with T-DNA or the Ti or Ri
plasmid present in the host. Introduction of the vector may be
performed via electroporation, tri-parental mating and other
techniques for transforming gram-negative bacteria which are known
to those skilled in the art. The manner of vector transformation
into the Agrobacterium host is not critical to this invention. The
Ti or Ri plasmid containing the T-DNA for recombination may be
capable or incapable of causing gall formation, and is not critical
to said invention so long as the vir genes are present in said
host.
[0174] In some cases where Agrobacterium is used for
transformation, the expression construct being within the T-DNA
borders will be inserted into a broad spectrum vector such as pRK2
or derivatives thereof as described in Ditta et al. (1980) and EPO
0 120 515. Included within the expression construct and the T-DNA
will be one or more markers as described herein which allow for
selection of transformed Agrobacterium and transformed plant cells.
The particular marker employed is not essential to this invention,
with the preferred marker depending on the host and construction
used.
[0175] For transformation of plant cells using Agrobacterium,
explants may be combined and incubated with the transformed
Agrobacterium for sufficient time to allow transformation thereof.
After transformation, the Agrobacteria are killed by selection with
the appropriate antibiotic and plant cells are cultured with the
appropriate selective medium. Once calli are formed, shoot
formation can be encouraged by employing the appropriate plant
hormones according to methods well known in the art of plant tissue
culturing and plant regeneration. However, a callus intermediate
stage is not always necessary. After shoot formation, said plant
cells can be transferred to medium which encourages root formation
thereby completing plant regeneration. The plants may then be grown
to seed and said seed can be used to establish future generations.
Regardless of transformation technique, the gene encoding a
bacterial protein is preferably incorporated into a gene transfer
vector adapted to express said gene in a plant cell by including in
the vector a plant promoter regulatory element, as well as 3'
non-translated transcriptional termination regions such as Nos and
the like.
[0176] In addition to numerous technologies for transforming
plants, the type of tissue that is contacted with the foreign genes
may vary as well. Such tissue would include but would not be
limited to embryogenic tissue, callus tissue types I, II, and III,
hypocotyl, meristem, root tissue, tissues for expression in phloem,
and the like. Almost all plant tissues may be transformed during
dedifferentiation using appropriate techniques described
herein.
[0177] As mentioned above, a variety of selectable markers can be
used, if desired. Preference for a particular marker is at the
discretion of the artisan, but any of the following selectable
markers may be used along with any other gene not listed herein
which could function as a selectable marker. Such selectable
markers include but are not limited to aminoglycoside
phosphotransferase gene of transposon Tn5 (Aph II) which encodes
resistance to the antibiotics kanamycin, neomycin and G418, as well
as those genes which encode for resistance or tolerance to
glyphosate; hygromycin; methotrexate; phosphinothricin (bialaphos
or glufosinate); imidazolinones, sulfonylureas and
triazolopyrimidine herbicides, such as chlorsulfuron; bromoxynil,
dalapon and the like.
[0178] In addition to a selectable marker, it may be desirous to
use a reporter gene. In some instances a reporter gene may be used
with or without a selectable marker. Reporter genes are genes that
are typically not present in the recipient organism or tissue and
typically encode for proteins resulting in some phenotypic change
or enzymatic property. Examples of such genes are provided in
Weising et al., 1988. Preferred reporter genes include the
beta-glucuronidase (GUS) of the uidA locus of E. coli, the
chloramphenicol acetyl transferase gene from Tn9 of E. coli, the
green fluorescent protein from the bioluminescent jellyfish
Aequorea victoria, and the luciferase genes from firefly Photinus
pyralis. An assay for detecting reporter gene expression may then
be performed at a suitable time after said gene has been introduced
into recipient cells. A preferred such assay entails the use of the
gene encoding beta-glucuronidase (GUS) of the uidA locus of E. coli
as described by Jefferson et al., (1987) to identify transformed
cells.
[0179] In addition to plant promoter regulatory elements, promoter
regulatory elements from a variety of sources can be used
efficiently in plant cells to express foreign genes. For example,
promoter regulatory elements of bacterial origin, such as the
octopine synthase promoter, the nopaline synthase promoter, the
mannopine synthase promoter; promoters of viral origin, such as the
cauliflower mosaic virus (35S and 19S), 35T (which is a
re-engineered 35S promoter, see U.S. Pat. No. 6,166,302, especially
Example 7E) and the like may be used. Plant promoter regulatory
elements include but are not limited to ribulose-1,6-bisphosphate
(RUBP) carboxylase small subunit (ssu), beta-conglycinin promoter,
beta-phaseolin promoter, ADH promoter, heat-shock promoters, and
tissue specific promoters. Other elements such as matrix attachment
regions, scaffold attachment regions, introns, enhancers,
polyadenylation sequences and the like may be present and thus may
improve the transcription efficiency or DNA integration. Such
elements may or may not be necessary for DNA function, although
they can provide better expression or functioning of the DNA by
affecting transcription, mRNA stability, and the like. Such
elements may be included in the DNA as desired to obtain optimal
performance of the transformed DNA in the plant. Typical elements
include but are not limited to Adh-intron 1, Adh-intron 6, the
alfalfa mosaic virus coat protein leader sequence, osmotin UTR
sequences, the maize streak virus coat protein leader sequence, as
well as others available to a skilled artisan. Constitutive
promoter regulatory elements may also be used thereby directing
continuous gene expression in all cells types and at all times
(e.g., actin, ubiquitin, CaMV 35S, and the like). Tissue specific
promoter regulatory elements are responsible for gene expression in
specific cell or tissue types, such as the leaves or seeds (e.g.,
zein, oleosin, napin, ACP, globulin and the like) and these may
also be used.
[0180] Promoter regulatory elements may also be active (or
inactive) during a certain stage of the plant's development as well
as active in plant tissues and organs. Examples of such include but
are not limited to pollen-specific, embryo-specific,
corn-silk-specific, cotton-fiber-specific, root-specific,
seed-endosperm-specific, or vegetative phase-specific promoter
regulatory elements and the like. Under certain circumstances it
may be desirable to use an inducible promoter regulatory element,
which is responsible for expression of genes in response to a
specific signal, such as: physical stimulus (heat shock genes),
light (RUBP carboxylase), hormone (Em), metabolites, chemical
(tetracycline responsive), and stress. Other desirable
transcription and translation elements that function in plants may
be used. Numerous plant-specific gene transfer vectors are known in
the art.
[0181] Plant RNA viral based systems can also be used to express
bacterial protein. In so doing, the gene encoding a protein can be
inserted into the coat promoter region of a suitable plant virus
which will infect the host plant of interest. The protein can then
be expressed thus providing protection of the plant from herbicide
damage. Plant RNA viral based systems are described in U.S. Pat.
No. 5,500,360 to Mycogen Plant Sciences, Inc. and U.S. Pat. Nos.
5,316,931 and 5,589,367 to Biosource, now Large Scale Biology.
[0182] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety to the extent they are not inconsistent
with the explicit teachings of this specification.
[0183] Following are examples that illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted.
EXAMPLE 1
Method for Identifying Genes that Impart Resistance to 2,4-D In
Planta
[0184] As a way to identify genes which possess herbicide degrading
activities in planta, it is possible to mine current public
databases such as NCBI (National Center for Biotechnology
Information). To begin the process, it is necessary to have a
functional gene sequence already identified that encodes a protein
with the desired characteristics (i.e., a-ketoglutarate dioxygenase
activity). This protein sequence is then used as the input for the
BLAST (Basic Local Alignment Search Tool) (Altschul et al., 1997)
algorithm to compare against available NCBI protein sequences
deposited. Using default settings, this search returns upwards of
100 homologous protein sequences at varying levels. These range
from highly identical (85-98%) to very low identity (23-32%) at the
amino acid level. Traditionally only sequences with high homology
would be expected to retain similar properties to the input
sequence. In this case we chose only those sequences with <50%
homology. We go on to exemplify that cloning and recombinantly
expressing homologues with as little as 27% amino acid conservation
can be used to impart commercial levels of resistance not only to
the intended herbicide, but also to substrates never previously
tested with these enzymes.
[0185] PCR and cloning of gene into pET. A single gene (rdpA) was
identified from the NCBI database (see the ncbi.nlm.nih.gov
website; accession #AF516752) as a homologue with only 28% amino
acid identity to tfdA from Ralstonia eutropha. Percent identity was
determined by first translating both the rdpA and tfdA DNA
sequences deposited in the database to proteins, then using
ClustalW in the VectorNTl software package to perform the multiple
sequence alignment.
[0186] The strain of Sphingobium herbicidovorans containing the
rdpA gene was obtained from ATCC (American Type Culture Collection
strain #700291). The lyophilized strain was revived according to
ATCC protocol and stored at -80.degree. C. in 20% glycerol for
internal use as Dow Bacterial strain DB 536. From this freezer
stock, a plate of Tryptic Soy Agar was then struck out with a
loopful of cells for isolation, and incubated at 28.degree. C. for
3 days.
[0187] A single colony was used to inoculate 100 ml of Tryptic Soy
Broth in a 500 ml tri-baffled flask, which was incubated overnight
at 28.degree. C. on a floor shaker at 150 rpm. From this, total DNA
was isolated with the gram negative protocol of Qiagen's DNeasy kit
(Qiagen cat. #69504). The following primers were designed to
amplify the target gene from genomic DNA, Forward: 5' TCT AGA AGG
AGA TAT ACC ATG CAT GCT GCA CTG TCC CCC CTC TCC CAG CG 3' [(SEQ ID
NO:1) (added Xba I restriction site and Ribosome Binding Site
(RBS))] and Reverse: 5' CTC GAG TTA CTA GCG CGC CGG GCG CAC GCC ACC
GAC CG 3' [(SEQ ID NO:2)(added extra stop codon and Xho I
site)].
[0188] Twenty microliter reactions were set up as follows:
MasterMix 8 ea. primer 1.mu.l (50 pmoles/.mu.l), gDNA 2.5 H.sub.2O
7.5 PCR was then carried out under the following conditions:
94.degree. C. 45 sec, 52.degree. C. 1.5 minute, 72.degree. C. 1.5
minute, for 30 cycles, followed by a final cycle of 72.degree. C. 5
minute, using Eppendorf's Master Taq kit (Eppendorf cat. #0032
002.250). The resulting .about.1 kb PCR product was cloned into pCR
2.1 (Invitrogen cat. # K4550-40) following the included protocol,
with chemically competent TOP1OF' E. coli as the host strain, for
verification of nucleotide sequence.
[0189] Ten of the resulting white colonies were picked into 4 ml
Luria Broth +50 .mu.g/ml Kanamycin (LB K), and grown overnight at
37.degree. C. with agitation. Plasmids were purified from each
culture using Promega Wizard Plus SV kit (Promega cat. #A1460) and
following the included protocol. Sequencing was carried out with
Beckman CEQ Quick Start Kit (Beckman Coulter cat. #608120) using
M13 Forward (5' GTA AAA CGA CGG CCA GT 3') (SEQ ID NO:16) and
Reverse (5' CAG GAA ACA GCT ATG AC 3') (SEQ ID NO:17) primers, per
manufacturers instructions. This gene sequence (SEQ ID NO:3), and
its corresponding protein (SEQ ID NO:9) was given a new general
designation for internal consistency AAD-1 (v1) (AryloxyAlkanoate
Dioxygenase).
[0190] Using the restriction enzymes corresponding to the sites
added with the primer linkers (Xba 1, Xho 1) AAD-1 (v1) was cut out
of the pCR2.1 vector and ligated into a pET 280
streptomycin/spectinomycin resistant vector. Ligated products were
then transformed into TOP1OF' E. coli, and plated on to Luria
Broth+50 .mu.g/ml Streptomycin & Spectinomycin (LB S/S) agar
plates. To differentiate between AAD-1 (v1):pET 280 and pCR2.1:pET
280 ligations, approximately 20 isolated colonies were picked into
6 ml of LB S/S, and grown at 37.degree. C. for 4 hours with
agitation.
[0191] Each culture was then spotted onto LB K plates, which were
incubated at 37.degree. C. overnight. Colonies that grew on the LB
K were assumed to have the pCR2.1 vector ligated in, and were
discarded. Plasmids were isolated from the remaining cultures as
before. This expression construct was given the designation pDAB
3203.
EXAMPLE 2
Expression and Testing
[0192] 2.1--HPLC Analysis.
[0193] Plasmid pDAB 3203 was maintained frozen at -80.degree. C. in
TOP1OF' cells (Invitrogen) as Dow Recombinant strain DR 1878. For
expression, plasmid DNA purified from TOP1OF' culture using
Promega's Wizard kit (Fisher cat. #PR-A1460) was transformed into
BL-21 Star (DE3) cells (Invitrogen cat. #C6010-03) following
manufacturer's protocol. After transformation, 50 .mu.l of the
cells were plated onto LB S/S agar plates and incubated overnight
at 37.degree. C.
[0194] The next morning, all colonies from the entire plate were
scraped into 100 mls LB in a 500 ml tri-baffled flask and incubated
at 37.degree. C/200 rpm for 1 hr. Gene expression was then induced
with 1 mM IPTG, and incubated for 4 hrs at 30.degree. C/200 rpm.
All 100 ml of culture was centrifuged at 4000 rpm for 20 mM. The
supernatants were then discarded, and the pellets were resuspended
in 10 ml of 50 mM MOPS. These were then subjected to three 45-sec
rounds of sonication to lyse the cells. Following this, lysates
were centrifuged at 15,000 rpm to remove cell debris. The
supernatant was pipetted off and stored at 4.degree. C. To check
for recombinant expression, a 20 .mu.l aliquot was run on a 4-20%
Tris Glycine gel (Invitrogen cat. #EC60255).
[0195] After expression was confirmed, enzyme activity was tested
as follows. First, an aliquot of the cell extract was desalted with
a PD-10 cartridge (Amersham cat. #17-0435-01). This was then used
for subsequent herbicide enzyme reactions.
[0196] For each reaction, the following were combined: 2,4-D (125
.mu.g/ml), [Ascorbate (1 mM), Ferrous ion (50 .mu.M),
.alpha.-ketoglutarate (1 mM), in 100 mM MOPS], cell extract (100
.mu.l). This reaction was then incubated at room temp for 30 mM,
after which the reaction was stopped with the addition of 0.1 N HC1
until pH was between 2 and 3. Half of the reaction volume
(.about.500 .mu.l) was set aside for bioassay, the remaining volume
was organically extracted using Solid Phase Extraction tubes
(Fisher cat. #11-131-6), eluting with 400 .mu.l of Acetonitrile
+0.05% TFA.
[0197] The extracts were then tested on HPLC for loss of the 2,4-D
peak or presence of any additional peaks resulting from the
degradation or modification of 2,4-D. Conditions for the HPLC were:
Luna 10 .mu., C18(2) 250.times.4.6mm (Phenomenex cat.
#00G-4253-E0), run at 50% ACN+0.05% TFA: 50% H.sub.2O+0.05% TFA to
100% ACN+0.05% TFA over 5 min.
[0198] 2.2--Plate Test Bioassays for Herbicide Degradation.
[0199] Plant bioassays were used to determine if in vitro enzymatic
herbicide transformation resulted in a concomitant loss in
herbicidal activity. Because of the selective nature of the
herbicides being tested (i.e., monocot plants controlled by AOPP
herbicides and dicot plants controlled by auxinic herbicides),
wildtype Agrostis palustris var. Pencross and Arabidopsis thaliana
var. Columbia were used as monocot and dicot test species,
respectively. Each species is amenable to germination and growth in
small Petri dishes.
[0200] Arabidopsis seeds were surface sterilized for 10 mM in 50%
commercial bleach/deionized water (v/v) with 1 .mu.L of Tween-20
added as a wetting agent with vigorous agitation (shaker table @
250 rpm). Bleach solution was decanted inside a sterile hood and
rinsed three times with sterile water. Bentgrass seeds were surface
sterilized for 20 minutes in a like manner
[0201] Twenty to thirty sterilized seeds for each test species used
were added onto a sterile, solidified agar Plate Test Medium (PTM)
[2.5 mM KNO.sub.3, 2.5 mM KH.sub.2PO.sub.4, 50 mM FeSO.sub.4, 10 mM
NaEDTA (pH 8.0), 2 mM MgSO.sub.4, 2 mM Ca(NO.sub.3).sub.2, 70 .mu.M
H.sub.3BO.sub.3, 14 .mu.M MnCl.sub.2, 0.5 .mu.M CuSO.sub.4, 1 .mu.M
ZnSO.sub.4, 0.2 .mu.M NaMoO.sub.4.2H.sub.2O, 10 .mu.M NaCl, 10 nM
CoCl2.H.sub.2O, 0.8% (w/v) sucrose, 0.4% agarose (w/v)] for
bioassay in 60.times.15-mm Petri dishes (Falcon 1007). PTM was
additionally modified by adding up to six rates of test herbicide
standards or herbicide-enzyme test solution dilutions such that the
four-fold concentration increments covered a rate range of three
orders of magnitude with the GR.sub.50 rate (50% growth reduction)
approximately in the center of the range.
[0202] For herbicide-enzyme test solutions, the maximal
concentration was determined based on the nominal concentration
before any subsequent enzymatic degradation would occur. Seeds were
evenly spread by adding 3 ml of melted PTM of the same composition,
swirling, and allowing to solidify. Plates were sealed and
maintained under sterile conditions in a low light growth chamber
(24 h day.sup.-1, 100 .mu.E/m.sup.2s.sup.1, 23.degree. C.) for 7
days. Root length or root+shoot length were measured for five
randomly chosen Arabidopsis and bentgrass plants, respectively,
average mean length (percent of untreated control) vs. nominal
herbicide concentration and GR50determined.
[0203] This bioassay was used to confirm the loss of herbicidal
activity as a result of AAD-1 (v1) degradation of the oxyalkanoate
side chain from various agronomically relevant herbicides. In
several instances, the anticipated phenol product co-eluted with
the parent acid on HPLC and the bioassay served as the primary
screen for herbicide degradation. Tables 6 and 7 represent
herbicidal substrates tested.
TABLE-US-00006 TABLE 6 Arabidopsis plate test bioassay for
commercial phenoxy and pyridinyloxyalkanoate auxin substrates.
GR.sub.50 (nM) Chemical + Chemical Chemical Blank Chemical +
GR.sub.50 tested alone Vector* AAD-1 v1 ratio** Structure 2,4-D 22
17 267 16 ##STR00055## DCP >1000 nd nd nd ##STR00056## Dichlor-
prop nd 30 1000 33 ##STR00057## Triclopyr 255 1000 1000 1
##STR00058## Fluroxy- pyr 2200 2250 1825 <1 ##STR00059## *Blank
vector represents of cell lysate treatment where E. coli pET vector
had no gene insert. **GR50 ratio is a measure of the loss of
herbicidal activity of enzyme-expressing lysate treatment vs blank
vector treatments. A number .gtoreq.2 is considered the threshold
for detecting herbicide activity loss with this assay.
TABLE-US-00007 TABLE 7 Bentgrass plate test bioassay for commercial
aryloxyphenoxyalkanoate ACCase-inhibiting substrates. GR.sub.50
(nM) Chemical + Chemical + Chemical Chemical Blank AAD-1 GR.sub.50
tested alone Vector* v1 ratio** Structure Haloxyfop- RS 28 21 520
25 ##STR00060## Diclofop- RS nd 20 130 7 ##STR00061## *Blank vector
represents of cell lysate treatment where E. coli pET vector had no
gene insert. **GR50 ratio is a measure of the loss of herbicidal
activity of enzyme-expressing lysate treatment vs blank vector
treatments. A number .gtoreq.2 is considered the threshold for
detecting herbicide activity loss with this assay.
[0204] 2.3--HPLC Results.
[0205] From the literature, it was known that dioxygenase enzymes
in this class require .alpha.-ketoglutarate as a co-substrate (for
a general scheme, see FIG. 1) and ferrous ion to bind in the active
site. Other experiments in the literature have shown that the
addition of ascorbate increased the enzymatic activity by
maintaining the iron in the reduced state, thus preventing the
enzyme for being degraded. Based on this previous work, initial
assays were set up under the assumption that the subject enzyme
would work in the same way as other members of this general class
of enzyme.
[0206] Surprisingly, the initial HPLC results showed the presence
of a new peak at 6.1 minute, in addition to a reduced 2,4-D peak at
5.5 min. This new peak was not present in the control assay. For an
initial identification of the peak at 6.1 minutes, a DCP control
was run under our assay conditions and predictably this also eluted
at 6.1 minutes. The formation of this product was confirmed using a
colorimetric assay to detect phenols (see example 3.1) as well as
mass spectrometry. As expected, AAD-1 (v1) carries out a similar
reaction as other members of this enzyme class. In the bioassay,
these same samples were also shown to have an almost complete loss
of 2,4-D herbicidal activity in the Arabidopsis plate assay (FIG.
2). Regardless of the specific conditions of the assay (i.e.,
longer incubations, more enzyme), only 50-75% of the 2,4-D could be
degraded to DCP as measured by HPLC. In fact, longer induction of
the BL-21 E. coli cells with IPTG only resulted in less active
enzyme, even though more total recombinant protein was
expressed.
[0207] After demonstrating degradation of 2,4-D, additional
substrates were tested with similar ring substitutions (i.e.,
oxyacetates and oxypropionates). The first compounds tested were
the pyridine analogs fluroxypyr and triclopyr, which are
pyridinyloxyacetates. No enzyme activity was detected on either of
these as substrates. Additional tests on various analogs of these
two pyridinyloxyacetates with either the fluorine or the amino
groups removed also were not degraded. Interestingly however,
adding a fluorine to the 5 position of 2,4-D resulted in an almost
total loss of enzyme degradation (see next section for additional
results).
[0208] ACCase inhibitors, haloxyfop and diclofop, were then tested
using the same conditions as with 2,4-D. (The corresponding phenol
metabolites co-eluted with the parent compound under the HPLC
conditions used.) The bioassay results from these samples showed
loss of herbicidal activity against both haloxyfop (FIG. 3) and
diclofop. These results were also confirmed by the colorimetric
assay, which was also used to test a wider sampling of these
compounds.
[0209] 2.4--Plate Test Bioassays for Herbicide Degradation.
[0210] Bioassay tests results corroborated initial HPLC results
that indicated loss of 2,4-D parent following incubation of 2,4-D
solutions with unpurified recombinant AAD-1 (v1) extracts (FIG. 2).
Additionally, the herbicidal activity of the phenoxypropionic acid,
dichlorprop, was also effectively degraded. The ratio of the
nominal GR50 for herbicide+enzyme solution versus the herbicide
solution alone served as measure of loss of parent herbicide
activity resulting from enzyme activity. A ratio of 2-3 typically
correlated with 50-75% loss of parent herbicide activity (Table 6).
Often a GR.sub.50 could not be determined following enzyme
treatment; de facto, no detectable herbicide activity remained.
[0211] The AOPP class of herbicides, too, served as excellent
substrates for AAD-1 (v1) as shown by near complete degradation of
graminicidal activity using the bentgrass plate bioassay (FIG. 3
and Table 7). These data are significant in that this is the first
reported observation for any members of this class of enzyme to be
active on herbicides outside the phenoxy auxins. The implications
are that this enzyme is promiscuous enough to utilize chemicals
with similar phenoxyalkanoate substructures even though they have
completely different modes of action as herbicides.
EXAMPLE 3
In vVitro Assay of AAD-1 (v1) Activity Via Colorimetric Phenol
Detection
[0212] 3.1--AAD-1 (v1) Assay.
[0213] AAD-1 (v1) enzyme activity was measured by colorimetric
detection of the product phenol using a protocol modified from that
of Fukumori and Hausinger (1993) (J. Biol. Chem. 268: 24311-24317)
to enable deployment in a 96-well microplate format. The
colorimetric assay has been described for use in measuring the
activity of dioxygenases cleaving 2,4-D and dichlorprop to release
the product 2,4-dichlorophenol. However, other phenols could
potentially be released from different aryloxyalkanoate herbicides
such as haloxyfop and cyhalofop (see FIG. 4). The color yield from
several phenols was compared to that of 2,4-dichlorophenol using
the detection method previously described to ascertain which phenol
products could be readily detected. Phenols and phenol analogs were
tested at a final concentration of 100 .mu.M in 0.15 ml 20 mM MOPS
pH 6.75 containing 200 .mu.M NH.sub.4(FeSO.sub.4).sub.2, 200 .mu.M
sodium ascorbate. The phenols derived from haloxyfop and cyhalofop
had equivalent color yields to that of 2,4-dichlorophenol and so
were readily detected. Pyridinols derived from fluroxypyr and
triclopyr produced no significant color. The color yield of
2,4-dichlorophenol and the haloxyfop phenol was linear and
proportional to the concentration of phenol in the assay up to
.about.500 .mu.M. A calibration curve performed under standard
assay conditions (160 .mu.1 final assay volume) indicated that an
absorbance at 510 nm of 1.0 was obtained from 172 .mu.M phenol.
[0214] Enzyme assays were performed in a total volume of 0.15 ml 20
mM MOPS pH 6.75 containing 200 .mu.M NH.sub.4FeSO.sub.4, 200 .mu.M
sodium ascorbate, 1 mM .alpha.-ketoglutarate, the appropriate
substrate (added from a 100 mM stock made up in DMSO), and enzyme.
Assays were initiated by addition of the aryloxyalkanoate
substrate, enzyme or a-ketoglutarate at time zero. After 15 minutes
of incubation at 25.degree. C., the reaction was terminated by
addition of 10 .mu.1 100 mM sodium EDTA. Color was developed by
addition of 15 .mu.1 pH 10 buffer (3.09 g boric acid+3.73 g KCl+44
ml 1 N KOH), 1.5 .mu.l 2% 4-aminoantipyrine and 1.5 .mu.l 8%
potassium ferricyanide. After 10 to 20 mM, the absorbance at 510 nm
was recorded in a spectrophotometric microplate reader. Blanks
contained all reagents except for enzyme to account for the
occasional slight contamination of some of the substrates by small
amounts of phenols. Later assays were made more convenient by
consolidating the additions as follows: the reaction was quenched
by addition of 30 .mu.l of a 1:1:1 mix of 50 mM Na EDTA; pH 10
buffer and 0.2% 4-aminoantipyrine, then adding 10 .mu.l 0.8%
potassium ferricyanide.
[0215] 3.2--Extraction.
[0216] Activity of recombinant AAD-1 (v1) expressed in Escherichia
coli. E. coli cell pellets were resuspended in 0.1 M Tris, pH 7.4+1
mg/ml lysozyme (5 ml/cells from 250 ml culture; 20 ml/cells from 1
liter) at room temperature. After about 15 minutes with occasional
shaking, the suspension was frozen in liquid nitrogen then thawed.
DNase was added to 0.02 mg/ml final concentration and MgCl.sub.2 to
1 mM. After the extract was no longer viscous, the extract was
centrifuged for 15 mM. The supernatant was passed over a BioRad
10DG column pre-equilibrated with 20 mM MOPS pH 6.75 and the eluant
stored in aliquots at -70.degree. C. Assays were either performed
with these unpurified desalted extracts or with purified
enzymes.
[0217] A cell pellet from a 250 ml culture of induced E. coli cells
expressing pDAB3203 containing the gene encoding AAD-1 (v1) was
extracted and assayed using the previously described protocols. The
2,4-D cleaving activity in the AAD-1 (v1) extract was compared to
that from E. coli cells expressing a vector without AAD-1 (v1)
using 1 mM 2,4-D and is shown in FIG. 5. The amount of
2,4-dichlorophenol formed is clearly proportional to the amount of
extract added to the assay whereas the control extract contains no
2,4-D cleaving activity.
[0218] The activity of this extract was tested on four additional
herbicides, (R,S)-dichlorprop, (R,S)-mecoprop, (R,S)-haloxyfop and
(R,S)-diclofop in comparison to 2,4-D (all at a final concentration
of 0.5 mM) using 4.mu.l of the E. coli extract per assay with a 15
mM assay period. FIG. 6A shows that AAD-1 (v1) cleaved all five
herbicides to yield a phenol with the relative activity on the
substrates being dichlorprop=mecoprop>diclofop>haloxyfop
2,4-D. Thus AAD-1 (v1) has activity on graminicidal
aryloxyphenoxypropionate herbicides as well as phenoxy auxins.
[0219] The AAD-1 (v1) extract was then tested using racemic
(R,S)-haloxyfop, the R enantiomer of haloxyfop and the S-enantiomer
of cyhalofop (all at 0.5 mM) as potential substrates to ascertain
the likely enantiomeric specificity of AAD-1 (v1). The results are
shown in FIG. 6B. The activity of the enzyme on (R)-haloxyfop was
equivalent to that on (R,S)-haloxyfop whereas no activity could be
seen on the S-enantiomer of cyhalofop indicating that AAD-1 (v1)
has R specificity on AOPPs.
EXAMPLE 4
Substrate Specificity of AAD-1 (v1)
[0220] 4.1--Additional Substrates of AAD-1 (v1).
[0221] The substrate specificity of AAD-1 (v1) toward a variety of
commercial and experimental herbicides was tested. Purified AAD-1
(v1) was used at either 1 or 10 .mu.g per 160 .mu.l assay and each
substrate was tested at 1 mM with an assay time of 15 minutes.
Table 3 shows the A510 detected after the action of AAD-1 (v1) on a
variety of aryloxyalkanoate auxinic herbicides and auxin analogs.
The best substrate tested was dichlorprop, with mecoprop also being
efficiently cleaved. Two other phenoxypropionates, the 4-fluoro and
3-amino analogs of dichlorprop, were also acted on effectively by
AAD-1 (v1). AAD-1 (v1) produced small amounts of phenol from a
variety of phenoxyacetates including 2,4-D. The relative rates on
these substrates are better gauged from the assays using the higher
amounts (10 .mu.g) of AAD-1 (v1). From these data, 2,4-D is cleaved
by AAD-1 (v1), as are two phenoxyalkylsulfonates, X188476 and
X398166 (sesone).
[0222] Table 4 shows data for a variety of AOPP graminicide
herbicides as AAD1 substrates, and also the safener cloquintocet.
All the commercial AOPP herbicides tested were effectively cleaved
by AAD-1 (v1). This is an unexpected discovery and greatly
increases the potential utility of this enzyme for conferring
resistance to a wide variety of graminicidal herbicides in
transgenic uses, in addition to auxins. AAD-1 (v1) had the highest
activity on quizalofop (76% of the dichlorprop rate) and lowest
activity on cyhalofop (27% of the quizalofop rate, 21% of the
dichlorprop rate). The aryloxyacetate analog of haloxyfop (X043865)
was cleaved very slowly with only a small increase in A510 using
the higher (10 .mu.g) amount of enzyme. This is consistent with
higher activity of AAD-1 (v1) seen on phenoxypropionates relative
to auxin phenoxyacetates. Minimal activity was detected on
(S)-cyhalofop indicating that AAD-1 (v1) has a significant
preference for the R enantiomers of aryloxypropionate substrates.
Similarly, no activity was noted against the quinolinoxyacetate
safener, cloquintocet, which is consistent with the observation
that AAD-1 (v1) prefers aryloxypropionate substrates over phenoxy
auxins.
[0223] Substrates X11115427, X124987 and MCPA were tested at 1 mM
using 27 .mu.g crude recombinant AAD-1 (v1) per assay. All three
compounds were substrates for AAD-1 (v1) but with different
relative effectiveness (Table 8). X11115427 was slightly better as
a substrate than 2,4-D (125% of the 2,4-D rate) in contrast to the
close analog 3-amino-dichlorprop, which is .about.7-fold better
than 2,4-D as a substrate (Table 3). The 5-F substitution appears
to decrease the effectiveness of X11115427 as a substrate for AAD-1
(v1). The rates of product formation from 5-F-phenoxyacetate and
MCPA were 32% and 55% that of 2,4-D respectively.
[0224] Table 8: Effect of AAD-1 (v1) on three substrates relative
to 2,4-D. Substrates were assayed as in Table 6 at 1 mM using a
crude recombinant AAD-1 (v1) extract from E. coli.
TABLE-US-00008 TABLE 8 Effect of AAD-1 (v1) on three substrates
relative to 2,4-D. Registry ID MOLSTRUCTURE Compound A510 % 2,4-D
195517 ##STR00062## 2,4-D 0.177 100 11115427 ##STR00063##
(R,S)-3-amino, 5- F-dichlorprop 0.221 125 124987 ##STR00064## 5-F,
2,4-D 0.056 32 192711 ##STR00065## MCPA 0.097 55
[0225] 4.2--Kinetic Characterization.
[0226] The K.sub.m and k.sub.cat values of purified AAD-1 (v1) (see
Example 10) were determined for four herbicide substrates,
(R)-dichlorprop, (R)-haloxyfop, (R)-quizalofop and 2,4-D under
standard assay conditions (25 mM MOPS, pH 6.8; 200 .mu.M Na
ascorbate; 200 .mu.M Fe.sup.0+; 1 mM .alpha.-ketoglutarate;
25.degree. C.). The dose response curves were fitted using Grafit
(Erithacus Software, UK), and the graphs and derived constants are
shown in FIG. 7 and Table 9 respectively. The K.sub.m values for
the four substrates were fairly similar (75-125 .mu.M), but the
k.sub.cat values varied significantly. (R)-dichlorprop had the
highest k.sub.cat value and 2,4-D the lowest (10% that of
(R)-dichlorprop). These k.sub.cat values were consistent with the
range of values seen in the substrate specificity tests shown in
Table 3 and Table 4 as these were performed at high (saturating)
substrate concentrations (1 mM).
TABLE-US-00009 TABLE 9 Kinetic Constants for AAD-1 (v1) Substrates.
Kinetic constants were derived from the data in FIG. 7 using Grafit
fitting to the Michaelis-Menten equation. Kinetic Constants for
AAD-1 (v1) Substrates. V.sub.max K.sub.m (.mu.mol (.mu.M)
min.sup.-1 mg.sup.-1 k.sub.cat k.sub.cat/K.sub.m Substrate .+-. SE
AAD1) .+-. SE (min.sup.-1) (min.sup.-1 mM.sup.-1) R-dichlorprop 75
.+-. 10 0.79 .+-. 0.03 26.1 348 R-quizalofop 125 .+-. 20 0.57 .+-.
0.03 18.9 151 R-haloxyfop 120 .+-. 54 0.34 .+-. 0.04 11.2 94 2,4-D
96 .+-. 8 0.57 .+-. 0.00 2.7 28
[0227] The relative K.sub.m and kcat values for dichlorprop and
2,4-D differ significantly from those published for the R-specific
dioxygenase from Delftia acidovorans by Westendorf et al. (2003)
(Acta Biotechnol. 23: 3-17). The published k.sub.cat/K.sub.m value
for 2,4-D is 0.6% that of dichlorprop, whereas in our studies, the
kcat/Km value for 2,4-D is 8% that of dichlorprop. Thus, in this
study, AAD-1 (v1) is unexpectedly effective at catalyzing the
cleavage of 2,4-D. This increases its potential utility for
conferring diverse herbicide tolerance traits in transgenic
applications.
[0228] 4.3--Additional Substrates for AAD-1 (v1).
[0229] Three additional substrates were tested at 1 mM using 27
.mu.g crude recombinant AAD-1 (v1) per assay; X11115427, X124987
and MCPA. The results are shown in Table 8. All three compounds
were substrates for AAD-1 (v1) but with different relative
effectiveness. X11115427 was only slightly better (125%) as a
substrate than 2,4-D. This is in contrast to 3-aminodichlorprop
which is >7 fold better than 2,4-D as a substrate (Table 3).
Thus, the 5-F substitution has significantly decreased the
effectiveness of X11115427 as a substrate for AAD-1 (v1). A similar
pattern is seen with 5-F-2,4-D which is only 32% as effective as a
substrate relative to 2,4-D. In this assay, MCPA was also less
effective as a substrate of AAD-1 (v1) (55% relative to 2,4-D).
EXAMPLE 5
Optimization of Sequence for Expression in Plants
[0230] 5.1--Background.
[0231] To obtain high expression of heterologous genes in plants,
it may be preferred to reengineer said genes so that they are more
efficiently expressed in (the cytoplasm of) plant cells. Maize is
one such plant where it may be preferred to re-design the
heterologous gene(s) prior to transformation to increase the
expression level thereof in said plant. Therefore, an additional
step in the design of genes encoding a bacterial protein is
reengineering of a heterologous gene for optimal expression.
[0232] One reason for the reengineering of a bacterial protein for
expression in maize is due to the non-optimal G+C content of the
native gene. For example, the very low G+C content of many native
bacterial gene(s) (and consequent skewing towards high A+T content)
results in the generation of sequences mimicking or duplicating
plant gene control sequences that are known to be highly A+T rich.
The presence of some A+T-rich sequences within the DNA of gene(s)
introduced into plants (e.g., TATA box regions normally found in
gene promoters) may result in aberrant transcription of the
gene(s). On the other hand, the presence of other regulatory
sequences residing in the transcribed mRNA (e.g., polyadenylation
signal sequences (AAUAAA), or sequences complementary to small
nuclear RNAs involved in pre-mRNA splicing) may lead to RNA
instability. Therefore, one goal in the design of genes encoding a
bacterial protein for maize expression, more preferably referred to
as plant optimized gene(s), is to generate a DNA sequence having a
higher G+C content, and preferably one close to that of maize genes
coding for metabolic enzymes. Another goal in the design of the
plant optimized gene(s) encoding a bacterial protein is to generate
a DNA sequence in which the sequence modifications do not hinder
translation.
[0233] Table 10 illustrates how high the G+C content is in maize.
For the data in Table 10, coding regions of the genes were
extracted from GenBank (Release 71) entries, and base compositions
were calculated using the MacVector.TM. program (Accelerys, San
Diego, Calif.). Intron sequences were ignored in the
calculations.
TABLE-US-00010 TABLE 10 Compilation of G + C contents of protein
coding regions of maize genes Range % Mean % Protein Class.sup.a G
+ C G + C.sup.b Metabolic Enzymes (76) 44.4-75.3 59.0 (.+ -.8.0)
Structural Proteins (18) 48.6-70.5 63.6 (.+ -.6.7) Regulatory
Proteins (5) 57.2-68.8 62.0 (.+ -.4.9) Uncharacterized Proteins (9)
41.5-70.3 64.3 (.+ -.7.2) All Proteins (108) 44.4-75.3 60.8 (.+
-.5.2) .sup.a Number of genes in class given in parentheses. .sup.b
Standard deviations given in parentheses. .sup.c Combined groups
mean ignored in mean calculation
[0234] Due to the plasticity afforded by the redundancy/degeneracy
of the genetic code (i.e., some amino acids are specified by more
than one codon), evolution of the genomes in different organisms or
classes of organisms has resulted in differential usage of
redundant codons. This "codon bias" is reflected in the mean base
composition of protein coding regions. For example, organisms with
relatively low G+C contents utilize codons having A or T in the
third position of redundant codons, whereas those having higher G+C
contents utilize codons having G or C in the third position. It is
thought that the presence of "minor" codons within an mRNA may
reduce the absolute translation rate of that mRNA, especially when
the relative abundance of the charged tRNA corresponding to the
minor codon is low. An extension of this is that the diminution of
translation rate by individual minor codons would be at least
additive for multiple minor codons. Therefore, mRNAs having high
relative contents of minor codons would have correspondingly low
translation rates. This rate would be reflected by subsequent low
levels of the encoded protein.
[0235] In engineering genes encoding a bacterial protein for maize
(or other plant, such as cotton or soybean) expression, the codon
bias of the plant has been determined. The codon bias for maize is
the statistical codon distribution that the plant uses for coding
its proteins and the preferred codon usage is shown in Table 11.
After determining the bias, the percent frequency of the codons in
the gene(s) of interest is determined. The primary codons preferred
by the plant should be determined, as well as the second, third,
and fourth choices of preferred codons when multiple choices exist.
A new DNA sequence can then be designed which encodes the amino
sequence of the bacterial protein, but the new DNA sequence differs
from the native bacterial DNA sequence (encoding the protein) by
the substitution of the plant (first preferred, second preferred,
third preferred, or fourth preferred) codons to specify the amino
acid at each position within the protein amino acid sequence. The
new sequence is then analyzed for restriction enzyme sites that
might have been created by the modification. The identified sites
are further modified by replacing the codons with first, second,
third, or fourth choice preferred codons. Other sites in the
sequence which could affect transcription or translation of the
gene of interest are the exon:intron junctions (5' or 3'), poly A
addition signals, or RNA polymerase termination signals. The
sequence is further analyzed and modified to reduce the frequency
of TA or GC doublets. In addition to the doublets, G or C sequence
blocks that have more than about four residues that are the same
can affect transcription of the sequence. Therefore, these blocks
are also modified by replacing the codons of first or second
choice, etc. with the next preferred codon of choice.
TABLE-US-00011 TABLE 11 Preferred amino acid codons for proteins
expressed in maize Amino Acid Codon* Alanine GCC/GCG Cysteine
TGC/TGT Asp artic Acid GAC/GAT Glutamic Acid GAG/GAA Phenyl alanine
TTC/TTT Glycine GGC/GGG Histidine CAC/CAT Isoleucine ATC/ATT Lysine
AAG/AAA Leucine CTG/CTC Methionine ATG Asparagine AAC/AAT Proline
CCG/CCA Glutamine CAG/CAA Arginine AGG/CGC Serine AGC/TCC Threonine
ACC/ACG Valine GTG/GTC Tryptophan TGG Tryrosine TAC/TAT Stop
TGA/TAG
[0236] It is preferred that the plant optimized gene(s) encoding a
bacterial protein contain about 63% of first choice codons, between
about 22% to about 37% second choice codons, and between about 15%
to about 0% third or fourth choice codons, wherein the total
percentage is 100%. Most preferred the plant optimized gene(s)
contains about 63% of first choice codons, at least about 22%
second choice codons, about 7.5% third choice codons, and about
7.5% fourth choice codons, wherein the total percentage is 100%.
The method described above enables one skilled in the art to modify
gene(s) that are foreign to a particular plant so that the genes
are optimally expressed in plants. The method is further
illustrated in PCT application WO 97/13402.
[0237] Thus, in order to design plant optimized genes encoding a
bacterial protein, a DNA sequence is designed to encode the amino
acid sequence of said protein utilizing a redundant genetic code
established from a codon bias table compiled for the gene sequences
for the particular plant. The resulting DNA sequence has a higher
degree of codon diversity, a desirable base composition, can
contain strategically placed restriction enzyme recognition sites,
and lacks sequences that might interfere with transcription of the
gene, or translation of the product mRNA. Thus, synthetic genes
that are functionally equivalent to the proteins/genes of the
subject invention can be used to transform hosts, including plants.
Additional guidance regarding the production of synthetic genes can
be found in, for example, U.S. Pat. No. 5,380,831.
[0238] 5.2--Rebuild Analysis.
[0239] Extensive analysis of the 888 base pairs (bp) of the DNA
sequence of the native AAD-1 (0) coding region (SEQ ID NO:3)
revealed the presence of several sequence motifs that are thought
to be detrimental to optimal plant expression, as well as a
non-optimal codon composition. To improve production of the
recombinant protein in monocots as well as dicots, a
"plant-optimized" DNA sequence (SEQ ID NO:5) was developed that
encodes SEQ ID NO:11, which is the same as the native SEQ ID NO:9
except for the addition of an alanine residue at the second
position. The additional alanine codon (GCT; underlined in SEQ ID
NO:5) was included to encode an Nco I site (CCATGG) spanning the
ATG start codon, to enable subsequent cloning operations. The
proteins encoded by the native (v1) and plant-optimized (v3) coding
regions are 99.3% identical, differing only at amino acid number 2.
In contrast, the native (v1) and plant-optimized (v3) DNA sequences
of the coding regions are only 77.7% identical. A sequence
alignment was made of the native and plant-optimized DNAs, and
Table 12 shows the differences in codon compositions of the native
and plant-optimized sequences.
TABLE-US-00012 TABLE 12 Codon composition comparison of native
AAD-1 (v1) coding region and Plant- Optimized version. Plant Plant
Plant Plant Amino Native Native Opt Opt Amino Native Opt Opt Acid
Codon Gene # Gene % Gene # Gene % Acid Codon Gene # Gene # Gene %
ALA (A) GCA 2 9.1 5 22 LEU (L) CTA 0 0.0 0 0 22 GCC 10 45.5 8 35 22
CTC 5 22.7 7 32 GCG 9 40.9 0 0 CTG 16 72.7 0 0 GCT 1 4.5 10 43 CTT
0 0.0 8 36 ARG (R) AGA 0 0.0 7 30 TTA 0 0.0 0 0 23 AGG 0 0.0 7 30
TTG 1 4.5 7 32 CGA 0 0.0 0 0 LYS (K) AAA 1 14.3 2 29 CGC 16 69.6 5
22 7 AAG 6 85.7 5 71 CGG 4 17.4 0 0 MET (M) ATG 8 100 8 100 CGT 3
13.0 4 17 PHE (F) TTC 10 76.9 9 69 ASN (N) AAC 8 100.0 4 50 13 TTT
3 23.1 4 31 8 AAT 0 0.0 4 50 PRO (P) CCA 1 5.9 8 47 ASP (D) GAC 15
78.9 10 53 17 CCC 9 52.9 1 6 19 GAT 4 21.1 9 47 CCG 7 41.2 0 0 CYS
(C) TGC 3 100.0 2 67 CCT 0 0.0 8 47 3 TGT 0 0.0 1 33 SER (S) AGC 11
73.3 4 27 END TAA 0 0.0 0 0 15 AGT 0 0.0 0 0 1 TAG 1 100.0 0 0 TCA
0 0.0 4 27 TGA 0 0.0 1 100 TCC 2 13.3 3 20 GLN (Q) CAA 0 0.0 7 54
TCG 2 13.3 0 0 13 CAG 13 100.0 6 46 TCT 0 0.0 4 27 GLU (E) GAA 8
50.0 5 31 THR (T) ACA 1 4.3 6 26 16 GAG 8 50.0 11 69 23 ACC 16 69.6
9 39 GLY (G) GGA 0 0.0 6 29 ACG 5 21.7 0 0 21 GGC 15 71.4 7 33 ACT
1 4.3 8 35 GGG 3 14.3 2 10 TRP (W) TGG 5 100 5 100 GGT 3 14.3 6 29
TYR (Y) TAC 7 70.0 6 60 HIS (H) CAC 6 60.0 5 50 10 TAT 3 30.0 4 40
10 CAT 4 40.0 5 50 VAL (V) GTA 2 7.1 0 0 ILE (I) ATA 0 0.0 3 25 28
GTC 11 39.3 8 29 12 ATC 12 100.0 5 42 GTG 15 53.6 10 36 ATT 0 0.0 4
33 GTT 0 0.0 10 36 Totals 148 149 Totals 148 148
[0240] 5.3--Completion of Binary Vectors.
[0241] 5.3.1--Rebuilt AAD-1 (v3). The Plant Optimized Gene AAD-1
(v3) was Received from Picoscript (the gene rebuild design was
completed (see above) and out-sourced to Picoscript for
construction) and sequence verified (SEQ ID NO:5) internally, to
confirm that no alterations of the expected sequence were present.
The sequencing reactions were carried out with M13 Forward (SEQ ID
NO:16) and M13 Reverse (SEQ ID NO:17) primers using the Beckman
Coulter "Dye Terminator Cycle Sequencing with Quick Start Kit"
reagents as before. Sequence data was analyzed and results
indicated that no anomalies were present in the plant optimized
AAD-1 (v3) DNA sequence. The AAD-1 (v3) gene was cloned into
pDAB726 as an Nco I--Sac I fragment. The resulting construct was
designated pDAB720, containing: [AtUbil0 promoter: Nt OSM 5'UTR:
AAD-1 (v3): Nt OSM3'UTR: ORF1 polyA3'UTR] (verified with Not I
restriction digests). A Not I-Not I fragment containing the
described cassette was then cloned into the Not I site of the
binary vector pDAB3038. The resulting binary vector, pDAB721,
containing the following cassette [AtUbil0 promoter: Nt OSMS'UTR:
AAD-1 (v3): Nt OSM 3'UTR: ORF1 polyA 3'UTR: CsVMV promoter: PAT:
ORF25/26 3'UTR] was restriction digested (with Bam HI, EcoR I, EcoR
V, HinD III, Pac I, and Xmn I) for verification of the correct
orientation. The verified completed construct (pDAB721) was used
for transformation into Agrobacterium (see section 6.2).
[0242] 5.3.2--Native AAD1 (v1) and Modified AAD-1 (v2). The AAD-1
(v1) gene (SEQ ID NO:3) was PCR amplified from pDAB3203. During the
PCR reaction, alterations were made within the primers to introduce
the Ncol and Sad restriction sites in the 5' primer and 3' primer,
respectively. The primers "rdpA(ncol)" [CCC ATG GCT GCT GCA CTG TCC
CCC CTC TCC] (SEQ ID NO:6) and "3'saci" [GAG CTC ACT AGC GCG CCG
GGC GCA CGC CAC CGA] (SEQ ID NO:7) were used to amplify a DNA
fragment using the Fail Safe PCR System (Epicenter).
[0243] The PCR amplicon was ligated into the pCR 2.1 TOPO TA
cloning vector (Invitrogen) and sequence verified with M13 Forward
(SEQ ID NO:16) and M13 Reverse (SEQ ID NO:17) primers using the
Beckman Coulter "Dye Terminator Cycle Sequencing with Quick Start
Kit" sequencing reagents.
[0244] Sequence data identified a clone with the correct sequence.
During analysis a superfluous Notl restriction site was identified
toward the 3' end of AAD-1 (v1). This site was removed to
facilitate cloning into pDAB3038. To remove the additional site a
PCR reaction was performed with an internal 5' primer. The NotI
site was altered by incorporating a new codon for an amino acid to
remove the spurious Nod site. This change would alter the arginine
at position 212 to a cysteine. The PCR primers "BstEII/Del NotI"
[TGG TGG TGA CCC ATC CGG GCA GCG GCT GCA AGG GCC] (SEQ ID NO:8) and
"3' saci" (SEQ ID NO:7) were used.
[0245] A PCR reaction was completed using the Fail Safe PCR System
(Epicenter) and the resulting fragment was cloned into the pCR 2.1
TOPO TA cloning kit (Invitrogen). Confirmation of the correct PCR
product was completed by DNA sequencing, and the "fixed" gene was
given the designation AAD-1 (v2) (SEQ ID NO:4).
[0246] A sequencing reaction using the M13 Reverse primer (SEQ ID
NO:17) and the Beckman Coulter "Dye Terminator Cycle Sequencing
with Quick Start Kit" sequencing reagents indicated that the
correct PCR fragment had been isolated. This construct was digested
with the BstEII and Sad enzymes. The resulting fragment was cloned
into the pCR2.1 AAD-1 (v2) construct (pCR2.1 Delta NotI) and
confirmed via restriction enzyme digestion.
[0247] The modified AAD-1 (v2) gene was then cloned into pDAB726 as
a NcoI/Sacl DNA fragment. The resulting construct (pDAB708) was
verified with restriction digests. This construct was then cloned
into the binary pDAB3038 as a NotI-NotI fragment. The final
resulting construct was given the designation pDAB766, containing
the [AtUbi10 promoter: Nt OSMS'UTR: AAD-1 (v2): Nt OSM 3'UTR: ORF1
polyA 3'UTR: CsVMV promoter: PAT: ORF25/26 3'UTR] and was
restriction digested for verification of the correct orientation.
The completed construct was then used for transformation into
Agrobacterium.
[0248] 5.3.3--Design of a soybean-codon-biased DNA sequence
encoding a soybean EPSPS having mutations that confer glyphosate
tolerance. This example teaches the design of a new DNA sequence
that encodes a mutated soybean 5-enolpyruvoylshikimate 3-phosphate
synthase (EPSPS), but is optimized for expression in soybean cells.
The amino acid sequence of a triply-mutated soybean EPSPS is
disclosed as SEQ ID NO:5 of WO 2004/009761. The mutated amino acids
in the so-disclosed sequence are at residue 183 (threonine of
native protein replaced with isoleucine), residue 186 (arginine in
native protein replaced with lysine), and residue 187 (proline in
native protein replaced with serine). Thus, one can deduce the
amino acid sequence of the native soybean EPSPS protein by
replacing the substituted amino acids of SEQ ID NO:5 of WO
2004/009761 with the native amino acids at the appropriate
positions. Such native protein sequence is presented herein as SEQ
ID NO:20. A doubly mutated soybean EPSPS protein sequence,
containing a mutation at residue 183 (threonine of native protein
replaced with isoleucine), and at residue 187 (proline in native
protein replaced with serine) is presented herein as SEQ ID
NO:21.
[0249] A codon usage table for soybean (Glycine max) protein coding
sequences, calculated from 362,096 codons (approximately 870 coding
sequences), was obtained from the "kazusa.or.jp/codon" World Wide
Web site. Those data were reformatted as displayed in Table 13.
Columns D and H of Table 13 present the distributions (in % of
usage for all codons for that amino acid) of synonymous codons for
each amino acid, as found in the protein coding regions of soybean
genes. It is evident that some synonymous codons for some amino
acids (an amino acid may be specified by 1, 2, 3, 4, or 6 codons)
are present relatively rarely in soybean protein coding regions
(for example, compare usage of GCG and GCT codons to specify
alanine). A biased soybean codon usage table was calculated from
the data in Table 13. Codons found in soybean genes less than about
10% of total occurrences for the particular amino acid were
ignored. To balance the distribution of the remaining codon choices
for an amino acid, a weighted average representation for each codon
was calculated, using the formula:
Weighted % of C1=1/(%C1+%C2+%C3+etc.).times.%C1.times.100
where C1 is the codon in question, C2, C3, etc. represent the
remaining synonymous codons, and the % values for the relevant
codons are taken from columns D and H of Table 13 (ignoring the
rare codon values in bold font). The Weighted % value for each
codon is given in Columns C and G of Table 13. TGA was arbitrarily
chosen as the translation terminator. The biased codon usage
frequencies were then entered into a specialized genetic code table
for use by the OptGene.TM. gene design program (Ocimum Biosolutions
LLC, Indianapolis, Ind.).
TABLE-US-00013 TABLE 13 Synonymous codon representation in soybean
protein coding sequences, and calculation of a biased codon
representation set for soybean-optimized synthetic gene design. A B
C D E F G H Amino Weighted Soybean Amino Weighted Soybean Acid
Codon % % Acid Codon % % ALA (A) GCA 33.1 30.3 LEU (L) CTA DNU 9.1
GCC 24.5 22.5 CTC 22.4 18.1 GCG DNU* 8.5 CTG 16.3 13.2 GCT 42.3
38.7 CTT 31.5 25.5 ARG (R) AGA 36.0 30.9 TTA DNU 9.8 AGG 32.2 27.6
TTG 29.9 24.2 CGA DNU 8.2 LYS (K) AAA 42.5 42.5 CGC 14.8 12.7 AAG
57.5 57.5 CGG DNU 6.0 MET (M) ATG 100.0 100 CGT 16.9 14.5 PHE (F)
TTC 49.2 49.2 ASN (N) AAC 50.0 50.0 TTT 50.8 50.8 AAT 50.0 50.0 PRO
(P) CCA 39.8 36.5 ASP (D) GAC 38.1 38.1 CCC 20.9 19.2 GAT 61.9 61.9
CCG DNU 8.3 CYS (C) TGC 50.0 50.0 CCT 39.3 36.0 TGT 50.0 50.0 SER
(S) AGC 16.0 15.1 END TAA DNU 40.7 AGT 18.2 17.1 TAG DNU 22.7 TCA
21.9 20.6 TGA 100.0 36.6 TCC 18.0 16.9 GLN (Q) CAA 55.5 55.5 TCG
DNU 6.1 CAG 44.5 44.5 TCT 25.8 24.2 GLU (E) GAA 50.5 50.5 THR (T)
ACA 32.4 29.7 GAG 49.5 49.5 ACC 30.2 27.7 GLY (G) GGA 31.9 31.9 ACG
DNU 8.3 GGC 19.3 19.3 ACT 37.4 34.3 GGG 18.4 18.4 TRP (W) TGG 100.0
100 GGT 30.4 30.4 TYR (Y) TAC 48.2 48.2 HIS (H) CAC 44.8 44.8 TAT
51.8 51.8 CAT 55.2 55.2 VAL (V) GTA 11.5 11.5 ILE (I) ATA 23.4 23.4
GTC 17.8 17.8 ATC 29.9 29.9 GTG 32.0 32.0 ATT 46.7 46.7 GTT 38.7
38.7 *DNU = Do Not Use
[0250] To derive a soybean-optimized DNA sequence encoding the
doubly mutated EPSPS protein, the protein sequence of SEQ ID NO:21
was reverse-translated by the OptGene.TM. program using the
soybean-biased genetic code derived above. The initial DNA sequence
thus derived was then modified by compensating codon changes (while
retaining overall weighted average representation for the codons)
to reduce the numbers of CG and TA doublets between adjacent
codons, increase the numbers of CT and TG doublets between adjacent
codons, remove highly stable intrastrand secondary structures,
remove or add restriction enzyme recognition sites, and to remove
other sequences that might be detrimental to expression or cloning
manipulations of the engineered gene. Further refinements of the
sequence were made to eliminate potential plant intron splice
sites, long runs of A/T or C/G residues, and other motifs that
might interfere with RNA stability, transcription, or translation
of the coding region in plant cells. Other changes were made to
eliminate long internal Open Reading Frames (frames other than+1).
These changes were all made within the constraints of retaining the
soybean-biased codon composition as described above, and while
preserving the amino acid sequence disclosed as SEQ ID NO:21.
[0251] The soybean-biased DNA sequence that encodes the EPSPS
protein of SEQ ID
[0252] NO:21 is given as bases 1-1575 of SEQ ID NO:22. Synthesis of
a DNA fragment comprising SEQ ID NO:22 was performed by a
commercial supplier (PicoScript, Houston Tex.).
[0253] 5.3.4--Cloning of additional binary constructs. The
completion of pDAB3295 and pDAB3757 incorporated the use of the
GateWay Cloning Technology (Invitrogen, cat #11791-043 and cat
#12535-019). The GateWay Technology uses lambda phage-based
site-specific recombination to insert a gene cassette into a
vector. For more information refer to Gateway Technology: A
universal technology to clone DNA sequence for functional analysis
and expression in multiple systems, 1999-2003, Invitrogen Corp.,
1600 Faraday Ave., Carlsbad, Calif. 92008 (printed--2003). All
other constructs created for transformation into appropriate plant
species were built using similar procedures as above and other
standard molecular cloning methods (Maniatis et al., 1982). Table
14 lists all the transformation constructs used with appropriate
promoters and features defined, as well as the crop
transformed.
[0254] The sacB gene was added to the binary vector pDAB3289 as a
bacterial negative selection marker to reduce the persistence of
Agrobacterium associated with transformed plant tissue. SacB is a
levan-sucrase enzyme produced by Bacillus spp. and is toxic to most
Gram negative bacteria when grown in the presence of sucrose (Gay
et al., 1983). The sacB gene was recovered on a Hind III fragment
from plasmid pRE112 (Edwards, et al., 1998) and cloned into the
unique Hind III site in pDAB3289.
TABLE-US-00014 TABLE 14 Binary constructs used in transformation of
various plant species. Species * Gene of pDAB pDAS Trans-formed
interest Feature Feature # # into (GOI) Promoter 1 2 GOI 2 721 A,
T, Ct, S, Ca AAD1 v3 AtUbi10 NtOsm -- -- 3230 A EPSPS AtUbi10 NtOsm
RB7 Mar -- v2 3289 S AAD1 v3 CsVMV NtOsm RB7 Mar EPSPS v2 3291 S
AAD1 v3 CsVMV NtOsm RB7 Mar EPSPS v2 3295 S AAD1 v3 CsVMV NtOsm RB7
Mar -- v2 3297 1270 A, T AAD1 v3 CsVMV NtOsm RB7 Mar -- v2 3403 Cn,
R AAD1 v3 ZmUbi1 -- RB7 Mar -- v2 3404 Cn AAD1 v3 ZmUbi1 -- RB7 Mar
-- v2 3415 1283 Cn AAD1 v3 ZmUbi1 -- RB7 Mar -- v2 3602 1421 Cn
AAD1 v3 ZmUbi1 -- RB7 Mar -- v2 3757 Ca AAD1 v3 CsVMV NtOsm RB7 Mar
EPSPS v2 3705 A AAD2 v2 AtUbi10 NtOsm RB7 Mar -- v2 Bacterial Plant
pDAB Bacterial Selection Selection # Promoter Selection gene gene 2
gene Promoter Trxn Method 721 -- Erythromycin -- pat CsVMV Agro
binary 3230 -- Spectinomycin -- AAD1 v3 CsVMV Agro binary 3289
AtUbi10 Spectinomycin sacB Hptll AtUbi3 Agro binary 3291 AtUbi10
Spectinomycin -- Hptll AtUbi3 Agro binary 3295 -- Spectinomycin --
pat AtUbi10 Aerosol beam 3297 -- Spectinomycin -- pat AtUbi10 Agro
binary 3403 -- Ampicillin -- Same as GOI Whiskers/Gun 3404 --
Ampicillin -- pat OsAct1 Whiskers 3415 -- Ampicillin -- AHAS v3
OsAct1 Whiskers 3602 -- Spectinomycin -- AHAS v3 OsAct1 Agro
Superbinary 3757 AtUbi10 Spectinomycin -- pat AtUbi11 Agro binary
3705 -- Erythromycin -- pat CsVMV Agro binary * A = Arabidopsis
CsVMV = Cassava Vein Mosaic Virus Promoter ZmUbi1 = Zea mays
Ubiquitin 1 Promoter T = Tobacco AtUbi10 = Arabidopsis thaliana
Ubiquitin 10 Promoter Hptll = hygromycin phosphotransferase S =
Soybean RB7 Mar v2 = Nicotiana tabacum matrix associated region
(MAR) Ct = Cotton Nt Osm = Nicotiana tabacum Osmotin 5'
Untranslated Region and the Nicotiana tabacum Osmotin 3'
Untranslated Region R = Rice Cn = Corn (721 and 793) Atu ORF1 3'
UTR = Agrobacterium tumefaciens Open Reading Frame 1 3'
Untranslated Region Ca = Canola (3295 and 3757) Atu 0RF24 3' UTR =
Agrobacterium tumefaciens Open Reading Frame 24 3' Untranslated
Region
EXAMPLE 6
Transformation into Arabidopsis and Selection
[0255] 6.1--Arabidopsis thaliana Growth Conditions.
[0256] Wildtype Arabidopsis seed was suspended in a 0.1% Agarose
(Sigma Chemical Co., St. Louis, Mo.) solution. The suspended seed
was stored at 4.degree. C. for 2 days to complete dormancy
requirements and ensure synchronous seed germination
(stratification).
[0257] Sunshine Mix LP5 (Sun Gro Horticulture, Bellevue, Wash.) was
covered with fine vermiculite and sub-irrigated with Hoagland's
solution until wet. The soil mix was allowed to drain for 24 hours.
Stratified seed was sown onto the vermiculite and covered with
humidity domes (KORD Products, Bramalea, Ontario, Canada) for 7
days.
[0258] Seeds were germinated and plants were grown in a Conviron
(models CMP4030 and CMP3244, Controlled Environments Limited,
Winnipeg, Manitoba, Canada) under long day conditions (16 hours
light/8 hours dark) at a light intensity of 120-150
.mu.mol/m.sup.2sec under constant temperature (22.degree. C.) and
humidity (40-50%). Plants were initially watered with Hoagland's
solution and subsequently with deionized water to keep the soil
moist but not wet.
[0259] 6.2--Agrobacterium Transformation.
[0260] An LB+agar plate with erythromycin (Sigma Chemical Co., St.
Louis, Mo.) (200 mg/L) or spectinomycin (100 mg/L) containing a
streaked DH5a colony was used to provide a colony to inoculate 4 ml
mini prep cultures (liquid LB+erythromycin). The cultures were
incubated overnight at 37.degree. C. with constant agitation.
Qiagen (Valencia, Calif.) Spin Mini Preps, performed per
manufacturer's instructions, were used to purify the plasmid
DNA.
[0261] Electro-competent Agrobacterium tumefaciens (strains Z707s,
EHA101s, and LBA4404s) cells were prepared using a protocol from
Weigel and Glazebrook (2002). The competent Agrobacterium cells
were transformed using an electroporation method adapted from
Weigel and Glazebrook (2002). 50 .mu.l of competent agro cells were
thawed on ice and 10-25 ng of the desired plasmid was added to the
cells. The DNA and cell mix was added to pre-chilled
electroporation cuvettes (2 mm). An Eppendorf Electroporator 2510
was used for the transformation with the following conditions,
Voltage: 2.4kV, Pulse length: 5msec.
[0262] After electroporation, 1 ml of YEP broth (per liter: 10 g
yeast extract, 10 g Bacto-peptone, 5 g NaCl) was added to the
cuvette, and the cell-YEP suspension was transferred to a 15 ml
culture tube. The cells were incubated at 28.degree. C. in a water
bath with constant agitation for 4 hours. After incubation, the
culture was plated on YEP+agar with erythromycin (200 mg/L) or
spectinomycin (100 mg/L) and streptomycin (Sigma Chemical Co., St.
Louis, Mo.) (250 mg/L). The plates were incubated for 2-4 days at
28.degree. C.
[0263] Colonies were selected and streaked onto fresh YEP+agar with
erythromycin (200 mg/L) or spectinomycin (100 mg/L) and
streptomycin (250 mg/L) plates and incubated at 28.degree. C. for
1-3 days. Colonies were selected for PCR analysis to verify the
presence of the gene insert by using vector specific primers.
Qiagen Spin Mini Preps, performed per manufacturer's instructions,
were used to purify the plasmid DNA from selected Agrobacterium
colonies with the following exception: 4 ml aliquots of a 15 ml
overnight mini prep culture (liquid YEP+erythromycin (200 mg/L) or
spectinomycin (100 mg/L)) and streptomycin (250 mg/L)) were used
for the DNA purification. An alternative to using Qiagen Spin Mini
Prep DNA was lysing the transformed Agrobacterium cells, suspended
in 10 .mu.l of water, at 100.degree. C. for 5 minutes. Plasmid DNA
from the binary vector used in the Agrobacterium transformation was
included as a control. The PCR reaction was completed using Taq DNA
polymerase from Takara Mims Bio Inc. (Madison, Wis.) per
manufacturer's instructions at 0.5.times. concentrations. PCR
reactions were carried out in a MJ Research Peltier Thermal Cycler
programmed with the following conditions; 1) 94.degree. C. for 3
minutes, 2) 94.degree. C. for 45 seconds, 3) 55.degree. C. for 30
seconds, 4) 72.degree. C. for 1 minute, for 29 cycles then 1 cycle
of 72.degree. C. for 10 minutes. The reaction was maintained at
4.degree. C. after cycling. The amplification was analyzed by 1%
agarose gel electrophoresis and visualized by ethidium bromide
staining. A colony was selected whose PCR product was identical to
the plasmid control.
[0264] 6.3--Arabidopsis Transformation
[0265] Arabidopsis was transformed using the floral dip method. The
selected colony was used to inoculate one or more 15-30 ml
pre-cultures of YEP broth containing erythromycin (200 mg/L) or
spectinomycin (100 mg/L) and streptomycin (250 mg/L). The
culture(s) was incubated overnight at 28.degree. C. with constant
agitation at 220 rpm. Each pre-culture was used to inoculate two
500 ml cultures of YEP broth containing erythromycin (200 mg/L) or
spectinomycin (100 mg/L) and streptomycin (250 mg/L) and the
cultures were incubated overnight at 28.degree. C. with constant
agitation. The cells were then pelleted at approx. 8700x g for 10
minutes at room temperature, and the resulting supernatant
discarded. The cell pellet was gently resuspended in 500 ml
infiltration media containing: 1/2.times. Murashige and Skoog
salts/Gamborg's B5 vitamins, 10% (w/v) sucrose, 0.04404 benzylamino
purine (10 pl/liter of 1 mg/ml stock in DMSO) and 300 .mu.l/liter
Silwet L-77. Plants approximately 1 month old were dipped into the
media for 15 seconds, being sure to submerge the newest
inflorescence. The plants were then laid down on their sides and
covered (transparent or opaque) for 24 hours, then washed with
water, and placed upright. The plants were grown at 22.degree. C.,
with a 16-hour light/8-hour dark photoperiod. Approximately 4 weeks
after dipping, the seeds were harvested.
[0266] 6.4--Selection of Transformed Plants.
[0267] Freshly harvested T.sub.1 seed (transformed with native
[AAD-1 (v2)] or plant optimized [AAD-1 (v3)] gene) was allowed to
dry for 7 days at room temperature. T.sub.1 seed was sown in
26.5.times.51-cm germination trays (T.O. Plastics Inc., Clearwater,
Minn.), each receiving a 200 mg aliquots of stratified T.sub.1 seed
(10,000 seed) that had previously been suspended in 40 ml of 0.1%
agarose solution and stored at 4.degree. C. for 2 days to complete
dormancy requirements and ensure synchronous seed germination.
[0268] Sunshine Mix LP5 (Sun Gro Horticulture Inc., Bellevue,
Wash.) was covered with fine vermiculite and subirrigated with
Hoagland's solution until wet, then allowed to gravity drain. Each
40 ml aliquot of stratified seed was sown evenly onto the
vermiculite with a pipette and covered with humidity domes (KORD
Products, Bramalea, Ontario, Canada) for 4-5 days. Domes were
removed 1 day prior to initial transformant selection using
glufosinate postemergence spray (selecting for the co-transformed
PAT gene).
[0269] Five to six days after planting (DAP) and again 10 DAP, Ti
plants (cotyledon and 2-4-1f stage, respectively) were sprayed with
a 0.2% solution of Liberty herbicide (200 g ai/L glufosinate, Bayer
Crop Sciences, Kansas City, Mo.) at a spray volume of 10 ml/tray
(703 L/ha) using a DeVilbiss compressed air spray tip to deliver an
effective rate of 280 g ai/ha glufosinate per application.
Survivors (plants actively growing) were identified 5-7 days after
the final spraying and transplanted individually into 3-inch pots
prepared with potting media (Metro Mix 360). Transplanted plants
were covered with humidity domes for 3-4 days and placed in a
22.degree. C. growth chamber as before. Domes were subsequently
removed and plants moved to the greenhouse (22.+-.5.degree. C.,
50.+-.30% RH, 14 h light:10 dark, minimum 500 pE/m.sup.2s.sup.1
natural+supplemental light) at least 1 day prior to testing for the
ability of AAD-1 (v3) (plant optimized gene) or AAD-1 (v2) (native
microbial gene) to provide phenoxy auxin herbicide resistance.
[0270] Random individual T.sub.1 plants selected for glufosinate
resistance above were confirmed for expression of the PAT protein
using a PAT ELISA kit (Part no. 7000045, Strategic Diagnostics,
Inc., Newark, DE) to non-destructively confirm fidelity of
selection process (manufacturer's protocol). Plants were then
randomly assigned to various rates of phenoxy herbicides
(dichlorprop or 2,4-D). Phenoxy rates initially applied were 12.5 g
ae/ha 2,4-D and 50 or 200 g ae/ha dichlorprop. GR.sub.99 for
Arabidopsis is about 50 g ae/ha 2,4-D and 200 g ae/ha dichlorprop.
Elevated rates were applied in subsequent trials (50, 200, 800, or
3200g ae/ha).
[0271] All auxin herbicide applications were made using the
DeVilbiss sprayer as described above to apply 703 L/ha spray volume
(0.4 ml solution/3-inch pot) or applied by track sprayer in a 187
L/ha spray volume. 2,4-D used was either technical grade (Sigma,
St. Louis, Mo.) dissolved in DMSO and diluted in water (<1% DMSO
final concentration) or the commercial dimethylamine salt
formulation (456 g ae/L, NuFarm, St Joseph, Mo.). Dichlorprop used
was technical grade (Sigma, St. Louis, Mo.) dissolved in DMSO and
diluted in water (<1% DMSO final concentration). As herbicide
rates increased beyond 800 g ae/ha, the pH of the spray solution
became exceedingly acidic, burning the leaves of young, tender
Arabidopsis plants and complicating evaluation of the primary
effects of the herbicides. It became standard practice to apply
these high rates of phenoxy herbicides in 200 mM Tris buffer (pH
9.0) to a final pH of .about.7-8.
[0272] Some T.sub.1 individuals were subjected to alternative
commercial herbicides instead of a phenoxy auxin. One point of
interest was determining whether haloxyfop could be effectively
degraded in planta.
[0273] Although Arabidopsis, being a dicot, is not an optimal
system for testing ACCase-inhibiting AOPP grass herbicides, AAD-1
(v3)-transformed Ti plants were subjected to elevated rates
(400-1600 g ae/ha) of RS-haloxyfop acid (internally synthesized)
that do cause growth abnormalities and death of wildtype
Arabidopsis using the DeVilbiss sprayer as described above Injury
ratings were taken 7 and 14 days after treatment. Likewise, T.sub.1
individuals were treated with the pyridyloxyacetate auxin
herbicide, fluroxypyr.
[0274] 6.5--Results of Selection of Transformed Plants.
[0275] The first Arabidopsis transformations were conducted using
AAD-1 (v3) (plant optimized gene). T.sub.1 transformants were first
selected from the background of untransformed seed using a
glufosinate selection scheme. Over 400,000 T.sub.1 seed were
screened and 493 glufosinate resistant plants were identified (PAT
gene), equating to a transformation/selection frequency of 0.12%.
Depending on the lot of seed tested, this ranged from 0.05-0.23%
(see Table 15). A small lot of AAD-1 (v2) (native)-transformed seed
were also selected using the glufosinate selection agent. Two
hundred seventy eight glufosinate-resistant T.sub.1 individuals
were identified out of 84,000 seed screened (0.33%
transformation/selection frequency).
TABLE-US-00015 TABLE 15 Selection of AAD-1 (v3) (plant optimized),
or AAD-1 (v2) (native), AAD-2 (v1) (native), or plant optimized
AAD-2 (v2)-transformed T.sub.1 individual plants using glufosinate
and 2,4-D. Total seed % of selected Selection Codon sown and Number
of Selection Selection rate plants expressing agent Gene bias
screened resistant T.sub.1 rate range PAT.sup.3 Glufosinate.sup.1
AAD-1 (v2) n 84,000 278 0.33% 0.33% nd.sup.4 Glufosinate.sup.1
AAD-1 (v3) p 400,500 493 0.12% 0.05 to 0.23% 97% 2,4-D.sup.2 AAD-1(
v3) p 70,000 53 0.08% 0.07 to 0.08% 96% Glufosinate.sup.1 AAD-2
(v1) n 1,301,500 228 0.018% 0.007 to 0.021% 100% Glufosinate.sup.1
AAD-2( v2) p 200,000 224 0.11% 0.11% nd.sup.4 .sup.1Glufosinate
selection scheme: 280 g ai/ha glufosinate applied 5-6 + 10 DAP
.sup.22,4-D selection scheme: 50 g ai/ha 2,4-D applied 5-7 + 10-14
DAP .sup.3PAT protein expression determined by PAT ELISA strips
.sup.4nd, not determined .sup.5codon bias, n-native microbial gene,
p = plant optimized
[0276] T.sub.1 plants selected above were subsequently transplanted
to individual pots and sprayed with various rates of commercial
aryloxyalkanoate herbicides. Table 16 compares the response of
native AAD-1 (v2) and plant optimized AAD-1 (v3) genes to impart
2,4-D resistance to Arabidopsis Ti transformants Both genes
imparted resistance to individual Ti Arabidopsis plants. Within a
given treatment, the level of plant response varied greatly and can
be attributed to the fact each plant represents an independent
transformation event. Of important note, at each 2,4-D rate tested,
there were individuals that were unaffected while some were
severely affected. An overall population injury average by rate is
presented in Table 16 simply to demonstrate the significant
difference between the plants transformed with AAD-1 (v2) or AAD-1
(v3) versus the wildtype or PAT/CryIF transformed controls. Also
evident is that tolerance appears to be significantly greater
(frequency and overall level of individual response) for the plant
optimized sequence AAD-1 (v3) versus the native sequence AAD-1 (v2)
(see Table 16). Higher rates of 2,4-D (up to 3,200 g ae/ha) have
been applied to additional T.sub.1 individuals expressing AAD-1
(v3). Injury levels tend to be greater and the frequency of highly
resistant plants is lower at these elevated rates (6x field rates).
Also at these high rates, the spray solution becomes highly acidic
unless buffered. Arabidopsis grown mostly in the growth chamber has
a very thin cuticle and severe burning effects can complicate
testing at these elevated rates. Nonetheless, some individuals have
survived 3,200 g ae/ha 2,4-D with little or no injury.
TABLE-US-00016 TABLE 16 AAD-1 v3 (plant optimized), or AAD-1 v2
(native), or AAD-2 (native)- transformed T.sub.1 Arabidopsis
response to a range of 2,4-D rates applied postemergence. Response
is presented in terms of % visual injury 2 WAT. Data are presented
as a histogram of individuals exhibiting little or no injury
(<20%), moderate injury (20-40%), or severe injury (>40%).
Since each T.sub.1 is an independent transformation event, one can
expect significant variation of individual T.sub.1 responses within
a given rate. An arithmetic mean and standard deviation is
presented for each treatment. The range in individual response is
also indicated in the last column for each rate and transformation.
PAT/C1F-transformed Arabidopsis served as an auxin-sensitive
transformed control. Wildtype Arabidopsis is untransformed. AAD-1
v3 (plant optimized), (native) or AAD-1 v2 (native), or
AAD-2-transformed T.sub.1 Arabidopsis response to a range of 2,4-D
rates applied postemergence. % Injury % Injury Std Averages <20%
20-40% >40% Ave Dev Native AAD-1 (v2) gene Untreated
control-buffer 20 6 7 25.3 34.7 50 g ae/ha 2,4-D 55 16 9 14.8 22.7
200 g ae/ha 2,4-D 45 11 24 34.1 39.3 800 g ae/ha 2,4-D 11 32 37
52.5 34.2 Native AAD-2 gene Untreated control-buffer 4 1 1 25.0
21.7 50 g ae/ha 2,4-D 1 2 11 68.2 30.2 200 g ae/ha 2,4-D 0 3 11
82.7 28.8 800 g ae/ha 2,4-D 0 0 14 99.8 0.8 Rebuilt AAD-1 (v3) gene
Untreated control-buffer 9 0 0 0.0 0.0 50 g ae/ha 2,4-D 10 1 5 24.3
35.9 200 g ae/ha 2,4-D 11 4 1 14.0 25.9 800 g ae/ha 2,4-D 11 4 1
14.7 26.1 Wildtype Untreated control-buffer 11 0 0 0.0 0.0 50 g
ae/ha 2,4-D 0 0 15 90.0 0.0 200 g ae/ha 2,4-D 0 0 15 95.1 0.5 800 g
ae/ha 2,4-D 0 0 15 100.0 0.0 PAT/Cry1F (transformed control)
Untreated control-buffer 11 0 0 0.0 0.0 50 g ae/ha 2,4-D 0 0 15
90.7 4.2 200 g ae/ha 2,4-D 0 0 15 97.2 1.7 800 g ae/ha 2,4-D 0 0 15
100.0 0.0
[0277] Table 17 shows a similar dose response of T.sub.1
Arabidopsis to the phenoxypropionic acid, dichlorprop. Similar
trends were seen as with 2,4-D, indicating the chiral propionic
side chain indeed serves as an acceptable substrate. Next, it was
determined that a degree of increased haloxyfop tolerance could be
imparted to transformed Arabidopsis at elevated rates of 400-1,600
g ae/ha (Table 18). Normal field use rate for haloxyfop (a
grass-specific herbicide) is around 50-70 g ae/ha. Dicots are
generally considered naturally tolerant to AOPP herbicides;
however, severe physiological effects do occur in Arabidopsis at
these elevated rates. Some AAD1 (v3) transformed individuals did
exhibit increased tolerance to haloxyfop. This provides the first
in planta data that AAD-1 (v3) will provide AOPP resistance. No
resistance was observed with fluroxypyr (a pryridyloxyacetate
auxin) in transformed Arabidopsis, consistent with in vitro work
using heterologously expressed enzyme.
TABLE-US-00017 TABLE 17 T.sub.1 Arabidopsis response to a range of
dichlroprop rates applied postemergence. Response is presented in
terms of % visual injury 2 WAT. Data are presented as a histogram
of individuals exhibiting little or no injury (<20%), moderate
injury (20-40%), or severe injury (>40%). Since each T.sub.1 is
an independent transformation event, one can expect significant
variation of individual T.sub.1 responses within a given rate. An
arithmetic mean and standard deviation is presented for each
treatment. The range in individual response is also indicated in
the last column for each rate and transformation.
PAT/Cry1F-transformed Arabidopsis served as an auxin-sensitive
transformed control. Wildtype Arabidopsis is untransformed. Table
17. T.sub.1 Arabidopsis response to a range of dichlroprop rates
applied postemergence. AAD-1 v3 % Injury % Injury Averages <20%
20-40% >40% Ave Std Dev Range Untreated control 3 0 0 0.0 0.0 0
12.5 g ae/ha RS-dichlorprop 7 1 0 5.0 7.6 0-20 50 g ae/ha
RS-dichlorprop 7 1 0 3.1 8.8 0-25 200 g ae/ha RS-dichlorprop 4 1 3
40.0 50.1 0-100 800 g ae/ha RS-dichlorprop 0 5 3 51.9 40.0 20-100
PAT/Cry1F % Injury % Injury Averages <20% 20-40% >40% Ave Std
Dev Range Untreated control 3 0 0 0.0 0.0 0 12.5 g ae/ha
RS-dichlorprop 0 6 2 38.1 25.3 20-95 50 g ae/ha RS-dichlorprop 0 0
8 80.0 25.3 50-100 200 g ae/ha RS-dichlorprop 0 0 8 98.3 2.2 95-100
800 g ae/ha RS-dichlorprop 0 0 8 100.0 0.0 100 Wildtype % Injury %
Injury Averages <20% 20-40% >40% Ave Std Dev Range Untreated
control 3 0 0 0.0 0.0 0 12.5 g ae/ha RS-dichlorprop 3 0 0 13.3 2.9
10-15 50 g ae/ha RS-dichlorprop 0 0 3 53.3 5.8 50-60 200 g ae/ha
RS-dichlorprop 0 0 3 95.0 5.0 90-100 800 g ae/ha RS-dichlorprop 0 0
3 100.0 0.0 100
TABLE-US-00018 TABLE 18 T1 Arabidopsis response to a range of
haloxyfop rates applied postemergence at artificially high rates
attempting to show tolerance of the dicot Arabidopsis to the
graminicide. Response is presented in terms of % visual injury 2
WAT. Data are presented as a histogram of individuals exhibiting
little or no injury (<20%), moderate injury (20-40%), or severe
injury (>40%). Since each T1 is an independent transformation
event, one can expect significant variation of individual T1
responses within a given rate. An arithmetic mean and standard
deviation is presented for each treatment. The range in individual
response is also indicated in the last column for each rate and
transformation. PAT/Cry1F-transformed Arabidopsis served as an
auxin-sensitive transformed control. Wildtype Arabidopsis is
untransformed. Table 18. T1 Arabidopsis response to a range of
haloxyfop rates applied postemergence at artificially high rates
attempting to show tolerance of the dicot Arabdopsis to the
graminicide. AAD-1 v3 % Injury % Injury Averages <20% 20-40%
>40% Ave Std Dev Range Untreated control 3 0 0 0.0 0.0 0 100 g
ae/ha haloxyfop 4 0 0 0.0 0.0 0 200 g ae/ha haloxyfop 4 0 0 0.0 0.0
0 400 g ae/ha haloxyfop 3 1 0 6.3 9.5 0-20 800 g ae/ha haloxyfop 1
1 2 46.3 42.7 0-85 1600 g ae/ha haloxyfop 1 0 3 65.0 47.3 0-100
PAT/Cry1F % Injury % Injury Averages <20% 20-40% >40% Ave Std
Dev Range Untreated control 3 0 0 0.0 0.0 0 100 g ae/ha haloxyfop 4
0 0 0.0 0.0 0 200 g ae/ha haloxyfop 4 0 0 10.0 0.0 10 400 g ae/ha
haloxyfop 0 4 0 27.5 5.0 20-30 800 g ae/ha haloxyfop 0 0 4 78.8 6.3
70-85 1600 g ae/ha haloxyfop 0 0 4 47.5 43.5 80-100 Wildtype %
Injury % Injury Averages <20% 20-40% >40% Ave Std Dev Range
Untreated control 3 0 0 0.0 0.0 0 100 g ae/ha haloxyfop 3 0 0 0.0
0.0 0 200 g ae/ha haloxyfop 3 0 0 0.0 0.0 0 400 g ae/ha haloxyfop 0
3 0 20.0 0.0 20 800 g ae/ha haloxyfop 0 0 3 73.3 10.4 70-85 1600 g
ae/ha haloxyfop 0 0 3 93.3 11.5 80-100
[0278] 6.6--AAD-1 (v3) as a selectable marker.
[0279] The ability to use AAD-1 (v3) as a selectable marker using
2,4-D as the selection agent was analyzed initially with
Arabidopsis transformed with as described above. Tiseed transformed
with PAT and AAD1 (v3) (pDAB 721) were sown into flats and
germinated as described above and compared to similar seed treated
with the normal glufosinate selection scheme (5 and 10 DAP). 2,4-D
(50g ae/ha) was applied to seedling Arabidopsis as previously done
with glufosinate. Variation in number of applications and timing of
application were tested. Each tray of plants received either one or
two application timings of 2,4-D in one of the following treatment
schemes: 5+10 DAP, 5+14 DAP, 10 DAP, 10+14 DAP, 14 DAP. Plants were
identified as Resistant or Sensitive 19 DAP and ELISA test strips
run to determine frequency of successfully co-transforming an
active PAT gene.
[0280] Fifty-three out of 70,000 seed planted were identified as
resistant to 2,4-D. ELISA was used to screen a subset of
44-individuals from this population for PAT protein expression.
Ninety six percent of the individuals were positive indicating the
presence of the co-transformed gene, PAT. The low number of
negative ELISA results (4%) is in line with a 3% error rate in
populations of glufosinate-resistant plants (Table 15). The
efficiency of selection appears to be somewhat less with 2,4-D
(0.08%) vs. glufosinate (0.12%); however, the range of selection
rates across all experiments would indicate both selection agents
are equally good for selecting Arabidopsis transformed with AAD-1
(v3) or PAT genes, respectively. Two successive applications most
accurately identify resistant individuals with both herbicides
tested.
[0281] 6.7--Heritability.
[0282] A variety of T.sub.1 events were self-pollinated to produce
T.sub.2 seed. These seed were progeny tested by applying 2,4-D (200
g ae/ha) to 100 random T2 siblings. Each individual T.sub.2 plant
was transplanted to 7.5-cm square pots prior to spray application
(track sprayer at 187 L/ha applications rate). More than 60% of the
T.sub.1 families (T.sub.2 plants) segregated in the anticipated 3
Resistant:1 Sensitive model for a dominantly inherited single locus
with Mendelian inheritance as determined by Chi square analysis
(P>0.05).
[0283] Seed were collected from 12 to 20 T.sub.2 individuals
(T.sub.3 seed). Twenty-five T.sub.3 siblings from each of eight
randomly-selected T.sub.2 families were progeny tested as
previously described. Approximately one-third of the T.sub.2
families anticipated to be homozygous (non-segregating populations)
have been identified in each line tested: ranging in frequency from
one to four out of the eight families tested. These data show AAD1
(v3) is stably integrated and inherited in a Mendelian fashion to
at least three generations.
[0284] 6.8--Additional Herbicide Resistance Attributable to AAD-1
in Arabidopsis.
[0285] The ability of AAD-1 (v3) to provide resistance to other
aryloxyphenoxyalkanoate herbicides in transgenic Arabidopsis was
determined using a modified in vitro plate assay. Seeds from
wild-type Arabidopsis thaliana as well as Arabidopsis thaliana
containing the plant optimized AAD-1 (v3) gene (T.sub.4 homozygous
plants id=PAAD1.315.064) were sterilized by agitating for 10 min in
a 50% bleach solution. These seeds were then rinsed four times with
sterile water to remove the bleach.
[0286] Dose response assays utilized a nutrient media (see below)
supplemented with various rates of test compounds. The test
compounds were added to the heated media (55.degree. C.) as
concentrated solutions in DMSO. Control wells had the appropriate
amount of DMSO without any additional compound. The final
concentration of DMSO never exceeded 1% (v/v). After thorough
mixing, a 6 mL aliquot of the warm media containing the appropriate
concentration of compound was added to each well of a 6-well, flat
bottom, polystyrene tissue culture tray (Falcon 353046, Becton
Dickson and Company, Franklin Lakes, N.J.). After the media
solidified, approximately 20 to 30 Arabidopsis seeds were applied
on top of the solidified media and the remaining 2 mL of media was
poured over the seeds. The plates were lightly agitated to disperse
the seeds, covered and allowed to cool until the media had
completely solidified. The plates were incubated for 7 days at
25.degree. C. under continual fluorescent lighting (75 .mu.E
m.sup.-2 s.sup.-1). Nutrient Media Composition was as described in
Example 2.2 and in Somerville and Orgen (1982).
[0287] Assessment of growth reduction. The apical portion of the
Arabidopsis plants grown in the treated media was assessed visually
relative to the apical portion of the plants grown in the media
containing only DMSO. Values were recorded as % growth reduction.
Assessments of root growth inhibition of the Arabidopsis plants
grown in the treated media were achieved by carefully extracting
the plants from the media and measuring the length of the root.
These root lengths were then compared to the root length of the
control plants to determine a % growth reduction. A minimum of five
plants were assessed for each treatment. The values recorded are an
average of all the plants assessed. The calculated concentration to
reach 50% inhibition effect (I.sub.50) were determined for both
root and shoots of wildtype and AAD-1-transformed Arabidopsis. The
ratios of resistant to sensitive biotypes are included in Table 19.
A ratio >2 for both root and shoot measurements generally
signifies significant resistance. The higher the ratio, the greater
the level of resistance. All commercial phenoxy auxins showed
significant levels of resistance including oxyacetic acids (2,4-D
and MCPA) as well as oxypropionic acids (dichlorprop and mecoprop).
In fact, the chronic root assessment shows resistance to the
oxypropionic acid is higher with AAD-1 (v3) than for the oxyacetic
acids, consistent with enzymatic characteristics of AAD-1 (v1).
Assessment of other auxins containing pyridine rings showed AAD-1
(v3) did not effectively protect Arabidopsis form
pyridyloxyacetates herbicides, triclopyr and fluroxypyr, or the
picolinic acid herbicide, picloram. The broad phenoxy auxin
resistance is the first reported in planta. The alternative auxins
to which AAD-1 does not protect would be viable tools for the
control and containment of AAD-1-transformed commercial crops or
experimental plant species.
TABLE-US-00019 TABLE 19 In vitro plate test assessment of herbicide
substrate cross resistance afforded by AAD-1 (v3) in homozygous
T.sub.4 Arabidopsis (ARBTH). wt wt ARBTH PAAD1.315.064 ARBTH
PAAD1.315.064 shoot T3 ARBTH root ARBTH 150 shoot 150 150 root 150
shoot root Compound Structure ppm ratio ratio Phenoxy auxins 2,4-D
##STR00066## 0.2 10 <0.01 0.04 50 >4 dichlorprop ##STR00067##
2 5 0.01 1.5 2.5 150 Mecoprop ##STR00068## 2 25 0.01 1.5 12.5 150
MCPA ##STR00069## 0.2 1 0.01 0.03 5 3 2,4,5-T ##STR00070## 1.5 10
<0.01 <0.01 6.67 NA Pyridine auxins Fluroxypyr ##STR00071## 2
1 0.2 0.2 0.5 1 Triclopyr ##STR00072## 0.2 0.04 0.02 0.02 0.2 1
Picloram ##STR00073## 1 0.5 0.3 0.15 0.5 0.5
[0288] 6.9--Foliar Applications Herbicide Resistance in AAD-1
Arabidopsis.
[0289] The ability of AAD-1 (v3) to provide resistance to other
aryloxyphenoxyalkanoate auxin herbicides in transgenic Arabidopsis
was determined by foliar application of various substrates
described in Example 6.8. T4 generation Arabidopsis seed,
homozygous for AAD-1 (v3) (line AAD1.01.315.076) was stratified,
and sown into selection trays much like that of Arabidopsis
(Example 6.4). A transformed-control line containing PAT and the
insect resistance gene Cry1F was planted in a similar manner
Seedlings were transferred to individual 3-inch pots in the
greenhouse. All plants were sprayed with the use of a track sprayer
set at 187 L/ha. The plants were sprayed from a range of phenoxy
auxin herbicides: 12.5-1600 g ae/ha 2,4-D dimethylamine salt (DMA)
(Riverside Chemicals), 12.5-1600 g ae/ha mecoprop (AH Marks),
50-3200 g ae/ha R-dichlorprop (AH Marks), 8.75-1120 g ae/ha 2,4,5-T
(technical grade); pyridyloxyacetates herbicides 50-3200 g ae/ha
triclopyr (Dow AgroSciences) and 50-3200 g ae/ha fluroxypyr (Dow
AgroSciences); and the 2,4-D metabolite resulting from AAD-1
activity, 2,4-dichlorophenol (DCP, Sigma) (at 50-3200 g ae/ha,
technical grade). All applications were formulated in 200 mM Hepes
buffer (pH 7.5). Each treatment was replicated 3-4 times. Plants
were evaluated at 3 and 14 days after treatment and are averaged
over two experiments.
[0290] These results (see Table 20) confirm that AAD-1 (v3) in
Arabidopsis provides robust resistance to the phenoxyacetic auxins,
phenoxypropionic auxins, but have not shown significant cross
resistance to the pyridyloxyacetic auxins tested and corroborates
the in vitro enzyme and whole plate substrate specificity data.
Additionally, there is no effect of the metabolite,
2,4-dichlorphohenol (DCP), on wildtype or transgenic
Arabidopsis.
TABLE-US-00020 TABLE 20 Comparison of homozygous T.sub.4 AAD-1 (v3)
and wildtype Arabidopsis plant response to various foliar-applied
auxinic herbicides. Ave % Injury 14DAT AAD1.01.315.076.T.sub.4
Herbicide Treatment homozygous AAD1 plants PatCry1f - Control
Phenoxypropionic auxins 50 g ae/ha R-Dichlorprop 3 31 200 g ae/ha
R-Dichlorprop 3 73 800 g ae/ha R-Dichlorprop 3 89 3200 g ae/ha
R-Dichlorprop 3 95 12.5 g ae/ha Mecoprop 3 0 25 g ae/ha Mecoprop 0
2 50 g ae/ha Mecoprop 0 17 100 g ae/ha Mecoprop 0 33 200 g ae/ha
Mecoprop 3 62 400 g ae/ha Mecoprop 0 78 800 g ae/ha Mecoprop 0 93
1600 g ae/ha Mecoprop 0 100 Phenoxyacetic auxins 12.5 g ae/ha 2,4-D
DMA 0 67 25 g ae/ha 2,4-D DMA 0 78 50 g ae/ha 2,4-D DMA 0 93 100 g
ae/ha 2,4-D DMA 0 100 200 g ae/ha 2,4-D DMA 0 100 400 g ae/ha 2,4-D
DMA 0 100 800 g ae/ha 2,4-D DMA 0 100 1600 g ae/ha 2,4-D DMA 0 100
8.75 g ae/ha 2,4,5-T 0 0 17.5 g ae/ha 2,4,5-T 3 20 35 g ae/ha
2,4,5-T 0 43 70 g ae/ha 2,4,5-T 3 85 140 g ae/ha 2,4,5-T 0 95 280 g
ae/ha 2,4,5-T 0 98 560 g ae/ha 2,4,5-T 17 100 1120 g ae/ha 2,4,5-T
3 100 Pyridyloxyacetic auxins 50 g ae/ha Triclopyr 31 36 200 g
ae/ha Triclopyr 58 65 800 g ae/ha Triclopyr 74 84 3200 g ae/ha
Triclopyr 97 95 50 g ae/ha Fluroxypyr 48 76 200 g ae/ha Fluroxypyr
75 85 800 g ae/ha Fluroxypyr 88 85 3200 g ae/ha Fluroxypyr 95 95
Inactive DCP metabolite 50 g ae/ha 2,4-DCP 0 0 200 g ae/ha 2,4-DCP
0 0 800 g ae/ha 2,4-DCP 0 0 3200 g ae/ha 2,4-DCP 0 0
[0291] 6.10--Relationship of Plant Growth to AAD-1 (v3) Expression
in Arabidopsis.
[0292] An experiment was designed to examine if the level of AAD-1
(v3) expression in Arabidopsis varies at different growth stages. A
high-tolerance, homozygous, AAD-1 (v3) T4 line
(id=PAAD1.01.345.163) was grown in greenhouse. Half of the plants
were treated with 800 g ae /ha of 2,4-D (as previously described)
while the other half were not treated. Two leaves, the 3rd leaf
from the top and the 5th leaf from the bottom, were harvested from
5 plants, both treated and untreated, and analyzed by ELISA and
Western Blotting (as described in Example 11) experiments at 4, 10,
14, 20 and 25 DAT. FIGS. 8A and 8B showed that there was
statistically no difference in AAD-1 (v3) expression between young
and old leaves. In addition, the herbicide 2,4-D had little impact
on the expression level of AAD-1 (v3) protein. The protein levels
accumulated in older plants with some significant protein
degradation at the later time points.
[0293] In a separate experiment, four different homozygous T4 lines
of Arabidopsis displaying different tolerance level to the
herbicide 2,4-D were sprayed with various levels (0, 200, 800 and
3200 g/ha) of 2,4-D and their herbicide injury and AAD-1 (v3)
expression were examined Four days after the herbicide treatment,
little injury was observed in three of the four lines, even at the
highest dose tested (FIG. 9A). These plants also expressed high
level of AAD-1 (v3), from 0.1 to 0.25% of (FIG. 9B). On the
contrary, the low tolerance line expressed less than 0.1% of AAD-1
(v3) in TSP and suffered observable injury. More importantly, they
recovered from the injury on 14 DAT (FIG. 9A), indicating that the
low level of AAD-1 (v3) expression was able to protect the plants
from the serious herbicide damage. All control plants suffered
serious injury and died 14 DAT at doses 800 g ae/ha 2,4-D and
above.
[0294] 6.11--Molecular Analysis of AAD-1 (v3) Arabidopsis.
[0295] Invader Assay (methods of Third Wave Agbio Kit Procedures)
for PAT gene copy number and/or Southern blot analysis was
performed with total DNA obtained from Qiagen DNeasy kit on
multiple AAD-1 (v3) homozygous lines to determine stable
integration of the plant transformation unit containing PAT and
AAD-1 (v3). Analysis assumed direct physical linkage of these genes
as they were contained on the same plasmid.
[0296] For Southern analysis, a total of 1.mu.g of DNA was
subjected to an overnight digest of
[0297] Nsi I for pDAB721 to obtain integration data. The samples
were run on a large 0.85% agarose gel overnight at 40 volts. The
gel was then denatured in 0.2 M NaOH, 0.6 M NaCl for 30 minutes.
The gel was then neutralized in 0.5 M Tris HCl, 1.5 M NaCl pH of
7.5 for 30 minutes. A gel apparatus containing 20SSC was then set
up to obtain a gravity gel to nylon membrane (Millipore INYC00010)
transfer overnight. After the overnight transfer the membrane was
then subjected to UV light via a crosslinker (stratagene UV
stratalinker 1800) at 1200.times.100 microjoules. The membrane was
then washed in 0.1% SDS, 0.1 SSC for 45 minutes. After the 45
minute wash, the membrane was baked for 3 hours at 80.degree. C.
and then stored at 4.degree. C. until hybridization. The
hybridization template fragment consisted of the prepared primers
(Pat 5-3 AGATACCCTTGGTTGGTTGC) (SEQ ID NO:23) and (Pat 3-3
CAGATGGATCGTTTGGAAGG) (SEQ ID NO:24) designed to obtain the coding
region of PAT. The product was run on a 1% agarose gel and excised
and then gel extracted using the Qiagen (28706) gel extraction
procedure. The membrane was then subjected to a pre-hybridization
at 60.degree. C. step for 1 hour in Perfect Hyb buffer (Sigma
H7033). The Prime it RmT dCTP-labeling rxn (Stratagene 300392)
procedure was used to develop the p32 based probe (Perkin Elmer).
The probe was cleaned up using the Probe Quant. G50 columns
(Amersham 27-5335-01). Two million counts CPM per ml of Perfect Hyb
buffer was used to hybridize the southern blots overnight. After
the overnight hybridization the blots were then subjected to two 20
minute washes at 65.degree. C. in 0.1% SDS, 0.1 SSC. The blots were
then exposed to film overnight, incubating at -80.degree. C.
[0298] Results showed all 2,4-D resistant plants assayed contained
PAT (and thus by inference, AAD-1 (v3)). Copy number analysis
showed total inserts ranged from 1 to >10 copies. This
correlates, too, with the AAD-1 (V3) protein expression data
indicating that the presence of the enzyme yields significantly
high levels of resistance (>>200 fold) to all commercially
available phenoxyacetic and phenoxypropionic acids.
[0299] 6.12--Arabidopsis transformed with molecular stack of AAD-1
(v3) and glyphosate resistance gene.
[0300] T.sub.1 Arabidopsis seed was produced, as previously
described, containing pDAB3230 plasmid (AAD-1 (v3)+EPSPS) coding
for a putative glyphosate resistance trait. T.sub.1 transformants
were selected using AAD-1 (v3) as the selectable marker as
described in example 6.6, except the 2,4-D rate used was 75 g
ae/ha. Twenty-four T.sub.1 individually transformed events were
recovered from the first selection attempt and transferred to
three-inch pots in the greenhouse as previously described. Three
different control Arabidopsis lines were also tested: wildtype
Columbia-0, AAD-1 (v3)+PAT T.sub.5 homozygous lines
(pDAB721-transformed), and PAT+CryIF homozygous line (transformed
control). Only pDAB3230 plants were pre-selected at the seedling
stage for 2,4-D tolerance. Four days after transplanting, plants
were evenly divided for foliar treatment by track sprayer as
previously described with 0, 26.25, 105, 420, or 1680 g ae/ha
glyphosate (Glyphomax Plus, Dow AgroSciences) in 200 mM Hepes
buffer (pH 7.5). All treatments were replicated 4 or 5 times.
Plants were evaluated 7 and 14 days after treatment. I.sub.50
values were calculated and show >14 fold level of tolerance
imparted by EPSPS molecularly stacked with AAD-1 (v3) (see FIG.
10). AAD-1 (v3) did not provide resistance to glyphosate itself
(re: pDAB721 response). These T.sub.1 plants will be grown to seed,
self-pollinated to yield T.sub.2 seed. The pDAB 3230 T.sub.1 plants
have demonstrated tolerance to lethal doses of 2,4-D and
glyphosate. T.sub.2 plants will be further tested to demonstrate
these co-transformed plants will withstand glyphosate+2,4-D
treatments applied in tankmix as described in Example 21 and shown
for AAD-1 (v3)-transformed corn in Example 8.
EXAMPLE 7
WHISKERS-Mediated Transformation Into Maize, and Use of AAD-1 (v3)
as a Selectable Marker
[0301] 7.1--Cloning of AAD-1 (v3).
[0302] The AAD-1 (v3) fragment was received on an NcoI/SacI
fragment. Construct pDAB4005 was digested with NcoI and Sad and the
5175 bp backbone fragment isolated. The two fragments were ligated
together using T4 DNA ligase and transformed into DHS a cells.
Minipreps were performed on the resulting colonies using Qiagen's
QIA Spin mini prep kit, and the colonies were digested to check for
orientation. The correct intermediate plasmid was named pDAB3403.
Both pDAB3403 and pDAB8505 (OsActl/PAT/ZmLip) were digested with
NotI. The 3442 bp band from pDAB3403 and the 11017 bp band from
pDAB8505 were isolated and purified. The fragments were ligated
together, transformed into DH5a, and the resulting plasmids were
screened for orientation. The final construct was designated
pDAB3404, which contains
ZmUbi1/po-aad1/ZmPer5:OsAct1/PAT/ZmLip.
[0303] 7.2--Callus/Suspension Initiation.
[0304] To obtain immature embryos for callus culture initiation,
F.sub.1 crosses between greenhouse-grown Hi-II parents A and B
(Armstrong et al. 1991) were performed. When embryos were 1.0-1.2
mm in size (approximately 9-10 days post-pollination), ears were
harvested and surface sterilized by scrubbing with Liqui-Nox.RTM.
soap, immersed in 70% ethanol for 2-3 minutes, then immersed in 20%
commercial bleach (0.1% sodium hypochlorite) for 30 minutes.
[0305] Ears were rinsed in sterile, distilled water, and immature
zygotic embryos were aseptically excised and cultured on 15Ag10
medium (N6 Medium (Chu et al., 1975), 1.0 mg/L 2,4-D, 20 g/L
sucrose, 100 mg/L casein hydrolysate (enzymatic digest), 25 mM
L-proline, 10 mg/L AgNO.sub.3, 2.5 g/L Gelrite, pH 5.8) for 2-3
weeks with the scutellum facing away from the medium. Tissue
showing the proper morphology (Welter et al., 1995) was selectively
transferred at biweekly intervals onto fresh 15Ag10 medium for
about 6 weeks, then transferred to 4 medium (N6 Medium, 1.0 mg/L
2,4-D, 20 g/L sucrose, 100 mg/L casein hydrolysate (enzymatic
digest), 6 mM L-proline, 2.5 g/L Gelrite, pH 5.8) at bi-weekly
intervals for approximately 2 months.
[0306] To initiate embryogenic suspension cultures, approximately 3
ml packed cell volume (PCV) of callus tissue originating from a
single embryo was added to approximately 30 ml of H9CP+liquid
medium (MS basal salt mixture (Murashige and Skoog, 1962), modified
MS Vitamins containing 10-fold less nicotinic acid and 5-fold
higher thiamine-HCl, 2.0 mg/L 2,4-D, 2.0 mg/L a-naphthaleneacetic
acid (NAA), 30 g/L sucrose, 200 mg/L casein hydrolysate (acid
digest), 100 mg/L myo-inositol, 6 mM L-proline, 5% v/v coconut
water (added just before subculture), pH 6.0). Suspension cultures
were maintained under dark conditions in 125 ml Erlenmeyer flasks
in a temperature-controlled shaker set at 125 rpm at 28.degree. C.
Cell lines typically became established within 2 to 3 months after
initiation. During establishment, suspensions were subcultured
every 3.5 days by adding 3 ml PCV of cells and 7 ml of conditioned
medium to 20 ml of fresh H9CP+liquid medium using a wide-bore
pipette. Once the tissue started doubling in growth, suspensions
were scaled-up and maintained in 500 ml flasks whereby 12 ml PCV of
cells and 28 ml conditioned medium was transferred into 80 ml
H9CP+medium. Once the suspensions were fully established, they were
cryopreserved for future use.
[0307] 7.3--Cryopreservation and Thawing of Suspensions.
[0308] Two days post-subculture, 4 ml PCV of suspension cells and 4
ml of conditioned medium were added to 8 ml of cryoprotectant
(dissolved in H9CP+ medium without coconut water, 1 M glycerol, 1 M
DMSO, 2 M sucrose, filter sterilized) and allowed to shake at 125
rpm at 4.degree. C. for 1 hour in a 125 ml flask. After 1 hour 4.5
ml was added to a chilled 5.0 ml Corning cryo vial. Once filled
individual vials were held for 15 minutes at 4.degree. C. in a
controlled rate freezer, then allowed to freeze at a rate of
-0.5.degree. C./minute until reaching a final temperature of
-40.degree. C. After reaching the final temperature, vials were
transferred to boxes within racks inside a Cryoplus 4 storage unit
(Forma Scientific) filled with liquid nitrogen vapors.
[0309] For thawing, vials were removed from the storage unit and
placed in a closed dry ice container, then plunged into a water
bath held at 40-45.degree. C. until "boiling" subsided. When
thawed, contents were poured over a stack of .about.8 sterile 70 mm
Whatman filter papers (No. 4) in covered 100.times.25 mm Petri
dishes. Liquid was allowed to absorb into the filters for several
minutes, then the top filter containing the cells was transferred
onto GN6 medium (N6 medium, 2.0 mg/L 2,4-D, 30 g/L sucrose, 2.5 g/L
Gelrite, pH 5.8) for 1 week. After 1 week, only tissue with
promising morphology was transferred off the filter paper directly
onto fresh GN6 medium. This tissue was subcultured every 7-14 days
until 1 to 3 grams was available for suspension initiation into
approximately 30 mL H9CP+ medium in 125 ml Erlenmeyer flasks. Three
milliliters PCV was subcultured into fresh H9CP+ medium every 3.5
days until a total of 12 ml PCV was obtained, at which point
subculture took place as described previously.
[0310] 7.4-Dose Response of Non-Transformed Tissue to Haloxyfop
Acid.
[0311] Non-transformed donor Hi-II cell lines were pooled together,
WHISKERS-treated without DNA, filtered onto GN6 medium at the rate
of 6 ml per filter, and allowed to callus for 2-3 weeks at
28.degree. C. Approximately 200 mg of callused suspension tissue
was transferred per treatment to selection media in 60.times.20 mm
plates containing 30, 100 or 300 nM R-haloxyfop acid. Three
replicates were used per concentration. A control medium containing
1 mg/L bialaphos (from Herbiace commercial formulation, Meiji
Seika, Japan), GN6 (1H), was also included for comparison. Callus
was removed after 2 weeks, weighed, and transferred to fresh media
of the same concentration for another 2 weeks. After a total of 4
weeks elapsed time, tissue was removed, weighed a final time, and
then discarded. Results are shown in FIG. 11.
[0312] Two separate dose response studies of callus tissue to the
phenol degradation products of haloxyfop and cyhalofop,
respectively, were also completed to confirm that this end product
would not be deleterious to callus growth. Data from a cyhalofop
phenol dose response (see FIG. 12) shows that at 1 .mu.M cyhalofop
phenol, growth is still 76% as high as the control without
cyhalofop phenol. Data from a haloxyfop phenol dose response showed
that even at 300 nM haloxyfop phenol, growth was equal to or
greater than the control lacking haloxyfop phenol (data not
shown).
[0313] 7.5--WHISKERS-Mediated Transformation using Bialaphos
Selection.
[0314] Approximately 24 hours prior to transformation, 12 ml PCV of
previously cryopreserved embryogenic maize suspension cells plus 28
ml of conditioned medium was subcultured into 80 ml of GN6 liquid
medium (GN6 medium lacking Gelrite) in a 500 ml Erlenmeyer flask,
and placed on a shaker at 125 rpm at 28.degree. C. This was
repeated 2 times using the same cell line such that a total of 36
ml PCV was distributed across 3 flasks. After 24 hours the GN6
liquid media was removed and replaced with 72 ml GN6 S/M osmotic
medium (N6 Medium, 2.0 mg/L 2,4-D, 30 g/L sucrose, 45.5 g/L
sorbitol, 45.5 g/L mannitol, 100 mg/L myo-inositol, pH 6.0) per
flask in order to plasmolyze the cells. The flasks were placed on a
shaker in the dark for 30-35 minutes, and during this time a 50
mg/ml suspension of silicon carbide whiskers was prepared by adding
the appropriate volume of GN6 S/M liquid medium to .about.405 mg of
pre-autoclaved, silicon carbide whiskers (Advanced Composite
Materials, Inc.).
[0315] After incubation in GN6 S/M, the contents of each flask were
pooled into a 250 ml centrifuge bottle. Once all cells settled to
the bottom, all but .about.14 ml of GN6 S/M liquid was drawn off
and collected in a sterile 1-L flask for future use. The pre-wetted
suspension of whiskers was vortexed for 60 seconds on maximum speed
and 8.1 ml was added to the bottle, to which 170 .mu.g DNA was
added as a last step. The bottle was immediately placed in a
modified Red Devil 5400 commercial paint mixer and agitated for 10
seconds. After agitation, the cocktail of cells, media, whiskers
and DNA was added to the contents of the 1-L flask along with 125
ml fresh GN6 liquid medium to reduce the osmoticant. The cells were
allowed to recover on a shaker for 2 hours before being filtered
onto Whatman #4 filter paper (5.5 cm) using a glass cell collector
unit that was connected to a house vacuum line.
[0316] Either 3 or 6 mL of dispersed suspension was pipetted onto
the surface of the filter as the vacuum was drawn. Filters were
placed onto 60.times.20 mm plates of GN6 medium. Plates were
cultured for 1 week at 28.degree. C. in a dark box that was loosely
sealed with a single layer of plastic (<2 mils thick) to
minimize evaporation of the individual plates.
[0317] After 1 week, filter papers were transferred to 60.times.20
mm plates of GN6 (1H) medium (N6 Medium, 2.0 mg/L 2,4-D, 30 g/L
sucrose, 100 mg/L myo-inositol, 1.0 mg/L bialaphos, 2.5 g/L
Gelrite, pH 5.8) or GN6D (1H) medium (same as GN6 (1H) except with
8.0 mg/L dicamba and 0.8 mg/L 2,4-D).
[0318] Plates were placed in boxes and cultured as before for an
additional week. Two weeks post-transformation, the tissue was
embedded by scraping either 1/2 the cells on the plate or else all
cells on the plate into 3.0 mL of melted GN6 agarose medium (N6
medium, 2.0 mg/L 2,4-D, 30 g/L sucrose, 100 mg/L myo-inositol, 7
g/L Sea Plaque agarose, pH 5.8, autoclaved for only 10 minutes at
121.degree. C.) containing 1 mg/L bialaphos. The tissue was broken
up and the 3 mL of agarose and tissue were evenly poured onto the
surface of a 100.times.15 mm plate of GN6 (1H) or GN6D (1H) medium.
This was repeated for all remaining plates. Once embedded, plates
were individually sealed with Nescofilm.RTM. or Parafilm M.RTM.,
and then cultured for 1 week at 28.degree. C. in dark boxes.
[0319] Putatively transformed isolates were typically first visible
5-8 weeks post-transformation. Any potential isolates were removed
from the embedded plate and transferred to fresh selection medium
of the same concentration in 60.times.20 mm plates. If sustained
growth was evident after approximately 2 weeks, an event was deemed
to be resistant and was submitted for molecular analysis.
[0320] Regeneration was initiated by transferring callus tissue to
a cytokinin-based induction medium, 28 (1H), containing 1 mg/L
bialaphos, MS salts and vitamins, 30.0 g/L sucrose, 5 mg/L BAP,
0.25 mg/L 2,4-D, 2.5 g/L Gelrite; pH 5.7. Cells were allowed to
grow in low light (13 .mu.Em.sup.-2s.sup.-1) for one week then
higher light (40 .mu.Em.sup.-2s.sup.-1) for another week before
being transferred to regeneration medium, 36 (1H), which was
identical to 28 (1H) except that it lacked plant growth regulators.
Small (3-5 cm) plantlets were removed and placed into
150.times.25-mm culture tubes containing selection-free SHGA medium
(Schenk and Hildebrandt basal salts and vitamins, 1972; 1 g/L
myo-inositol, 10 g/L sucrose, 2.0 g/L Gelrite, pH 5.8). Once
plantlets developed a sufficient root and shoot system, they were
transplanted to soil in the greenhouse.
[0321] 7.6--WHISKERS-Mediated Transformation using Haloxyfop
Selection.
[0322] DNA delivery parameters for direct selection on "fops" were
identical to the bialaphos selection procedure except that 85 .mu.g
of pDAB3403 and 85 .mu.g of construct containing a GFP (Green
Fluorescent Protein) reporter gene were co-transformed together,
and only 3 mL of suspension was filtered onto GN6 medium following
the 2-hour recovery.
[0323] After 0-7 days on GN6 selection-free medium, filter papers
were transferred to 60.times.20-mm plates of GN6 medium containing
2 mg/L 2,4-D plus 50, 100, or 200 nM R-haloxyfop acid. Plates were
placed in boxes and cultured for one additional week. After one
week, the tissue was embedded by scraping all cells from the plate
into 3.0 mL of melted GN6 agarose medium containing the same
concentration of selection agent as in the previous transfer. All
steps afterward were identical to the PAT selection/regeneration
protocol except that 100 nM R-haloxyfop acid was included in the
regeneration media instead of 1 mg/L bialaphos.
[0324] 7.7--Results. Multiple experiments testing various levels of
haloxyfop and cyhalofop were initiated, and 47 isolates were
recovered from direct selection. A subset of the callus events were
submitted for screening using PCR and Western analyses. Following
these expression data, 21 lead events were submitted for Southern
analysis. Results using Ncol, a unique cutter, to obtain
integration data following probing with AAD-1 (v3), unequivocally
demonstrate stable integration of AAD-1 (v3) following
Whiskers-mediated transformation coupled with "fop" selection.
[0325] 7.8--Quantitative Demonstration of In Vitro Tolerance from
AAD-1 (v3) Expressing Callus Events from bialaphos Selection
[0326] Ninety-seven callus isolates recovered from bialaphos
selection were submitted for PAT copy number via Invader analysis
and AAD-1 (v3) PTU analysis via PCR (see Example 7.10). AAD-1 (v3)
protein expression using Western blot/Sandwich ELISA (Example 11)
was completed on a subset of the events. A summary is described in
Table 21 below. At least 15 To plants were regenerated from each of
these events and sent for spray testing and seed production.
TABLE-US-00021 TABLE 21 PCR for AAD-1 (v3) Callus Event PAT Copy #
PTU Western 3404-001 2 + + 3404-006 2 + + 3404-013 3 + + 3404-017 1
+ + 3404-020 3 + nd 3404-022 2 + + 3404-025 2 + + 3404-027 3 + +
3404-031 1 + + 3404-033 2 + nd 3404-036 3 + + 3404-044 3 + +
3404-050 3 + + 3404-053 3 + + 3404-074 2 + + 3404-082 2 + +
[0327] A smaller subset of these events was assessed in
dose-response studies in comparison to a non-transformed control. A
range of concentrations of haloxyfop (from Gallant Super
formulation), up to 400 nM, were tested. Dose response data for one
event, 3404-006, was generated using the general methods of Example
7.4, and is shown in FIG. 13. This demonstrates that event 3404-006
showed no significant reduction in callus growth at haloxyfop
concentrations up to 400 nM whereas non-transgenic corn callus
tissue growth was inhibited at this rate. These data have not been
normalized to account for inherent growth differences that are not
related to the expression of the transgene.
[0328] 7.9--WHISKERS-Mediated Transformation using imazethapyr
Selection.
[0329] The ZmUbi1/AAD-1(v3)/ZmPer5 cassette was removed from
pDAB3404 with AscI/SwaI and inserted into pDAB2212 to create the
AAD-1 (v3) and AHAS Whiskers transformation vector pDAB3415, which
is also referred to as pDAS1283) Once completed, this construct was
transformed into maize via silicon carbide whiskers-mediated
transformation as described in Example 7.4 except 2 mL of cells
were filtered, followed by a 7-day recovery on GN6 medium followed
by selection on media containing 3 .mu.M imazethapyr from
Pursuit.RTM. DG herbicide. Following Invader analysis, 36 events
were identified that contained the AAD-1 (v3) and the AHAS
genes.
[0330] Fifty-three corn calli events from these transformations
were tested in both ELISA and Western Blotting experiments for
their AAD-1 (v3) expression--a subset of the data is shown below.
For the events listed in Table 22, the expression levels of those
detected positive ranged from 90 to over 1000 ppm of total soluble
proteins.
TABLE-US-00022 TABLE 22 AAD-1 Event ID Expression Western Number
Level (ppm) Blot 1283[1]-001 2 - 1283[1]-002 206 +++ 1283[1]-003 1
- 1283[1]-004 90 +++ 1283[1]-005 1 - 1283[1]-006 105 +++
1283[1]-007 212 ++ 1283[1]-008 114 + 1283[1]-009 305 + 1283[1]-010
2 - 1283[1]-011 4 - 1283[1]-012 200 +++ 1283[1]-013 134 +++
1283[1]-014 4 - 1283[1]-015 194 +++ 1283[1]-016 4 - 1283[1]-017 196
+++ 1283[1]-018 3 - 1283[1]-019 178 + 1283[1]-020 260 ++
1283[1]-021 144 +++ 1283[1]-022 140 +++ 1283[1]-023 191 +++
1283[1]-024 392 ++ 1283[1]-025 368 ++ 1283[1]-026 14 - 1283[1]-027
1006 ++ Neg Control 3 - Neg Control 3 - Standard (0.5 .mu.g/mL) ++
Standard (5 .mu.g/mL) ++++
[0331] 7.10--Molecular Analysis: Maize Materials and Methods.
[0332] 7.10.1--Tissue harvesting DNA isolation and quantification.
Fresh tissue was placed into tubes and lyophilized at 4.degree. C.
for 2 days. After the tissue was fully dried, a tungsten bead
(Valenite) was placed in the tube and the samples were subjected to
1 minute of dry grinding using a Kelco bead mill. The standard
DNeasy DNA isolation procedure was then followed (Qiagen, DNeasy
69109). An aliquot of the extracted DNA was then stained with Pico
Green (Molecular Probes P7589) and read in the fluorometer (BioTek)
with known standards to obtain the concentration in ng/.mu.l.
[0333] 7.10.2--Invader assay analysis. The DNA samples are diluted
to 20 ng/.mu.l then denatured by incubation in a thermocycler at
95.degree. C. for 10 minutes. Signal Probe mix was then prepared
using the provided oligo mix and MgCl.sub.2 (Third Wave
Technologies). An aliquot of 7.5 .mu.l was placed in each well of
the Invader assay plate followed by an aliquot of 7.5 .mu.l of
controls, standards, and 20 ng/.mu.l diluted unknown samples. Each
well was overlaid with 15 .mu.l of mineral oil (Sigma). The plates
are then incubated at 63.degree. C. for 1 hour and read on the
fluorometer (Biotek). Calculation of % signal over background for
the target probe divided by the % signal over background internal
control probe will calculate the ratio. The ratio of known copy
standards developed and validated with Southern blot analysis was
used to identify the estimated copy of the unknown events.
[0334] 7.10.3--Polymerase chain reaction. A total of 100 ng of
total DNA was used as the template. 20 mM of each primer was used
with the Takara Ex Taq PCR Polymerase kit (Mirus TAKRR001A).
Primers for the AAD-1 (v3) PTU were (Forward--ATAATGCCAGC
CTGTTAAACGCC) (SEQ ID NO:25) and (Reverse--CTCAAGCATATGAATGACCT
CGA) (SEQ ID NO:26). The PCR reaction was carried out in the 9700
Geneamp thermocycler (Applied Biosystems), by subjecting the
samples to 94.degree. C. for 3 minutes and 35 cycles of 94.degree.
C. for 30 seconds, 63.degree. C. for 30 seconds, and 72.degree. C.
for 1 minute and 45 seconds followed by 72.degree. C. for 10
minutes. Primers for Coding Region PCR AAD-1 (v3) were
(Forward--ATGGCTCATGCTGCCCTCAGCC) (SEQ ID NO:27) and
(Reverse--CGGGC AGGCCTAACTCCACCAA) (SEQ ID NO:28). The PCR reaction
was carried out in the 9700 Geneamp thermocycler (Applied
Biosystems), by subjecting the samples to 94.degree. C. for 3
minutes and 35 cycles of 94.degree. C. for 30 seconds, 65.degree.
C. for 30 seconds, and 72.degree. C. for 1 minute and 45 seconds
followed by 72.degree. C. for 10 minutes. PCR products were
analyzed by electrophoresis on a 1% agarose gel stained with
EtBr.
[0335] 7.10.4--Southern blot analysis. Southern blot analysis was
performed with total
[0336] DNA obtained from Qiagen DNeasy kit. A total of 2 .mu.g of
DNA was subjected to an overnight digestion of Afl II and also
EcoRV for pDAB3404, Ncol for pDAB3403, and Spel for pDAB1421 to
obtain integration data. After the overnight digestion an aliquot
of .about.100 ng was run on a 1% gel to ensure complete digestion.
After this assurance the samples were run on a large 0.85 agarose
gel overnight at 40 volts. The gel was then denatured in 0.2 M
NaOH, 0.6 M NaCl for 30 minutes. The gel was then neutralized in
0.5 M Tris HCl, 1.5 M NaCl pH of 7.5 for 30 minutes. A gel
apparatus containing 20.times.SSC was then set up to obtain a
gravity gel to nylon membrane (Millipore INYC00010) transfer
overnight. After the overnight transfer the membrane was then
subjected to UV light via a crosslinker (Stratagene UV stratalinker
1800) at 1200.times.100 microjoules. The membrane was then washed
in 0.1% SDS, 0.1 SSC for 45 minutes. After the 45 minute wash, the
membrane was baked for 3 hours at 80.degree. C. and then stored at
4.degree. C. until hybridization. The hybridization template
fragment was prepared using the above coding region PCR using
plasmid pDAB3404. The product was run on a 1% agarose gel and
excised and then gel extracted using the Qiagen (28706) gel
extraction procedure. The membrane was then subjected to a
pre-hybridization at 60.degree. C. step for 1 hour in Perfect Hyb
buffer (Sigma H7033). The Prime it RmT dCTP-labeling rxn
(Stratagene 300392) procedure was used to develop the p32 based
probe (Perkin Elmer). The probe was cleaned up using the Probe
Quant. G50 columns (Amersham 27-5335-01). Two million counts CPM
were used to hybridize the southern blots overnight. After the
overnight hybridization the blots were then subjected to two 20
minute washes at 65.degree. C. in 0.1% SDS, 0.1 SSC. The blots were
then exposed to film overnight, incubating at -80.degree. C.
EXAMPLE 8
In Vivo Tolerance and Field Tolerance Data Generated from
PAT-Selected (pDAB3404) AAD-1 (v3) Events
[0337] 8.1--Tolerance of T.sub.0 Corn Plants to AOPP
Herbicides.
[0338] If more than 15 clone plants per event were successfully
regenerated, then extra plants were transferred to the greenhouse
for preliminary tolerance screening with postemergence-applied AOPP
herbicides on To corn plants. Greenhouse-acclimated plants were
allowed to grow until 2-4 new, normal looking leaves had emerged
from the whorl (i.e., plants had transitioned from tissue culture
to greenhouse growing conditions). Plants were grown at 27.degree.
C. under 16 hour light: 8 hour dark conditions in the greenhouse.
Plants were then treated with commercial formulations of one of
three AOPP herbicides: Assure.RTM. II (DuPont), Clincher* (Dow
AgroSciences), or Gallant Super* (Dow AgroSciences) for quizalofop,
cyhalofop, or haloxyfop, respectively. Herbicide applications were
made with a track sprayer at a spray volume of 187 L/ha, 50-cm
spray height, and all sprays contained 1% v/v Agridex crop oil
concentrate adjuvant. The number of clones of each event varied
from week to week due to the rate of regeneration and acclimation
of each event. Overall, an attempt was made to treat representative
clones of each event with a range of herbicide doses ranging from
1.times. lethal dose (.about.1/8.times. field dose) up to 8.times.
field doses (64.times. lethal dose). A lethal dose is defined as
the rate that causes >95% injury to the Hi-II inbred. Hi-II is
the genetic background of the transformants of the present
invention.
[0339] AOPP's are generally very potent corn-killing herbicides.
Three to four leaf Hi-II corn grown from seed is effectively killed
(>95% injury) with 8.8, 62.5, and 4.4 g ae/ha of haloxyfop,
cyhalofop, and quizalofop, respectively. Each AAD-1
(v3)-transformed line tested survived a minimally lethal dose of
each AOPP herbicide tested. In fact, most lines tested survived
with no visible injury (14 DAT) even when treated with an 8.times.
field dose (64.times. lethal dose) of quizalofop. Several
individual clones from events "017" and "038," however, did incur
significant injury at elevated rates. This could be a function of
lower gene expression due to how or where the gene was
inserted.
[0340] The high level of AOPP tolerance was demonstrated in most
events, even when applications were made to plants just coming out
of tissue culture (T.sub.0 stage). Significantly, this tolerance
was shown for all three AOPP herbicides and likely will extend to
all AOPP herbicides as previously shown for AAD-1 in vitro.
[0341] FIG. 14 shows the responses of several AAD-1
(v3)--transformed and non-transformed event clones to lethal doses
of two AOPP herbicides (haloxyfop and quizalofop) applied 1 week
prior.
[0342] Table 23 shows data for the responses of selected AAD-1
(v3)-transformed To corn events to three AOPP herbicides applied
postemergence.
TABLE-US-00023 TABLE 23 Selected AAD-1 (v3)-transformed T0 corn
events response to three AOPP herbicides applied postemergence.
Haloxyfog* Cyhalofop** Quizalofop*** % Injury Construct Event Clone
g ae/ha g ae/ha g ae/ha 14 DAT 3404 001 016 8.8 0 3404 001 018 62.5
0 3404 001 017 8.8 0 3404 001 019 8.8 30 3404 001 020 35 0 3404 017
018 35 0 3404 017 019 35 0 3404 017 020 70 0 3404 017 021 70 0 3404
017 022 140 0 3404 017 023 140 30 3404 017 024 280 30 3404 017 025
280 20 3404 022 019 8.8 0 3404 022 020 17.5 0 3404 022 016 17.5 0
3404 022 024 62.5 0 3404 022 018 125 0 3404 022 021 8.8 0 3404 022
017 17.5 0 3404 022 022 35 0 3404 022 023 70 0 3404 033 012 35 0
3404 033 013 70 0 3404 033 014 70 0 3404 033 015 140 0 3404 033 016
280 0 3404 038 016 8.8 0 3404 038 018 62.5 0 3404 038 017 8.8 0
3404 038 019 35 70 3404 038 020 35 80 3404 038 021 70 80 3404 038
022 70 80 (lethal dose = (lethal dose = (lethal dose = 8.8 g ae/ha)
62.5 ae/ha) 4.4 g ae/ha) *Gallant super.sup.# + 1% COC (v/v)
**Clincher .sup.# + 1% COC (v/v) ***Assure II + 1% COC (v/v)
*Trademark of Dow AgroSciences, LLC
[0343] 8.2--Field Tolerance of pDAB3404 T.sub.1 Corn Plants to
Quizalofop 2,4-D, and Glufosinate Herbicides.
[0344] Two field trials were established at field stations in
Hawaii and Indiana. Corn seed from inbred T.sub.1 plants were
utilized to evaluate sixteen AAD1 event lines for tolerance against
quizalofop and 2,4-D. Three non-transformed hybrids were included
for comparison purposes. The hybrid Hi-II.times.5XH571 is of the
same parentage as the AAD-1 (v3) event lines. The hybrid Croplan
585SR is a sethoxydim resistant line.
[0345] The experimental design was a split-plot with four
replications. The main plot was herbicide treatment and the
sub-plot was AAD-1 (v3) event or comparison hybrid. Plots were one
row by 3.7 meters with approximately twenty-five seeds planted in
each row. For AAD1 events, seeds from a different lineage within
the event were planted in each replicate.
[0346] Glufosinate at 560 g ai/ha was applied to AAD-1 (v3) plots
at the V2 stage to eliminate non-transformed plants. Experimental
treatments included commercial formulations of quizalofop applied
at 70 and 140 g ae/ha, 2,4-D (dimethylamine salt) at 560 and 1120 g
ae/ha, and an untreated control. Treatments were applied using
backpack broadcast boom equipment delivering 187 L/ha carrier
volume at 130-200 kpa pressure. Quizalofop treatments were applied
at the V3-V4 corn stage and 2,4-D treatments were applied at the
V5-V6 stage.
[0347] Quizalofop treated plots were visually assessed for crop
injury at one and three weeks after application (WAA) using a
0-100% scale, where 0 equals no injury and 100 equals complete
death. 2,4-D treated plots were visually assessed for plant leaning
at 2 days after application (DAA) using 0-100% scale where 0 equals
no leaning from any plant and 100 equals all plants prone.
Additionally, 2,4-D plots were visually assessed at 3-4 WAA for
brace root deformation using a 0-10 scale.
[0348] 8.2.1--Results.
[0349] AAD-1 (v3) event response to the highest rates tested of
quizalofop and 2,4-D are shown in Table 24. These rates represent
approximately twice the normal commercial use rates.
Non-transformed hybrids were severely injured (80-100%) by
quizalofop at 70 g ae/ha including the sethoxydim resistant line,
although it displayed slightly better tolerance than the other two
hybrids. All AAD-1 (v3) events except one lineage of event 3404.001
displayed excellent tolerance to quizalofop at 70 g ae/ha. No
visible symptoms were observed on the AAD-1 (v3) events except with
the events noted above.
TABLE-US-00024 TABLE 24 2,4-D Resistance amine 2,4-D amine
Segregation Ratios Treatment Quizalofop (1120 g (1120 g following
Liberty (rate) = (140 g ae/ha) ae/ha) ae/ha) Analysis Results Spray
AAD-1 % copy Leaning Braceroot number AAD-1 AAD-1 % Injury 3 2 DAA
Deformation leaf Leaf Leaf T2 Evaluation WAA (0-100 3-4 WAA
(Southern Western ELISA T1 Population Population = (0-100 scale)
scale) (0-10 scale) analysis) Blot T0 T0 Average Average Event or
Hybrid IN HI IN HI IN HI IN HI HI 3404.001 25 25 0 5 1 2 3 +++ +++
40% 47% X 3404.006 0 0 0 0 0 0 1 ++ +++ 33% 26% X 3404.013 0 0 0 0
0 0 1 + +++ 63% 58% X 3404.017 0 0 1 0 0 0 2 +/- ++ 48% 47% X
3404.020 0 0 0 0 0 0 2 ++ +++ 50% 51% X 3404.022 0 0 1 0 0 0 1 + ++
51% 57% 76%* 3404.025 0 0 0 0 0 0 2 +++ ++++ 55% 59% X 3404.027 0 0
3 0 0 0 5 + ++ 51% 50% X 3404.031 0 0 1 0 0 0 1 ++ ++++ 47% 43%
61%* 3404.033 0 0 0 0 0 0 2 or 3 + ++ 52% 49% X 3404.036 0 0 0 0 0
0 4 ++ +++ 52% 48% X 3404.044 0 0 1 0 0 1 1 + +/- 50% 48% X
3404.050 0 0 0 1 0 1 2 nd nd 38% 28% X 3404.053 0 0 1 0 0 0 2 +++
+++ 48% 56% X 3404.074 0 0 0 0 0 0 1 ++ +++ 53% 52% 73%* 3404.082 0
0 0 0 0 1 3 nd nd 38% 36% X DK493 100 100 20 23 8 9 HI-II X 5XH571
100 100 13 34 7 9 CROPLAN 585SR 80 96 11 33 9 9 *Fits single locus
dominant trait segregation as determined by chi square analysis (P
> 0.05)
[0350] 2,4-D at the 1120 g ae/ha rate caused significant levels
(11-33%) of epinastic leaning in the non-transformed hybrids, a
normal response when applied beyond the V4 growth stage. Little or
no leaning was observed with all AAD-1 (v3) events except one
lineage of 3404.001 (Indiana location only) where moderate levels
(5-13%) of leaning occurred.
[0351] Brace roots of non-transformed hybrids were severely
deformed (rating of 9 on a 0-10 scale) by 2,4-D at the 1120 g ae/ha
rate. Again, this is a normal response to 2,4-D applied beyond the
V4 growth stage. As with the leaning response, little or no brace
root injury was observed with all AAD-1 (v3) events except one
lineage of 3404.001.
[0352] Similar trends occurred with lower tested rates of
quizalofop and 2,4-D although at reduced but still significant
response levels in the non-transformed hybrids (data not
shown).
[0353] These results indicate that most AAD-1 (v3) transformed
event lines displayed a high level of resistance to quizalofop and
2,4-D at rates that were lethal or caused severe epinastic
malformations to non-transformed corn hybrids. See also FIG.
16.
[0354] 8.2.2- Expected Mendelian segregation ratios on three
T.sub.2 events. Plants from individual lineages of each event were
randomly self-pollinated in the field. T.sub.2 seed were hand
harvested at physiological maturity. Based on single gene copy
number (see Table 24 above) and overall performance in Ti
generation (segregation, herbicide tolerance, and vigor), three
events (022, 031, and 074) were chosen for further evaluation in
the field. Breeding rows of each event were planted using a
precision cone planter each consisting of 2500-3000 seeds. At the
V2 growth stage, all AAD-1 (v3) lines were sprayed with 140 g ae/ha
quizalofop (Assure.RTM. II) using a backpack sprayer as previously
described. This rate rapidly killed all "null" (untransformed)
segregants. Each event had a segregation ratio consistent with
Mendelian inheritance of a single locus, dominant gene (3
resistant:1 sensitive, or 75% survival) (see Table 24). Homozygotes
and hemizygotes from event 74 were identified by zygosity testing
(refer to AAD-1 (v3) Invader assay description for corn).
Hemizygous plants were removed and homozygous AAD-1 (v3) plants
were crossed with BE1146 corn inbred introgressed and homozygous
for glyphosate resistance trait, NK603, creating a homogeneous Fi
hybrid seed that is hemizygous for glyphosate resistance, AAD-1
(v3), and glufosinate resistance.
[0355] 8.3--Stacking of AAD-1 (v3) and PAT with Glyphosate
Resistance Genes in Corn.
[0356] Homozygous T.sub.2 AAD-1 (v3)/PAT corn plants were crossed
with glyphosate resistant corn plants producing Fi seed containing
AAD-1 (v3), PAT, and glyphosate resistance genes as described in
the previous example.
[0357] F.sub.1 seeds were planted individually into 3-inch pots
prepared with Metro-Mix.RTM. 360 growing medium (Sun Gro
Horticulture). The pots were initially subirrigated with Hoagland's
solution until wet, then allowed to gravity drain, and grown at
27.degree. C. under 16 hour light:8 hour dark conditions in the
greenhouse. For the remainder of the study the plants were
subirrigated with deionized water.
[0358] Plants were allowed to grow until 2-4 leaves had emerged
from the whorl. At this point herbicide applications were made with
a track sprayer at a spray volume of 187 L/ha, 50-cm spray height.
The plants were sprayed with rates of 2,4-D DMA, glyphosate,
glufosinate, and various combinations of the three. All
applications were formulated in 200 mM Hepes buffer (pH 7.5). In
spray applications where glufosinate was present the treatment was
formulated with the addition of 2% w/v ammonium sulfate.
[0359] At 3 and 14 days after treatment (DAT) plants were
evaluated. Plants were assigned injury rating with respect to
stunting, chlorosis, and necrosis. Plants assigned an injury rating
of 90% or above are considered dead. Results of the study at 14 DAT
can be seen in Table 25.
TABLE-US-00025 TABLE 25 % Injury at 14 DAT Hi II X Field 5XH751
RR/PAT/AAD1 Rate Ave Ave Untreated control -- 0 0 840 g ae/ha
glyphosate 1X 98 0 1680 g ae/ha glyphosate 2X 100 0 3360 g ae/ha
glyphosate 4X 100 0 560 g ae/ha 2,4-D DMA 1X 10 0 1120 g ae/ha
2,4-D DMA 2X 14 0 2240 g ae/ha 2,4-D DMA 4X 29 0 470 g ae/ha
glufosinate 1X 80 0 940 g ae/ha glufosinate 2X 90 3 1880 g ae/ha
glufosinate 4X 96 15 840 g ae/ha glyphosate + 1X + 1X 96 1 560 g
ae/ha 2,4-D DMA 1680 g ae/ha glyphosate + 2X + 2X 100 2 1120 g
ae/ha 2,4-D DMA 3360 g ae/ha glyphosate + 4X + 4X 100 1 2240 g
ae/ha 2,4-D DMA 470 g ae/ha glufosinate + 1X + 1X 89 5 560 g ae/ha
2,4-D DMA 940 g ae/ha glufosinate + 2X + 2X 91 10 1120 g ae/ha
2,4-D DMA 1880 g ae/ha glufosinate + 4X + 4X 97 13 2240 g ae/ha
2,4-D DMA 840 g ae/ha glyphosate + 1X + 1X 90 5 470 g ae/ha
glufosinate 1680 g ae/ha glyphosate + 2X + 2X 98 15 940 g ae/ha
glufosinate 3360 g ae/ha glyphosate + 4X + 4X 100 15 1880 g ae/ha
glufosinate
[0360] This study demonstrated that the AAD-1 (v3) gene in corn can
be stacked with a glyphosate resistance gene and a glufosinate
resistance gene to provide robust field-level tolerance to 2,4-D,
glyphosate, and glufosinate alone or in tank mix combinations.
[0361] 8.3.1--Resistance of AAD-1 (v3) corn using a tank mix of
2,4-D DMA and quizalofop. T.sub.2BC.sub.1 seeds of hemizygous event
number 3404-025.001R/R001 Bulked.001.S058 were planted individually
into 3-inch pots prepared with Metro-Mix.RTM. 360 growing medium.
The pots were initially sub-irrigated with Hoagland's solution
until wet, then allowed to gravity drain, and grown at 27.degree.
C. under 16 hour light:8 hour dark conditions in the greenhouse.
For the remainder of the study the plants were sub-irrigated with
de-ionized water.
[0362] Plants were allowed to grow until V1 stage in the
greenhouse. At this point the plants were selected with 560 g ae/ha
Assure.RTM. II with the addition of 1% Agridex crop oil concentrate
in 200 mM Hepes buffer with the research track sprayer set at 187
L/ha. Plants were allowed 4 days to show symptoms of the selection.
All plants were uninjured. Herbicide applications were made with a
track sprayer at a spray volume of 187 L/ha, 18-in spray height.
All applications were formulated in 200 mM Hepes buffer (pH 7.5)
with the addition of 1% v/v Agridex.
[0363] At 3 and 14 days after treatment (DAT) plants were
evaluated. Plants were assigned injury rating with respect to
stunting, chlorosis, and necrosis. Plants assigned an injury rating
of 90% or above are considered dead. Plants from this particular
lineage had 0% injury at 14 DAT for all tank mixed combinations,
while the wild-type had 100% injury. These results indicate AAD-1
(v3) not only provides robust field level resistance to 2,4-D and
quizalofop individually, but also to exaggerated rates of multiple
combinations of the two chemistries. One could logically expect to
implement novel weed control measures with combinations of phenoxy
auxins and AOPP graminicides in corn (or other crops transformed
with AAD-1) not previously enabled by a single gene HTC.
[0364] 8.3.2--Tolerance of (pDAB3403) To corn plants to quizalofop
herbicide. A target of approximately eight To plant clones from
each of 17 events were regenerated and transferred to the
greenhouse for preliminary tolerance screening with
postemergence-applied discriminating rate of quizalofop herbicide
applied by track sprayer at 35 g ae/ha (1.times. field rate,
4.times. lethal dose) to 3-leaf, greenhouse-adapted To corn plants
using track sprayer conditions previously described. Plants were
rated as Resistant or Sensitive 7 days after treatment. Control,
non-transgenic corn was included with each spray application. Two
events, Event 014 and 047, had two or more To clones sensitive to
35 g ae/ha quizalofop, indicating an unexpected level of
sensitivity for this event. The 15 other events showed stable
integration, protein expression, and the ability to tolerate a
4.times. lethal dose of quizalofop at the whole plant level.
[0365] 8.3.3 Expression of AAD-1 (v3) with respect to quizalofop
tolerance.
[0366] Three different T2 lineages from 3404 transformations that
were pre-screened with Liberty.RTM. (as described previously) to
remove nulls were chosen to compare their tolerance to quizalofop
with respect to their AAD-1 (v3) expression. Expression was
measured at 14 DAT (data not shown) and at 30 DAT (see FIG. 15.).
The highest tolerance line, event 3404-074, always expressed with a
higher amount of AAD-1 (v3) than the other two events at 1X and
higher field rates. This data concludes that corn expressing AAD-1
(v3) can be protected from quizalofop injury at the highest level
tested (2,240 g/ha), which is 16 times the 1X field dose of 35
g/ha. In addition, the expression level was consistent throughout
the period of the experiment.
EXAMPLE 9
Agrobacterium-Mediated Transformation of Maize with AAD-1 (v3)
[0367] 9.1--Plant Material.
[0368] Seeds of a "High II" (i.e., Parent A and B) Fi cross
(Armstrong et al., 1991) are planted directly into 5 gallon-pots
containing 95:5 Metro-Mix.RTM. 360: Mineral soil. The plants are
grown in the greenhouse with a 16 hour photoperiod supplemented by
a combination of high pressure sodium and metal halide lamps.
[0369] 9.2--Tissue Source.
[0370] For obtaining immature Hi-II (F2) embryos, controlled
sib-pollinations were performed. On the day of pollination,
actively shedding tassels are bagged, and fresh pollen is collected
and applied carefully onto the silks. Immature embryos were
isolated as described in Example 7.2.
[0371] 9.3--Preparation of a Superbinary Vector.
[0372] Construction of an Agrobacterium construct, pDAB2272,
containing the AAD-1 (v3) gene in combination with the AHAS
selectable marker gene was accomplished by isolating the 3443 base
pair NotI fragment from pDAB3404 containing ZmUbil v2/ AAD-1
(v3)/ZmPer5 v2 and inserting it into the Notl site of pDAB8549. The
resulting plasmid contains the ZmUbil v2/ AAD-1 (v3)/ ZmPer5 v2 and
the OsAct1 v2/AHAS v3/ZmLip v1 cassettes flanked by non-identical
MAR regions in the direct orientation. This was subsequently
transformed into LBA4404/pSB1 to create the superbinary vector,
which was named pDAB3602 but was also referred to as pDAS1421.
[0373] 9.4--Bacterial Supply.
[0374] All transformations use the "Super Binary" vector from Japan
Tobacco described in U.S. Pat. No. 5,591,616 ("Method for
Transforming Monocotyledons"). To prepare the Agrobacterium
suspension for treatment, 1-2 loops of pDAS1421 recombinant
bacteria from a YP streak plate was put into 5 ml of LS-inf. Mod
medium (LS Basal Medium (Linsmaier and Skoog, 1965), N6 vitamins,
1.5 mg/L 2,4-D, 68.5 g/L sucrose, 36.0 g/L glucose, 6 mM L-proline,
pH 5.2). The mixture was vortexed until a uniform suspension was
achieved. The bacterial concentration was taken using a
Klett-Summerson Photoelectric Colorimeter by reading the density of
the solution. The solution was adjusted to a concentration of Klett
200 (.about.1.times.10.sup.9 cfu/ml) and 100 .mu.M actetosyringone
added to the solution.
[0375] 9.5--Infection and Cocultivation.
[0376] The immature embryos are isolated directly into a microfuge
tube containing 2 ml LS-inf. Mod liquid medium. Each tube,
containing .about.100 embryos, is vortexed for 3-5 sec. The medium
is removed and replaced with fresh liquid medium and the vortex is
repeated. The liquid medium is again removed and this time replaced
with an Agrobacterium solution at the Klett 200 concentration. The
Agrobacterium and embryo mixture is vortexed for 30 sec. Following
a 5 minute incubation at room temperature, the embryos were
transferred to LS-As Mod medium (LS Basal Medium, N6 vitamins, 1.5
mg/L 2,4-D, 30.0 g/L sucrose, 6 mM L-proline, 0.85 mg/L AgNO.sub.3,
1, 100 .mu.M actetosyringone, 3.0 g/L Gelrite, pH 5.8) for a 5-day
co-cultivation at 25.degree. C.
[0377] 9.6--Dose Response Using Immature Embryos.
[0378] Dose response studies were initiated using immature embryos
treated with
[0379] Agrobacterium strain LBA4404 lacking a plasmid as described
previously. Once treated, embryos were allowed to co-cultivate for
5 days at 25.degree. C. and were then transferred to selection
media containing various levels of R-haloxyfop or R-cyhalofop.
Embryos were also transferred to media containing 1 mg/L bialaphos
and 100 nM imazethapyr as negative controls. Embryos were scored
for % embryogenic callus formation after 2 weeks, and then again
after 4 weeks. Embryos were tested on R-haloxyfop levels up to 30
nM; however, insufficient reduction of callus formation was seen at
the highest levels, so higher concentrations (50-100 nM) were used
during transformation experiments. Data from embryos grown on
cyhalofop-containing media is shown in FIG. 17.
[0380] 9.7--Selection.
[0381] After co-cultivation, the embryos were moved through a
2-step selection scheme after which transformed isolates were
obtained. For selection, LSD Mod medium (LS Basal Medium, N6
vitamins, 1.5 mg/L 2,4-D, 0.5 g/L MES, 30.0 g/L sucrose, 6 mM
L-proline, 1.0 mg/L AgNO3 , 250 mg/L cephotaxime, 2.5 g/L Gelrite,
pH 5.7) was used along with one of two selection levels of either
haloxyfop, cyhalofop, or imazethapyr. Throughout the selection
phase, the embryos are cultured in the dark at 28.degree. C. The
embryos were first transferred to an initial level of selection
(50-100 nM R-haloxyfop or 300 nM R-cyhalofop) for 14 days, then
moved up to a higher selection level (250-500 nM R-haloxyfop acid
or 1.5 .mu.M cyhalofop) at a rate of 5 embryos/plate. A subset of
embryos were similarly stepped-up from 100 to 500 nM imazethapyr
from Pursuit.RTM. DG as a positive control. Pursuit.degree. is used
as the chemical selection agent when the AHAS gene is used, based
on U.S. Pat. No. 5,731,180. Tissue was transferred at biweekly
intervals on the same medium until embryogenic colonies were
obtained. These colonies were maintained on the high selection
pressure for the remainder of the culture period. The recovered
transgenic colonies were bulked up by transferring to fresh
selection medium at 2-week intervals for regeneration and further
analysis.
[0382] 9.8.--Regeneration and Seed Production.
[0383] For regeneration, the cultures are transferred to 28
"induction" medium and 36 "regeneration" medium as described
previously containing either 100 nM R-haloxyfop or 1.5 M cyhalofop
for differentiation of plantlets. When plantlets were established,
they were transferred to SHGA tubes to allow for further growth and
development of the shoot and roots as described previously.
Controlled pollinations for seed production were conducted as
described previously.
[0384] 9.9--Event Recovery and Analysis Particulars; Whole Plant
Screening of T.sub.0 Corn Lineages Containing AAD-1 (v3) and AHAS
(pDAS1421).
[0385] Seventy-two Agrobacterium-transformed events were selected
on various levels of R-haloxyfop acid and R-cyhalofop acid in
vitro. Twenty-two callus samples were analyzed by Southern blot
analysis for stable integration of AAD-1 (v3) into the genome as
described previously. Ten single copy events, as indicated in Table
26, were chosen to be regenerated.
TABLE-US-00026 TABLE 26 Southern Assessment of T.sub.0 lineages In
vitro Copy # Western Blot resistant to 35 g ae/ha Selection agent
(callus) (AAD-1) T.sub.0 quizalofop Corn event (nM) AAD-1 Callus
Resistant Sensitive 1421[21]-016 50 Haloxyfop 1 + 8 0 1421[22]-020
100 Haloxyfop 1 ++ 8 0 1421[22]-022 100 Haloxyfop 1 + 8 0
1421[22]-023 100 Haloxyfop 1 ++ 8 0 1421[3]-036 100 Haloxyfop 1 ++
8 0 1421[4]-031 300 Cyhalofop 1 ++ 9 0 1421[4]-032 300 Cyhalofop 1
++ 8 0 1421[4]-033 300 Cyhalofop 1 ++ 12 0 1421[4]-034 300
Cyhalofop 1 ++ 8 0 1421[4]-035 300 Cyhalofop 1 ++ 6 0 Events with
more than 1 copy were not taken to the greenhouse
[0386] A minimum six regenerated clonal lineages per event were
moved to soil in the greenhouse and screened using a track sprayer
as previously described to apply 35 g ae/ha quizalofop when 2-4
new, normal leaves had emerged (see section 8.3.3). Presence of
AAD-1 protein on a Western blot correlated perfectly with herbicide
resistance in the To generation regardless of which AOPP herbicide
was used for selection. There is no negative impact of the second
HTC gene (AHAS) on the function of the AAD-1 (v3).
Example 10
Purification of AAD-1 (v1) for Antibody Creation and Biochemical
Characterization
[0387] All operations during purification were carried out at
4.degree. C. Frozen or fresh E. coli cells from approximately 1 L
culture, grown and induced as in Example 3, were re-suspended in
200 ml of extraction buffer containing 20 mM Tris-HC1, 1 mM EDTA,
and 2 ml of Protease Inhibitor Cocktail (Sigma), and disrupted by
ultrasonication treatment on ice using a Branson sonifier. The
soluble extract was obtained by centrifugation in a GSA rotor
(Sorvall) at 12,000 rpm (24,000g) for 20 minutes. The supernatant
was then loaded onto a Mono Q ion exchange column (Pharmacia HR
10/10) equilibrated with 20 mM Tris-HC1, 1 mM EDTA, pH 8.0, and the
column was washed with same buffer for 10 CV (80 ml). The protein
was eluted with 80 ml of a 0 to 0.25 M NaCl linear gradient in
column buffer, while 2 ml fractions were collected. The fractions
containing AAD-1 (v1) were pooled and concentrated using MWCO 30
kDa membrane centrifugation spin columns (Millipore). The sample
was then further separated on a Superdex 200 size exclusion column
(Pharmacia, XK 16/60) with buffer containing 20 mM Tris-HCl, 0.15 M
NaCl, and 1 mM DTT, pH 8.0 at a flow rate of 1 ml/min. Purification
procedures were analyzed by SDS-PAGE, and protein concentration was
determined by Bradford assay using bovine serum albumin as
standard.
Example 11
Recombinant AAD1 Purification and Antibody Production
[0388] Plasmid pDAB3203 containing the AAD-1 (v1) gene was
maintained frozen at -80.degree. C. in TOP1OF' cells (Invitrogen)
as Dow Recombinant strain DR1878. For expression, plasmid DNA
purified from TOP1OF' cell culture using Promega's Wizard Kit
(Fisher cat. #PR-A1460) was transformed into BL-21 Star (DE3) cells
(Invitrogen cat. #C6010-03) following manufacturer's protocol.
After transformation, 50 .mu.L of the cells were plated onto LB S/S
agar plates, and incubated overnight at 37.degree. C. All colonies
from the entire agar plate were scraped into 100 mL LB in a 500 mL
tribaffled flask and incubated at 37.degree. C. with 200 rpm
shaking for 1 hr. Gene expression was then induced with 1 mM IPTG,
and incubated for 4 hrs at 30.degree. C. with 200 rpm shaking. All
100 mL of culture was centrifuged at 4000 rpm for 20 min. The
supernatant were then discarded, and the pellets were resuspended
in 200 mL of extraction containing 20 mM Tris-HC1 (pH 8.0), 1 mM
EDTA, and 2 mL of Protease Inhibitor Cocktail (Sigma), and
disrupted by ultrasonication treatment on ice using a Branson
sonifier. The lysate was centrifuged at 24,000.times.g for 20 mM to
remove cell debris. The supernatant containing the AAD-1 protein
was then subjected to purification protocol.
[0389] All AAD-1 (v1) purifications were conducted at 4.degree. C.
as discussed in Example 10, unless otherwise stated. The cell
lysate was loaded onto a Mono Q ion exchange column (Pharmacia Cat.
#HR 10/10) equilibrated with 20 mM Tris-HCl (pH 8.0) 1 mM EDTA,
followed by 80 mL of washing with the same buffer. The proteins
were eluted with 80 mL of a 0 to 0.25 M NaCl linear gradient in
column buffer, while 2 mL fractions were collected. The fractions
containing AAD-1 were pooled and concentrated using MWCO 30 kDa
membrane centrifugation spin columns (Millipore). The sample was
then further separated on a Superdex 200 size exclusion column
(Pharmacia, XK 16/60) with buffer containing 20 mM Tris-HCl (pH
8.0), 0.15 M NaCl and 1 mM DTT. Protein concentration was
determined by Bradford assay using bovine serum albumin as
standard.
[0390] Five milligrams purified AAD-1 (v1) was delivered to Zymed
Laboratories, Inc. (South San Francisco, Calif.) for rabbit
polyclonal antibody production. The rabbit received 5 injections in
the period of 5 weeks with each injection containing 0.5 mg of the
purified protein suspended in 1 mL of Incomplete Freund's Adjuvant.
Sera were tested in both ELISA and Western blotting experiments to
confirm specificity and affinity before affinity purification and
horseradish peroxidase (HRP) conjugation (Zymed Lab Inc).
[0391] 11.1--Extracting AAD-1 (v3) from Plant Leaves.
[0392] Approximately 50 to 100 mg of leaf tissue was cut into small
pieces and put into microfuge tubes containing 2 stainless steel
beads (4.5 mm; Daisy Co., cat. #145462-000) and 300 .mu.L plant
extraction buffer (PBS containing 0.1% Triton X-100 and 10 mM DTT).
The tubes were shaken for 4 mM with a bead beater at maximum speed
followed by centrifugation for 10 mM at 5,000.times.g. The
supernatant containing the plant soluble proteins were analyzed for
both total soluble protein (TSP) and AAD-1 (v3) concentrations.
[0393] 11.2--Bradford Assay.
[0394] Total soluble protein concentration from plant leaf tissues
were determined by Bradford assay using bovine serum albumin (BSA)
as standard. Five micro-liter of serially diluted BSA in PBS or
plant extract was transferred to 96-well microtiter plate in
triplicates. For standards, concentrations were ranged from 2000 to
15.6 .mu.g/mL. The protein assay concentrate was first diluted 5
fold in PBS and 250 .mu.L was added to each well and incubated at
room temp for 5 mM Each optical density (OD) was measured at 595 nm
using a microplate reader. The protein concentration of each sample
was extrapolated from standard curve using the Softmax.RTM. Pro
(ver. 4.0) (Molecular Devices).
[0395] 11.3--Enzyme Linked Immuno-Sorbent Assay (ELISA).
[0396] The assay was conducted at room temperature unless otherwise
stated. One hundred micro-liter of purified anti-AAD-1 antibody
(0.5 .mu.g/mL) was coated on 96-well microtiter well and incubated
at 4.degree. C. for 16 hours. The plate was washed four times with
washing buffer (100 mM phosphate buffered saline (PBS; pH 7.4)
containing 0.05% Tween 20) using a plate washer, followed by
blocking with 4% skim milk dissolved in PBS for 1 hour. After
washing, 100 .mu.L standard AAD-1 of known concentrations or plant
extract (see previous section) was incubated in the wells. For
standard curve, purified AAD-1 concentrations ranged from 100 to
1.6 ng/mL in triplicates. Plant extracts were diluted 5, 10, 20,
and 40 fold in PBS and analyzed in duplicates. After 1 hour
incubation, the plate was washed as above. One hundred micro-liter
anti-AAD-1 antibody-HRP conjugate (0.25 ug/mL) was incubated in
each well for 1 hour before washing. One hundred micro-liter HRP
substrate, 1-StepTM Ultra TMB-ELISA (Pierce), was incubated in each
well for 10 minutes before the reaction was stopped by adding 100
.mu.L 0.4N H.sub.2SO.sub.4. The OD of each well was measured using
a microplate reader at 450 nm. To determine the concentrations of
AAD-1 in plant extract, the OD value of duplicates were averaged
and extrapolated from the standard curve using the Softmax.RTM. Pro
ver. 4.0 (Molecular Devices).
[0397] For comparison, each sample was normalized with its TSP
concentration and percent expression to TSP was calculated.
[0398] 11.4--Western Blotting Analysis
[0399] Plant extracts or AAD-1 standards (5 and 0.5 .mu.g/mL) were
incubated with Laemmli sample buffer at 95.degree. C. for 10
minutes and electrophoretically separated in 8-16% Tris-Glycine
Precast gel. Proteins were then electro-transferred onto
nitrocellulose membrane using standard protocol. After blocking in
4% skim milk in PBS, AAD-1 protein was detected by anti-AAD1
antiserum followed by goat anti-rabbit/HRP conjugates. The detected
protein was visualized by chemiluminescence substrate ECL Western
Analysis Reagent (Amersham cat. #RPN 21058).
EXAMPLE 12
Tobacco Transformation
[0400] Tobacco transformation with Agrobacterium tumefaciens was
carried out by a method similar, but not identical, to published
methods (Horsch et al., 1988). To provide source tissue for the
transformation, tobacco seed (Nicotiana tabacum cv. Kentucky 160)
was surface sterilized and planted on the surface of TOB-medium,
which is a hormone-free Murashige and Skoog medium (Murashige and
Skoog, 1962) solidified with agar. Plants were grown for 6-8 weeks
in a lighted incubator room at 28-30.degree. C. and leaves
collected sterilely for use in the transformation protocol. Pieces
of approximately one square centimeter were sterilely cut from
these leaves, excluding the midrib. Cultures of the Agrobacterium
strains (EHA101S containing pDAB721, AAD-1 (v3)+PAT), grown
overnight in a flask on a shaker set at 250 rpm at 28.degree. C.,
were pelleted in a centrifuge and resuspended in sterile Murashige
& Skoog salts, and adjusted to a final optical density of 0.5
at 600 nm. Leaf pieces were dipped in this bacterial suspension for
approximately 30 seconds, then blotted dry on sterile paper towels
and placed right side up on TOB+ medium (Murashige and Skoog medium
containing 1 mg/L indole acetic acid and 2.5 mg/L benzyladenine)
and incubated in the dark at 28.degree. C. Two days later the leaf
pieces were moved to TOB+ medium containing 250 mg/L cefotaxime
(Agri-Bio, North Miami, Fla.) and 5 mg/L glufosinate ammonium
(active ingredient in Basta, Bayer Crop Sciences) and incubated at
28-30.degree. C. in the light. Leaf pieces were moved to fresh TOB+
medium with cefotaxime and Basta twice per week for the first two
weeks and once per week thereafter. Four to six weeks after the
leaf pieces were treated with the bacteria, small plants arising
from transformed foci were removed from this tissue preparation and
planted into medium TOB-containing 250 mg/L cefotaxime and 10 mg/L
Basta in PhytatrayTM II vessels (Sigma). These plantlets were grown
in a lighted incubator room. After 3 weeks, stem cuttings were
taken and re-rooted in the same media. Plants were ready to send
out to the greenhouse after 2-3 additional weeks.
[0401] Plants were moved into the greenhouse by washing the agar
from the roots, transplanting into soil in 13.75 cm square pots,
placing the pot into a Ziploc.RTM. bag (SC Johnson & Son,
Inc.), placing tap water into the bottom of the bag, and placing in
indirect light in a 30.degree. C. greenhouse for one week. After
3-7 days, the bag was opened; the plants were fertilized and
allowed to grow in the open bag until the plants were
greenhouse-acclimated, at which time the bag was removed. Plants
were grown under ordinary warm greenhouse conditions (30.degree.
C., 16 hour day, 8 hour night, minimum natural+supplemental
light=500 .mu.E/m.sup.2s.sup.1).
[0402] Prior to propagation, To plants were sampled for DNA
analysis to determine the insert copy number. The PAT gene which
was molecularly linked to AAD-1 (v3) was assayed for convenience.
Fresh tissue was placed into tubes and lyophilized at 4.degree. C.
for 2 days. After the tissue was fully dried, a tungsten bead
(Valenite) was placed in the tube and the samples were subjected to
1 minute of dry grinding using a Kelco bead mill. The standard
DNeasy DNA isolation procedure was then followed (Qiagen, DNeasy
69109). An aliquot of the extracted DNA was then stained with Pico
Green (Molecular Probes P7589) and read in the florometer (BioTek)
with known standards to obtain the concentration in ng/.mu.l.
[0403] The DNA samples were diluted to 9 ng/.mu.l, then denatured
by incubation in a thermocycler at 95.degree. C. for 10 minutes.
Signal Probe mix was then prepared using the provided oligo mix and
MgCl.sub.2 (Third Wave Technologies). An aliquot of 7.5 was placed
in each well of the Invader assay plate followed by an aliquot of
7.5 .mu.l of controls, standards, and 20 ng/.mu.l diluted unknown
samples. Each well was overlaid with 15 .mu.l of mineral oil
(Sigma). The plates were then incubated at 63.degree. C. for 1.5
hours and read on the florometer (Biotek). Calculation of % signal
over background for the target probe divided by the % signal over
background internal control probe will calculate the ratio. The
ratio of known copy standards developed and validated with southern
blot analysis was used to identify the estimated copy of the
unknown events (Table 27).
TABLE-US-00027 TABLE 27 Tobacco T0 events transformed with pDAB721
(AAD-1(v3) + PAT). Relative PAT copy Coding ELISA (.mu.g tolerance
T0 number Region PCR AAD-1/ml spray with Event (Southern) for AAD-1
plant extract) 2,4-D * 721(1)1 1 + 0.9 Medium 721(2)1 nd nd 0.6
Medium 721(2)2 5 + 0.3 Low 721(2)3 3 + 2.6 Medium 721(2)5 5 + 4.1
Variable 721(2)6 3 + 0.5 Variable 721(2)8 5 + 0.3 High 721(2)11 3 +
n/a High 721(2)12 3 + 4.1 Medium 721(2)13 2 + 0.5 Medium 721(2)14 5
+ 0.2 High 721(2)16 4 + 3.2 Medium 721(2)17 3 + nd High 721(2)18 5
+ nd High 721(2)19 >10 + nd Low 721(2)20 5 + nd Medium 721(2)21
4 + nd High 721(2)22 7 + nd Medium 721(2)23 >10 + nd Variable
721(3)003 3 + nd Variable 721(3)008 2 + nd High 721(3)012 1 + nd
High 721(3)4 2 + 0.5 High 721(3)5 9 + 3.3 High 721(3)6 4 + 7.1
Variable 721(3)9 2 + 1 Low 721(3)10 3 + 0.6 High 721(3)11 7 + 6 Low
721(3)13 4 + 0.1 High 721(3)014 2 + 0.1 Medium nd = not done
Legend: Relative tolerance * Injury at 3200 g ae/ha 2,4-D (14DAT)
Low >50% injury Medium 20-50% injury High <20% injury
Variable inconsistent
[0404] Copy number estimations were confirmed by Southern Analysis
on several events. Southern blot analysis was performed with total
DNA obtained from Qiagen DNeasy kit. A total of 2 .mu.gs of DNA was
subjected to an overnight digest of NsiI and also HindIII for
pDAB721 to obtain integration data. After the overnight digestion
an aliquot of .about.100 ngs was run on a 1% gel to ensure complete
digestion. After this assurance the samples were processed using
same protocol as in Example 6 section 11.
[0405] All events were also assayed for the presence of the AAD-1
(v3) gene by PCR using the same extracted DNA samples. A total of
100 ng of total DNA was used as template. 20 mM of each primer was
used with the Takara Ex Taq PCR Polymerase kit (Minis TAKRROO1A).
Primers for the Coding Region PCR AAD-1 were (RdpAcodF ATGGCTCA
TGCTGCCCTCAGCC) (SEQ ID NO:27) and (RdpAcodR CGGGCAGGCCTAACTCCACC
AA) (SEQ ID NO:28). The PCR reaction was carried out in the 9700
Geneamp thermocycler (Applied Biosystems), by subjecting the
samples to 94.degree. C. for 3 minutes and 35 cycles of 94.degree.
C. for 30 seconds, 64.degree. C. for 30 seconds, and 72.degree. C.
for 1 minute and 45 seconds followed by 72.degree. C. for 10
minutes. PCR products were analyzed by electrophoresis on a 1%
agarose gel stained with EtBr. Four to 12 clonal lineages from each
of 30 PCR positive events were regenerated and moved to the
greenhouse.
[0406] A representative plant from each of 19 events was assayed
for AAD-1 (v3) expression by ELISA methods previously described.
All events assayed showed detectable levels of AAD-1 (v3) (Table
27). Protein expression varied across events.
[0407] T.sub.0 plants from each of the 30 events were challenged
with a wide range of 2,4-D sprayed on plants that were 3-4 inches
tall. Spray applications were made as previously described using a
track sprayer at a spray volume of 187 L/ha. 2,4-D dimethylamine
salt (Riverside Corp) was applied at 0, 50, 200, 800, or 3200 g
ae/ha to representative clones from each event mixed in deionized
water. Each treatment was replicated 1-3 times. Injury ratings were
recorded 3 and 14 DAT. Every event tested was more tolerant to
2,4-D than the untransformed control line KY160. In several events,
some initial auxinic herbicide-related epinasty occurred at doses
of 800 g ae/ha or less. Some events were uninjured at this rate
(equivalent to 1.5.times. field rate). All events suffered some
level temporary auxinic damage 3 DAT when treated with 3200 g
ae/ha. Some leaf burning also occurred at this high rate due to the
acidity of the spray solution. Future trials at high 2,4-D rates
were buffered. Response of T.sub.0 plants treated with 3200 g ae/ha
2,4-D (.about.6.times. field rate) was used to discern relative
tolerance of each event into "low" (>50% injury 14 DAT),
"medium" (20-50% injury), "high" (<20% injury). Some events were
inconsistent in response among replicates and were deemed
"variable" (Table 27).
[0408] Verification of high 2,4-D tolerance.
[0409] Two to four T.sub.0 individuals surviving high rates of
2,4-D were saved from each event and allowed to self fertilize in
the greenhouse to give rise to T.sub.1 seed. Two AAD-1 (v3) tobacco
lines (event 721(2)-013.010 and 721(3)-008.005) were chosen from
the To generation. The T.sub.1 seed was stratified, and sown into
selection trays much like that of Arabidopsis (Example 6.4),
followed by selective removal of untransformed nulls in this
segregating population with 280 g ai/ha glufosinate (PAT
selection). Survivors were transferred to individual 3-inch pots in
the greenhouse. These lines provided medium and high levels of
robustness to 2,4-D in the To generation. Improved consistency of
response is anticipated in Ti plants not having come directly from
tissue culture. These plants were compared against wildtype KY 160
tobacco. All plants were sprayed with the use of a track sprayer
set at 187L/ha. The plants were sprayed from a range of 70-4480 g
ae/ha 2,4-D dimethylamine salt (DMA), R-Dichlorprop, and a 50/50
mix of the two herbicides. All applications were formulated in
200mM Hepes buffer (pH 7.5). Each treatment was replicated 4 times.
Plants were evaluated at 3 and 14 days after treatment. Plants were
assigned injury rating with respect to stunting, chlorosis, and
necrosis. The T.sub.1 generation is segregating, so some variable
response is expected due to difference in zygosity. (Table 28). No
injury was observed at rates below 1X field rates (560 g ae/ha) for
2,4-D or R-dichlorprop in either event. Very little injury was
observed even up to 8 times field rates (4480 g ae/ha) and this was
exhibited as stunting, not auxinic herbicide damage. These results
indicated commercial level tolerance can be provided by AAD-1 (v3),
even in a very auxin-sensitive dicot crop like tobacco. These
results also show resistance can be imparted to both chiral
(2,4-dichlorophenoxypropionic acid) and achiral
(2,4-dichlorophenoxyacetic acid) phenoxy auxin herbicides alone or
in tank mix combination.
TABLE-US-00028 TABLE 28 Segregating AAD-1 T.sub.1 tobacco plants'
response to phenoxy auxin herbicides. 721(2)013.010 721(3)008.005
KY160 - (medium tolerance (high tolerance in Wildtype in T.sub.0
generation) T.sub.0 generation) Herbicide Average % Injury of
Replicates 14 DAT 560 g ae/ha 2,4-D DMA 75 5 0 1120 g ae/ha 2,4-D
DMA 80 5 2 2240 g ae/ha 2,4-D DMA 90 5 0 4480 g ae/ha 2,4-D DMA 95
5 5 560 g ae/ha R-dichlorprop 70 5 0 1120 g ae/ha R-dichlorprop 75
5 0 2240 g ae/ha R-dichlorprop 88 5 0 4480 g ae/ha R-dichlorprop 95
10 5 560 g ae/ha 2,4-D DMA/R-dichlorprop 80 5 5 1120 g ae/ha 2,4-D
DMA/R-dichlorprop 80 10 10 2240 g ae/ha 2,4-D DMA/R-dichlorprop 95
15 15 4480 g ae/ha 2,4-D DMA/R-dichlorprop 95 15 15
[0410] A 100 plant progeny test was also conducted on each of the
two AAD-1 (v3) lines (events 721(2)-013.010 and 721(3)-008.005).
The seeds were stratified, sown, and transplanted with respect to
the procedure above except null plants were not removed by Liberty
selection. All plants were then sprayed with 560 g ae/ha 2,4-D DMA
as previously described. After 14 DAT, resistant and sensitive
plants were counted. Both event `013` and `008` segregated as a
single locus, dominant Mendelian trait (3R:1S) as determined by Chi
square analysis. AAD-1 is heritable as a robust phenoxy auxin
resistance gene in multiple species.
[0411] Field level tolerance will be demonstrated by planting
T.sub.1 or T.sub.2 seed in the greenhouse, selectively removing the
null plants by Liberty selection as previously described, and
rearing individual seedlings in 72-well transplant flats (Hummert
International) in Metro 360 media according to growing conditions
indicated above. Individual plants will be transplanted into the
field plots using an industrial vegetable planter. Drip or overhead
irrigation will be used to keep plants growing vigorously. Once
plants reach 6-12 inches in height, tobacco plants will be sprayed
with a broad range of phenoxy auxins and rated as shown above.
Environmental stresses are more significant under field conditions;
however, based on previous experience with AAD-1 (v3)-transformed
corn, robust translation of resistance from the greenhouse to the
field is expected.
Example 13
Soybean Transformation
[0412] Soybean improvement via gene transfer techniques has been
accomplished for such traits as herbicide tolerance (Padgette et
al., 1995), amino acid modification (Falco et al., 1995), and
insect resistance (Parrott et al., 1994). Introduction of foreign
traits into crop species requires methods that will allow for
routine production of transgenic lines using selectable marker
sequences, containing simple inserts. The transgenes should be
inherited as a single functional locus in order to simplify
breeding. Delivery of foreign genes into cultivated soybean by
microprojectile bombardment of zygotic embryo axes (McCabe et al.,
1988) or somatic embryogenic cultures (Finer and McMullen, 1991),
and Agrobacterium-mediated transformation of cotyledonary explants
(Hinchee et al., 1988) or zygotic embryos (Chee et al., 1989) have
been reported.
[0413] Transformants derived from Agrobacterium-mediated
transformations tend to possess simple inserts with low copy number
(Birch, 1991). There are benefits and disadvantages associated with
each of the three target tissues investigated for gene transfer
into soybean, zygotic embryonic axis (Chee et al., 1989; McCabe et
al., 1988), cotyledon (Hinchee et al., 1988) and somatic
embryogenic cultures (Finer and McMullen, 1991). The latter have
been extensively investigated as a target tissue for direct gene
transfer. Embryogenic cultures tend to be quite prolific and can be
maintained over a prolonged period. However, sterility and
chromosomal aberrations of the primary transformants have been
associated with age of the embryogenic suspensions (Singh et al.,
1998) and thus continuous initiation of new cultures appears to be
necessary for soybean transformation systems utilizing this tissue.
This system needs a high level of 2,4-D, 40 mg/L concentration, to
initiate the embryogenic callus and this poses a fundamental
problem in using the AAD-1 (v3) gene since the transformed locus
could not be developed further with 2,4-D in the medium. So, the
meristem based transformation is ideal for the development of 2,4-D
resistant plant using AAD-1 (v3).
[0414] 13.1--Transformation Method 1: cotyledonary Node
Transformation of Soybean Mediated by Agrobacterium
tumefaciens.
[0415] The first reports of soybean transformation targeted
meristematic cells in the cotyledonary node region (Hinchee et al.,
1988) and shoot multiplication from apical meristems (McCabe et
al., 1988). In the A. tumefaciens-based cotyledonary node method,
explant preparation and culture media composition stimulate
proliferation of auxiliary meristems in the node (Hinchee et al.,
1988). It remains unclear whether a truly dedifferentiated, but
totipotent, callus culture is initiated by these treatments. The
recovery of multiple clones of a transformation event from a single
explant and the infrequent recovery of chimeric plants (Clemente et
al., 2000; Olhoft et al., 2003) indicates a single cell origin
followed by multiplication of the transgenic cell to produce either
a proliferating transgenic meristem culture or a uniformly
transformed shoot that undergoes further shoot multiplication. The
soybean shoot multiplication method, originally based on
microprojectile bombardment (McCabe et al., 1988) and, more
recently, adapted for Agrobacterium-mediated transformation
(Martinell et al., 2002), apparently does not undergo the same
level or type of dedifferentiation as the cotyledonary node method
because the system is based on successful identification of germ
line chimeras. The range of genotypes that have been transformed
via the Agrobacterium-based cotyledonary node method is steadily
growing (Olhoft and Somers, 2001). This de novo meristem and shoot
multiplication method is less limited to specific genotypes. Also,
this is a non 2,4-D based protocol which would be ideal for 2,4-D
selection system. Thus, the cotyledonary node method may be the
method of choice to develop 2,4-D resistant soybean cultivars.
Though this method was described as early as 1988 (Hinchee et al.,
1988), only very recently has it been optimized for routine high
frequency transformation of several soybean genotypes (Zhang et
al., 1999; Zeng et al., 2004).
[0416] 13.1.1--Agrobacterium preparation. The plasmid, pDAB721,
contains the AAD-1 (v3) gene under the control of the Arabidopsis
Ubi10 promoter. This plasmid also carries the PAT gene under the
control of rice actin promoter coding for an enzyme that degrades
glufosinate which can be used as a selection agent for
transformants. This vector can be used in the transformation
experiment described below. The construct pDAB721 was mobilized
into the Agrobacterium strain EHA101S by electroporation.
[0417] Agrobacterium cultures harboring pDAB721 used in the
transformations can be grown in YEP medium (10 g/L peptone, 5 g/L
yeast extract and 5 g/L NaCl, pH 7.0). Agrobacterium cultures are
pelleted at low speed and resuspended in SCM liquid medium (see
below) to OD660 of 0.6 for use in the inoculations.
[0418] 13.1.2--Plant transformation. Seeds of "Thorne,"
"Williams82," or "NE3001," public genotypes of soybean, can be
disinfected by a 20-minute wash in 20% (v/v) commercial bleach
(NaClO) amended with 2 drops of Liqui-Nox.RTM.. The seeds should be
rinsed five times with sterile water on hormone-free SHGA medium
and allowed to germinate for 5 days at 24.degree. C., with an 18/6
hour light/dark regime. B5 medium consists of macro and micro
nutrients and vitamins described by Gamborg et al. (1968) (Sigma,
cat. #G 5893, St. Louis). All media are solidified with 0.8% (w/v)
washed agar (Sigma cat. #A 8678). Alternatively, certain genotypes
of soybean can undergo a dry surface sterilization procedure using
chlorine gas (Cl.sub.2) whereby mature seeds are placed into
100.times.25 mm Petri plates in a single layer using about 130
seeds per plate. Approximately 3-4 plates are placed into a
desiccator within a fume hood in such a way that all the plates are
half open and that there is enough space for a 250 ml beaker in the
middle of the desiccator. The beaker is filled with 95 ml of
commercial bleach, to which 5 ml of concentrated (12N) HC1 is added
dropwise along the side of the beaker. The desiccator is
immediately closed and allowed to stand for at least 16 hours in a
fume hood. Following sterilization, the plates are closed, then
brought to a laminar flow hood and left open for about 30 minutes
to remove any excessive Cl.sub.2 gas. Seeds are then germinated as
described previously. The dry surface sterilized seeds will remain
sterile at room temperature for about 2 weeks. Explants are
prepared for inoculation as previously described (Hinchee et al.,
1988).
[0419] Explants should be inoculated for 30 minutes. Cocultivation
and Agrobacterium re-suspension medium consists of B5 medium
supplemented with 1 mg/L BAP, 1 mg/L GA3, 3% (w/v) sucrose, 20 mM
MES(2-1N-morpholinolethane sulfonic acid), 400 mg/L L-cysteine
(Olhoft and Somers, 2001) pH 5.7, 0.99 mM dithiothreitol (DTT), and
200 .mu.M acetosyringone (AS). Explants are cocultivated for 5 days
at 25.degree. C. Following cocultivation, explants were washed in
the cocultivation medium containing 100 mg/L timentin and 200 mg/L
cefotaxime, without MES and AS.
[0420] Explants are to be placed on shoot induction medium and
transferred every 14 days for a total of 28 days prior to herbicide
selection. The shoot induction medium consists of full-strength B5
medium supplemented with 1.7 mg/L BAP, 100 mg/L timentin , 200 mg/L
cefotaxime pH 5.7 and 3% (w/v) sucrose. Cotyledons should be placed
adaxial side up with the cotyledonary nodal region flush to the
medium, amended with increasing levels of Basta (2, 5, 6 then 7
mg/L glufosinate ammonium) or sublethal levels of 2,4-D ranging
from 10 mg to 400 mg/L every 2 weeks for a total of 8 weeks.
[0421] Differentiating explants are subsequently transferred to
shoot elongation medium for an additional 4 to 10 weeks under the
same glufosinate selection or under decreased 2,4-D selection
pressure ranging from 10 mg to 100 mg/L. The elongation medium
consisted of B5 medium (Sigma cat. #M0404) amended with 1 mg/L
zeatin riboside, 0.1 mg/L IAA (indole-3-acetic acid), 0.5 mg/L GA3,
50 mg/L glutamine, 50 mg/L asparagine, and 3% (w/v) sucrose, pH
5.8. Elongated shoots should be rooted, without further selection,
on half-strength MS/B5 medium with full-strength vitamins plus 0.5
mg/L NAA (.alpha.-naphthaleneacetic acid) or 0.1 mg/L IAA and 2%
(w/v) sucrose.
[0422] The antibiotics, timentin and cefotaxime, are maintained
within the media throughout selection. Cultures are transferred to
fresh medium every 2 weeks. Plantlets are acclimated for 1 to 2
weeks prior to transfer to the greenhouse.
[0423] 13.1.3--Progeny evaluation. T.sub.0 plants will be allowed
to self fertilize in the greenhouse to give rise to T.sub.1 seed.
T.sub.1 plants (and to the extent enough T.sub.0 plant clones are
produced) will be sprayed with a range of herbicide doses to
determine the level of herbicide protection afforded by AAD-1 (v3)
and PAT genes in transgenic soybean. Rates of 2,4-D used on T.sub.0
plants will typically use one or two selective rates in the range
of 100-400 g ae/ha. T.sub.1 seed will be treated with a wider
herbicide dose ranging from 50-3200 g ae/ha 2,4-D. Likewise,
T.sub.0 and T.sub.1 plants can be screened for glufosinate
resistance by postemergence treatment with 200-800 and 50-3200 g
ae/ha glufosinate, respectively. Analysis of protein expression
will occur as described in Example 9 for Arabidopsis and corn.
Determination of the inheritance of AAD-1 (v3) will be made using
T.sub.1 and T.sub.2 progeny segregation with respect to herbicide
tolerance.
[0424] 13.2--Transformation Method 2: "No-Shake" Agrobacterium
Mediated Trans-Formation of Non-Regenerable Soybean Suspension
Cells.
[0425] The DAS Soybean cell suspensions were cultured on a 3d cycle
with 10 ml of settled suspension volume transferred to 115 ml of
fresh liquid medium. Settled cell volume was determined by allowing
the cell suspension to settle for 2 min in the 125-mL flask after
vigorous swirling and then drawing cells from the bottom of the
flask with a wide bore 10 ml pipette. The flasks were then
transferred to orbital shakers at 140 rpm.
[0426] Aliquots of 4 ml of the suspension cells at 0.72
OD.sup.60.degree. was transferred along with 200 .mu.M
acetosyringone (AS) onto a 100.times.25 sterile Petri plate. EHA105
Agrobacterium suspension at a density of 1.2 0D.sup.65.degree. in a
100 .mu.L volume was added and mixed well. The Agrobacterium and
suspension cell mixture was swirled well and the plate was
transferred to dark growth chamber where the temperature was
maintained at 25.degree. C.
[0427] 13.2.1--Selection of Soybean Suspension Cells and Isolation
of Transformed Colonies.
[0428] After 4 days of co-cultivation the plate was swirled again
to mix the suspension well and a 1.5 ml aliquot was transferred to
the selection medium and spread on the gel medium on a 100.times.15
ml Petri-plate. The selection medium consisted of full-strength B5
medium supplemented with 1.7 mg/L BAP, 100 mg/L timentin, 200 mg/L
cefotaxime pH 5.7 and 3% (w/v) sucrose and the medium was amended
with glufosinate ammonium at 5 mg/L level. After a 10 min drying in
the hood the plates were incubated for 4 weeks in dark at
28.degree. C. Colonies appeared in selection and a total of 11
colonies were transferred to fresh medium from 3 different
experiments and maintained for 3-4 months. All the 11 resistant
colonies produced calli that were growing on the 5 mg/L glufosinate
containing selection medium. The non-transformed suspension cells
were sensitive when plated on to 0.5 mg/l glufosinate ammonium
medium. However, the transformed colonies were resistant to
5.times. concentration of glufosinate ammonium and were maintained
for up to 4 months.
[0429] Callus events were sampled for analyses when they reached 2
to 3 cm in diameter. At least two of colony isolates, one each from
two different experiments, were analyzed for AAD1 protein
expression. The ELISA and Western analysis carried out on these two
isolates showed positive expression of AAD1 proteins. Both ELISA
(Table 29) and Western Blotting (FIG. 18) analysis on two separate
soybean calli transformed with AAD-1 (v3) gene indicated that the
callus cells are expressing AAD-1 (v3) protein. The sandwich ELISA
detected 0.0318% and 0.0102% total soluble protein of AAD-1 (v3) in
two different callus tissue samples. Due to the sensitivity and
cross reactivity of the antiserum, multiple bands were observed in
the western blot. However, AAD-1 (v3) specific band was observed in
both callus samples but not in the wild type (negative) tissue.
Coding region PCR analyses showed the expected size products of the
AAD1 and the PAT coding regions in these colonies indicating that
they were transformed.
TABLE-US-00029 TABLE 29 PCR and ELISA data for transgenic soybean
events. AAD-1 PAT Coding Coding T SP AAD-1 % Event region PCR
region PCR (.mu.g/mL) (ng/mL) Expression 1-1 + + 1995.13 633.89
0.0318% 2-1 + + 2018.91 205.92 0.0102% Negative - - 2074.63 -1.22
-0.0001% Control
[0430] 13.3--Transformation Method 3: Aerosol-Beam Mediated
Transformation of Embryogenic Soybean Callus Tissue.
[0431] Culture of embryogenic soybean callus tissue and subsequent
beaming were as described in U.S. Patent No. 6,809,232 (Held et
al.).
[0432] Embryogenic calluses of several Stine elite varieties,
including 96E750, 96E94, 97E986, 96E144 and 96E692, were separately
collected into the center of plates of B1-30 3Co5My or B1-30
3Co5My0.25PA0.5K three days after transfer to fresh medium. The
tissue was then beamed with pDAB3295 using linearized DNA at a
concentration of approximately 0.2 .mu.g/ml. After beaming, the
embryogenic callus was transferred to fresh B1-30 3Co5My or B1-30
3Co5My0.25PA0.5K for one passage of a month. The tissue was then
transferred to selective medium containing 1 mg/l bialaphos. With
bialaphos, selection typically was maintained at 1 mg/l for the
first two one-month passages and then increased to 2 mg/l for the
following three to seven months. Transgenic events were identified
when callus tissue generated by transformation experiments began to
organize and develop into embryogenic structures while still on
selective media containing 2,4-D plus bialaphos. Once identified,
the maturing structures were regenerated into plants according to
the following protocol: Embryogenic structures were transferred off
B1-30 3Co5My or B1-30 3/co5My0.25PA0.5K to B3 medium. After 3 to 4
weeks' growth on B3 medium, individual structures were transferred
to fresh medium. After another 3 to 4 weeks, maturing embryos were
transferred to B5G medium and placed in the light. Embryos that had
elongated and produced roots were transferred to tubes containing
1/2 B5G medium where they continued development into plantlets; and
these plantlets were removed from the tubes and placed into
pots.
[0433] Variations of media referred to in Table 30 were tested,
e.g., B1-30 3Co5My, which was made by adding 3% coconut water and 5
gm/l myo-inositol to B1-30. Other variations included: B1-30
3Co5My0.25 PA0.5K which contained B1-30 basal medium plus 3%
coconut water, 5 gm/l myo-inositol, 0.25 gm/l phytic acid, and 0.5
gm/l additional KH2PO4 and 1/2 B5G which contained all ingredients
of B5G medium at half strength.
TABLE-US-00030 TABLE 30 Growth Media for Soybean Ingredients in 1
Liter B1-30 B3 B5G Ms Salts 4.43 g 4.43 g B5 Salts 3.19 g NaEDTA
37.3 mg 37.3 mg 37.3 mg 2,4-D 30 mg Activated charcoal 5 g Phytagar
8 g 8 g Gelrite 2 g pH 5.8 5.8 5.8
EXAMPLE 14
AAD-1 (v3) Enablement in Cotton
[0434] 14.1--Cotton Transformation Protocol.
[0435] Cotton seeds (Co310 genotype) are surface-sterilized in 95%
ethanol for 1 minute, rinsed, sterilized with 50% commercial bleach
for twenty minutes, and then rinsed 3 times with sterile distilled
water before being germinated on G-media (Table 31) in Magenta GA-7
vessels and maintained under high light intensity of 40-60
.mu.E/m2, with the photoperiod set at 16 hours of light and 8 hours
dark at 28.degree. C.
[0436] Cotyledon segments (.about.5mm) square are isolated from
7-10 day old seedlings into liquid M liquid media (Table 31) in
Petri plates (Nunc, item #0875728). Cut segments are treated with
an Agrobacterium solution (for 30 minutes) then transferred to
semi-solid M-media (Table 31) and undergo co-cultivation for 2-3
days. Following co-cultivation, segments are transferred to MG
media (Table 31). Carbenicillin is the antibiotic used to kill the
Agrobacterium and glufosinate-ammonium is the selection agent that
would allow growth of only those cells that contain the transferred
gene.
[0437] Agrobacterium preparation. Inoculate 35 ml of Y media (Table
31) (containing streptomycin (100 mg/ml stock) and erythromycin
(100 mg/ml stock)), with one loop of bacteria to grow overnight in
the dark at 28.degree. C., while shaking at 150 rpm. The next day,
pour the Agrobacterium solution into a sterile oakridge tube
(Nalge-Nunc, 3139-0050), and centrifuge for in Beckman J2-21 at
8,000 rpm for 5 minutes. Pour off the supernatant and resuspend the
pellet in 25 ml of M liquid (Table 31) and vortex. Place an aliquot
into a glass culture tube (Fisher, 14-961-27) for Klett reading
(Klett-Summerson, model 800-3). Dilute the new suspension using M
liquid media to a Klett-meter reading of 10.sup.8 colony forming
units per mL with a total volume of 40 ml.
[0438] After three weeks, callus from the cotyledon segments is
isolated and transferred to fresh MG media. The callus is
transferred for an additional 3 weeks on MG media. Callus is then
transferred to CG-media (Table 31), and transferred again to fresh
selection medium after three weeks. After another three weeks the
callus tissue is transferred to D media (Table 31) lacking plant
growth regulators for embryogenic callus induction. After 4-8 weeks
on this media, embryogenic callus is formed, and can be
distinguished from the non-embryogenic callus by its
yellowish-white color and granular cells. Embryos start to
regenerate soon after and are distinct green in color.
[0439] Larger, well-developed embryos are isolated and transferred
to DK media (Table 31) for embryo development. After 3 weeks (or
when the embryos have developed), germinated embryos are
transferred to fresh media for shoot and root development. After
4-8 weeks, any well-developed plants are transferred into soil and
grown to maturity. Following a couple of months, the plant has
grown to a point that it can be sprayed to determine if it has
resistance to 2,4-D.
TABLE-US-00031 TABLE 31 Media for Cotton Transformation Ingredients
in 1 liter G M liquid M MG CG D DK Y LS Salts (5.times.) 200 ml 200
ml 200 ml 200 ml 200 ml Glucose 30 grams 30 grams 30 grams 30 grams
20 grams modified B5 vit (1000.times.) 1 ml 1 ml 1 ml 1 ml 1 ml 10
ml 1 ml kinetin (1 mM) 1 ml 1 ml 1 ml 4.6 ml 0.5 ml 2,4-D (1 mM) 1
ml 1 ml 1 ml agar 8 grams 8 grams 8 grams 8 grams 8 grams 8 grams
DKW salts (D190) 1 package 1 package MYO-Inositol (100.times.) 1 ml
10 ml Sucrose 3% 30 grams 30 grams 10 grams NAA Carbenicillin (250
mg/ml) 2 ml 0.4 ml GLA (10 mg/ml) 0.5 ml 0.3 ml Peptone 10 grams
Yeast Extract 10 grams NaCl 5 grams
[0440] 14.2--Experiment Specifics.
[0441] For this experiment 500 cotyledon segments were treated with
pDAB721. Of the 500 segments treated, 475 had callus isolated while
on selection (95% transformation frequency). The callus was
selected on glufosinate-ammonium, due to the inclusion of the PAT
gene in the construct, since there was already a selection scheme
developed. Callus line analysis in the form of PCR and Invader were
initiated to determine the insertion patterns and to be sure the
gene was present at the callus stage, then callus lines that were
embryogenic were sent for Western analysis.
[0442] 14.3--Callus Analysis Results.
[0443] The object of the analysis is to eliminate any lines that do
not have the complete PTU, show no expression or that have high
copy number, so those lines are not regenerated. Of the 475 pDAB721
transformed callus lines, 306 were sent for PCR analysis and
Invader assay (Table 32). Very few lines were PCR negative. The
Invader results are not complete at this time, because some samples
had low DNA amounts when extracted, and these have been
resubmitted. However, the current Invader data shows a few of the
lines submitted have high copy number (copy number of >2) (Table
32). Due to the large number of lines that passed analysis, it was
necessary to decrease the number of embryogenic callus lines being
maintained due to the volume. Ninety lines have been sent for
Western analysis, and eight of those were negative. The western
analysis showed high expression from the majority of the lines
(Table 32). Eighty-two embryogenic callus lines are being
maintained for plant regeneration based on analysis results (and
results pending).
TABLE-US-00032 TABLE 32 Analysis of cotton callus Line Copy # PTU
Western 1 2 pos *** 2 1 pos *** 3 1 pos *** 4 2 pos *** 5 1 pos ***
6 1 pos neg 7 1 pos **** 8 1 pos ***** 9 2 pos * 10 1 pos * 11 1
pos ***** 12 1 pos * 13 1 pos * 14 1 pos ** 15 1 pos **** 16 1 pos
***** 17 1 pos * 18 1 pos *** 19 1 pos ** 20 1 pos ***** 21 2 pos
***** 22 1 pos ***** 23 1 pos ***** 24 4 pos * or neg 25 1 pos ****
26 1 pos **** 27 low DNA pos ***** 28 low DNA pos ** 29 low DNA pos
***** 30 17 pos * 31 low DNA pos ***** 32 low DNA pos ***** 33 low
DNA pos **** 34 low DNA faint pos ***** 35 low DNA pos **** 36 low
DNA pos neg 37 low DNA pos **** 38 low DNA neg ***** 39 1 pos ****
40 low DNA pos * 41 low DNA pos * 42 low DNA pos ** 43 1 pos *****
44 low DNA pos ***** 45 1 pos ***** 46 2 pos ***** 47 1 pos *** 48
1 pos *** 49 3 faint pos neg 50 1 pos **** 51 4 pos neg 52 2 pos
**** 53 1 pos *** 54 1 pos **** 55 1 pos neg 56 5 pos * 57 2 faint
pos neg 58 8 pos **** 59 2 pos **** 60 5 pos ***** 61 1 pos ** 62 1
pos ** 63 1 pos *** 64 1 pos *** 65 3 pos **** 66 5 pos * 67 6 pos
* 68 Low DNA pos neg 69 Low DNA pos **** 70 low DNA pos ** 71 low
DNA faint pos ** 72 low DNA pos **** 73 low DNA neg *** 74 low DNA
faint pos *** 75 Low DNA pos *** 76 Low DNA pos neg 77 low DNA
faint pos **** 78 low DNA pos **** 79 1 pos ** 80 low DNA pos ***
81 low DNA pos *** 82 low DNA pos *** 83 low DNA neg **** 84 low
DNA pos *** 85 low DNA pos ** 86 low DNA pos * standard AAD1 5
ug/ml ***** standard AAD1 0.5 ug/ml **
[0444] 14.4--Plant Regeneration.
[0445] Two AAD-1 (v3) cotton lines have produced plants according
to the above protocol that have been sent to the greenhouse. To
demonstrate the AAD-1 (v3) gene provides resistance to 2,4-D in
cotton, both the AAD-1 (v3) cotton plant and wild-type cotton
plants were sprayed with a track sprayer set at 187 L/ha. The
plants were sprayed at 560 g ae/ha 2,4-DMA formulated in 200 mM
Hepes buffer (pH 7.5). The plants were evaluated at 3, 7, and 14
days after treatment. Plants were assigned injury ratings with
respect to stunting, chlorosis, and necrosis. Plants assigned an
injury rating of 90% or above are considered dead. Three days after
treatment (DAT) the wild-type plant began showing epinasy and
received a rating of 15%; in contrast, the AAD-1 (v3) plant showed
0% injury. By 7 DAT epinasy continued on the wild-type and the new
growth shoots began turning brown. It received a rating of 50% at
this time. At 14 DAT the AAD-1 (v3) plant was still uninjured,
whereas the wild-type was severely stunted, and the new growth
areas were brown and shriveled. Thus, the wild-type received a
rating of 90% at 14 DAT.
[0446] This study demonstrates that the AAD-1 (v3) gene in cotton
provides substantial tolerance to 2,4-D up to at least 560 g
ae/ha.
EXAMPLE 15
Agrobacterium Transformation of Other Crops
[0447] In light of the subject disclosure, additional crops can be
transformed according to the subject invention using techniques
that are known in the art. For Agrobacterium-mediated
trans-formation of rye, see, e.g., Popelka and Altpeter (2003). For
Agrobacterium-mediated transformation of soybean, see, e.g.,
Hinchee et al., 1988. For Agrobacterium-mediated transformation of
sorghum, see, e.g., Zhao et al., 2000. For Agrobacterium-mediated
transformation of barley, see, e.g., Tingay et al., 1997. For
Agrobacterium-mediated transformation of wheat, see, e.g., Cheng et
al., 1997. For Agrobacterium-mediated transformation of rice, see,
e.g., Hiei et al., 1997.
[0448] The Latin names for these and other plants are given below.
It should be clear that these and other (non-Agrobacterium)
transformation techniques can be used to transform AAD-1 (v3), for
example, into these and other plants, including but not limited to
Maize (Gramineae Zea mays), Wheat (Pooideae Triticum spp.), Rice
(Gramineae Oryza spp. and Zizania spp.), Barley (Pooideae Hordeum
spp.), Cotton (Abroma Dicotyledoneae Abroma augusta, and Malvaceae
Gossypium spp.), Soybean (Soya Leguminosae Glycine max), Sugar beet
(Chenopodiaceae Beta vulgaris altissima), Sugar cane (Arenga
pinnata), Tomato (Solanaceae Lycopersicon esculentum and other
spp., Physalis ixocarpa, Solanum incanum and other spp., and
Cyphomandra betacea), Potato, Sweet potato, Rye (Pooideae Secale
spp.), Peppers (Solanaceae Capsicum annuum, sinense, and
frutescens), Lettuce (Compositae Lactuca sativa, perennis, and
pulchella), Cabbage, Celery (Umbelliferae Apium graveolens),
Eggplant (Solanaceae Solanum melongena), Sorghum (all Sorghum
species), Alfalfa (Leguminosae Medicago sativum), Carrot
(Umbelliferae Daucus carota sativa), Beans (Leguminosae Phaseolus
spp. and other genera), Oats (Avena Sativa and Strigosa), Peas
(Leguminosae Pisum, Vigna, and Tetragonolobus spp.), Sunflower
(Compositae Helianthus annuus), Squash (Dicotyledoneae Cucurbita
spp.), Cucumber (Dicotyledoneae genera), Tobacco (Solanaceae
Nicotiana spp.), Arabidopsis (Cruciferae Arabidopsis thaliana),
Turfgrass (Lolium, Agrostis, and other families), and Clover
(Leguminosae). Such plants, with AAD-1 (v3) genes, for example, are
included in the subject invention.
EXAMPLE 16
Stacking AAD-1 (v3) with AHAS Herbicide Resistance Gene
[0449] Stacking AAD-1 (v3) with an AHAS herbicide resistance gene
is described in Example 7.9.
Example 17
Further Evidence of Surprising Results: AAD-1 vs. AAD-2
[0450] 17.1--AAD-2 (v1) Initial Cloning.
[0451] Another gene was identified from the NCBI database (see the
ncbi.nlm nih.gov website; accession #AP005940) as a homologue with
only 44% amino acid identity to This gene is referred to herein as
AAD-2 (v1) for consistency. Percent identity was determined by
first translating both the AAD-2 and tfdA DNA sequences (SEQ ID
NO:12 and GENBANK Accession No. M16730, respectively) to proteins
(SEQ ID NO:13 and GENBANK Accession No. M16730, respectively), then
using ClustalW in the VectorNTl software package to perform the
multiple sequence alignment.
[0452] The strain of Bradyrhizobium japonicum containing the AAD-2
(v1) gene was obtained from Northern Regional Research Laboratory
(NRRL, strain #B4450). The lyophilized strain was revived according
to NRRL protocol and stored at -80.degree. C. in 20% glycerol for
internal use as Dow Bacterial strain DB 663. From this freezer
stock, a plate of Tryptic Soy Agar was then struck out with a
loopful of cells for isolation, and incubated at 28.degree. C. for
3 days. A single colony was used to inoculate 100 ml of Tryptic Soy
Broth in a 500 ml tri-baffled flask, which was incubated overnight
at 28.degree. C. on a floor shaker at 150 rpm. From this, total DNA
was isolated with the gram negative protocol of Qiagen's DNeasy kit
(Qiagen cat. #69504). The following primers were designed to
amplify the target gene from genomic DNA, Forward: 5' ACT AGT AAC
AAA GAA GGA GAT ATA CCA TGA CGA T 3' [(brjap 5'(spel) SEQ ID NO:14
(added Spe I restriction site and Ribosome Binding Site (RBS))] and
Reverse: 5' TTC TCG AGC TAT CAC TCC GCC GCC TGC TGC TGC 3' [(br jap
3' (xhoI) SEQ ID NO:15 (added a Xho I site)].
[0453] Fifty microliter reactions were set up as follows: Fail Safe
Buffer 25 .mu.l, ea. primer 1 .mu.l (50 ng/.mu.l), gDNA 1 .mu.l
(200 ng/.mu.l), H.sub.2O 21 .mu.l, Taq polymerase 1 .mu.l (2.5
units/.mu.l). Three Fail Safe Buffers--A, B, and C--were used in
three separate reactions. PCR was then carried out under the
following conditions: 95.degree. C. 3.0 minutes heat denature
cycle; 95.degree. C. 1.0 minute, 50.degree. C. 1.0 minute,
72.degree. C. 1.5 minutes, for 30 cycles; followed by a final cycle
of 72.degree. C. 5 minutes, using the FailSafe PCR System
(Epicenter cat. #FS99100). The resulting -1 kb PCR product was
cloned into pCR 2.1 (Invitrogen cat. #K4550-40) following the
included protocol, with chemically competent TOP10F' E. coli as the
host strain, for verification of nucleotide sequence.
[0454] Ten of the resulting white colonies were picked into 3.mu.l
Luria Broth +1000 .mu.g/ml Ampicillin (LB Amp), and grown overnight
at 37.degree. C. with agitation. Plasmids were purified from each
culture using Nucleospin Plus Plasmid Miniprep Kit (BD Biosciences
cat. #K3063-2) and following included protocol. Restriction
digestion of the isolated DNA's was completed to confirm the
presence of the PCR product in the pCR2.1 vector. Plasmid DNA was
digested with the restriction enzyme EcoRI (New England Biolabs
cat. #R0101S). Sequencing was carried out with Beckman CEQ Quick
Start Kit (Beckman Coulter cat. #608120) using M13 Forward [5' GTA
AAA CGA CGG CCA GT 3'] (SEQ ID NO:16) and Reverse [5' CAG GAA ACA
GCT ATG AC 3'] (SEQ ID NO:17) primers, per manufacturers
instructions. This gene sequence and its corresponding protein was
given a new general designation AAD-2 (v1) for internal
consistency.
[0455] 17.2--Completion of AAD-2 (v1) Binary Vector.
[0456] The AAD-2 (v1) gene was PCR amplified from pDAB3202. During
the PCR reaction alterations were made within the primers to
introduce the AfIIII and Sad restriction sites in the 5' primer and
3' primer, respectively. The primers "Ncol of Brady" [5' TAT ACC
ACA TGT CGA TCG CCA TCC GGC AGC TT 3'] (SEQ ID NO:18) and "SacI of
Brady" [5' GAG CTC CTA TCA CTC CGC CGC CTG CTG CTG CAC 3'] (SEQ ID
NO:19) were used to amplify a DNA fragment using the Fail Safe PCR
System (Epicentre). The PCR product was cloned into the pCR 2.1
TOPO TA cloning vector (Invitrogen) and sequence verified with M13
Forward and M13 Reverse primers using the Beckman Coulter "Dye
Terminator Cycle Sequencing with Quick Start Kit" sequencing
reagents. Sequence data identified a clone with the correct
sequence (pDAB716). The AfIIII/SacI AAD-2 (v1) gene fragment was
then cloned into the Ncol/Sacl pDAB726 vector. The resulting
construct (pDAB717); AtUbil0 promoter: Nt OSM 5'UTR: AAD-2 (v1): Nt
OSM3'UTR: ORF1 polyA 3'UTR was verified with restriction digests
(with NcoI/SacI). This construct was cloned into the binary
pDAB3038 as a NotI--NotI DNA fragment. The resulting construct
(pDAB767); AtUbil0 promoter: Nt OSMS'UTR: AAD-2 (v1): Nt OSM 3'UTR:
ORF1 polyA 3'UTR: CsVMV promoter: PAT: ORF25/26 3'UTR was
restriction digested (with NotI, EcoRI, HinDIII, NcoI, PvuII, and
Sall) for verification of the correct orientation. The completed
construct (pDAB767) was then used for transformation into
Agrobacterium.
[0457] 17.3--Comparison of Substrate Specificities of AAD-2 (v1)
and AAD-1 (v1)
[0458] The activity of an extract from E. coli expressing AAD-2
(v1) (pDAB3202) prepared as in Example 11 was tested on four
herbicides, 2,4-D, (R,S)-dichlorprop, (R,S)-haloxyfop and
(R)-haloxyfop (all at a final concentration of 0.5 mM) using 3
.mu.l (42 .mu.g) of E. coli extract per assay with a 15 min assay
period. FIG. 22 shows that the relative AAD-2 (v1) activity on the
substrates was 2,4-D=dichlorprop>(R,S)-haloxyfop
>>(R)-haloxyfop. Thus AAD-2 (v1) differs from AAD-1 (v1) in
that it has a similar level of activity on 2,4-D as on dichlorprop
(whereas the activity of AAD-1 (v1) on 2,4-D is .about.10% that of
dichlorprop). AAD-2 (v1) also differs from AAD-1 (v1) in that it is
unable to act on (R)-haloxyfop. Table 33 shows data from additional
substrates tested with AAD-1 (v1) and AAD-2 (v1) that confirm that
AAD-2 (v1) is specific for (S)-enantiomer substrates, in contrast
to AAD-1 (v1) which is specific for (R)-enantiomers. In another
test, AAD-2 (v1) was found to differ from AAD-1 (v1) in that it
releases little or no detectable phenol from 2,4-D sulfonate (in
which a sulfonate group replaces the carboxylate of 2,4-D) whereas
AAD-1 (v1) produces significant levels of phenol from this compound
(.about.25% of 2,4-D.)
TABLE-US-00033 TABLE 33 Comparison of AAD1 and AAD2 activity on
various substrates. Substrates were assayed at 0.5 mM for 15 mM in
25 mM MOPS pH 6.8, 200 .mu.M Fe.sup.2+, 200 .mu.M Na ascorbate, 1
mM .alpha.-ketoglutarate using 4 .mu.l AAD1 (32 .mu.g protein)
extract or 3 .mu.l AAD2 extract (42 .mu.g protein). A510 STRUCTURE
Reg ID Compound Enantiomer AAD1 AAD2 ##STR00074## 18706 quizalofop
R 0.27 0.01 ##STR00075## 8671 haloxyfop R 0.12 0 ##STR00076## 66905
haloxyfop R,S 0.1 0.3 ##STR00077## 14623 cyhalofop R,S 0.12 0.1
##STR00078## 14603 cyhalofop R 0.14 0 ##STR00079## 7466 cyhalofop S
0 0.15 ##STR00080## 11044492 fenoxaprop R 0.14 0 ##STR00081## 43865
haloxyfop-acetate -- 0 0.22
[0459] The enzyme kinetics of partially purified AAD-1 (v1) and
AAD-2 (v1) were compared using 2,4-D as substrate. See FIG. 19. The
K.sub.m values for 2,4-D were 97 and 423 .mu.M for AAD-1 (v1) and
AAD-2 (v1) respectively and the apparent V. values were 0.11 and
0.86 A.sub.510 units, respectively (Table 34). Because equivalent
amounts of enzyme were used in the assays (as determined by
SDS-PAGE analysis), it can be concluded that the k.sub.cat of AAD-2
(v1) for 2,4-D is almost 8-fold higher than AAD-1 (v1) and the
k.sub.cat/K.sub.m is 2-fold higher. Thus AAD-2 (v1) is
significantly more efficient at cleaving 2,4-D in vitro than AAD-1
(v1). This is in surprising contrast to the in planta findings
reported below, where plants expressing AAD-1 (v1) are
significantly better in conferring 2,4-D resistance relative to
AAD-2 (v1).
TABLE-US-00034 TABLE 34 Comparison of the Km and "Vmax" values for
the aryloxyalkanoate dioxygenases (AADs) from pDAB3202 (AAD-2) and
pDAB3203 [AAD-1 (v1)] with different herbicide substrates: Vmax
Km/Vmax Enzyme Compound Km (.mu.M) .+-. SE (A510 units) (arbitrary
units) AAD-2 2,4-D 423 (.+-.1) 0.86 2.03 AAD-1 (v1) 2,4-D 97
(.+-.21) 0.11 1.16 NOTES: Assays were performed in MOPS pH 6.75 + 1
mM .alpha.-ketoglutarate + 0.1 mM Na ascorbate + 0.1 mM Fe2+ and
the released phenols colorimetrically detected using
4-aminoantipyrine/ferricyanide.
[0460] 17.4--Evaluation of Transformed Arabidopsis.
[0461] Freshly harvested Ti seed transformed with a native [AAD-1
(v2)], plant optimized AAMD-1 (v3)1, or native AAD-2 (v1) gene was
allowed to dry for 7 days at room temperature. T.sub.1 seed was
sown in 26.5.times.51-cm germination trays (T.O. Plastics Inc.,
Clearwater, Minn.), each receiving a 200 mg aliquots of stratified
Ti seed (40,000 seed) that had previously been suspended in 40 ml
of 0.1% agarose solution and stored at 4.degree. C. for 2 days to
complete dormancy requirements and ensure synchronous seed
germination.
[0462] Sunshine Mix LP5 (Sun Gro Horticulture Inc., Bellevue,
Wash.) was covered with fine vermiculite and subirrigated with
Hoagland's solution until wet and then allowed to gravity drain.
Each 40 ml aliquot of stratified seed was sown evenly onto the
vermiculite with a pipette and covered with humidity domes (KORD
Products, Bramalea, Ontario, Canada) for 4-5 days. Domes were
removed 1 day prior to initial transformant selection using
glufosinate postemergence spray (selecting for the co-transformed
PAT gene).
[0463] Five to six days after planting (DAP) and again 10 DAP, Ti
plants (cotyledon and 2-4-1f stage, respectively) were sprayed with
a 0.2% solution of Liberty herbicide (200 g ai/L glufosinate, Bayer
Crop Sciences, Kansas City, Mo.) at a spray volume of 10 ml/tray
(703 L/ha) using a DeVilbiss compressed air spray tip to deliver an
effective rate of 280 g ai/ha glufosinate per application.
Survivors (plants actively growing) were identified 5-7 days after
the final spraying and transplanted individually into 3-inch pots
prepared with potting media (Metro Mix 360). Transplanted plants
were covered with humidity domes for 3-4 days and placed in a
22.degree. C. growth chamber as before. Domes were subsequently
removed and plants moved to the greenhouse (22.+-.5.degree. C.,
50.+-.30% RH, 14 hours light:10 dark, minimum 500
.mu.E/m.sup.2s.sup.1 natural+supplemental light) at least 1 day
prior to testing for the ability of AAD-1 (v3), AAD-1 (v2),or AAD-2
(v1) to provide phenoxy auxin herbicide resistance.
[0464] Random individual T.sub.1 plants selected for glufosinate
resistance above were confirmed for expression of the PAT protein
using a PAT ELISA kit (Part no. 7000045, Strategic Diagnostics,
Inc., Newark, Del.) to non-destructively confirm fidelity of
selection process (manufacturer's protocol). Plants were then
randomly assigned to various rates of 2,4-D (50-800 g ae/ha).
[0465] Herbicide applications were applied by track sprayer in a
187 L/ha spray volume. 2,4-D used was the commercial dimethylamine
salt formulation (456 g ae/L, NuFarm, St Joseph, Mo.) mixed in 200
mM Tris buffer (pH 9.0).
[0466] 17.5--Results of Selection of Transformed Plants.
[0467] The first Arabidopsis transformations were conducted using
AAD-1 (v3). Ti transformants were first selected from the
background of untransformed seed using a glufosinate selection
scheme. Over 400,000 Ti seed were screened and 493 glufosinate
resistant plants were identified (PAT gene), equating to a
transformation/ selection frequency of 0.12%. Depending on the lot
of seed tested, this ranged from 0.05-0.23% (see Table 15 in
Example 6.5 above). A small lot of native AAD-1 (v2) transformed
seed were also selected using the glufosinate selection agent. Two
hundred seventy eight glufosinate-resistant Ti individuals were
identified out of 84,000 seed screened (0.33%
transformation/selection frequency). Surprisingly, Arabidopsis
transformations using the native AAD-2(v1) gene provided a very low
transformation frequency when selected for glufosinate tolerance
(PAT selectable marker function). Approximately 1.3 million seed
have been screened and only 228 glufosinate transformants were
recovered, equating to a transformation/selection frequency of
0.018% (see Table 15). Transformation frequency for native AAD-2
(v1) was only 6% of that of native AAD-1 (v2). The native AAD2 (v1)
gene was subsequently synthetically optimized, cloned, and
transformed as pDAB3705, into Arabidopsis using methods previously
described (see Example 5). The plant optimized AAD2 (v2) (SEQ ID
NO:29, which encodes SEQ ID NO:30) yielded a normal Ti Arabidopsis
selection frequency using Liberty herbicide of approximately 0.11%
(see Table 15).
[0468] T.sub.1 plants selected above were subsequently transplanted
to individual pots and sprayed with various rates of commercial
aryloxyalkanoate herbicides. Table 16 (in Example 6.5 above)
compares the response of AAD-1 (v2), AAD-1 (v3), AAD-2 (v1) and
AAD-1(v2) genes to impart 2,4-D resistance to Arabidopsis T.sub.1
transformants. All genes did provide some significant 2,4-D
resistance versus the transformed and untransformed control lines;
however, individual constructs were widely variable in their
ability to impart 2,4-D resistance to individual T.sub.1
Arabidopsis plants. Within a given treatment, the level of plant
response varied greatly and can be attributed to the fact each
plant represents an independent transformation event. Of important
note, at each 2,4-D rate tested, there were individuals that were
unaffected while some were severely affected. An overall population
injury average by rate is presented in Table 16 simply to
demonstrate the significant difference between the plants
transformed with AAD-1 (v2), AAD-1 (v3), AAD-2 (v1) or AAD-2 (v2)
versus the wildtype or PAT/Cry1F transformed controls.
[0469] Surprisingly, AAD-2 (v1) transformants were far less
resistant to 2,4-D than either AAD-1 (v2) or AAD-1 (v3) genes
(Table 16) both from a frequency of highly tolerant plants as well
as overall average injury. No plants transformed with AAD-2 (v1)
survived 200 g ae/ha 2,4-D relatively uninjured (<20% visual
injury), and overall population injury was about 83%. Conversely,
56% (45 of 80) AAD-1 (v2)-transformed T.sub.1 plants survived 200 g
ae/ha 2,4-D uninjured (population injury average=34%), and >73%
(11 of 15) AAD-1 (v3) T.sub.1 plants were uninjured (population
injury average=14%). See FIG. 20. Tolerance improved slightly for
plant-optimized AAD2 (v2) versus the native gene; however,
comparison of both AAD-1 and -2 plant optimized genes indicates a
significant advantage for AAD-1 (v3) in planta (see Table 16).
[0470] These results are unexpected given that in vitro comparison
of native AAD-1 (v2) and AAD-2 (v1) indicated 2,4-D was better
degraded by AAD-2 (v1). AAD-2 (v1) is expressed in individual Ti
plants to varying levels with the anticipated size; however, little
protection from 2,4-D injury is afforded by this expressed protein.
Little correlation exists between expression level noted on the
Western blot and the level of injury from 2,4-D on the same plants.
See FIG. 21. No large difference was evident in protein expression
level (in planta) for the native and plant optimized AAD-2 genes.
These data corroborate earlier findings that make the functional
expression of AAD-1 (v3) in planta for imparting herbicide
resistance to 2,4-D and AOPP herbicides is unexpected.
Example 18
Preplant Burndown Applications
[0471] This and the following Examples are specific examples of
novel herbicide uses made possible by the subject AAD-1
invention.
[0472] Preplant burndown herbicide applications are intended to
kill weeds that have emerged over winter or early spring prior to
planting a given crop. Typically these applications are applied in
no-till or reduced tillage management systems where physical
removal of weeds is not completed prior to planting. An herbicide
program, therefore, must control a very wide spectrum of broadleaf
and grass weeds present at the time of planting. Glyphosate,
gramoxone, and glufosinate are examples of non-selective,
non-residual herbicides widely used for preplant burndown herbicide
applications. Some weeds, however, are difficult to control at this
time of the season due to one or more of the following: inherent
insensitivity of the weed species or biotype to the herbicide,
relatively large size of winter annual weeds, and cool weather
conditions limiting herbicide uptake and activity. Several
herbicide options are available to tankmix with these herbicides to
increase spectrum and activity on weeds where the non-selective
herbicides are weak. An example would be 2,4-D tankmix applications
with glyphosate to assist in the control of Conyza canadensis
(horseweed). Glyphosate can be used from 420 to 1680 g ae/ha, more
typically 560 to 840 g ae/ha, for the preplant burndown control of
most weeds present; however, 280-1120 g ae/ha of 2,4-D can be
applied to aid in control of many broadleaf weed species (e.g.,
horseweed).
[0473] 2,4-D is an herbicide of choice because it is effective on a
very wide range of broadleaf weeds, effective even at low
temperatures, and extremely inexpensive. However, if the subsequent
crop is a sensitive dicot crop, 2,4-D residues in the soil
(although short-lived) can negatively impact the crop. Soybeans are
a sensitive crop and require a minimum time period of 7 days (for
280 g ae/ha 2,4-D rate) to at least 30 days (for 2,4-D applications
of 1120 g ae/ha) to occur between burndown applications and
planting. 2,4-D is prohibited as a burndown treatment prior to
cotton planting (see federal labels, most are available through
CPR, 2003 or online at cdms.net/manuf/manuf.asp). With AAD-1 (v3)
transformed cotton or soybeans, these crops should be able to
survive 2,4-D residues in the soil from burndown applications
applied right up to and even after planting before emergence of the
crop. The increased flexibility and reduced cost of tankmix (or
commercial premix) partners will improve weed control options and
increase the robustness of burndown applications in important
no-till and reduced tillage situations. This example is one of many
options that will be available. Those skilled in the art of weed
control will note a variety of other applications including, but
not limited to gramoxone+2,4-D or glufosinate+2,4-D by utilizing
products described in federal herbicide labels (CPR, 2003) and uses
described in Agriliance Crop Protection Guide (2003), as examples.
Those skilled in the art will also recognize that the above example
can be applied to any 2,4-D-sensitive (or other phenoxy auxin
herbicide) crop that would be protected by the AAD-1 (v3) gene if
stably transformed.
Example 19
In-Crop Use of Phenoxy Auxins Herbicides in AAD-1 (v3) Only
Transformed Soybeans, Cotton, and Other Dicot Crops
[0474] AAD-1 (v3) can enable the use of phenoxy auxin herbicides
(e.g., 2,4-D, dichlorprop, MCPA, et al.) for the control of a wide
spectrum of broadleaf weeds directly in crops normally sensitive to
2,4-D. Application of 2,4-D at 280 to 2240 g ae/ha would control
most broadleaf weed species present in agronomic environments. More
typically, 560-1120 g ae/ha is used. For a complete weed control
system, grass weeds must be controlled. A variety of broad spectrum
graminicide herbicides, including but not limited to haloxyfop,
quizalofop, fenoxaprop, fluazifop, sethoxydim, and clethodim are
currently registered for use in most dicot crops which are
naturally tolerant to these herbicides. A combination of quizalofop
(20-100 g ae/ha) plus 2,4-D (420-840 g ae/ha) could provide two
herbicide modes of action in an AAD-1 (v3)-transformed dicot crop
(i.e., soybean or cotton) that would control most agronomic weeds
in a similar fashion to glyphosate in glyphosate tolerant crops
(see weed control spectra by reference to Agriliance Crop
Protection Guide performance ratings).
[0475] An advantage to this additional tool is the extremely low
cost of the broadleaf herbicide component and potential short-lived
residual weed control provided by higher rates of 2,4-D and/or AOPP
herbicides when used at higher rates, whereas a non-residual
herbicide like glyphosate would provide no control of later
germinating weeds. This tool also provides a mechanism to rotate
herbicide modes of action with the convenience of HTC as an
integrated herbicide resistance and weed shift management strategy
in a glyphosate tolerant crop/AAD-1 (v3) HTC rotation strategy,
whether one rotates crops species or not. Additionally, the grass
and broadleaf weed control components of this system are
independent of one another, thus allowing one skilled in the are of
weed control to determine the most cost effective and efficacious
ratio of auxin and AOPP herbicide. For example, if broadleaf weeds
were the only significant weeds present when herbicide applications
were needed, an herbicide application of 560 to 1120 g ae/ha 2,4-D
could be made without another herbicide. This would reduce
unnecessary herbicide applications, provide flexibility to reduce
input costs and reduce environmental loads of pesticides, and
reduce unnecessary selection pressure for the development of
herbicide resistant weeds.
[0476] Further benefits could include tolerance to 2,4-D drift or
volatilization as mechanisms for off-site 2,4-D injury to dicot
crops; no interval required before planting following 2,4-D
application (see previous example); and fewer problems from
contamination injury to dicot crops resulting from incompletely
cleaned bulk tanks that had contained 2,4-D. Dicamba (and other
herbicides) can still be used for the subsequent control of AAD-1
(v3)-transformed dicot crop volunteers.
[0477] Those skilled in the art will also recognize that the above
example can be applied to any 2,4-D-sensitive (or other phenoxy
auxin herbicide) crop that would be protected by the AAD-1 (v3)
gene if stably transformed. One skilled in the art of weed control
will now recognize that use of various commercial phenoxy auxin
herbicides alone or in combination with any commercial AOPP
herbicide is enabled by AAD-1 (v3) transformation. Specific rates
of other herbicides representative of these chemistries can be
determined by the herbicide labels compiled in the CPR (Crop
Protection Reference) book or similar compilation or any commercial
or academic crop protection references such as the Crop Protection
Guide from Agriliance (2003). Each alternative herbicide enabled
for use in HTCs by AAD-1 (v3), whether used alone, tank mixed, or
sequentially, is considered within the scope of this invention.
Example 20
In-Crop Use of Phenoxy Auxins and AOPP Herbicides in AAD-1 (v3)
Only Transformed Corn, Rice, and Other Monocot Species
[0478] In an analogous fashion, transformation of grass species
(such as, but not limited to, corn, rice, wheat, barley, or turf
and pasture grasses) with AAD-1 (v3) would allow the use of highly
efficacious AOPP graminicides in crops normally sensitive to these
herbicides. Most grass species have a natural tolerance to auxinic
herbicides such as the phenoxy auxins (i.e., 2,4-D, dichlorprop, et
al.). However, a relatively low level of crop selectivity has
resulted in diminished utility in these crops due to a shortened
window of application timing and alternative broadleaf weeds. AAD-1
(v3)-transformed monocot crops would, therefore, enable the use of
a similar combination of treatments described for dicot crops such
as the application of 2,4-D at 280 to 2240 g ae/ha to control most
broadleaf weed species. More typically, 560-1120 g ae/ha would be
used. A variety of broad spectrum AOPP graminicide herbicides
(including but not limited to haloxyfop, quizalofop, fenoxaprop,
and fluazifop) could be utilized for effectively controlling a wide
selection of grass weeds. Cyclohexanedione graminicidal herbicides
like sethoxydim, clethodim, et al. could not be used in this system
as shown for dicot crops since AAD-1 would not protect from this
chemistry, and grass crops will be naturally sensitive to the
cyclohexanedione chemistries. However, this attribute would enable
the use of cyclohexanedione herbicides for the subsequent control
of AAD-1 (v3)-transformed grass crop volunteers. Similar weed
control strategies are now enabled by AAD-1 for the dicot crop
species. A combination of quizalofop (20-100 g ae/ha) plus 2,4-D
(420-840 g ae/ha) could provide two herbicide modes of action in an
AAD-1 (v3) transformed monocot crop (e.g., corn and rice) that
would control most agronomic weeds in a similar fashion to
glyphosate in glyphosate tolerant crops (see weed control spectra
by reference to Agriliance Crop Protection Guide performance
ratings).
[0479] An advantage to this additional tool is the extremely low
cost of the broadleaf herbicide component and potential short-lived
residual weed control provided by higher rates of 2,4-D and/or AOPP
herbicides when used at higher rates. In contrast, a non-residual
herbicide like glyphosate would provide no control of
later-germinating weeds. This tool would also provide a mechanism
to rotate herbicide modes of action with the convenience of HTC as
an integrated-herbicide-resistance and weed-shift-management
strategy in a glyphosate tolerant crop/AAD-1 (v3) HTC rotation
strategy, whether one rotates crops species or not. Additionally,
the grass and broadleaf weed control components of this system are
independent of one another, thus allowing one skilled in the are of
weed control to determine the most cost effective and efficacious
ratio of auxin and AOPP herbicide. For example, if broadleaf weeds
were the only significant weeds present when herbicide applications
were needed, an herbicide application of 560 to 1120 g ae/ha 2,4-D
could be made without another herbicide. This would reduce
unnecessary herbicide applications, provide flexibility to reduce
input costs and reduce environmental loads of pesticides, and
reduce unnecessary selection pressure for the development of
herbicide resistant weeds. The increased tolerance of corn, and
other monocots to the phenoxy auxins shall enable use of these
herbicides in-crop without growth stage restrictions or the
potential for crop leaning, unfurling phenomena such as
"rat-tailing," crop leaning, growth regulator-induced stalk
brittleness in corn, or deformed brace roots.
[0480] Those skilled in the art will now also recognize that the
above example can be applied to any monocot crop that would be
protected by the AAD-1 (v3) gene from injury by any AOPP herbicide.
One skilled in the art of weed control will now recognize that use
of various commercial phenoxy auxin herbicides alone or in
combination with any commercial AOPP herbicide is enabled by AAD-1
(v3) transformation. Specific rates of other herbicides
representative of these chemistries can be determined by the
herbicide labels compiled in the CPR (Crop Protection Reference)
book or similar compilation, labels compiled online (e.g.,
cdms.net/manuf/manuf.asp), or any commercial or academic crop
protection guides such as the Crop Protection Guide from Agriliance
(2003). Each alternative herbicide enabled for use in HTCs by AAD-1
(v3), whether used alone, tank mixed, or sequentially, is
considered within the scope of this invention.
Example 21
AAD-1 (v3) Stacked With Glyphosate Tolerance Trait in Any Crop
[0481] The vast majority of cotton, canola, and soybean acres
planted in North America contain a glyphosate tolerance (GT) trait,
and adoption of GT corn is on the rise. Additional GT crops (e.g.,
wheat, rice, sugar beet, and turf) have been under development but
have not been commercially released to date. Many other glyphosate
resistant species are in experimental to development stage (e.g.,
alfalfa, sugar cane, sunflower, beets, peas, carrot, cucumber,
lettuce, onion, strawberry, tomato, and tobacco; forestry species
like poplar and sweetgum; and horticultural species like marigold,
petunia, and begonias; isb.vt.edu/cfdocs/fieldtestsl.cfm, 2005 on
the World Wide Web). GTC's are valuable tools for the sheer breadth
of weeds controlled and convenience and cost effectiveness provided
by this system. However, glyphosate's utility as a now-standard
base treatment is selecting for glyphosate resistant weeds.
Furthermore, weeds that glyphosate is inherently less efficacious
on are shifting to the predominant species in fields where
glyphosate-only chemical programs are being practiced. By stacking
AAD-1 (v3) with a GT trait, either through conventional breeding or
jointly as a novel transformation event, weed control efficacy,
flexibility, and ability to manage weed shifts and herbicide
resistance development could be improved. As mentioned in previous
examples, by transforming crops with AAD-(v3), one can selectively
apply AOPP herbicides in monocot crops, monocot crops will have a
higher margin of phenoxy auxin safety, and phenoxy auxins can be
selectively applied in dicot crops. Several scenarios for improved
weed control options can be envisioned where AAD-1 (v3) and a GT
trait are stacked in any monocot or dicot crop species: [0482] a)
Glyphosate can be applied at a standard postemergent application
rate (420 to 2160 g ae/ha, preferably 560 to 840 g ae/ha) for the
control of most grass and broadleaf weed species. For the control
of glyphosate resistant broadleaf weeds like Conyza canadensis or
weeds inherently difficult to control with glyphosate (e.g.,
Commelina spp), 280-2240 g ae/ha (preferably 560-1120 g ae/ha)
2,4-D can be applied sequentially, tank mixed, or as a premix with
glyphosate to provide effective control. [0483] b) Glyphosate can
be applied at a standard postemergent application rate (420 to 2160
g ae/ha, preferably 560 to 840 g ae/ha) for the control of most
grass and broadleaf weed species. For the control of glyphosate
resistant grass species like Lolium rigidum or Eleusine indica,
10-200 g ae/ha (preferably 20-100 g ae/ha) quizalofop can be
applied sequentially, tank mixed, or as a premix with glyphosate to
provide effective control. [0484] c) Currently, glyphosate rates
applied in GTC's generally range from 560 to 2240 g ae/ha per
application timing. Glyphosate is far more efficacious on grass
species than broadleaf weed species. AAD-1 (v3)+GT stacked traits
would allow grass-effective rates of glyphosate (105-840 g ae/ha,
more preferably 210-420 g ae/ha). 2,4-D (at 280-2240 g ae/ha, more
preferably 560-1120 g ae/ha) could then be applied sequentially,
tank mixed, or as a premix with grass-effective rates of glyphosate
to provide necessary broadleaf weed control. An AOPP herbicide like
quizalofop at 10-200 g ae/ha (preferably 20-100 g ae/ha and more
preferably 20-35 g ae/ha), could be for more robust grass weed
control and/or for delaying the development of glyphosate resistant
grasses. The low rate of glyphosate would also provide some benefit
to the broadleaf weed control; however, primary control would be
from the 2,4-D.
[0485] One skilled in the art of weed control will recognize that
use of one or more commercial phenoxy auxin herbicides alone or in
combination (sequentially or independently) with one or more
commercial AOPP herbicide is enabled by AAD-1 (v3) transformation
into crops. Specific rates of other herbicides representative of
these chemistries can be determined by the herbicide labels
compiled in the CPR (Crop Protection Reference) book or similar
compilation, labels compiled online (e.g.,
cdms.net/manuf/manuf.asp), or any commercial or academic crop
protection guides such as the Crop Protection Guide from Agriliance
(2003). Each alternative herbicide enabled for use in HTCs by AAD-1
(v3), whether used alone, tank mixed, or sequentially, is
considered within the scope of this invention.
Example 22
AAD-1 (v3) Stacked with Glufosinate Tolerance Trait in Any Crop
[0486] Glufosinate tolerance (PAT or bar) is currently present in a
number of crops planted in North America either as a selectable
marker for an input trait like insect resistance proteins or
specifically as an HTC trait. Crops include, but are not limited
to, glufosinate tolerant canola, corn, and cotton. Additional
glufosinate tolerant crops (e.g., rice, sugar beet, soybeans, and
turf) have been under development but have not been commercially
released to date. Glufosinate, like glyphosate, is a relatively
non-selective, broad spectrum grass and broadleaf herbicide.
Glufosinate's mode of action differs from glyphosate. It is faster
acting, resulting in desiccation and "burning" of treated leaves
24-48 hours after herbicide application. This is advantageous for
the appearance of rapid weed control. However, this also limits
translocation of glufosinate to meristematic regions of target
plants resulting in poorer weed control as evidenced by relative
weed control performance ratings of the two compounds in many
species (Agriliance, 2003).
[0487] By stacking AAD-1 (v3) with a glufosinate tolerance trait,
either through conventional breeding or jointly as a novel
transformation event, weed control efficacy, flexibility, and
ability to manage weed shifts and herbicide resistance development
could be improved. As mentioned in previous examples, by
transforming crops with AAD-1 (v3), one can selectively apply AOPP
herbicides in monocot crops, monocot crops will have a higher
margin of phenoxy auxin safety, and phenoxy auxins can be
selectively applied in dicot crops. Several scenarios for improved
weed control options can be envisioned where AAD-1 (v3) and a
glufosinate tolerance trait are stacked in any monocot or dicot
crop species: [0488] a) Glufosinate can be applied at a standard
postemergent application rate (200 to 1700 g ae/ha, preferably 350
to 500 g ae/ha) for the control of many grass and broadleaf weed
species. To date, no glufosinate-resistant weeds have been
confirmed; however, glufosinate has a greater number of weeds that
are inherently more tolerant than does glyphosate. [0489] i)
Inherently tolerant grass weed species (e.g., Echinochloa spp or
Sorghum spp) could be controlled by tank mixing 10-200 g ae/ha
(preferably 20-100 g ae/ha) quizalofop. [0490] ii) Inherently
tolerant broadleaf weed species (e.g., Cirsium arvensis and
Apocynum cannabinum) could be controlled by tank mixing 280-2240 g
ae/ha, more preferably 560-2240 g ae/ha, 2,4-D for effective
control of these more difficult-to-control perennial species and to
improve the robustness of control on annual broadleaf weed
species.
[0491] b) A three-way combination of glufosinate (200-500 g
ae/ha)+2,4-D (280-1120 g ae/ha)+quizalofop (10-100 g ae/ha), for
example, could provide more robust, overlapping weed control
spectrum. Additionally, the overlapping spectrum provides an
additional mechanism for the management or delay of herbicide
resistant weeds.
[0492] One skilled in the art of weed control will recognize that
use of one or more commercial phenoxy auxin herbicides alone or in
combination (sequentially or independently) with one or more
commercial AOPP herbicide is enabled by AAD-1 (v3) transformation
into crops. Specific rates of other herbicides representative of
these chemistries can be determined by the herbicide labels
compiled in the CPR (Crop Protection Reference) book or similar
compilation, labels compiled online (e.g.,
cdms.net/manuf/manuf.asp), or any commercial or academic crop
protection guides such as the Crop Protection Guide from Agriliance
(2003). Each alternative herbicide enabled for use in HTCs by AAD-1
(v3), whether used alone, tank mixed, or sequentially, is
considered within the scope of this invention.
Example 23
AAD-1 (v3) Stacked with AHAS Trait in Any Crop
[0493] Imidazolinone herbicide tolerance (AHAS, et al.) is
currently present in a number of crops planted in North America
including, but not limited to, corn, rice, and wheat. Additional
imidazolinone tolerant crops (e.g., cotton and sugar beet) have
been under development but have not been commercially released to
date. Many imidazolinone herbicides (e.g., imazamox, imazethapyr,
imazaquin, and imazapic) are currently used selectively in various
conventional crops. The use of imazethapyr, imazamox, and the
non-selective imazapyr has been enabled through imidazolinone
tolerance traits like AHAS et al. Imidazolinone tolerant HTCs to
date have the advantage of being non-transgenic. This chemistry
class also has significant soil residual activity, thus being able
to provide weed control extended beyond the application timing,
unlike glyphosate or glufosinate-based systems. However, the
spectrum of weeds controlled by imidazolinone herbicides is not as
broad as glyphosate (Agriliance, 2003). Additionally, imidazolinone
herbicides have a mode of action (inhibition of acetolactate
synthase, ALS) to which many weeds have developed resistance (Heap,
2004). By stacking AAD-1 (v3) with an imidazolinone tolerance
trait, either through conventional breeding or jointly as a novel
transformation event, weed control efficacy, flexibility, and
ability to manage weed shifts and herbicide resistance development
could be improved. As mentioned in previous examples, by
transforming crops with AAD-1 (v3), one can selectively apply AOPP
herbicides in monocot crops, monocot crops will have a higher
margin of phenoxy auxin safety, and phenoxy auxins can be
selectively applied in dicot crops. Several scenarios for improved
weed control options can be envisioned where AAD-1 (v3) and an
imidazolinone tolerance trait are stacked in any monocot or dicot
crop species: [0494] a) Imazethapyr can be applied at a standard
postemergent application rate of (35 to 280 g ae/ha, preferably
70-140 g ae/ha) for the control of many grass and broadleaf weed
species. [0495] i) ALS-inhibitor resistant broadleaf weeds like
Amaranthus rudis, Ambrosia trifida, Chenopodium album (among
others, Heap, 2004) could be controlled by tank mixing 280-2240 g
ae/ha, more preferably 560-1120 g ae/ha, 2,4-D. [0496] ii)
Inherently more tolerant broadleaf species to imidazolinone
herbicides like Ipomoea spp. can also be controlled by tank mixing
280-2240 g ae/ha, more preferably 560-1120 g ae/ha, 2,4-D. [0497]
iii) ALS-inhibitor resistant grass weeds like Sorghum halepense and
Lolium spp. can be controlled by tank mixing 10-200 g ae/ha
(preferably 20-100 g ae/ha) quizalofop. [0498] iv) Inherently
tolerant grass weed species (e.g., Agropyron repens) could also be
controlled by tank mixing 10-200 g ae/ha (preferably 20-100 g
ae/ha) quizalofop. [0499] b) A three-way combination of imazethapyr
(35 to 280 g ae/ha, preferably 70-140 g ae/ha)+2,4-D (280-1120 g
ae/ha)+quizalofop (10-100 g ae/ha), for example, could provide more
robust, overlapping weed control spectrum. Additionally, the
overlapping spectrum provides an additional mechanism for the
management or delay of herbicide resistant weeds.
[0500] One skilled in the art of weed control will recognize that
use of any of various commercial imidazolinone herbicides, phenoxy
auxin herbicides, or AOPP herbicide, alone or in multiple
combinations, is enabled by AAD-1 (v3) transformation and stacking
with any imidazolinone tolerance trait either by conventional
breeding or genetic engineering. Specific rates of other herbicides
representative of these chemistries can be determined by the
herbicide labels compiled in the CPR (Crop Protection Reference)
book or similar compilation, labels compiled online (e.g.,
cdms.net/manuf/manuf.asp), or any commercial or academic crop
protection guides such as the Crop Protection Guide from Agriliance
(2003). Each alternative herbicide enabled for use in HTCs by AAD-1
(v3), whether used alone, tank mixed, or sequentially, is
considered within the scope of this invention.
EXAMPLE 24
AAD-1 (v3) in Rice
[0501] 24.1--Media Description.
[0502] Culture media employed were adjusted to pH 5.8 with 1 M KOH
and solidified with 2.5 g/l Phytagel (Sigma). Embryogenic calli
were cultured in 100.times.20 mm Petri dishes containing 40 ml
semi-solid medium. Rice plantlets were grown on 50 ml medium in
Magenta boxes. Cell suspensions were maintained in 125-ml conical
flasks containing 35 ml liquid medium and rotated at 125 rpm.
Induction and maintenance of embryogenic cultures took place in the
dark at 25-26.degree. C., and plant regeneration and whole-plant
culture took place in a 16-h photoperiod (Zhang et al. 1996).
[0503] Induction and maintenance of embryogenic callus took place
on NB basal medium as described previously (Li et al. 1993), but
adapted to contain 500 mg/l glutamine Suspension cultures were
initiated and maintained in SZ liquid medium (Zhang et al. 1998)
with the inclusion of 30 g/l sucrose in place of maltose. Osmotic
medium (NBO) consisted of NB medium with the addition of 0.256 M
each of mannitol and sorbitol. Hygromycin-B-resistant callus was
selected on NB medium supplemented with 50 mg/l hygromycin B for
3-4 weeks. Pre-regeneration took place on medium (PRH50) consisting
of NB medium without 2,4-dichlorophenoxyacetic acid (2,4-D), but
with the addition of 2 mg/l 6-benzylaminopurine (BAP), 1 mg/l
.alpha.-naphthaleneacetic acid (NAA), 5 mg/l abscisic acid (ABA)
and 50 mg/l hygromycin B for 1 week. Regeneration of plantlets
followed via culture on regeneration medium (RNH50) comprising NB
medium without 2,4-D, and supplemented with 3 mg/l BAP, 0.5 mg/l
NAA, and 50 mg/l hygromycin B until shoots regenerated. Shoots were
transferred to rooting medium with half-strength Murashige and
Skoog basal salts and Gamborg's B5 vitamins, supplemented with 1%
sucrose and 50 mg/l hygromycin B (1/2MSH50).
[0504] 24.2--Tissue Culture Development.
[0505] Mature desiccated seeds of Oryza sativa L. japonica cv.
Taipei 309 were sterilized as described in Zhang et al. 1996.
Embryogenic tissues were induced by culturing sterile mature rice
seeds on NB medium in the dark. The primary callus approximately 1
mm in diameter, was removed from the scutellum and used to initiate
cell suspension in SZ liquid medium. Suspensions were then
maintained as described in Zhang 1995. Suspension-derived
embryogenic tissues were removed from liquid culture 3-5 days after
the previous subculture and placed on NBO osmotic medium to form a
circle about 2.5 cm across in a Petri dish and cultured for 4 h
prior to bombardment. Sixteen to 20 h after bombardment, tissues
were transferred from NBO medium onto NBH50 hygromycin B selection
medium, ensuring that the bombarded surface was facing upward, and
incubated in the dark for 14-17 days. Newly formed callus was then
separated from the original bombarded explants and placed nearby on
the same medium. Following an additional 8-12 days, relatively
compact, opaque callus was visually identified, and transferred to
PRH50 pre-regeneration medium for 7 days in the dark. Growing
callus, which became more compact and opaque was then subcultured
onto RNH50 regeneration medium for a period of 14-21 days under a
16-h photoperiod. Regenerating shoots were transferred to Magenta
boxes containing 1/2 MSH50 medium. Multiple plants regenerated from
a single explant are considered siblings and were treated as one
independent plant line. A plant was scored as positive for the hph
gene if it produced thick, white roots and grew vigorously on 1/2
MSH50 medium. Once plantlets had reached the top of Magenta boxes,
they were transferred to soil in a 6-cm pot under 100% humidity for
a week, then moved to a growth chamber with a 14-h light period at
30.degree. C. and in the dark at 21.degree. C. for 2-3 weeks before
transplanting into 13-cm pots in the greenhouse. Seeds were
collected and dried at 37.degree. C. for one week prior to storage
at 4.degree. C.
[0506] 24.3--Microprojectile Bombardment.
[0507] All bombardments were conducted with the Biolistic
PDS-1000/HeTM system (Bio-Rad, Laboratories, Inc.). Three
milligrams of 1.0 micron diameter gold particles were washed once
with 100% ethanol, twice with sterile distilled water and
resuspended in 50 .mu.l water in a siliconized Eppendorf tube. Five
micrograms plasmid DNA representing a 1:6 molar ratio of
pDOW3303(Hpt-containing vector) to pDAB3403, 20 .mu.l spermidine
(0.1 M) and 50 .mu.l calcium chloride (2.5 M) were added to the
gold suspension. The mixture was incubated at room temperature for
10 min, pelleted at 10000 rpm for 10 s, resuspended in 60 .mu.l
cold 100% ethanol and 8-9 .mu.l was distributed onto each
macrocarrier. Tissue samples were bombarded at 1100 psi and 27 in
of Hg vacuum as described by Zhang et al. (1996).
[0508] 24.4--Tolerance Testing.
[0509] Rice plantlets at the 3-5 leaf stage were sprayed with a
0.3% (v/v) solution of DuPont.TM. Assure.RTM. II containing 1%
(v/v) Agridex crop oil concentrate using a DeVilbiss bulb sprayer
(model 15-RD glass atomizer). This concentration corresponds to
approximately 140 g ae/ha. Each plant was spayed in a fume hood at
a distance of 8-12 inches with 6 full squirts of the sprayer
directed so that the entire plant was covered with an equal portion
of herbicide. Each squirt delivered approximately 100 .mu.l
solution to the plantlet. Once sprayed, plantlets were allowed to
dry for one hour before being moved out of the fume hood. Rating
for sensitivity or resistance was done at 10-14 days after
treatment (DAT) and is shown in Table 35 below.
TABLE-US-00035 TABLE 35 Sample 140 g ae/ha Name quizalofop Control
Dead 63-1A No injury 63-1F No injury 63-4B No injury 63-4D No
injury 63-6C Dead
[0510] 24.5--Tissue Harvesting, DNA Isolation and
Quantification.
[0511] Fresh tissue was placed into tubes and lyophilized at
4.degree. C. for 2 days. After the tissue was fully dried, a
tungsten bead (Valenite) was placed in the tube and the samples
were subjected to 1 minute of dry grinding using a Kelco bead mill.
The standard DNeasy DNA isolation procedure was then followed
(Qiagen, Dneasy 69109). An aliquot of the extracted DNA was then
stained with Pico Green (Molecular Probes P7589) and scanned in the
florometer (BioTek) with known standards to obtain the
concentration in ng/.mu.1.
[0512] 24.6--Southern Blot Analysis.
[0513] Southern blot analysis was performed with total DNA obtained
from the Qiagen DNeasy kit. A total of 2 .mu.g of DNA was subjected
to an overnight digest of HindIII for pDAB3403 to obtain
integration data. Likewise a total of 2 ug of DNA was subjected to
an overnight digest of MfeI to obtain the PTU data. After the
overnight digestion an aliquot of .about.100 ng was run on a 1% gel
to ensure complete digestion. After this assurance the samples were
run on a large 0.85% agarose gel overnight at 40 volts. The gel was
then denatured in 0.2 M NaOH, 0.6 M NaCl for 30 minutes. The gel
was then neutralized in 0.5 M Tris HCl, 1.5 M NaCl pH of 7.5 for 30
minutes. A gel apparatus containing 20.times.SSC was then set-up to
obtain a gravity gel to nylon membrane (Millipore INYC00010)
transfer overnight. After the overnight transfer the membrane was
then subjected to UV light via a crosslinker (Stratagene UV
stratalinker 1800) at 1200.times.100 microjoules. The membrane was
then washed in 0.1% SDS, 0.1 SSC for 45 minutes. After the 45
minute wash, the membrane was baked for 3 hours at 80.degree. C.
and then stored at 4.degree. C. until hybridization. The
hybridization template fragment was prepared using coding region
PCR using plasmid pDAB3404. A total of 100 ng of total DNA was used
as template. 20mM of each primer was used with the Takara Ex Taq
PCR Polymerase kit (Mirus TAKRROO1A). Primers for Southern fragment
PCR AAD-1 were (Forward--ATGGCTCATGCTGCCCTCAGCC) (SEQ ID NO:31) and
(Reverse--GGGCAGGCCTAACTCCACCAA) (SEQ ID NO:32). The PCR reaction
was carried out in the 9700 Geneamp thermocycler (Applied
Biosystems), by subjecting the samples to 94.degree. C. for 3
minutes and 35 cycles of 94.degree. C. for 30 seconds, 65.degree.
C. for 30 seconds, and 72.degree. C. for 1 minute and 45 seconds
followed by 72.degree. C. for 10 minutes.
[0514] The product was run on a 1% agarose gel and excised then gel
extracted using the Qiagen (28706) gel extraction procedure. The
membrane was then subjected to a pre-hybridization at 60.degree. C.
step for 1 hour in Perfect Hyb buffer (Sigma H7033). The Prime it
RmT dCTP-labeling reaction (Stratagene 300392) procedure was used
to develop the p32 based probe (Perkin Elmer). The probe was
cleaned-up using the Probe Quant. G50 columns (Amersham
27-5335-01). Two million counts CPM were used to hybridize the
Southern blots overnight. After the overnight hybridization the
blots were then subjected to two 20 minute washes at 65.degree. C.
in 0.1% SDS, 0.1 SSC. The blots were then exposed to a phosphor
image screen overnight and scanned using a storm scanner (MOLECULAR
DEVICES). A summary of the results is presented in Table 36.
TABLE-US-00036 TABLE 36 Southern Results. Integration Southern data
PTU Southern data Event Number of bands Expected size 3049 bp 63-1
A 8 yes, 7 distinct bands 63-1 F 5 yes, 9 distinct bands 63-4 A 20
yes, 20 distinct bands 63-4 D 20 yes, 19 distinct bands 63-6 C 2
Insufficient DNA yield for both cuts
[0515] Plants 63-1 A and 63-1 F are not the same event; Plants 63-4
A and 63-4 D are the same event. These events have the expected
size PTU's, but they are very complex. This Southern blot PTU data
correlates with expression data and the spray data. Sample 63-6 C
did not have enough DNA present to perform both Integration and PTU
southern blots.
[0516] 24.7--Western Data
[0517] Sample preparation and analysis conditions were as described
previously. Five transgenic rice lines and 1 non-transgenic control
were analyzed for AAD-1 expression using ELISA and Western blot.
AAD-1 was detected in four lines (63-1A, 63-1F, 63-4B and 63-4D)
but not in line 63-1C or the control plant. Expression levels
ranged from 15.6 to 183 ppm of total soluble protein. A summary of
the results is presented in Table 37.
TABLE-US-00037 TABLE 37 ELISA TSP [AAD1] Expression Lane Sample
Name (.mu.g/mL) (ng/mL) (ppm) Western 1 Control 6719.58 0.00 0.00 -
2 63-1A 8311.87 351.17 42.25 .+-. 3 63-1F 11453.31 2092.35 182.69
++ 4 63-4B 13835.09 216.00 15.61 + 5 36-4D 13656.49 717.05 52.51 ++
6 63-6C 5343.63 0.00 0.00 - 7 AAD1 Standard (0.5 .mu.g/mL) +++ 8
AAD1 Standard (5.0 .mu.g/mL) +++++
Example 25
Turf Grass Transformation Procedures
[0518] Genetic transformation, with AAD-1 (v3) substituted for the
"bar" gene as described below, of creeping bentgrass mediated by
Agrobacterium tumefaciens could be achieved through embryogenic
callus initiated from seeds (cv. Penn-A-4), as described generally
below. See "Efficiency of Agrobacterium tumefaciens-mediated
turfgrass (Agrostis stolonifera L) transformation" (Luo et. al.,
2004).
[0519] Callus is infected with an A. tumefaciens strain (LBA4404)
harboring a super-binary vector that contained an
herbicide-resistant bar gene driven either by a rice ubiquitin
promoter. The overall stable transformation efficiency ranged from
18% to 45%. Southern blot and genetic analysis confirmed transgene
integration in the creeping bentgrass genome and normal
transmission and stable expression of the transgene in the T.sub.1
generation. All independent transformation events carried one to
three copies of the transgene, and a majority (60-65%) contained
only a single copy of the foreign gene with no apparent
rearrangements.
[0520] 25.1--Seed Preparation for Embryogenic Callus Induction.
[0521] Mature seeds were dehusked with sand paper and surface
sterilized in 10% (v/v) Clorox bleach (6% sodium hypochlorite) plus
0.2% (v/v) Tween 20 (Polysorbate 20) with vigorous shaking for 90
min Following rinsing five times in sterile distilled water, the
seeds were placed onto callus-induction medium (MS basal salts and
vitamins, 30 g/l sucrose, 500 mg/l casein hydrolysate, 6.6 mg/l
3,6-dichloro-o-anisic acid (dicamba), 0.5 mg/l 6-benzylaminopurine
(BAP) and 2 g/l Phytagel. The pH of the medium was adjusted to 5.7
before autoclaving at 120.degree. C. for 20 min).
[0522] 25.2--Embryogenic Callus Induction.
[0523] The culture plates containing prepared seed explants were
kept in the dark at room temperature for 6 weeks. Embryogenic calli
were visually selected and subcultured on fresh callus-induction
medium in the dark at room temperature for 1 week before
co-cultivation.
[0524] 25.3--Agrobacterium Infection and Co-Cultivation.
[0525] One day before agro-infection, the embryogenic callus was
divided into 1- to 2-mm pieces and placed on callus-induction
medium containing 100 .mu.M acetosyringone. A 10-ul aliquot of
Agrobacterium (LBA4404) suspension (OD=1.0 at 660 nm) was then
applied to each piece of callus, followed by 3 days of
co-cultivation in the dark at 25.degree. C.
[0526] 25.4--Resting Stage and Agrobacterium Control.
[0527] For the antibiotic treatment step, the callus was then
transferred and cultured for 2 weeks on callus-induction medium
plus 125 mg/l cefotaxime and 250 mg/l carbenicillin to suppress
bacterial growth.
[0528] 25.5--Selection and Potential Transgenic Colony
Identification.
[0529] Subsequently, for selection, the callus was moved to
callus-induction medium containing 250 mg/l cefotaxime and 10 mg/l
phosphinothricin (PPT) for 8 weeks. Antibiotic treatment and the
entire selection process were performed at room temperature in the
dark. The subculture interval during selection was typically 3
weeks.
[0530] 25.6--Regeneration of Transgenic Plants.
[0531] For plant regeneration, the PPT-resistant proliferating
callus events are first moved to regeneration medium (MS basal
medium, 30 g/l sucrose, 100 mg/l myo-inositol, 1 mg/l BAP and 2 g/l
Phytagel) supplemented with cefotaxime, PPT or hygromycin. These
calli were kept in the dark at room temperature for 1 week and then
moved into the light for 2-3 weeks to develop shoots. No Albino
plants with PPT selection (Hygromycin if used as a selection agent,
produces a high level of albino plants).
[0532] 25.7--Root Induction and Transfer to Greenhouse.
[0533] Small shoots were then separated and transferred to
hormone-free regeneration medium containing PPT and cefotaxime to
promote root growth while maintaining selection pressure and
suppressing any remaining Agrobacterium cells. Plantlets with
well-developed roots (3-5 weeks) were then transferred to soil and
grown either in the greenhouse or in the field.
[0534] 25.8--Vernalization and Out-Crossing of Transgenic
Plants.
[0535] Transgenic plants were maintained out of doors in a
containment nursery (3-6 months) until the winter solstice in
December. The vernalized plants were then transferred to the
greenhouse and kept at 25.degree. C. under a 16/8 h photoperiod and
surrounded by non-transgenic wild-type plants that physically
isolated them from other pollen sources. The plants started
flowering 3-4 weeks after being moved back into the greenhouse.
They were out-crossed with the pollen from the surrounding wild
type plants. The seeds collected from each individual transgenic
plant were germinated in soil at 25.degree. C., and T1 plants were
grown in the greenhouse for further analysis.
[0536] 25.9--Other Target Grasses.
[0537] Other grasses that can be targeted for AAD-1 transformation
according to the subject invention include Annual meadowgrass (Poa
annua), Bahiagrass, Bentgrass, Bermudagrass, Bluegrass, Bluestems,
Bromegrass, Browntop bent (Agrostis capillaries), Buffalograss,
Canary Grass, Carpetgrass, Centipedegrass, Chewings fescue (Festuca
rubra commutate), Crabgrass, Creeping bent (Agrostis stolonifera),
Crested hairgrass (Koeleria macrantha), Dallisgrass, Fescue,
Festolium, Hard/sheeps fescue (Festuca ovina), Gramagrass,
Indiangrass, Johnsongrass, Lovegrass, mixes (Equine, Pasture,
etc.), Native Grasses, Orchardgrass, Perennial ryegrass (Lolium
perenne), Redtop, Rescuegrass, annual and perennial Ryegrass,
Slender creeping red fescue (Festuca rubra trichophylla),
Smooth-stalked meadowgrass (Poa pratensis), St. Augustine, Strong
creeping red fescue (Festuca rubra rubra), Sudangrass, Switchgrass,
Tall fescue (Festuca arundinacea), Timothy, Tufted hairgrass
(Deschampsia caespitosa), Turfgrasses, Wheatgrass, and
Zoysiagrass.
EXAMPLE 26
AAD-1 (v3) in Canola
[0538] 26.1--Canola Transformation.
[0539] The AAD-1 (v3) gene conferring resistance to 2,4-D was used
to transform Brassica napus var. Nexera* 710 with
Agrobacterium-mediated transformation. The construct contained
AAD-1 (v3) gene driven by CsVMV promoter and Pat gene driven by
AtUbi10 promoter.
[0540] Seeds were surface-sterilized with 10% commercial bleach for
10 minutes and rinsed 3 times with sterile distilled water. The
seeds were then placed on one half concentration of MS basal medium
(Murashige and Skoog, 1962) and maintained under growth regime set
at 25.degree. C., and a photoperiod of 16 hrs light/8 hrs dark.
[0541] Hypocotyl segments (3-5 mm) were excised from 5-7 day old
seedlings and placed on callus induction medium K1D1 (MS medium
with 1 mg/l kinetin and 1 mg/1 2,4-D) for 3 days as pre-treatment.
The segments were then transferred into a petri plate, treated with
Agrobacterium Z7075 or LBA4404 strain containing pDAB721. The
Agrobacterium was grown overnight at 28.degree. C. in the dark on a
shaker at 150 rpm and subsequently re-suspended in the culture
medium.
[0542] After 30 min treatment of the hypocotyl segments with
Agrobacterium, these were placed back on the callus induction
medium for 3 days. Following co-cultivation, the segments were
placed K1D1TC (callus induction medium containing 250 mg/l
Carbenicillin and 300 mg/l Timentin) for one week of recovery.
Alternately, the segments were placed directly on selection medium
K1D1H1 (above medium with 1 mg/l Herbiace). Carbenicillin and
Timentin were the antibiotics used to kill the Agrobacterium. The
selection agent Herbiace allowed the growth of the transformed
cells.
[0543] Callus samples from 35 independent events were tested by
PCR. All the 35 samples tested positive for the presence of AAD-1
(v3),whereas the non-transformed controls were negative (section on
PCR assay). Ten callus samples were confirmed to express the AAD-1
protein as determined by ELISA (section on protein analysis).
[0544] Callused hypocotyl segments were then placed on B3Z1H1 (MS
medium, 3 mg/l benzylamino purine, 1 mg/l Zeatin, 0.5 gm/l MES
[2-(N-morpholino) ethane sulfonic acid], 5 mg/l silver nitrate, 1
mg/l Herbiace, Carbenicillin and Timentin) shoot regeneration
medium. After 3 weeks shoots started regenerating. Hypocotyl
segments along with the shoots are transferred to B3Z1H3 medium (MS
medium, 3 mg/l benzylamino purine, 1 mg/l Zeatin, 0.5 gm/l MES
[2-(N-morpholino) ethane sulfonic acid], 5 mg/l silver nitrate, 3
mg/l Herbiace, Carbenicillin and Timentin) for another 3 weeks.
[0545] Shoots were excised from the hypocotyl segments and
transferred to shoot elongation medium MESH10 (MS, 0.5 gm/l MES, 10
mg/l Herbiace, Carbenicillin, Timentin) for 2-4 weeks. The
elongated shoots are cultured for root induction on MSI.1 (MS with
0.1 mg/l Indolebutyric acid). Once the plants had a well
established root system, these were transplanted into soil. The
plants were acclimated under controlled environmental conditions in
the Conviron for 1-2 weeks before transfer to the greenhouse.
[0546] The transformed T0 plants were self-pollinated in the
greenhouse to obtain T1 seed. The T0 plants and T1 progeny were
sprayed with a range of herbicide concentrations to establish the
level of protection by the AAD-1 (v3) gene.
[0547] 26.2--"Molecular Analysis": Canola Materials and Methods
[0548] 26.2.1--Tissue harvesting DNA isolation and quantification.
Fresh tissue was placed into tubes and lyophilized at 4.degree. C.
for 2 days. After the tissue was fully dried, a tungsten bead
(Valenite) was placed in the tube and the samples were subjected to
1 minute of dry grinding using a Kelco bead mill. The standard
DNeasy DNA isolation procedure was then followed (Qiagen, DNeasy
69109). An aliquot of the extracted DNA was then stained with Pico
Green (Molecular Probes P7589) and read in the florometer (BioTek)
with known standards to obtain the concentration in ng/ul.
[0549] 26.2.2--Polymerase chain reaction. A total of 100 ng of
total DNA was used as the template. 20mM of each primer was used
with the Takara Ex Taq PCR Polymerase kit (Mirus TAKRROO1A).
Primers for Coding Region PCR AAD-1 (v3) were (Forward--ATGGCTCATG
CTGCCCTCAGCC) (SEQ ID NO:27) and (Reverse--CGGGCAGGCCTAACTCCACCAA)
(SEQ ID NO:28). The PCR reaction was carried out in the 9700
Geneamp thermocycler (Applied Biosystems), by subjecting the
samples to 94.degree. C. for 3 minutes and 35 cycles of 94.degree.
C. for 30 seconds, 65.degree. C. for 30 seconds, and 72.degree. C.
for 2 minutes followed by 72.degree. C. for 10 minutes. PCR
products were analyzed by electrophoresis on a 1% agarose gel
stained with EtBr. 35 samples from 35 plants with AAD-1 (v3) events
tested positive. Three negative control samples tested
negative.
[0550] 26.3--ELISA.
[0551] Using established ELISA described in previous section, AAD-1
protein was detected in 10 different canola transformation events.
Expression levels ranged from 150 to over 1000 ppm of total soluble
protein (TSP). Three different untransformed calli samples were
tested in parallel with little signal detected, indicating that the
antibodies used in the assay have minimal cross reactivity to the
canola cell matrix. A summary of the results is presented in Table
38.
TABLE-US-00038 TABLE 38 Expression of AAD1 in Canola calli. [TSP]
[AAD1] Expression PCR for Sample # Weight (mg) (.mu.g/mL) (ng/mL)
(ppm TSP) AAD1 1 114 757.02 119.36 157.67 + 2 55 839.79 131.84
156.99 + 3 53 724.41 202.12 279.01 + 4 52 629.01 284.89 452.92 + 5
55 521.75 175.88 337.08 + 6 61 707.69 74.24 153.71 + 7 51 642.02
559.11 1026.73 + 8 65 707.69 270.73 382.56 + 9 51 642.02 197.90
308.25 + 10 51 1417.42 220.63 156.66 + Control 1 53 2424.67 18.67
7.70 - Control 2 61 2549.60 35.00 13.73 - Control 3 59 2374.41
22.79 9.60 -
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Sequence CWU 1
1
35150DNAArtificial SequenceForward primer used to amplify the
rdpA/AAD-1 (v1) gene 1tctagaagga gatataccat gcatgctgca ctgtcccccc
tctcccagcg 50238DNAArtificial SequenceReverse primer used to
amplify the rdpA/AAD-1 (v1) gene 2ctcgagttac tagcgcgccg ggcgcacgcc
accgaccg 383915DNASphingobium
herbicidovoransmisc_feature(1)..(18)Primer
linkermisc_feature(907)..(915)Primer linker 3tctagaagga gatataccat
gcatgctgca ctgtcccccc tctcccagcg ctttgagcgc 60atcgcggtcc agccgctgac
cggcgtcctg ggcgccgaga tcaccggcgt cgacctgcgc 120gagccgctcg
acgacagcac ctggaacgaa atcctcgacg cgttccacac ttaccaggtc
180atctattttc ccggccaggc gatcaccaac gaacagcaca tcgccttcag
ccggcgcttc 240ggccccgtcg atcccgtgcc cctgctcaag agcatcgaag
ggtatccaga ggtgcagatg 300atccgccgcg aagccaacga aagcgggcgt
gtgatcggtg aygactggca caccgacagc 360accttcctgg acgcaccgcc
ggccgccgtg gtgatgcgcg cgatcgacgt gcccgagcat 420ggcggcgaca
ccggttttct gagcatgtac accgcgtggg agacgctgtc gcccaccatg
480caggccacca tcgaagggtt gaacgtagtg cacagcgcca cgcgtgtgtt
cggctcgctc 540taccaggccc agaaccggcg cttcagcaac accagcgtca
aggtgatgga cgtcgacgcg 600ggcgaccgtg aaaccgtgca ccccctggtg
gtgacccatc cgggcagcgg ccgcaagggc 660ctgtacgtga accaggtcta
ttgccagcgc atcgagggca tgaccgatgc cgaaagcaaa 720ccgctgctgc
agttcctgta cgagcatgcg acacggttcg atttcacctg ccgcgtgcgc
780tggaagaagg accaggtcct ggtctgggac aacctgtgca cgatgcaccg
ggccgtaccc 840gactacgcgg gcaagttccg ctacctgacg cgcaccacgg
tcggtggcgt gcgcccggcg 900cgctagtaac tcgag 9154897DNAArtificial
SequenceAAD-1 (v2) primary sequencemisc_feature(1)..(2)primer
linkermisc_feature(891)..(897)primer linker 4ccatggctgc tgcactgtcc
cccctctccc agcgctttga gcgcatcgcg gtccagccgc 60tgaccggcgt cctgggcgcc
gagatcaccg gcgtcgacct gcgcgagccg ctcgacgaca 120gcacctggaa
cgaaatcctc gacgcgttcc acacttacca ggtcatctat tttcccggcc
180aggcgatcac caacgaacag cacatcgcct tcagccggcg cttcggcccc
gtcgatcccg 240tgcccctgct caagagcatc gaagggtatc cagaggtgca
gatgatccgc cgcgaagcca 300acgaaagcgg gcgtgtgatc ggtgatgact
ggcacaccga cagcaccttc ctggacgcac 360cgccggccgc cgtggtgatg
cgcgcgatcg acgtgcccga gcatggcggc gacaccggtt 420ttctgagcat
gtacaccgcg tgggagacgc tgtcgcccac catgcaggcc accatcgaag
480ggttgaacgt agtgcacagc gccacgcgtg tgttcggctc gctctaccag
gcccagaacc 540ggcgcttcag caacaccagc gtcaaggtga tggacgtcga
cgcgggcgac cgtgaaaccg 600tgcaccccct ggtggtgacc catccgggca
gcggctgcaa gggcctgtac gtgaaccagg 660tctattgcca gcgcatcgag
ggcatgaccg atgccgaaag caaaccgctg ctgcagttcc 720tgtacgagca
tgcgacacgg ttcgatttca cctgccgcgt gcgctggaag aaggaccagg
780tcctggtctg ggacaacctg tgcacgatgc accgggccgt acccgactac
gcgggcaagt 840tccgctacct gacgcgcacc acggtcggtg gcgtgcgccc
ggcgcgctag tgagctc 8975919DNAArtificial SequenceAAD-1 (v3) primary
sequencemisc_feature(1)..(2)primer
linkermisc_feature(6)..(8)additional alanine codon
(GCT)misc_feature(894)..(919)primer linker 5ccatggctca tgctgccctc
agccctctct cccaacgctt tgagagaata gctgtccagc 60cactcactgg tgtccttggt
gctgagatca ctggagtgga cttgagggaa ccacttgatg 120acagcacctg
gaatgagata ttggatgcct tccacactta ccaagtcatc tactttcctg
180gccaagcaat caccaatgag cagcacattg cattctcaag aaggtttgga
ccagttgatc 240cagtgcctct tctcaagagc attgaaggct atccagaggt
tcagatgatc cgcagagaag 300ccaatgagtc tggaagggtg attggtgatg
actggcacac agactccact ttccttgatg 360cacctccagc tgctgttgtg
atgagggcca tagatgttcc tgagcatggc ggagacactg 420ggttcctttc
aatgtacaca gcttgggaga ccttgtctcc aaccatgcaa gccaccatcg
480aagggctcaa cgttgtgcac tctgccacac gtgtgttcgg ttccctctac
caagcacaga 540accgtcgctt cagcaacacc tcagtcaagg tgatggatgt
tgatgctggt gacagagaga 600cagtccatcc cttggttgtg actcatcctg
gctctggaag gaaaggcctt tatgtgaatc 660aagtctactg tcagagaatt
gagggcatga cagatgcaga atcaaagcca ttgcttcagt 720tcctctatga
gcatgccacc agatttgact tcacttgccg tgtgaggtgg aagaaagacc
780aagtccttgt ctgggacaac ttgtgcacca tgcaccgtgc tgttcctgac
tatgctggca 840agttcagata cttgactcgc accacagttg gtggagttag
gcctgcccgc tgagtagtta 900gcttaatcac ctagagctc 919630DNAArtificial
SequencerdpA(ncoI) 5' primer 6cccatggctg ctgcactgtc ccccctctcc
30733DNAArtificial Sequence3'saci primer 7gagctcacta gcgcgccggg
cgcacgccac cga 33836DNAArtificial SequenceBstEII/ Del NotI 5'
primer 8tggtggtgac ccatccgggc agcggctgca agggcc
369295PRTSphingobium herbicidovorans 9Met His Ala Ala Leu Ser Pro
Leu Ser Gln Arg Phe Glu Arg Ile Ala1 5 10 15Val Gln Pro Leu Thr Gly
Val Leu Gly Ala Glu Ile Thr Gly Val Asp 20 25 30Leu Arg Glu Pro Leu
Asp Asp Ser Thr Trp Asn Glu Ile Leu Asp Ala 35 40 45Phe His Thr Tyr
Gln Val Ile Tyr Phe Pro Gly Gln Ala Ile Thr Asn 50 55 60Glu Gln His
Ile Ala Phe Ser Arg Arg Phe Gly Pro Val Asp Pro Val65 70 75 80Pro
Leu Leu Lys Ser Ile Glu Gly Tyr Pro Glu Val Gln Met Ile Arg 85 90
95Arg Glu Ala Asn Glu Ser Gly Arg Val Ile Gly Asp Asp Trp His Thr
100 105 110Asp Ser Thr Phe Leu Asp Ala Pro Pro Ala Ala Val Val Met
Arg Ala 115 120 125Ile Asp Val Pro Glu His Gly Gly Asp Thr Gly Phe
Leu Ser Met Tyr 130 135 140Thr Ala Trp Glu Thr Leu Ser Pro Thr Met
Gln Ala Thr Ile Glu Gly145 150 155 160Leu Asn Val Val His Ser Ala
Thr Arg Val Phe Gly Ser Leu Tyr Gln 165 170 175Ala Gln Asn Arg Arg
Phe Ser Asn Thr Ser Val Lys Val Met Asp Val 180 185 190Asp Ala Gly
Asp Arg Glu Thr Val His Pro Leu Val Val Thr His Pro 195 200 205Gly
Ser Gly Arg Lys Gly Leu Tyr Val Asn Gln Val Tyr Cys Gln Arg 210 215
220Ile Glu Gly Met Thr Asp Ala Glu Ser Lys Pro Leu Leu Gln Phe
Leu225 230 235 240Tyr Glu His Ala Thr Arg Phe Asp Phe Thr Cys Arg
Val Arg Trp Lys 245 250 255Lys Asp Gln Val Leu Val Trp Asp Asn Leu
Cys Thr Met His Arg Ala 260 265 270Val Pro Asp Tyr Ala Gly Lys Phe
Arg Tyr Leu Thr Arg Thr Thr Val 275 280 285Gly Gly Val Arg Pro Ala
Arg 290 29510295PRTArtificial SequenceAAD-1 v2
TranslationMISC_FEATURE(2)..(2)Different from
v1MISC_FEATURE(212)..(212)Different from v1 10Met Ala Ala Ala Leu
Ser Pro Leu Ser Gln Arg Phe Glu Arg Ile Ala1 5 10 15Val Gln Pro Leu
Thr Gly Val Leu Gly Ala Glu Ile Thr Gly Val Asp 20 25 30Leu Arg Glu
Pro Leu Asp Asp Ser Thr Trp Asn Glu Ile Leu Asp Ala 35 40 45Phe His
Thr Tyr Gln Val Ile Tyr Phe Pro Gly Gln Ala Ile Thr Asn 50 55 60Glu
Gln His Ile Ala Phe Ser Arg Arg Phe Gly Pro Val Asp Pro Val65 70 75
80Pro Leu Leu Lys Ser Ile Glu Gly Tyr Pro Glu Val Gln Met Ile Arg
85 90 95Arg Glu Ala Asn Glu Ser Gly Arg Val Ile Gly Asp Asp Trp His
Thr 100 105 110Asp Ser Thr Phe Leu Asp Ala Pro Pro Ala Ala Val Val
Met Arg Ala 115 120 125Ile Asp Val Pro Glu His Gly Gly Asp Thr Gly
Phe Leu Ser Met Tyr 130 135 140Thr Ala Trp Glu Thr Leu Ser Pro Thr
Met Gln Ala Thr Ile Glu Gly145 150 155 160Leu Asn Val Val His Ser
Ala Thr Arg Val Phe Gly Ser Leu Tyr Gln 165 170 175Ala Gln Asn Arg
Arg Phe Ser Asn Thr Ser Val Lys Val Met Asp Val 180 185 190Asp Ala
Gly Asp Arg Glu Thr Val His Pro Leu Val Val Thr His Pro 195 200
205Gly Ser Gly Cys Lys Gly Leu Tyr Val Asn Gln Val Tyr Cys Gln Arg
210 215 220Ile Glu Gly Met Thr Asp Ala Glu Ser Lys Pro Leu Leu Gln
Phe Leu225 230 235 240Tyr Glu His Ala Thr Arg Phe Asp Phe Thr Cys
Arg Val Arg Trp Lys 245 250 255Lys Asp Gln Val Leu Val Trp Asp Asn
Leu Cys Thr Met His Arg Ala 260 265 270Val Pro Asp Tyr Ala Gly Lys
Phe Arg Tyr Leu Thr Arg Thr Thr Val 275 280 285Gly Gly Val Arg Pro
Ala Arg 290 29511296PRTArtificial SequenceAAD-1 v3
TranslationMISC_FEATURE(2)..(3)Different from V1 11Met Ala His Ala
Ala Leu Ser Pro Leu Ser Gln Arg Phe Glu Arg Ile1 5 10 15Ala Val Gln
Pro Leu Thr Gly Val Leu Gly Ala Glu Ile Thr Gly Val 20 25 30Asp Leu
Arg Glu Pro Leu Asp Asp Ser Thr Trp Asn Glu Ile Leu Asp 35 40 45Ala
Phe His Thr Tyr Gln Val Ile Tyr Phe Pro Gly Gln Ala Ile Thr 50 55
60Asn Glu Gln His Ile Ala Phe Ser Arg Arg Phe Gly Pro Val Asp Pro65
70 75 80Val Pro Leu Leu Lys Ser Ile Glu Gly Tyr Pro Glu Val Gln Met
Ile 85 90 95Arg Arg Glu Ala Asn Glu Ser Gly Arg Val Ile Gly Asp Asp
Trp His 100 105 110Thr Asp Ser Thr Phe Leu Asp Ala Pro Pro Ala Ala
Val Val Met Arg 115 120 125Ala Ile Asp Val Pro Glu His Gly Gly Asp
Thr Gly Phe Leu Ser Met 130 135 140Tyr Thr Ala Trp Glu Thr Leu Ser
Pro Thr Met Gln Ala Thr Ile Glu145 150 155 160Gly Leu Asn Val Val
His Ser Ala Thr Arg Val Phe Gly Ser Leu Tyr 165 170 175Gln Ala Gln
Asn Arg Arg Phe Ser Asn Thr Ser Val Lys Val Met Asp 180 185 190Val
Asp Ala Gly Asp Arg Glu Thr Val His Pro Leu Val Val Thr His 195 200
205Pro Gly Ser Gly Arg Lys Gly Leu Tyr Val Asn Gln Val Tyr Cys Gln
210 215 220Arg Ile Glu Gly Met Thr Asp Ala Glu Ser Lys Pro Leu Leu
Gln Phe225 230 235 240Leu Tyr Glu His Ala Thr Arg Phe Asp Phe Thr
Cys Arg Val Arg Trp 245 250 255Lys Lys Asp Gln Val Leu Val Trp Asp
Asn Leu Cys Thr Met His Arg 260 265 270Ala Val Pro Asp Tyr Ala Gly
Lys Phe Arg Tyr Leu Thr Arg Thr Thr 275 280 285Val Gly Gly Val Arg
Pro Ala Arg 290 29512888DNABradyrhizobium japonicum USDA 110
12atgacgatcg ccatccggca gcttcagacg cattttgtcg gccaggtttc cggcctcgat
60ttgcgaaagc cgctcacgcc gggcgaggcc cgcgaggtcg agtccgccat ggacaaatac
120gcggtgctcg ttttccacga ccaggacatc accgacgagc agcagatggc
tttcgcgctg 180aacttcggcc agcgcgagga cgcgcgcggc ggcacggtca
ccaaggagaa ggactaccgg 240ctgcaatccg gcctgaacga cgtctccaat
ctcggcaagg acggcaagcc gctggccaag 300gacagccgca cgcacctgtt
caatctcggc aactgcctct ggcactccga cagctcgttc 360cgtcccattc
ccgcaaaatt ctcgctgctg tcggcgcgcg tggtgaaccc gacgggcggc
420aacaccgaat tcgcggacat gcgcgccgcc tatgacgcgc tcgacgacga
gaccaaggcc 480gaaatcgagg acctcgtctg cgagcactcg ctgatgtatt
cgcgcggctc gctcggcttc 540accgagtaca ccgacgaaga gaagcagatg
ttcaagccgg tcctgcaacg cctcgtgcgc 600acccatccgg tccaccgccg
caagtcgctg tatctctcgt cgcatgccgg caagatcgcc 660agcatgagcg
tgccggaggg gcggctgctg ttgcgcgatc tcaacgagca cgcgacgcag
720ccggaattcg tctacgtcca caaatggaag ctgcatgacc tcgtgatgtg
ggacaaccgc 780cagaccatgc accgcgtccg ccgctacgac cagtcccagc
cccgcgacat gcgccgcgcg 840acggtggcgg ggacggagcc gacggtgcag
cagcaggcgg cggagtag 88813289PRTBradyrhizobium japonicum USDA 110
13Met Thr Ile Ala Ile Arg Gln Leu Gln Thr His Phe Val Gly Gln Val1
5 10 15Ser Gly Leu Asp Leu Arg Lys Pro Leu Thr Pro Gly Glu Ala Arg
Glu 20 25 30Val Glu Ser Ala Met Asp Lys Tyr Ala Val Leu Val Phe His
Asp Gln 35 40 45Asp Ile Thr Asp Glu Gln Gln Met Ala Phe Ala Leu Asn
Phe Gly Gln 50 55 60Arg Glu Asp Ala Arg Gly Gly Thr Val Thr Lys Glu
Lys Asp Tyr Arg65 70 75 80Leu Gln Ser Gly Leu Asn Asp Val Ser Asn
Leu Gly Lys Asp Gly Lys 85 90 95Pro Leu Ala Lys Asp Ser Arg Thr His
Leu Phe Asn Leu Gly Asn Cys 100 105 110Leu Trp His Ser Asp Ser Ser
Phe Arg Pro Ile Pro Ala Lys Phe Ser 115 120 125Leu Leu Ser Ala Arg
Val Val Asn Pro Thr Gly Gly Asn Thr Glu Phe 130 135 140Ala Asp Met
Arg Ala Ala Tyr Asp Ala Leu Asp Asp Glu Thr Lys Ala145 150 155
160Glu Ile Glu Asp Leu Val Cys Glu His Ser Leu Met Tyr Ser Arg Gly
165 170 175Ser Leu Gly Phe Thr Glu Tyr Thr Asp Glu Glu Lys Gln Met
Phe Lys 180 185 190Pro Val Leu Gln Arg Leu Val Arg Thr His Pro Val
His Arg Arg Lys 195 200 205Ser Leu Tyr Leu Ser Ser His Ala Gly Lys
Ile Ala Ser Met Ser Val 210 215 220Pro Glu Gly Arg Leu Leu Leu Arg
Asp Leu Asn Glu His Ala Thr Gln225 230 235 240Pro Glu Phe Val Tyr
Val His Lys Trp Lys Leu His Asp Leu Val Met 245 250 255Trp Asp Asn
Arg Gln Thr Met His Arg Val Arg Arg Tyr Asp Gln Ser 260 265 270Gln
Pro Arg Asp Met Arg Arg Ala Thr Val Ala Gly Thr Glu Pro Thr 275 280
285Val1434DNAArtificial Sequencebrjap 5'(speI) 14actagtaaca
aagaaggaga tataccatga cgat 341533DNAArtificial Sequencebr jap 3'
(xhoI) 15ttctcgagct atcactccgc cgcctgctgc tgc 331617DNAArtificial
SequenceM13 forward primer 16gtaaaacgac ggccagt 171717DNAArtificial
SequenceM13 reverse primer 17caggaaacag ctatgac 171832DNAArtificial
SequenceNcoI of Brady 18tataccacat gtcgatcgcc atccggcagc tt
321933DNAArtificial SequenceSacI of Brady 19gagctcctat cactccgccg
cctgctgctg cac 3320525PRTGlycine max 20Met Ala Gln Val Ser Arg Val
His Asn Leu Ala Gln Ser Thr Gln Ile1 5 10 15Phe Gly His Ser Ser Asn
Ser Asn Lys Leu Lys Ser Val Asn Ser Val 20 25 30Ser Leu Arg Pro Arg
Leu Trp Gly Ala Ser Lys Ser Arg Ile Pro Met 35 40 45His Lys Asn Gly
Ser Phe Met Gly Asn Phe Asn Val Gly Lys Gly Asn 50 55 60Ser Gly Val
Phe Lys Val Ser Ala Ser Val Ala Ala Ala Glu Lys Pro65 70 75 80Ser
Thr Ser Pro Glu Ile Val Leu Glu Pro Ile Lys Asp Phe Ser Gly 85 90
95Thr Ile Thr Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile Leu Leu
100 105 110Leu Ala Ala Leu Ser Glu Gly Thr Thr Val Val Asp Asn Leu
Leu Tyr 115 120 125Ser Glu Asp Ile His Tyr Met Leu Gly Ala Leu Arg
Thr Leu Gly Leu 130 135 140Arg Val Glu Asp Asp Lys Thr Thr Lys Gln
Ala Ile Val Glu Gly Cys145 150 155 160Gly Gly Leu Phe Pro Thr Ser
Lys Glu Ser Lys Asp Glu Ile Asn Leu 165 170 175Phe Leu Gly Asn Ala
Gly Thr Ala Met Arg Pro Leu Thr Ala Ala Val 180 185 190Val Ala Ala
Gly Gly Asn Ala Ser Tyr Val Leu Asp Gly Val Pro Arg 195 200 205Met
Arg Glu Arg Pro Ile Gly Asp Leu Val Ala Gly Leu Lys Gln Leu 210 215
220Gly Ala Asp Val Asp Cys Phe Leu Gly Thr Asn Cys Pro Pro Val
Arg225 230 235 240Val Asn Gly Lys Gly Gly Leu Pro Gly Gly Lys Val
Lys Leu Ser Gly 245 250 255Ser Val Ser Ser Gln Tyr Leu Thr Ala Leu
Leu Met Ala Ala Pro Leu 260 265 270Ala Leu Gly Asp Val Glu Ile Glu
Ile Val Asp Lys Leu Ile Ser Val 275 280 285Pro Tyr Val Glu Met Thr
Leu Lys Leu Met Glu Arg Phe Gly Val Ser 290 295 300Val Glu His Ser
Gly Asn Trp Asp Arg Phe Leu Val His Gly Gly Gln305 310 315 320Lys
Tyr Lys Ser Pro Gly Asn Ala Phe Val Glu Gly Asp Ala Ser Ser 325 330
335Ala Ser Tyr Leu Leu Ala Gly Ala Ala Ile Thr Gly Gly Thr Ile Thr
340 345 350Val Asn Gly Cys Gly Thr Ser Ser Leu Gln Gly Asp Val Lys
Phe Ala 355 360 365Glu Val Leu Glu Lys Met Gly
Ala Lys Val Thr Trp Ser Glu Asn Ser 370 375 380Val Thr Val Ser Gly
Pro Pro Arg Asp Phe Ser Gly Arg Lys Val Leu385 390 395 400Arg Gly
Ile Asp Val Asn Met Asn Lys Met Pro Asp Val Ala Met Thr 405 410
415Leu Ala Val Val Ala Leu Phe Ala Asn Gly Pro Thr Ala Ile Arg Asp
420 425 430Val Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met Ile Ala
Ile Cys 435 440 445Thr Glu Leu Arg Lys Leu Gly Ala Thr Val Glu Glu
Gly Pro Asp Tyr 450 455 460Cys Val Ile Thr Pro Pro Glu Lys Leu Asn
Val Thr Ala Ile Asp Thr465 470 475 480Tyr Asp Asp His Arg Met Ala
Met Ala Phe Ser Leu Ala Ala Cys Gly 485 490 495Asp Val Pro Val Thr
Ile Lys Asp Pro Gly Cys Thr Arg Lys Thr Phe 500 505 510Pro Asp Tyr
Phe Glu Val Leu Glu Arg Leu Thr Lys His 515 520
52521525PRTArtificial sequenceDoubly mutated Soybean EPSPS protein
threonine 183 converted to isoleucine; proline 187 converted to
serine 21Met Ala Gln Val Ser Arg Val His Asn Leu Ala Gln Ser Thr
Gln Ile1 5 10 15Phe Gly His Ser Ser Asn Ser Asn Lys Leu Lys Ser Val
Asn Ser Val 20 25 30Ser Leu Arg Pro Arg Leu Trp Gly Ala Ser Lys Ser
Arg Ile Pro Met 35 40 45His Lys Asn Gly Ser Phe Met Gly Asn Phe Asn
Val Gly Lys Gly Asn 50 55 60Ser Gly Val Phe Lys Val Ser Ala Ser Val
Ala Ala Ala Glu Lys Pro65 70 75 80Ser Thr Ser Pro Glu Ile Val Leu
Glu Pro Ile Lys Asp Phe Ser Gly 85 90 95Thr Ile Thr Leu Pro Gly Ser
Lys Ser Leu Ser Asn Arg Ile Leu Leu 100 105 110Leu Ala Ala Leu Ser
Glu Gly Thr Thr Val Val Asp Asn Leu Leu Tyr 115 120 125Ser Glu Asp
Ile His Tyr Met Leu Gly Ala Leu Arg Thr Leu Gly Leu 130 135 140Arg
Val Glu Asp Asp Lys Thr Thr Lys Gln Ala Ile Val Glu Gly Cys145 150
155 160Gly Gly Leu Phe Pro Thr Ser Lys Glu Ser Lys Asp Glu Ile Asn
Leu 165 170 175Phe Leu Gly Asn Ala Gly Ile Ala Met Arg Ser Leu Thr
Ala Ala Val 180 185 190Val Ala Ala Gly Gly Asn Ala Ser Tyr Val Leu
Asp Gly Val Pro Arg 195 200 205Met Arg Glu Arg Pro Ile Gly Asp Leu
Val Ala Gly Leu Lys Gln Leu 210 215 220Gly Ala Asp Val Asp Cys Phe
Leu Gly Thr Asn Cys Pro Pro Val Arg225 230 235 240Val Asn Gly Lys
Gly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly 245 250 255Ser Val
Ser Ser Gln Tyr Leu Thr Ala Leu Leu Met Ala Ala Pro Leu 260 265
270Ala Leu Gly Asp Val Glu Ile Glu Ile Val Asp Lys Leu Ile Ser Val
275 280 285Pro Tyr Val Glu Met Thr Leu Lys Leu Met Glu Arg Phe Gly
Val Ser 290 295 300Val Glu His Ser Gly Asn Trp Asp Arg Phe Leu Val
His Gly Gly Gln305 310 315 320Lys Tyr Lys Ser Pro Gly Asn Ala Phe
Val Glu Gly Asp Ala Ser Ser 325 330 335Ala Ser Tyr Leu Leu Ala Gly
Ala Ala Ile Thr Gly Gly Thr Ile Thr 340 345 350Val Asn Gly Cys Gly
Thr Ser Ser Leu Gln Gly Asp Val Lys Phe Ala 355 360 365Glu Val Leu
Glu Lys Met Gly Ala Lys Val Thr Trp Ser Glu Asn Ser 370 375 380Val
Thr Val Ser Gly Pro Pro Arg Asp Phe Ser Gly Arg Lys Val Leu385 390
395 400Arg Gly Ile Asp Val Asn Met Asn Lys Met Pro Asp Val Ala Met
Thr 405 410 415Leu Ala Val Val Ala Leu Phe Ala Asn Gly Pro Thr Ala
Ile Arg Asp 420 425 430Val Ala Ser Trp Arg Val Lys Glu Thr Glu Arg
Met Ile Ala Ile Cys 435 440 445Thr Glu Leu Arg Lys Leu Gly Ala Thr
Val Glu Glu Gly Pro Asp Tyr 450 455 460Cys Val Ile Thr Pro Pro Glu
Lys Leu Asn Val Thr Ala Ile Asp Thr465 470 475 480Tyr Asp Asp His
Arg Met Ala Met Ala Phe Ser Leu Ala Ala Cys Gly 485 490 495Asp Val
Pro Val Thr Ile Lys Asp Pro Gly Cys Thr Arg Lys Thr Phe 500 505
510Pro Asp Tyr Phe Glu Val Leu Glu Arg Leu Thr Lys His 515 520
525221604DNAArtificial sequenceSoybean-biased DNA sequence encoding
doubly- mutated EPSPS disclosed in SEQ ID NO21, with added
sequencemisc_feature(1)..(1575)Coding sequence of soybean-biased
DNA sequence encoding the EPSPS protien of SEQ ID
NO21misc_feature(1576)..(1578)Translation terminator
tgamisc_feature(1579)..(1604)Sequence included to introduce
translation stop codons in all six open reading frames, and to
introduce a SacI restriction enzyme recognition site for cloning
purposes 22atggctcaag tctcccgtgt tcacaatctt gctcagtcaa cccaaatctt
tggacattca 60agcaactcaa acaaactgaa gtctgtgaat tctgtctcac ttcgcccacg
cctttgggga 120gcatccaaga gtcgcatacc aatgcacaag aatgggagtt
tcatgggcaa cttcaatgtt 180gggaaaggca attctggtgt cttcaaagtt
tcagcttctg ttgcagccgc agagaaaccc 240agcacttccc ctgagattgt
tcttgaaccc attaaggact tcagtggaac aatcactctg 300cctggatcaa
agagtctttc aaacagaata cttctcttgg cagctctgag tgaaggaacc
360actgtagttg acaacctttt gtactctgaa gatattcatt acatgttggg
tgctctcaga 420actcttgggt tgagagttga agatgacaag accacaaaac
aagccatagt tgaaggatgt 480ggtgggttgt ttccaacaag caaagaatcc
aaagatgaga tcaacttgtt tcttggcaat 540gctggaattg caatgagaag
cctcactgct gcagtagttg cagctggtgg gaatgcaagt 600tatgtccttg
atggtgtccc cagaatgagg gaaaggccca tcggtgacct tgtggctggc
660ctgaaacagc ttggagcaga tgttgattgc ttcttgggca caaactgccc
tccagtgaga 720gtgaatggga agggaggttt gcctggtgga aaggtcaaac
tgagtggatc agtctcttcc 780cagtatctga ctgccttgct catggctgcc
cctctggctt tgggtgatgt ggagattgaa 840atagtggaca agttgatttc
tgttccatat gtggaaatga ccctcaaact catggagagg 900tttggagttt
ctgttgaaca ttctggcaac tgggatcgtt tccttgtaca tggaggtcag
960aagtacaaaa gccctggcaa tgcctttgtt gaaggggatg caagctctgc
ttcctatctc 1020ttggctgggg ctgccatcac tggtgggacc atcactgtga
atggctgtgg cacctcatcc 1080cttcaaggtg atgtaaagtt tgcagaggtc
ttggagaaaa tgggtgccaa ggtcacctgg 1140tctgagaaca gtgtaactgt
gtctggacct cccagagact tcagtggcag aaaggttctc 1200cgtggaattg
atgtgaacat gaacaagatg ccagatgtgg ccatgaccct cgctgttgta
1260gccctgtttg caaatggacc aactgcaatc cgtgatgttg cttcatggag
ggtgaaggag 1320acagagagga tgattgccat ttgcacagaa ctccgcaaac
ttggtgcaac agttgaagag 1380ggaccagatt actgtgtgat aaccccacct
gagaagctca atgtgacagc cattgacacc 1440tatgatgacc acagaatggc
aatggctttc tcccttgctg cctgtggtga tgtgcctgtg 1500actatcaaag
accctgggtg cacaaggaag acatttccag actactttga agttttggag
1560aggttgacaa agcactgagt agttagctta atcacctaga gctc
16042320DNAArtificial sequencePrimer Pat 5-3 23agataccctt
ggttggttgc 202420DNAArtificial sequencePrimer Pat 3-3 24cagatggatc
gtttggaagg 202523DNAArtificial sequenceAAD-1 PTU forward primer
25ataatgccag cctgttaaac gcc 232623DNAArtificial sequenceAAD-1 PTU
reverse primer 26ctcaagcata tgaatgacct cga 232722DNAArtificial
sequenceForward primer for Coding Region PCR AAD-1 (RdpAcodF)
27atggctcatg ctgccctcag cc 222822DNAArtificial sequenceReverse
primer for Coding Region PCR AAD-1 (RdpAcodR) 28cgggcaggcc
taactccacc aa 2229932DNAArtificial sequenceAAD-2 v2 plant-optimized
nucleotide 29ccatggctac catagcaatc agacagctcc agacccactt tgtgggtcaa
gtttctggat 60tggacctcag aaagccactc actcctggag aagccagaga agttgaatca
gctatggaca 120agtacgcagt tcttgtcttc catgaccaag acatcacaga
tgagcaacag atggcctttg 180ccctcaactt tggtcagagg gaggatgcac
gtggtggcac tgtcaccaaa gagaaggatt 240accgtcttca gtctggcctc
aatgatgttt ccaacttggg caaagatgga aagccacttg 300ccaaggacag
ccgcacccat ttgttcaacc ttggaaactg cttgtggcat tctgactcca
360gcttcagacc aatcccagcc aagttcagcc tcctttctgc tcgtgttgtg
aacccaactg 420gtgggaacac tgagtttgct gacatgagag ctgcctatga
tgctcttgac gatgaaacca 480aagctgagat tgaggacctt gtgtgtgagc
actctctcat gtactcaagg ggctcacttg 540gcttcactga gtacacagat
gaagagaagc aaatgttcaa gcccgtcttg cagcgcttgg 600tccgcacaca
ccctgtgcac cgtcgcaaat cactctacct ctccagccat gccggaaaga
660ttgccagcat gtccgtccct gaagggaggc tccttttgag ggatttgaat
gaacatgcta 720ctcagcctga gttcgtctat gttcacaaat ggaagttgca
tgatcttgtg atgtgggaca 780ataggcaaac catgcacaga gtgaggagat
atgaccagtc ccaacccaga gacatgcgcc 840gtgcaacagt tgctgggacc
gagcccacag tgcaacagca agcagcagag tgagtagtta 900gcttaatcac
ctagagctcg gtcaccagat ct 93230296PRTArtificial sequenceTranslated
AAD-2 v2 protein 30Met Ala Thr Ile Ala Ile Arg Gln Leu Gln Thr His
Phe Val Gly Gln1 5 10 15Val Ser Gly Leu Asp Leu Arg Lys Pro Leu Thr
Pro Gly Glu Ala Arg 20 25 30Glu Val Glu Ser Ala Met Asp Lys Tyr Ala
Val Leu Val Phe His Asp 35 40 45Gln Asp Ile Thr Asp Glu Gln Gln Met
Ala Phe Ala Leu Asn Phe Gly 50 55 60Gln Arg Glu Asp Ala Arg Gly Gly
Thr Val Thr Lys Glu Lys Asp Tyr65 70 75 80Arg Leu Gln Ser Gly Leu
Asn Asp Val Ser Asn Leu Gly Lys Asp Gly 85 90 95Lys Pro Leu Ala Lys
Asp Ser Arg Thr His Leu Phe Asn Leu Gly Asn 100 105 110Cys Leu Trp
His Ser Asp Ser Ser Phe Arg Pro Ile Pro Ala Lys Phe 115 120 125Ser
Leu Leu Ser Ala Arg Val Val Asn Pro Thr Gly Gly Asn Thr Glu 130 135
140Phe Ala Asp Met Arg Ala Ala Tyr Asp Ala Leu Asp Asp Glu Thr
Lys145 150 155 160Ala Glu Ile Glu Asp Leu Val Cys Glu His Ser Leu
Met Tyr Ser Arg 165 170 175Gly Ser Leu Gly Phe Thr Glu Tyr Thr Asp
Glu Glu Lys Gln Met Phe 180 185 190Lys Pro Val Leu Gln Arg Leu Val
Arg Thr His Pro Val His Arg Arg 195 200 205Lys Ser Leu Tyr Leu Ser
Ser His Ala Gly Lys Ile Ala Ser Met Ser 210 215 220Val Pro Glu Gly
Arg Leu Leu Leu Arg Asp Leu Asn Glu His Ala Thr225 230 235 240Gln
Pro Glu Phe Val Tyr Val His Lys Trp Lys Leu His Asp Leu Val 245 250
255Met Trp Asp Asn Arg Gln Thr Met His Arg Val Arg Arg Tyr Asp Gln
260 265 270Ser Gln Pro Arg Asp Met Arg Arg Ala Thr Val Ala Gly Thr
Glu Pro 275 280 285Thr Val Gln Gln Gln Ala Ala Glu 290
2953122DNAArtificial sequenceSouthern fragment PCR AAD-1 forward
primer 31atggctcatg ctgccctcag cc 223221DNAArtificial
sequenceSouthern fragment PCR AAD-1 reverse primer 32gggcaggcct
aactccacca a 213384PRTArtificial
Sequencealpha-ketoglutarate-dependent dioxygenase
motifmisc_feature(2)..(2)Xaa can be any naturally occurring amino
acidMISC_FEATURE(3)..(3)Xaa at position 3 is aspartic acid or
glutamic acidmisc_feature(4)..(7)Xaa can be any naturally occurring
amino acidMISC_FEATURE(8)..(8)Xaa at position 8 is threonine or
serinemisc_feature(9)..(78)Xaa can be any naturally occurring amino
acidmisc_feature(80)..(83)Xaa can be any naturally occurring amino
acid 33His Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa His Xaa65 70 75 80Xaa Xaa Xaa Arg34171PRTArtificial
SequenceAAD-1 motifmisc_feature(2)..(2)Xaa can be any naturally
occurring amino acidmisc_feature(4)..(27)Xaa can be any naturally
occurring amino acidmisc_feature(29)..(159)Xaa can be any naturally
occurring amino acidmisc_feature(161)..(170)Xaa can be any
naturally occurring amino acid 34His Xaa Asp Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Thr Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105
110Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
115 120 125Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 130 135 140Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa His145 150 155 160Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Arg 165 17035175PRTArtificial SequenceAAD-1 motif
extendedmisc_feature(2)..(2)Xaa can be any naturally occurring
amino acidmisc_feature(4)..(27)Xaa can be any naturally occurring
amino acidmisc_feature(29)..(159)Xaa can be any naturally occurring
amino acidmisc_feature(161)..(170)Xaa can be any naturally
occurring amino acidmisc_feature(172)..(174)Xaa can be any
naturally occurring amino acid 35His Xaa Asp Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Thr Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105
110Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
115 120 125Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 130 135 140Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa His145 150 155 160Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Arg Xaa Xaa Xaa Arg 165 170 175
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