U.S. patent application number 14/254793 was filed with the patent office on 2014-08-07 for protein mixtures for maize insect control.
This patent application is currently assigned to E I DU PONT DE NEMOURS AND COMPANY. The applicant listed for this patent is E I DU PONT DE NEMOURS AND COMPANY, PIONEER HI BRED INTERNATIONAL INC. Invention is credited to John Lindsey Flexner, Deirdre Kapka-Kitzman, Lisa Procyk, Bruce H. Stanley, Jianzhou Zhao.
Application Number | 20140223609 14/254793 |
Document ID | / |
Family ID | 42352589 |
Filed Date | 2014-08-07 |
United States Patent
Application |
20140223609 |
Kind Code |
A1 |
Flexner; John Lindsey ; et
al. |
August 7, 2014 |
PROTEIN MIXTURES FOR MAIZE INSECT CONTROL
Abstract
Embodiments of the present invention relate to insecticidal
Bacillus thuringiensis Cry1 and Cry2 polypeptides. Methods for
using the polypeptides and nucleic acids of embodiments of the
invention to synergistically enhance resistance of plants to insect
predation are encompassed in embodiments of the present
invention.
Inventors: |
Flexner; John Lindsey;
(Landenberg, PA) ; Kapka-Kitzman; Deirdre;
(Ankeny, IA) ; Procyk; Lisa; (Ankeny, IA) ;
Stanley; Bruce H.; (Wilmington, DE) ; Zhao;
Jianzhou; (Johnston, IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
E I DU PONT DE NEMOURS AND COMPANY
PIONEER HI BRED INTERNATIONAL INC |
Wilmington
Johnston |
DE
IA |
US
US |
|
|
Assignee: |
E I DU PONT DE NEMOURS AND
COMPANY
Wilmington
DE
PIONEER HI BRED INTERNATIONAL INC
Johnston
IA
|
Family ID: |
42352589 |
Appl. No.: |
14/254793 |
Filed: |
April 16, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12691841 |
Jan 22, 2010 |
8729336 |
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14254793 |
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61146875 |
Jan 23, 2009 |
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Current U.S.
Class: |
800/300 ;
424/115; 504/116.1; 514/143; 514/229.2; 514/355; 514/359; 514/406;
514/408; 514/478; 514/531; 514/634; 800/302 |
Current CPC
Class: |
Y02A 40/146 20180101;
C12N 15/8274 20130101; C12N 15/8286 20130101; Y02A 40/162 20180101;
A01N 63/10 20200101; A01N 63/10 20200101; A01N 63/10 20200101; A01N
63/10 20200101; A01N 2300/00 20130101; A01N 63/10 20200101; A01N
63/10 20200101; A01N 63/10 20200101; A01N 2300/00 20130101 |
Class at
Publication: |
800/300 ;
800/302; 514/531; 504/116.1; 514/229.2; 514/355; 424/115; 514/406;
514/359; 514/634; 514/478; 514/143; 514/408 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Claims
1. A method of reducing pest damage in a transgenic plant
comprising: planting a first transgenic plant seed, wherein the
first transgenic plant seed comprises a first transgene and a
second transgene, wherein the first transgene causes expression of
a Cry1 protein in a plant, wherein the Cry1 protein is selected
from the group consisting of (a) SEQ ID NO:1, (b) SEQ ID NO:4, and
(c) a polypeptide that is at least 90% identical to the amino acid
sequence of SEQ ID NO:1 or SEQ ID NO:4; and the second transgene
causes expression of a Cry2 protein in a plant, wherein the Cry2
protein is selected from the group consisting of (a) SEQ ID NO:2
and (b) a polypeptide that is at least 90% identical to the amino
acid sequence of SEQ ID NO:2; thereby reducing damage caused by a
first target pest to a plant grown from the first transgenic plant
seed.
2. The method of claim 1 wherein the transgenic plant is maize.
3. The method of claim 1 wherein the first target pest is a member
of order Lepidoptera.
4. The method of claim 3 wherein the first target pest is selected
from the group consisting of European corn borer and corn
earworm.
5. The method of claim 1 further comprising treating the first
transgenic plant seed with a pesticidal agent.
6. The method of claim 5 wherein the pesticidal agent is selected
from the group consisting of: an insecticide, an acaricide, a
nematicide, a fungicide, a bactericide, a herbicide, or a
combination thereof.
7. The method of claim 6 wherein the pesticidal agent is an
insecticide.
8. The method of claim 7 wherein the insecticide is selected from
the group consisting of: a pyrethrin, a synthetic pyrethrin, an
oxadizine, a chloronicotinyl, a nitroguanidine, a triazole, an
organophosphate, a pyrrol, a pyrazole, a phenol pyrazole, a
diacylhydrazine, a biological/fermentation product, a carbamate, or
a combination thereof.
9. The method of claim 1 wherein the first transgenic plant seed
further comprises a herbicide resistance gene.
10. The method of claim 9 wherein the herbicide resistance gene is
selected from the group consisting of: glyphosate
N-acetyltransferase (GAT), 5-enolpyruvylshikimate-3-phosphate
synthase (EPSPS), phosphinothricin N-acetyltransferase (PAT) or a
combination thereof.
11. A method for providing synergistic insecticidal activity
against at least one pest comprising: providing a first transgenic
plant, wherein the first transgenic plant expresses a Cry1-derived
insecticidal polypeptide and a Cry2-derived insecticidal
polypeptide, wherein the Cry1-derived insecticidal polypeptide
comprises a polypeptide selected from the group consisting of: (a)
SEQ ID NO:1 and (b) SEQ ID NO:4, and (c) a polypeptide that is at
least 90% identical to the amino acid sequence of SEQ ID NO:1 or
SEQ ID NO:4; and wherein the Cry2-derived insecticidal polypeptide
comprises a polypeptide selected from the group consisting of (a)
SEQ ID NO:2 and (b) a polypeptide that is at least 90% identical to
the amino acid sequence of SEQ ID NO:2; thereby resulting in
synergistic insect resistance against a first target pest.
12. The method of claim 11 wherein the transgenic plant is
maize.
13. The method of claim 11 wherein the first target pest is a
member of order Lepidoptera.
14. The method of claim 13 wherein the first target pest is
selected from the group consisting of European corn borer and corn
earworm.
15. The method of claim 11 further comprising treating the first
transgenic plant seed with a pesticidal agent.
16. The method of claim 15 wherein the pesticidal agent is selected
from the group consisting of: an insecticide, an acaricide, a
nematicide, a fungicide, a bactericide, a herbicide, or a
combination thereof.
17. The method of claim 16 wherein the pesticidal agent is an
insecticide.
18. The method of claim 17 wherein the insecticide is selected from
the group consisting of: a pyrethrin, a synthetic pyrethrin, an
oxadizine, a chloronicotinyl, a nitroguanidine, a triazole, an
organophosphate, a pyrrol, a pyrazole, a phenol pyrazole, a
diacylhydrazine, a biological/fermentation product, a carbamate, or
a combination thereof.
19. The method of claim 11 wherein the first transgenic plant
further comprises a herbicide resistance gene.
20. The method of claim 19 wherein the herbicide resistance gene is
selected from the group consisting of: glyphosate
N-acetyltransferase (GAT), 5-enolpyruvylshikimate-3-phosphate
synthase (EPSPS), phosphinothricin N-acetyltransferase (PAT) or a
combination thereof.
21. A method of reducing pest damage in a transgenic plant
comprising: planting a first transgenic plant seed, wherein the
first transgenic plant seed comprises a first transgene and a
second transgene, wherein the first transgene causes expression of
a Cry1 protein in a plant and the second transgene causes
expression of a Cry2 protein in a plant, the Cry1 protein selected
from the group consisting of the polypeptide of SEQ ID NO:1 and the
polypeptide of SEQ ID NO:4; and the Cry2 protein comprising the
polypeptide of SEQ ID NO: 2, thereby reducing damage caused by a
first target pest to a plant grown from the first transgenic plant
seed.
22. The method of claim 21 wherein the transgenic plant is
maize.
23. The method of claim 21 wherein the first target pest is a
member of order Lepidoptera.
24. The method of claim 23 wherein the first target pest is
selected from the group consisting of European corn borer and corn
earworm.
25. A transgenic plant comprising a first transgene and a second
transgene, wherein the first transgene causes expression of a Cry1
protein in a plant and the second transgene causes expression of a
Cry2 protein in a plant.
26. The transgenic plant of claim 25, wherein the Cry1 protein
selected from the group consisting of the polypeptide of SEQ ID
NO:1 and the polypeptide of SEQ ID NO:4; and wherein the Cry2
protein comprises the polypeptide of SEQ ID NO: 2.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn.119
to provisional application Ser. No. 61/146,875 filed Jan. 23, 2009,
herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] Embodiments of the present invention relate generally to the
field of pest control, providing insecticidal polypeptides related
to combinations of Bacillus thuringiensis (B. t.) Cry1 and Cry2
polypeptides and the polynucleotides that encode them. Embodiments
of the present invention also relate to methods and compositions
for improved resistance of plants to insect predation, including,
but not limited to, transgenic plant production. The Cry1 and Cry2
polypeptide mixtures provide improved insecticidal activity and
synergism against key plant pests, including maize pests.
BACKGROUND OF THE INVENTION
[0003] Numerous commercially valuable plants, including common
agricultural crops, are susceptible to attack by insect and
nematode pests, causing substantial reductions in crop yield and
quality. For example, growers of maize (Zea mays), commonly
referred to as corn in the United States, face a major problem with
combating pest infestations. Insects, nematodes, and related
arthropods annually destroy an estimated 15% of agricultural crops
in the United States and an even greater percentage in developing
countries. In addition, competition with weeds and parasitic and
saprophytic plants account for even more potential yield losses.
Yearly, such pests cause over $100 billion in crop damage in the
United States alone.
[0004] In an effort to combat pest infestations, various methods
have been employed in order to reduce or eliminate pests in a
particular plot. These efforts include rotating corn with other
crops that are not a host for a particular pest and applying
pesticides to the above-ground portion of the crop, applying
pesticides to the soil in and around the root systems of the
affected crop. Traditionally, farmers have relied heavily on
chemical pesticides to combat pest damage. However, the use of
chemical pesticides is costly, as farmers apply billions of gallons
of synthetic pesticides to combat these pests each growing season,
costing nearly $8 billion. In addition, such pesticides are
inconvenient for farmers, result in the emergence of
insecticide-resistant pests, and they raise significant
environmental and health concerns.
[0005] Because of concern about the impact of pesticides on public
health and the health of the environment, significant efforts have
been made to find ways to reduce the amount of chemical pesticides
that are used. Recently, much of this effort has focused on the
development of transgenic crops that are engineered to express
insect toxicants derived from microorganisms. For example, U.S.
Pat. No. 5,877,012 to Estruch et al. discloses the cloning and
expression of proteins from such organisms as Bacillus,
Pseudomonas, Clavibacter and Rhizobium into plants to obtain
transgenic plants with resistance to such pests as black cutworms,
armyworms, several borers and other insect pests. Publication
WO/EP97/07089 by Privalle et al. teaches the transformation of
monocotyledons, such as corn, with a recombinant DNA sequence
encoding peroxidase for the protection of the plant from feeding by
corn borers, earworms and cutworms. Jansens et al., Crop Sci.,
37(5):1616-1624 (1997), reported the production of transgenic corn
containing a gene encoding a crystalline protein from Bt that
controlled both generations of Eastern Corn Borer (ECB). U.S. Pat.
Nos. 5,625,136 and 5,859,336 to Koziel et al. reported that the
transformation of corn with a gene from Bt that encoded for a
.delta.-endotoxin provided the transgenic corn with improved
resistance to ECB. Additionally, a comprehensive report of field
trials of transgenic corn that expresses an insecticidal protein
from Bt has been provided by Armstrong et al., Crop Science,
35(2):550-557 (1995).
[0006] For these and other reasons, there is a demand for
alternative insecticidal agents for agricultural crops. For
example, maize plants incorporating transgenic genes which cause
the maize plant to produce insecticidal proteins providing
protection from the target pest(s) is a more environmentally
friendly approach to controlling pests. The use of pesticidal
crystal proteins derived from the soil bacterium Bt commonly
referred to as "Cry proteins" have been utilized. Cry proteins are
globular protein molecules which accumulate as protoxins in
crystalline form during late stage of the sporulation of Bt. After
ingestion by the pest, the crystals are solubilized to release
protoxins in the alkaline midgut environment of the larvae.
Protoxins (.about.130 kDa) are converted into mature toxic
fragments (.about.66 kDa N terminal region) by gut proteases. Many
of these proteins are quite toxic to specific target insects, but
harmless to plants and other non-targeted organisms. Some Cry
proteins have been recombinantly expressed in crop plants to
provide pest-resistant transgenic plants. Among those,
Bt-transgenic cotton and corn have been widely cultivated.
[0007] A large number of Cry proteins have been isolated,
characterized and classified based on amino acid sequence homology.
See Crickmore et al., Microbiol. Mol. Biol. Rev., 62:807-813
(1998). This classification scheme provides a systematic mechanism
for naming and categorizing newly discovered Cry proteins. Bt
toxins have traditionally been categorized by their specific
toxicity towards specific insect categories. For example, the Cry1
group of toxins is toxic to Lepidoptera, and includes, but is not
limited to, Cry1Aa, Cry1Ab and Cry1Ac. See Hofte et al., Microbiol.
Rev., 53:242-255 (1989). The Cry1 classification is the best known
and contains the highest number of cry genes, currently totals over
130. Cry1 and Cry2 proteins share a minimal amount of sequence
homology. See, e.g., Crickmore et al. (1998) indicating that Cry1A
and Cry2A classes are among the most divergent.
[0008] It has generally been found that individual Cry proteins
possess relatively narrow activity spectra. For example, Cry1Ac was
the first toxin to be deployed in transgenic cotton for control of
H. virescens and H. zea insect pests. This toxin is known for its
high level toxicity to H. virescens. However, it is slightly
deficient in its ability to control H. zea and has almost no
activity on Spodoptera species. Additionally, Cry1Ab toxin has
slightly less activity on H. zea than Cry1Ac but has far superior
activity against S. exigua.
[0009] Cry2A is an exception as it is unusual in that this subset
of Cry proteins possesses a broader effective range that includes
toxicity to both the Lepidoptera and Diptera orders of insects. The
Cry2A protein was discovered to be a toxin showing a dual activity
against Trichoplusia ni (cabbage looper) and Aedes taeniorhynchus
(mosquito) (Yamamoto & McLaughlin, Biochem. Biophys. Res.
Comm., 130:414-421 (1982)). The nucleic acid molecule encoding the
Cry2A protein (termed Cry2Aa) was cloned and expressed in B.
megaterium and found to be active against both Lepidoptera and
Diptera insects (Donovan et al., J. Bacteriol., 170:4732-4738
(1988)). An additional coding sequence homologous to Cry2Aa was
cloned (termed Cry2Ab) and was found to be active only against
Lepidoptera larvae (Widner & Whiteley, J. Bacteriol.,
171(2):965-974 (1989)).
[0010] Second generation transgenic crops could be more resistant
to insects if they are able to express multiple, novel and/or
synergistic Bt genes.
[0011] Accordingly, it is an objective of embodiments of the
present invention to provide synergistic resistance to plant
insects.
[0012] Another objective of embodiments of the invention includes
methods for incorporating multiple Cry proteins into transgenic
plants, namely maize.
SUMMARY OF THE INVENTION
[0013] Embodiments of the present invention relate to Cry
polypeptides derived from Bt Cry1 and Cry2 polypeptides and
provides a novel means for improved and synergistic insecticidal
resistance against key crop pests. Embodiments of the present
invention also relate to transgenic plants expressing such a
nucleic acid and/or polypeptide. The transgenic plants can express
the transgene in any way known in the art, including, but not
limited to, constitutive expression, developmentally regulated
expression, tissue specific expression, etc. Additionally, seed
obtained from a transgenic plant of the invention is also
encompassed.
BRIEF DESCRIPTION OF THE DRAWING
[0014] FIG. 1 shows the sequences for the IP1-88 and IP2-127 Cry
proteins, as well as the DNA and amino acid sequence for a Cry1 (h)
protein relevant to the disclosure.
DETAILED DESCRIPTION
[0015] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which embodiments of this invention
belong. Unless mentioned otherwise, the techniques employed or
contemplated herein are standard methodologies well known to one of
ordinary skill in the art. The materials, processes and examples
described in the description are illustrative only and not intended
to be limiting to the scope of the claims in any manner. Many
modifications and other embodiments of the inventions set forth
herein will come to mind to one skilled in the art to which these
inventions pertain having the benefit of the teachings presented in
the foregoing descriptions and the associated drawings. Therefore,
it is to be understood that the disclosure is not to be limited to
the specific embodiments disclosed and that modifications and other
embodiments are intended to be included within the scope of the
appended claims.
[0016] As used herein, "pesticidal agent" or "pesticide" includes
any organism, organic substance, or inorganic substance that has
pesticidal activity. As used herein, the term "pesticidal activity"
refers to activity of an organism or a substance (such as, for
example, a protein) that can be measured by, but is not limited to,
pest mortality, pest weight loss, pest repellency, and other
behavioral and physical changes of a pest after feeding and
exposure for an appropriate length of time. Thus, an organism or
substance having pesticidal activity adversely impacts at least one
measurable parameter of pest fitness. Preferably, pesticidal
activity results in reduced damage to a plant with such a
pesticidal agent as compared with plants lacking such pesticidal
agent. If the pesticidal agent's target pest is an insect, it is
referred to as an "insecticide." If the pesticidal agent's target
pest is a mite, it is referred to as an "acaricide." If the
pesticidal agent's target pest is a mite, it is referred to as a
"nematicide". If the pesticidal agent's target pest is a fungus, it
is referred to as a "fungicide." If the pesticidal agent's target
pest is a bacterium, it is referred to as a "bactericide." If the
pesticidal agent's target pest is a plant, it is referred to as a
"herbicide."
[0017] Combinations of pesticidal agents can have one of three
effects on pesticidal activity: antagonistic, additive, or
synergistic. If the observed pesticidal activity of the two
pesticidal agents together is approximately the expected pesticidal
activity of the combination, the combination is said to be
"additive." If the observed pesticidal activity of the two
pesticidal agents together is less than the expected pesticidal
activity of the combination, the combination is said to be
"antagonistic." If the observed pesticidal activity of the two
pesticidal agents together is greater than the expected pesticidal
activity of the combination, the combination is said to be
"synergistic." The expected pesticidal activity for a given
combination of pesticidal agents is determined by the following
method. If X is the observed level of pesticidal activity of
pesticidal agent A alone and Y is the observed level of pesticidal
activity of pesticidal agent B alone, the expected pesticidal
activity of pesticidal agents A and B in combination (assuming the
level of pesticidal activity is measured on a scale from 0 to 100)
is X+Y-(X*Y)/100.
[0018] Embodiments of the invention may show an increase of
pesticidal activity of a given combination of 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% or
greater against the insect target as compared to the expected
pesticidal activity of the combination.
[0019] As used herein, "nucleic acid" includes reference to a
deoxyribonucleotide or ribonucleotide polymer in either single- or
double-stranded form, and unless otherwise limited, encompasses
known analogues (e.g., peptide nucleic acids) having the essential
nature of natural nucleotides in that they hybridize to
single-stranded nucleic acids in a manner similar to that of
naturally occurring nucleotides.
[0020] As used herein, the terms "encoding" or "encoded" when used
in the context of a specified nucleic acid mean that the nucleic
acid comprises the requisite information to direct translation of
the nucleotide sequence into a specified protein. The information
by which a protein is encoded is specified by the use of codons. A
nucleic acid encoding a protein may comprise non-translated
sequences (e.g., introns) within translated regions of the nucleic
acid or may lack such intervening non-translated sequences (e.g.,
as in cDNA).
[0021] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residues is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers.
[0022] The terms "residue" or "amino acid residue" or "amino acid"
are used interchangeably herein to refer to an amino acid that is
incorporated into a protein, polypeptide, or peptide (collectively
"protein"). The amino acid may be a naturally occurring amino acid
and, unless otherwise limited, may encompass known analogues of
natural amino acids that can function in a similar manner as
naturally occurring amino acids.
[0023] Polypeptides of the embodiments can be produced either from
a nucleic acid disclosed herein, or by the use of standard
molecular biology techniques. For example, a protein of the
embodiments can be produced by expression of a recombinant nucleic
acid of the embodiments in an appropriate host cell, or
alternatively by a combination of ex vivo procedures.
[0024] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides:
(a) "reference sequence," (b) "comparison window," (c) "sequence
identity," (d) "percentage of sequence identity," and (e)
"substantial identity." [0025] (a) As used herein, "reference
sequence" is a defined sequence used as a basis for sequence
comparison. A reference sequence may be a subset or the entirety of
a specified sequence; for example, as a segment of a full-length
cDNA or gene sequence, or the complete cDNA or gene sequence.
[0026] (b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. Generally, the
comparison window is at least 20 contiguous nucleotides in length,
and optionally can be 30, 40, 50, 100, or longer. Those of skill in
the art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide sequence a
gap penalty is typically introduced and is subtracted from the
number of matches.
[0027] Methods of alignment of sequences for comparison are well
known in the art. Thus, the determination of percent sequence
identity between any two sequences can be accomplished using a
mathematical algorithm. Non-limiting examples of such mathematical
algorithms are the algorithm of Myers and Miller (1988) CABIOS
4:11-17; the local alignment algorithm of Smith et al. (1981) Adv.
Appl. Math. 2:482; the global alignment algorithm of Needleman and
Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local
alignment method of Pearson and Lipman (1988) Proc. Natl. Acad.
Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)
Proc. Natl. Acad. Sci. USA 872264, as modified in Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
[0028] Computer implementations of these mathematical algorithms
can be utilized for comparison of sequences to determine sequence
identity. Such implementations include, but are not limited to:
CLUSTAL in the PC/Gene program (available from Intelligenetics,
Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP,
BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics
Software Package, Version 10 (available from Accelrys Inc., 9685
Scranton Road, San Diego, Calif., USA). Alignments using these
programs can be performed using the default parameters. The CLUSTAL
program is well described by Higgins et al. (1988) Gene 73:237-244
(1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al.
(1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS
8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.
The ALIGN program is based on the algorithm of Myers and Miller
(1988) supra. A PAM120 weight residue table, a gap length penalty
of 12, and a gap penalty of 4 can be used with the ALIGN program
when comparing amino acid sequences. The BLAST programs of Altschul
et al. (1990) J. Mol. Biol. 215:403 are based on the algorithm of
Karlin and Altschul (1990) supra. BLAST nucleotide searches can be
performed with the BLASTN program, score=100, wordlength=12, to
obtain nucleotide sequences homologous to a nucleotide sequence
encoding a protein of the embodiments. BLAST protein searches can
be performed with the BLASTX program, score=50, wordlength=3, to
obtain amino acid sequences homologous to a protein or polypeptide
of the embodiments. To obtain gapped alignments for comparison
purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described
in Altschul et al. (1997) Nucleic Acids Res. 25:3389.
Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an
iterated search that detects distant relationships between
molecules. See Altschul et al. (1997) supra. When utilizing BLAST,
Gapped BLAST, PSI-BLAST, the default parameters of the respective
programs (e.g., BLASTN for nucleotide sequences, BLASTX for
proteins) can be used, as described on the National Center for
Biotechnology Information website. Alignment may also be performed
manually by inspection.
[0029] Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using GAP Version 10
using the following parameters: % identity and % similarity for a
nucleotide sequence using GAP Weight of 50 and Length Weight of 3,
and the nwsgapdna.cmp scoring matrix; % identity and % similarity
for an amino acid sequence using GAP Weight of 8 and Length Weight
of 2, and the BLOSUM62 scoring matrix; or any equivalent program
thereof. The term "equivalent program" as used herein refers to any
sequence comparison program that, for any two sequences in
question, generates an alignment having identical nucleotide or
amino acid residue matches and an identical percent sequence
identity when compared to the corresponding alignment generated by
GAP Version 10.
[0030] GAP uses the algorithm of Needleman and Wunsch (1970) supra,
to find the alignment of two complete sequences that maximizes the
number of matches and minimizes the number of gaps. GAP considers
all possible alignments and gap positions and creates the alignment
with the largest number of matched bases and the fewest gaps. It
allows for the provision of a gap creation penalty and a gap
extension penalty in units of matched bases. GAP must make a profit
of gap creation penalty number of matches for each gap it inserts.
If a gap extension penalty greater than zero is chosen, GAP must,
in addition, make a profit for each gap inserted of the length of
the gap times the gap extension penalty. Default gap creation
penalty values and gap extension penalty values in Version 10 of
the GCG Wisconsin Genetics Software Package for protein sequences
are 8 and 2, respectively. For nucleotide sequences the default gap
creation penalty is 50 while the default gap extension penalty is
3. The gap creation and gap extension penalties can be expressed as
an integer selected from the group of integers consisting of from 0
to 200. Thus, for example, the gap creation and gap extension
penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65 or greater.
[0031] GAP presents one member of the family of best alignments.
There may be many members of this family, but no other member has a
better quality. GAP displays four figures of merit for alignments:
Quality, Ratio, Identity, and Similarity. The Quality is the metric
maximized in order to align the sequences. Ratio is the quality
divided by the number of bases in the shorter segment. Percent
Identity is the percent of the symbols that actually match. Percent
Similarity is the percent of the symbols that are similar. Symbols
that are across from gaps are ignored. A similarity is scored when
the scoring matrix value for a pair of symbols is greater than or
equal to 0.50, the similarity threshold. The scoring matrix used in
Version 10 of the GCG Wisconsin Genetics Software Package is
BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci.
USA 89:10915). [0032] (c) As used herein, "sequence identity" or
"identity" in the context of two nucleic acid or polypeptide
sequences makes reference to the residues in the two sequences that
are the same when aligned for maximum correspondence over a
specified comparison window. When percentage of sequence identity
is used in reference to proteins it is recognized that residue
positions which are not identical often differ by conservative
amino acid substitutions, where amino acid residues are substituted
for other amino acid residues with similar chemical properties
(e.g., charge or hydrophobicity) and therefore do not change the
functional properties of the molecule. When sequences differ in
conservative substitutions, the percent sequence identity may be
adjusted upwards to correct for the conservative nature of the
substitution. Sequences that differ by such conservative
substitutions are said to have "sequence similarity" or
"similarity". Means for making this adjustment are well known to
those of skill in the art. Typically this involves scoring a
conservative substitution as a partial rather than a full mismatch,
thereby increasing the percentage sequence identity. Thus, for
example, where an identical amino acid is given a score of 1 and a
non-conservative substitution is given a score of zero, a
conservative substitution is given a score between zero and 1. The
scoring of conservative substitutions is calculated, e.g., as
implemented in the program PC/GENE (Intelligenetics, Mountain View,
Calif.). [0033] (d) As used herein, "percentage of sequence
identity" means the value determined by comparing two optimally
aligned sequences over a comparison window, wherein the portion of
the polynucleotide sequence in the comparison window may comprise
additions or deletions (i.e., gaps) as compared to the reference
sequence (which does not comprise additions or deletions) for
optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison, and multiplying the result by 100 to yield
the percentage of sequence identity. [0034] (e)(i) The term
"substantial identity" of polynucleotide sequences means that a
polynucleotide comprises a sequence that has at least 70%, 80%,
90%, or 95% or more sequence identity when compared to a reference
sequence using one of the alignment programs described using
standard parameters. One of skill in the art will recognize that
these values can be appropriately adjusted to determine
corresponding identity of proteins encoded by two nucleotide
sequences by taking into account codon degeneracy, amino acid
similarity, reading frame positioning, and the like. Substantial
identity of amino acid sequences for these purposes generally means
sequence identity of at least 60%, 70%, 80%, 90%, or 95% or more
sequence identity.
[0035] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each other
under stringent conditions. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the Tm for the
specific sequence at a defined ionic strength and pH. However,
stringent conditions encompass temperatures in the range of about
1.degree. C. to about 20.degree. C. lower than the Tm, depending
upon the desired degree of stringency as otherwise qualified
herein. Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides they encode are substantially identical. This may
occur, e.g., when a copy of a nucleic acid is created using the
maximum codon degeneracy permitted by the genetic code. One
indication that two nucleic acid sequences are substantially
identical is when the polypeptide encoded by the first nucleic acid
is immunologically cross reactive with the polypeptide encoded by
the second nucleic acid.
[0036] (e)(ii) The term "substantial identity" in the context of a
peptide indicates that a peptide comprises a sequence with at least
70%, 80%, 85%, 90%, 95%, or more sequence identity to a reference
sequence over a specified comparison window. Optimal alignment for
these purposes can be conducted using the global alignment
algorithm of Needleman and Wunsch (1970) supra. An indication that
two peptide sequences are substantially identical is that one
peptide is immunologically reactive with antibodies raised against
the second peptide. Thus, a peptide is substantially identical to a
second peptide, for example, where the two peptides differ only by
a conservative substitution. Peptides that are "substantially
similar" share sequences as noted above except that residue
positions that are not identical may differ by conservative amino
acid changes.
[0037] The term "toxin" as used herein refers to a polypeptide
showing pesticidal activity or insecticidal activity or improved
pesticidal activity or improved insecticidal activity. "Bt" or
"Bacillus thuringiensis" toxin is intended to include the broader
class of Cry toxins found in various strains of Bt, which includes
such toxins as, for example, Cry1s, Cry2s, or Cry3s.
[0038] The terms "proteolytic site" or "cleavage site" refer to an
amino acid sequence which confers sensitivity to a class of
proteases or a particular protease such that a polypeptide
containing the amino acid sequence is digested by the class of
proteases or particular protease. A proteolytic site is said to be
"sensitive" to the protease(s) that recognize that site. It is
appreciated in the art that the efficiency of digestion will vary,
and that a decrease in efficiency of digestion can lead to an
increase in stability or longevity of the polypeptide in an insect
gut. Thus, a proteolytic site may confer sensitivity to more than
one protease or class of proteases, but the efficiency of digestion
at that site by various proteases may vary. Proteolytic sites
include, for example, trypsin sites, chymotrypsin sites, and
elastase sites.
[0039] Research has shown that the insect gut proteases of
Lepidopterans include trypsins, chymotrypsins, and elastases. See,
e.g., Lenz et al. (1991) Arch. Insect Biochem. Physiol. 16:
201-212; and Hedegus et al. (2003) Arch. Insect Biochem. Physiol.
53: 30-47. For example, about 18 different trypsins have been found
in the midgut of Helicoverpa armigera larvae (see Gatehouse et al.
(1997) Insect Biochem. Mol. Biol. 27: 929-944). The preferred
proteolytic substrate sites of these proteases have been
investigated. See, e.g., Peterson et al. (1995) Insect Biochem.
Mol. Biol. 25: 765-774.
[0040] Efforts have been made to understand the mechanism of action
of Bt toxins and to engineer toxins with improved properties. It
has been shown that insect gut proteases can affect the impact of
Bt Cry proteins on the insect. Some proteases activate the Cry
proteins by processing them from a "protoxin" form into a toxic
form, or "toxin." See Oppert (1999) Arch. Insect Biochem. Phys. 42:
1-12; Carroll et al. (1997) J. Invertebrate Pathology 70: 41-49.
This activation of the toxin can include the removal of the N- and
C-terminal peptides from the protein and can also include internal
cleavage of the protein. Other proteases can degrade the Cry
proteins. See Oppert, ibid.
[0041] A comparison of the amino acid sequences of Cry toxins of
different specificities reveals five highly-conserved sequence
blocks. Structurally, the toxins comprise three distinct domains
which are, from the N- to C-terminus: a cluster of seven
alpha-helices implicated in pore formation (referred to as "domain
1"), three anti-parallel beta sheets implicated in cell binding
(referred to as "domain 2"), and a beta sandwich (referred to as
"domain 3"). The location and properties of these domains are known
to those of skill in the art. See, e.g., Li et al. (1991) Nature,
305:815-821; Morse et al. (2001) Structure, 9:409-417. When
reference is made to a particular domain, such as domain 1, it is
understood that the exact endpoints of the domain with regard to a
particular sequence are not critical so long as the sequence or
portion thereof includes sequence that provides at least some
function attributed to the particular domain. Thus, for example,
when referring to "domain 1," it is intended that a particular
sequence includes a cluster of seven alpha-helices, but the exact
endpoints of the sequence used or referred to with regard to that
cluster are not critical. One of skill in the art is familiar with
the determination of such endpoints and the evaluation of such
functions.
[0042] In an effort to better characterize and improve Bt toxins,
strains of the bacterium Bt have been studied. An effort was
undertaken to identify the nucleotide sequences encoding the
crystal proteins from the selected strains, and the wild-type
(i.e., naturally occurring) nucleic acids of the embodiments were
isolated from these bacterial strains, cloned into an expression
vector, and transformed into E coli. Depending upon the
characteristics of a given preparation, it was recognized that the
demonstration of pesticidal activity sometimes required trypsin
pretreatment to activate the pesticidal proteins. Thus, it is
understood that some pesticidal proteins require protease digestion
(e.g., by trypsin, chymotrypsin, and the like) for activation,
while other proteins are biologically active (e.g., pesticidal) in
the absence of activation.
[0043] Such molecules may be altered by means described, for
example, in U.S. application Ser. No. 10/606,320, filed Jun. 25,
2003, and Ser. No. 10/746,914, filed Dec. 24, 2003. In addition,
nucleic acid sequences may be engineered to encode polypeptides
that contain additional mutations that confer improved or altered
pesticidal activity relative to the pesticidal activity of the
naturally occurring polypeptide. The nucleotide sequences of such
engineered nucleic acids comprise mutations not found in the wild
type sequences.
[0044] The mutant polypeptides of the embodiments are generally
prepared by a process that involves the steps of: obtaining a
nucleic acid sequence encoding a Cry family polypeptide; analyzing
the structure of the polypeptide to identify particular "target"
sites for mutagenesis of the underlying gene sequence based on a
consideration of the proposed function of the target domain in the
mode of action of the toxin; introducing one or more mutations into
the nucleic acid sequence to produce a desired change in one or
more amino acid residues of the encoded polypeptide sequence; and
assaying the polypeptide produced for pesticidal activity.
[0045] Many of the Bt insecticidal toxins are related to various
degrees by similarities in their amino acid sequences and tertiary
structure and means for obtaining the crystal structures of Bt
toxins are well known. Exemplary high-resolution crystal structure
solution of both the Cry3A and Cry3B polypeptides are available in
the literature. The solved structure of the Cry3A gene (Li et al.
(1991) Nature 353:815-821) provides insight into the relationship
between structure and function of the toxin. A combined
consideration of the published structural analyses of Bt toxins and
the reported function associated with particular structures,
motifs, and the like indicates that specific regions of the toxin
are correlated with particular functions and discrete steps of the
mode of action of the protein. For example, many toxins isolated
from Bt are generally described as comprising three domains: a
seven-helix bundle that is involved in pore formation, a
three-sheet domain that has been implicated in receptor binding,
and a beta-sandwich motif (Li et al. (1991) Nature 305:
815-821).
[0046] As reported in U.S. Pat. No. 7,105,332, and pending U.S.
application Ser. No. 10/746,914, filed Dec. 24, 2003, the toxicity
of Cry proteins can be improved by targeting the region located
between alpha helices 3 and 4 of domain 1 of the toxin. This theory
was premised on a body of knowledge concerning insecticidal toxins,
including: 1) that alpha helices 4 and 5 of domain 1 of Cry3A
toxins had been reported to insert into the lipid bilayer of cells
lining the midgut of susceptible insects (Gazit et al. (1998) Proc.
Natl. Acad. Sci. USA 95: 12289-12294); 2) the inventors' knowledge
of the location of trypsin and chymotrypsin cleavage sites within
the amino acid sequence of the wild-type protein; 3) the
observation that the wild-type protein was more active against
certain insects following in vitro activation by trypsin or
chymotrypsin treatment; and 4) reports that digestion of toxins
from the 3' end resulted in decreased toxicity to insects.
[0047] A series of mutations may be created and placed in a variety
of background sequences to create novel polypeptides having
enhanced or altered pesticidal activity. See, e.g., U.S.
application Ser. No. 10/606,320, filed Jun. 25, 2003, now
abandoned, and Ser. No. 10/746,914, filed Dec. 24, 2003. These
mutants include, but are not limited to: the addition of at least
one more protease-sensitive site (e.g., trypsin cleavage site) in
the region located between helices 3 and 4 of domain 1; the
replacement of an original protease-sensitive site in the wild-type
sequence with a different protease-sensitive site; the addition of
multiple protease-sensitive sites in a particular location; the
addition of amino acid residues near protease-sensitive site(s) to
alter folding of the polypeptide and thus enhance digestion of the
polypeptide at the protease-sensitive site(s); and adding mutations
to protect the polypeptide from degradative digestion that reduces
toxicity (e.g., making a series of mutations wherein the wild-type
amino acid is replaced by valine to protect the polypeptide from
digestion). Mutations may be used singly or in any combination to
provide polypeptides of the embodiments.
[0048] In this manner, the embodiments provide sequences comprising
a variety of mutations, such as, for example, a mutation that
comprises an additional, or an alternative, protease-sensitive site
located between alpha-helices 3 and 4 of domain 1 of the encoded
polypeptide. A mutation which is an additional or alternative
protease-sensitive site may be sensitive to several classes of
proteases such as serine proteases, which include trypsin and
chymotrypsin, or enzymes such as elastase. Thus, a mutation which
is an additional or alternative protease-sensitive site may be
designed so that the site is readily recognized and/or cleaved by a
category of proteases, such as mammalian proteases or insect
proteases. A protease-sensitive site may also be designed to be
cleaved by a particular class of enzymes or a particular enzyme
known to be produced in an organism, such as, for example, a
chymotrypsin produced by the corn earworm Heliothis zea (Lenz et
al. (1991) Arch. Insect Biochem. Physiol. 16: 201-212). Mutations
may also confer resistance to proteolytic digestion, for example,
to digestion by chymotrypsin at the C-terminus of the peptide.
[0049] The presence of an additional and/or alternative
protease-sensitive site in the amino acid sequence of the encoded
polypeptide can improve the pesticidal activity and/or specificity
of the polypeptide encoded by the nucleic acids of the embodiments.
Accordingly, the nucleotide sequences of the embodiments can be
recombinantly engineered or manipulated to produce polypeptides
having improved or altered insecticidal activity and/or specificity
compared to that of an unmodified wild-type toxin. In addition,
mutations may be placed in or used in conjunction with other
nucleotide sequences to provide improved properties. For example, a
protease-sensitive site that is readily cleaved by insect
chymotrypsin, e.g., a chymotrypsin found in the bertha armyworm or
the corn earworm (Hegedus et al. (2003) Arch. Insect Biochem.
Physiol. 53: 30-47; and Lenz et al. (1991) Arch. Insect Biochem.
Physiol. 16: 201-212), may be placed in a Cry background sequence
to provide improved toxicity to that sequence. In this manner, the
embodiments provide toxic polypeptides with improved
properties.
[0050] For example, a mutagenized Cry nucleotide sequence can
comprise additional mutants that comprise additional codons that
introduce a second trypsin-sensitive amino acid sequence (in
addition to the naturally occurring trypsin site) into the encoded
polypeptide. An alternative addition mutant of the embodiments
comprises additional codons designed to introduce at least one
additional different protease-sensitive site into the polypeptide,
for example, a chymotrypsin-sensitive site located immediately 5'
or 3' of the naturally occurring trypsin site. Alternatively,
substitution mutants may be created in which at least one codon of
the nucleic acid that encodes the naturally occurring
protease-sensitive site is destroyed and alternative codons are
introduced into the nucleic acid sequence in order to provide a
different (e.g., substitute) protease-sensitive site. A replacement
mutant may also be added to a Cry sequence in which the
naturally-occurring trypsin cleavage site present in the encoded
polypeptide is destroyed and a chymotrypsin or elastase cleavage
site is introduced in its place.
[0051] It is recognized that any nucleotide sequence encoding the
amino acid sequences that are proteolytic sites or putative
proteolytic sites (for example, sequences such as NGSR, RR, or LKM)
can be used and that the exact identity of the codons used to
introduce any of these cleavage sites into a variant polypeptide
may vary depending on the use, i.e., expression in a particular
plant species. It is also recognized that any of the disclosed
mutations can be introduced into any polynucleotide sequence of the
embodiments that comprises the codons for amino acid residues that
provide the native trypsin cleavage site that is targeted for
modification. Accordingly, variants of either full-length toxins or
fragments thereof can be modified to contain additional or
alternative cleavage sites, and these embodiments are intended to
be encompassed by the scope of the embodiments disclosed
herein.
[0052] It will be appreciated by those of skill in the art that any
useful mutation may be added to the sequences of the embodiments so
long as the encoded polypeptides retain pesticidal activity. Thus,
sequences may also be mutated so that the encoded polypeptides are
resistant to proteolytic digestion by chymotrypsin. More than one
recognition site can be added in a particular location in any
combination, and multiple recognition sites can be added to or
removed from the toxin. Thus, additional mutations can comprise
three, four, or more recognition sites. It is to be recognized that
multiple mutations can be engineered in any suitable polynucleotide
sequence; accordingly, either full-length sequences or fragments
thereof can be modified to contain additional or alternative
cleavage sites as well as to be resistant to proteolytic digestion.
In this manner, the embodiments provide Cry toxins containing
mutations that improve pesticidal activity as well as improved
compositions and methods for impacting pests using other Bt
toxins.
[0053] Mutations may protect the polypeptide from protease
degradation, for example by removing putative proteolytic sites
such as putative serine protease sites and elastase recognition
sites from different areas. Some or all of such putative sites may
be removed or altered so that proteolysis at the location of the
original site is decreased. Changes in proteolysis may be assessed
by comparing a mutant polypeptide with wild-type toxins or by
comparing mutant toxins which differ in their amino acid sequence.
Putative proteolytic sites and proteolytic sites include, but are
not limited to, the following sequences: RR, a trypsin cleavage
site; LKM, a chymotrypsin site; and NGSR, a trypsin site. These
sites may be altered by the addition or deletion of any number and
kind of amino acid residues, so long as the pesticidal activity of
the polypeptide is increased. Thus, polypeptides encoded by
nucleotide sequences comprising mutations will comprise at least
one amino acid change or addition relative to the native or
background sequence, or 2, 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, 32,
35, 38, 40, 45, 47, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,
150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, or
280 or more amino acid changes or additions. Pesticidal activity of
a polypeptide may also be improved by truncation of the native or
full-length sequence, as is known in the art.
[0054] Embodiments of the present invention provide insecticidal
polypeptides related to Bt Cry1 and Cry2 polypeptides. Nucleic acid
molecules encoding the polypeptides are also provided. Methods for
using the polypeptides and nucleic acids to enhance resistance of
plants to insect predation are encompassed.
[0055] The combination of a Cry1 and Cry2 protein yields a
synergistic effect against a plurality of target pests, providing
greater than expected mortality and/or resistance to a plurality of
target pests. Prior art has in fact taught away from the disclosed
embodiments, indicating that synergism is not an expected outcome
for insecticidal activity. Others skilled in the art have indicated
that such combinations result in the antagonism, rather than
synergism, effect on Helicoverpa armigera (Liao, C. et al.,
Toxicity of B. thuringiensis insecticidal proteins of Helicoverpa
armigera and H. punctigera, major pests of cotton, J. Invertebrate
Pathology 80:55-63 (2002)). Others have found reported tests using
Cry1 and Cry2 proteins with reported synergism effects (Ding et
al., Expression and synergism of two cry insecticidal protein genes
in P. fluorescens, Chinese J. of Microbiol., 40:573-578
(2000)).
Methods of Enhancing Insect Resistance in Plants
[0056] Embodiments of the present invention provide methods of
enhancing plant resistance to insect pests including, but not
limited to, members of order Lepidoptera, the Helicoverpa ssp.
(e.g., Helicoverpa Zea and Heliothis virescens), and/or Spodoptera
ssp. (e.g., Spodoptera exigua, Spodoptera frugiperda) through the
use of Cry1-derived insecticidal polypeptides combined with
Cry2-derived insecticidal polypeptides to produce a synergistic
effect. Any method known in the art can be used to cause the insect
pests to ingest one or more polypeptides during the course of
feeding on the plant. As such, the insect pest will ingest
insecticidal amounts of the one or more polypeptides of embodiments
of the invention and may discontinue feeding on the plant. In some
embodiments, the insect pest is killed by ingestion of the one or
more polypeptides. In other embodiments, the insect pests are
inhibited or discouraged from feeding on the plant without being
killed.
[0057] In one embodiment, transgenic plants can be made to express
one or more polypeptides. The transgenic plant may express the one
or more polypeptides in all tissues (e.g., global expression).
Alternatively, the one or more polypeptides may be expressed in
only a subset of tissues (e.g., tissue specific expression),
preferably those tissues consumed by the insect pest. Polypeptides
that are embodiments of the invention can be expressed
constitutively in the plant or be under the control of an inducible
promoter. Polypeptides that are embodiments of the invention may be
expressed in the plant cytosol or in the plant chloroplast either
by protein targeting or by transformation of the chloroplast
genome.
[0058] In another embodiment, a composition comprising one or more
polypeptides of embodiments of the invention can be applied
externally to a plant susceptible to the insect pests. External
application of the composition includes direct application to the
plant, either in whole or in part, and/or indirect application,
e.g., to the environment surrounding the plant such as the soil.
The composition can be applied by any method known in the art
including, but not limited to, spraying, dusting, sprinkling, or
the like. In general, the composition can be applied at any time
during plant growth. One skilled in the art can use methods known
in the art to determine empirically the optimal time for
administration of the composition. Factors that affect optimal
administration time include, but are not limited to, the type of
susceptible plant, the type of insect pest, which one or more
polypeptides are administered in the composition.
[0059] The composition comprising one or more polypeptides may be
substantially purified polypeptides, a cell suspension, a cell
pellet, a cell supernatant, a cell extract, or a spore-crystal
complex of Bt cells. The composition comprising one or more
polypeptides embodying the invention may be in the form of a
solution, an emulsion, a suspension, or a powder. Liquid
formulations may be aqueous or non-aqueous based and may be
provided as foams, gels, suspensions, emulsifiable concentrates, or
the like. The formulations may include agents in addition to the
one or more polypeptides embodying the invention. For example,
compositions may further comprise spreader-sticker adjuvants,
stabilizing agents, other insecticidal additives, diluents, agents
that optimize the rheological properties or stability of the
composition, such as, for example, surfactants, emulsifiers,
dispersants, or polymers.
[0060] In another embodiment, recombinant hosts that express one or
more polypeptides that are embodiments of the invention are applied
on or near a plant susceptible to attack by an insect pest. The
recombinant hosts include, but are not limited to, microbial hosts
and insect viruses that have been transformed with and express one
or more nucleic acid molecules (and thus polypeptides) of
embodiments of the invention. In some embodiments, the recombinant
host secretes the polypeptide into its surrounding environment so
as to contact an insect pest. In other embodiments, the recombinant
hosts colonize one or more plant tissues susceptible to insect
infestation.
[0061] The nucleotide sequences of the embodiments can also be used
to isolate corresponding sequences from other organisms,
particularly other bacteria, and more particularly other Bacillus
strains. In this manner, methods such as PCR, hybridization, and
the like can be used to identify such sequences based on their
sequence homology to the sequences set forth herein. Sequences that
are selected based on their sequence identity to the entire
sequences set forth herein or to fragments thereof are encompassed
by the embodiments. Such sequences include sequences that are
orthologs of the disclosed sequences. The term "orthologs" refers
to genes derived from a common ancestral gene and which are found
in different species as a result of speciation. Genes found in
different species are considered orthologs when their nucleotide
sequences and/or their encoded protein sequences share substantial
identity as defined elsewhere herein. Functions of orthologs are
often highly conserved among species.
[0062] In a PCR approach, oligonucleotide primers can be designed
for use in PCR reactions to amplify corresponding DNA sequences
from cDNA or genomic DNA extracted from any organism of interest.
Methods for designing PCR primers and PCR cloning are generally
known in the art and are disclosed in Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor
Laboratory Press, Plainview, N.Y.), hereinafter "Sambrook". See
also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods
and Applications (Academic Press, New York); Innis and Gelfand,
eds. (1995) PCR Strategies (Academic Press, New York); and Innis
and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New
York). Known methods of PCR include, but are not limited to,
methods using paired primers, nested primers, single specific
primers, degenerate primers, gene-specific primers, vector-specific
primers, partially-mismatched primers, and the like.
[0063] In hybridization techniques, all or part of a known
nucleotide sequence is used as a probe that selectively hybridizes
to other corresponding nucleotide sequences present in a population
of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or
cDNA libraries) from a chosen organism. The hybridization probes
may be genomic DNA fragments, cDNA fragments, RNA fragments, or
other oligonucleotides, and may be labeled with a detectable group
such as 32P or any other detectable marker. Thus, for example,
probes for hybridization can be made by labeling synthetic
oligonucleotides based on the sequences of the embodiments. Methods
for preparation of probes for hybridization and for construction of
cDNA and genomic libraries are generally known in the art and are
disclosed in Sambrook.
[0064] For example, an entire sequence disclosed herein, or one or
more portions thereof, may be used as a probe capable of
specifically hybridizing to corresponding sequences and messenger
RNAs. To achieve specific hybridization under a variety of
conditions, such probes include sequences that are unique to the
sequences of the embodiments and are generally at least about 10 or
20 nucleotides in length. Such probes may be used to amplify
corresponding Cry sequences from a chosen organism by PCR. This
technique may be used to isolate additional coding sequences from a
desired organism or as a diagnostic assay to determine the presence
of coding sequences in an organism. Hybridization techniques
include hybridization screening of plated DNA libraries (either
plaques or colonies; see, for example, Sambrook).
[0065] Hybridization of such sequences may be carried out under
stringent conditions. The term "stringent conditions" or "stringent
hybridization conditions" as used herein refers to conditions under
which a probe will hybridize to its target sequence to a detectably
greater degree than to other sequences (e.g., at least 2-fold,
5-fold, or 10-fold over background). Stringent conditions are
sequence-dependent and will be different in different
circumstances. By controlling the stringency of the hybridization
and/or washing conditions, target sequences that are 100%
complementary to the probe can be identified (homologous probing).
Alternatively, stringency conditions can be adjusted to allow some
mismatching in sequences so that lower degrees of similarity are
detected (heterologous probing). Generally, a probe is less than
about 1000 or 500 nucleotides in length.
[0066] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide. Exemplary low stringency conditions
include hybridization with a buffer solution of 30 to 35%
formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37.degree.
C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M
NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary
moderate stringency conditions include hybridization in 40 to 45%
formamide, 1.0 M NaCl, 1% SDS at 37.degree. C., and a wash in
0.5.times. to 1.times.SSC at 55 to 60.degree. C. Exemplary high
stringency conditions include hybridization in 50% formamide, 1 M
NaCl, 1% SDS at 37.degree. C., and a final wash in 0.1.times.SSC at
60 to 65.degree. C. for at least about 20 minutes. Optionally, wash
buffers may comprise about 0.1% to about 1% SDS. The duration of
hybridization is generally less than about 24 hours, usually about
4 to about 12 hours.
[0067] Specificity is typically the function of post-hybridization
washes, the critical factors being the ionic strength and
temperature of the final wash solution. For DNA-DNA hybrids, the Tm
(thermal melting point) can be approximated from the equation of
Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:
Tm=81.5.degree. C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L;
where M is the molarity of monovalent cations, % GC is the
percentage of guanosine and cytosine nucleotides in the DNA, "%
form" is the percentage of formamide in the hybridization solution,
and L is the length of the hybrid in base pairs. The Tm is the
temperature (under defined ionic strength and pH) at which 50% of a
complementary target sequence hybridizes to a perfectly matched
probe. Washes are typically performed at least until equilibrium is
reached and a low background level of hybridization is achieved,
such as for 2 hours, 1 hour, or 30 minutes.
[0068] Tm is reduced by about 1.degree. C. for each 1% of
mismatching; thus, Tm, hybridization, and/or wash conditions can be
adjusted to hybridize to sequences of the desired identity. For
example, if sequences with >90% identity are sought, the Tm can
be decreased 10.degree. C. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the Tm for the
specific sequence and its complement at a defined ionic strength
and pH. However, severely stringent conditions can utilize a
hybridization and/or wash at 1, 2, 3, or 4.degree. C. lower than
the Tm; moderately stringent conditions can utilize a hybridization
and/or wash at 6, 7, 8, 9, or 10.degree. C. lower than the Tm; low
stringency conditions can utilize a hybridization and/or wash at
11, 12, 13, 14, 15, or 20.degree. C. lower than the Tm.
[0069] Using the equation, hybridization and wash compositions, and
desired Tm, those of ordinary skill will understand that variations
in the stringency of hybridization and/or wash solutions are
inherently described. If the desired degree of mismatching results
in a Tm of less than 45.degree. C. (aqueous solution) or 32.degree.
C. (formamide solution), the SSC concentration can be increased so
that a higher temperature can be used. An extensive guide to the
hybridization of nucleic acids is found in Tijssen (1993)
Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2
(Elsevier, New York); and Ausubel et al., eds. (1995) Current
Protocols in Molecular Biology, Chapter 2 (Greene Publishing and
Wiley-Interscience, New York). See also Sambrook. Thus, isolated
sequences that encode a Cry protein of the embodiments and
hybridize under stringent conditions to the Cry sequences disclosed
herein, or to fragments thereof, are encompassed by the
embodiments.
[0070] Preferably, a Cry1 and Cry2 polypeptide are produced by a
transgenic plant, thereby making the plant resistant to attack from
a target pest and providing synergistic resistance to at least one
target pest. A discussion of production of such transgenic plants
is provided below.
Production of Transgenic Plants
[0071] Any method known in the art can be used for transforming a
plant or plant cell with a nucleic acid molecule of an embodiment
of the present invention. Nucleic acid molecules can be
incorporated into plant DNA (e.g., genomic DNA or chloroplast DNA)
or be maintained without insertion into the plant DNA (e.g.,
through the use of artificial chromosomes). Suitable methods of
introducing nucleic acid molecules into plant cells include
microinjection (Crossway et al., Biotechniques 4:320-334 (1986));
electroporation (Riggs et al., Proc. Natl. Acad. Sci., 83:5602-5606
(1986); D'Halluin et al., Plant Cell, 4:1495-1505 (1992));
Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and
5,981,840; Osjoda et al., Nature Biotechnology, 14:745-750 (1996);
Horsch et al., Science, 233:496-498 (1984); Fraley et al., Proc.
Natl. Acad. Sci., 80:4803 (1983); Futterer et al., Gene transfer to
plants, 213-263 (Potrykus 1995); direct gene transfer (Paszkowski
et al., EMBO J. 3:2717-2722 (1984)); ballistic particle
acceleration (U.S. Pat. Nos. 4,945,050, 5,879,918, 5,886,244, and
5,932,782; Tomes et al., Direct DNA Transfer into Intact Plant
Cells via Microprojectile Bombardment, Plant Cell, Tissue, and
Organ Culture: Fundamental Methods (Gamborg & Phillips 1995);
and McCabe et al., Biotechnology, 6:923-926 (1988)); virus-mediated
transformation (U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785,
5,589,367 and 5,316,931); pollen transformation (De Wet et al.,
Experimental Manipulation of Ovule Tissues, 197-209 (Chapman et al.
1985)); Lec 1 transformation (U.S. patent application Ser. No.
09/435,054; International Publication No. WO 00/28058);
whisker-mediated transformation (Kaeppler et al., Plant Cell
Reports, 9:415-418 (1990); Kaeppler et al., Theor. Appl. Genet.,
84:560-566 (1992)); and chloroplast transformation technology
(Bogorad, Trends in Biotechnology, 18:257-263 (2000); Ramesh et
al., Methods Mol Biol., 274:301-7 (2004); Hou et al., Transgenic
Res., 12:111-4 (2003); Kindle et al., Proc. Natl. Acad. Sci.,
88:1721-5 (1991); Bateman & Purton, Mol Gen Genet., 263:404-10
(2000); Sidorov et al., Plant J., 19:209-216 (1999)).
[0072] The choice of transformation protocols used for generating
transgenic plants and plant cells can vary depending on the type of
plant or plant cell, i.e., monocot or dicot, targeted for
transformation. Examples of transformation protocols particularly
suited for a particular plant type include those for: potato (Tu et
al., Plant Molecular Biology, 37:829-838 (1998); Chong et al.,
Transgenic Research, 9:71-78 (2000)); soybean (Christou et al.,
Plant Physiol., 87:671-674 (1988); McCabe et al., BioTechnology,
6:923-926 (1988); Finer & McMullen, In Vitro Cell Dev. Biol.,
27P:175-182 (1991); Singh et al., Theor. Appl. Genet., 96:319-324
(1998)); maize (Klein et al., Proc. Natl. Acad. Sci., 85:4305-4309
(1988); Klein et al., Biotechnology, 6:559-563 (1988); Klein et
al., Plant Physiol., 91:440-444 (1988); Fromm et al.,
Biotechnology, 8:833-839 (1990); Tomes et al., Direct DNA Transfer
into Intact Plant Cells via Microprojectile Bombardment, Plant
Cell, Tissue, and Organ Culture: Fundamental Methods (Gamborg &
Phillips 1995)); and cereals (Hooykaas-Van Slogteren et al., Nature
311:763-764 (1984); U.S. Pat. No. 5,736,369).
[0073] In some embodiments, more than one construct is used for
transformation in the generation of transgenic plants and plant
cells. Multiple constructs may be included in cis or trans
positions. In preferred embodiments, each construct has a promoter
and other regulatory sequences. Embodiments of the invention relate
to combinations of different Cry1 and Cry2 proteins resulting in a
synergistic effect against target pests such as those disclosed
herein. By way of example, the Cry1 protein may be the polypeptide
disclosed in SEQ ID NO:1 or 4, or a polypeptide that is at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.75%
identical to the polypeptide of SEQ ID NO:1 or 4. By way of further
example, the Cry2 protein may be the polypeptide disclosed in SEQ
ID NO:2, or a polypeptide that is at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, 99.5%, or 99.75% identical to the
polypeptide of SEQ ID NO:2. As a result, a nucleic acid encoding
such a Cry1 (such as, for example, the nucleic acid disclosed in
SEQ ID NO:3) or Cry2 protein may be used in such a construct.
[0074] Transformed plant cells which are derived by any of the
above transformation techniques can be cultured to regenerate a
whole plant which possesses the transformed genotype and thus the
desired phenotype. Such regeneration techniques rely on
manipulation of certain phytohormones in a tissue culture growth
medium, typically relying on a biocide and/or herbicide marker that
has been introduced together with the desired nucleotide sequences.
Plant regeneration from cultured protoplasts is described in the
art (e.g., Evans et al., Protoplasts Isolation and Culture,
Handbook of Plant Cell Culture, 124-176 (MacMillilan Publishing Co.
1983); and Binding, Regeneration of Plants, Plant Protoplasts,
21-73 (CRC Press, 1985). Regeneration can also be obtained from
plant callus, explants, organs, or parts thereof. Such regeneration
techniques are also described in the art (e.g., Klee et al., Ann.
Rev. of Plant Phys., 38:467-486 (1987)).
[0075] The term "plant" includes whole plants, shoot vegetative
organs/structures (e.g., leaves, stems and tubers), roots, flowers
and floral organs/structures (e.g., bracts, sepals, petals,
stamens, carpels, anthers and ovules), seed (including embryo,
endosperm, and seed coat) and fruit (the mature ovary), plant
tissue (e.g., vascular tissue, ground tissue, and the like) and
cells (e.g. guard cells, egg cells, trichomes and the like), and
progeny of same. The class of plants that can be used in
embodiments of the present invention includes the class of higher
and lower plants amenable to transformation techniques, including
angiosperms (monocotyledonous and dicotyledonous plants),
gymnosperms, ferns, and multicellular algae. Plants of a variety of
ploidy levels, including aneuploid, polyploid, diploid, haploid and
hemizygous plants are also included.
[0076] Embodiments of the invention may use nucleic acid molecules
to confer desired traits on essentially any plant. Thus,
embodiments of the invention have use over a broad range of plants,
including species from the genera Allium, Ananas, Anacardium,
Apium, Arachis, Asparagus, Athamantha, Atropa, Avena, Bambusa,
Beta, Brassica, Bromus, Browallia, Camellia, Cannabis, Carica,
Ceratonia, Cicer, Chenopodium, Chicorium, Citrus, Citrullus,
Capsicum, Carthamus, Cocos, Coffea, Coix, Cucumis, Cucurbita,
Cynodon, Dactylis, Datura, Daucus, Dianthus, Digitalis, Dioscorea,
Elaeis, Eliusine, Euphorbia, Festuca, Ficus, Fragaria, Geranium,
Glycine, Graminae, Gossypium, Helianthus, Heterocallis, Hevea,
Hibiscus, Hordeum, Hyoscyamus, Ipomoea, Lactuca, Lathyrus, Lens,
Lilium, Linum, Lolium, Lotus, Lupinus, Lycopersicon, Macadamia,
Macrophylla, Malus, Mangifera, Manihot, Majorana, Medicago, Musa,
Narcissus, Nemesia, Nicotiana, Onobrychis, Olea, Olyreae, Oryza,
Panicum, Panieum, Pannisetum, Petunia, Pelargonium, Persea,
Pharoideae, Phaseolus, Phleum, Picea, Poa, Pinus, Pistachia, Pisum,
Populus, Pseudotsuga, Pyrus, Prunus, Pseutotsuga, Psidium, Quercus,
Ranunculus, Raphanus, Ribes, Ricinus, Rhododendron, Rosa,
Saccharum, Salpiglossis, Secale, Senecio, Setaria, Sequoia,
Sinapis, Solanum, Sorghum, Stenotaphrum, Theobromus, Trigonella,
Trifolium, Triticum, Tsuga, Tulipa, Vicia, Vitis, Vigna, and
Zea.
[0077] In specific embodiments, transgenic plants are maize,
potato, rice, soybean, alfalfa, sunflower, canola, or cotton
plants.
[0078] Transgenic plants may be grown and pollinated with either
the same transformed strain or different strains. Two or more
generations of the plants may be grown to ensure that expression of
the desired nucleic acid molecule, polypeptide and/or phenotypic
characteristic is stably maintained and inherited. One of ordinary
skill in the art will recognize that after the nucleic acid
molecule of embodiments of the present invention is stably
incorporated in transgenic plants and confirmed to be operable, it
can be introduced into other plants by sexual crossing. Any of a
number of standard breeding techniques can be used, depending upon
the species to be crossed.
[0079] In certain embodiments the polynucleotides of the
embodiments can be stacked with any combination of polynucleotide
sequences of interest in order to create plants with a desired
trait. For example, the polynucleotides of the embodiments may be
stacked with any other polynucleotides encoding polypeptides having
pesticidal and/or insecticidal activity, such as other Bt toxic
proteins (described in, for example, U.S. Pat. Nos. 5,366,892;
5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al.,
Gene, 48: 109 (1986)), lectins (Van Damme et al., Plant Mol. Biol.,
24: 825 (1994)), pentin (described in U.S. Pat. No. 5,981,722), and
the like. The combinations generated can also include multiple
copies of any one of the polynucleotides of interest. The
polynucleotides of the embodiments can also be stacked with any
other gene or combination of genes to produce plants with a variety
of desired trait combinations including, but not limited to, traits
desirable for animal feed such as high oil genes (e.g., U.S. Pat.
No. 6,232,529); balanced amino acids (e.g., hordothionins (U.S.
Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409); barley
high lysine (Williamson et al., Eur. J. Biochem., 165: 99-106
(1987); and WO 98/20122) and high methionine proteins (Pedersen et
al., J. Biol. Chem., 261: 6279 (1986); Kirihara et al., Gene, 71:
359 (1988); and Musumura et al., Plant Mol. Biol., 12: 123 (1989));
increased digestibility (e.g., modified storage proteins (U.S. Pat.
No. 6,858,778); and thioredoxins (U.S. Pat. No. 7,009,087)); the
disclosures of which are herein incorporated by reference in their
entirety.
[0080] The polynucleotides of the embodiments can also be stacked
with traits desirable for disease or herbicide resistance (e.g.,
fumonisin detoxification genes (U.S. Pat. No. 5,792,931);
avirulence and disease resistance genes (Jones et al., Science,
266: 789 (1994); Martin et al., Science, 262: 1432 (1993);
Mindrinos et al., Cell, 78:1089 (1994)); acetolactate synthase
(ALS) mutants that lead to herbicide resistance such as the S4
and/or Hra mutations; genes encoding resistance to inhibitors of
glutamine synthase such as phosphinothricin or basta (e.g., bar or
PAT genes); and glyphosate resistance (EPSPS and GAT (glyphosate
acetyl transferase) genes (Castle et al., Science, 304:1151
(2004))); and traits desirable for processing or process products
such as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils
(e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO
94/11516)); modified starches (e.g., ADPG pyrophosphorylases
(AGPase), starch synthases (SS), starch branching enzymes (SBE),
and starch debranching enzymes (SDBE)); and polymers or bioplastics
(e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase,
polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase
(Schubert et al., J. Bacteriol., 170:5837-5847 (1988)) facilitate
expression of polyhydroxyalkanoates (PHAs)); the disclosures of
which are herein incorporated by reference. One could also combine
the polynucleotides of the embodiments with polynucleotides
providing agronomic traits such as male sterility (see, e.g., U.S.
Pat. No. 5,583,210), stalk strength, flowering time, or
transformation technology traits such as cell cycle regulation or
gene targeting (e.g., WO 99/61619, WO 00/17364, and WO 99/25821);
the disclosures of which are herein incorporated by reference.
[0081] These stacked combinations can be created by any method
including, but not limited to, cross-breeding plants by any
conventional or TopCross methodology, or genetic transformation. If
the sequences are stacked by genetically transforming the plants,
the polynucleotide sequences of interest can be combined at any
time and in any order. For example, a transgenic plant comprising
one or more desired traits can be used as the target to introduce
further traits by subsequent transformation. The traits can be
introduced simultaneously in a co-transformation protocol with the
polynucleotides of interest provided by any combination of
transformation cassettes. For example, if two sequences will be
introduced, the two sequences can be contained in separate
transformation cassettes (trans) or contained on the same
transformation cassette (cis). Expression of the sequences can be
driven by the same promoter or by different promoters. In certain
cases, it may be desirable to introduce a transformation cassette
that will suppress the expression of the polynucleotide of
interest. This may be combined with any combination of other
suppression cassettes or over expression cassettes to generate the
desired combination of traits in the plant. It is further
recognized that polynucleotide sequences can be stacked at a
desired genomic location using a site-specific recombination
system. See, e.g., WO 99/25821, WO 99/25854, WO 99/25840, WO
99/25855, and WO 99/25853, all of which are herein incorporated by
reference.
Determination of Expression in Transgenic Plants
[0082] Any method known in the art can be used for determining the
level of expression in a plant of a nucleic acid molecule of
embodiments of the invention or polypeptide encoded therefrom. For
example, the expression level in a plant of a polypeptide encoded
by a nucleic acid molecule of embodiments of the invention can be
determined by immunoassay, quantitative gel electrophoresis, etc.
Expression of nucleic acid molecules of embodiments of the
invention can be measured directly by reverse transcription
quantitative PCR (qRT-PCR) of isolated RNA from the plant.
Additionally, the expression level in a plant of a polypeptide
encoded by a nucleic acid molecule of embodiments of the invention
can be determined by the degree to which the plant phenotype is
altered. In one embodiment, enhanced insect resistance is the
phenotype to be assayed.
[0083] As used herein, "enhanced insect resistance" refers to
increased resistance of a transgenic plant expressing a polypeptide
of an embodiment of the invention to consumption and/or infestation
by an insect pest as compared to a plant not expressing a
polypeptide of an embodiment of the invention. Enhanced resistance
can be measured in a number of ways. In one embodiment, enhanced
resistance is measured by decreased damage to a plant expressing a
polypeptide of an embodiment of the invention as compared to a
plant not expressing a polypeptide of an embodiment of the
invention after the same period of insect incubation. Insect damage
can be assessed visually. For example in cotton plants, damage
after infestation can be measured by looking directly at cotton
plant bolls for signs of consumption by insects. In another
embodiment, enhanced resistance is measured by increased crop yield
from a plant expressing a polypeptide of an embodiment of the
invention as compared to a plant not expressing a polypeptide of an
embodiment of the invention after the same period of insect
incubation.
[0084] Insect pests include insects selected from the orders
Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga,
Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera,
Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly
Lepidoptera.
[0085] Larvae of the order Lepidoptera include, but are not limited
to, armyworms, cutworms, loopers, and heliothines in the family
Noctuidae Spodoptera frugiperda JE Smith (fall armyworm); S. exigua
Hubner (beet armyworm); S. litura Fabricius (tobacco cutworm,
cluster caterpillar); Mamestra configurata Walker (bertha
armyworm); M. brassicae Linnaeus (cabbage moth); Agrotis ipsilon
Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm);
A. subterranea Fabricius (granulate cutworm); Alabama argillacea
Hubner (cotton leaf worm); Trichoplusia ni Hubner (cabbage looper);
Pseudoplusia includens Walker (soybean looper); Anticarsia
gemmatalis Hubner (velvetbean caterpillar); Hypena scabs Fabricius
(green cloverworm); Heliothis virescens Fabricius (tobacco
budworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindara
Barnes and Mcdunnough (rough skinned cutworm); Euxoa messoria
Harris (darksided cutworm); Earias insulana Boisduval (spiny
bollworm); E. vittella Fabricius (spotted bollworm); Helicoverpa
armigera Hubner (American bollworm); H. zea Boddie (corn earworm or
cotton bollworm); Melanchra picta Harris (zebra caterpillar); Egira
(Xylomyges) curialis Grote (citrus cutworm); borers, casebearers,
webworms, coneworms, and skeletonizers from the family Pyralidae
Ostrinia nubilalis Hubner (European corn borer); Amyelois
transitella Walker (naval orangeworm); Anagasta kuehniella Zeller
(Mediterranean flour moth); Cadra cautella Walker (almond moth);
Chilo suppressalis Walker (rice stem borer); C. partellus, (sorghum
borer); Corcyra cephalonica Stainton (rice moth); Crambus
caliginosellus Clemens (corn root webworm); C. teterrellus Zincken
(bluegrass webworm); Cnaphalocrocis medinalis Guenee (rice leaf
roller); Desmia funeralis Hubner (grape leaffolder); Diaphania
hyalinata Linnaeus (melon worm); D. nitidalis Stoll (pickleworm);
Diatraea grandiosella Dyar (southwestern corn borer), D.
saccharalis Fabricius (surgarcane borer); Eoreuma loftini Dyar
(Mexican rice borer); Ephestia elutella Hubner (tobacco (cacao)
moth); Galleria mellonella Linnaeus (greater wax moth);
Herpetogramma licarsisalis Walker (sod webworm); Homoeosoma
electellum Hulst (sunflower moth); Elasmopalpus lignosellus Zeller
(lesser cornstalk borer); Achroia grisella Fabricius (lesser wax
moth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga
thyrisalis Walker (tea tree web moth); Maruca testulalis Geyer
(bean pod borer); Plodia interpunctella Hubner (Indian meal moth);
Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis
Guenee (celery leaftier); and leafrollers, budworms, seed worms,
and fruit worms in the family Tortricidae Acleris gloverana
Walsingham (Western blackheaded budworm); A. variana Fernald
(Eastern blackheaded budworm); Archips argyrospila Walker (fruit
tree leaf roller); A. rosana Linnaeus (European leaf roller); and
other Archips species, Adoxophyes orana Fischer von Rosslerstamm
(summer fruit tortrix moth); Cochylis hospes Walsingham (banded
sunflower moth); Cydia latiferreana Walsingham (filbertworm); C.
pomonella Linnaeus (coding moth); Platynota flavedana Clemens
(variegated leafroller); P. stultana Walsingham (omnivorous
leafroller); Lobesia botrana Denis & Schiffermuller (European
grape vine moth); Spilonota ocellana Denis & Schiffermuller
(eyespotted bud moth); Endopiza viteana Clemens (grape berry moth);
Eupoecilia ambiguella Hubner (vine moth); Bonagota salubricola
Meyrick (Brazilian apple leafroller); Grapholita molesta Busck
(oriental fruit moth); Suleima helianthana Riley (sunflower bud
moth); Argyrotaenia spp.; Choristoneura spp.
[0086] Selected other agronomic pests in the order Lepidoptera
include, but are not limited to, Alsophila pometaria Harris (fall
cankerworm); Anarsia lineatella Zeller (peach twig borer); Anisota
senatoria J. E. Smith (orange striped oakworm); Antheraea pernyi
Guerin-Meneville (Chinese Oak Silkmoth); Bombyx mori Linnaeus
(Silkworm); Bucculatrix thurberiella Busck (cotton leaf
perforator); Colias eurytheme Boisduval (alfalfa caterpillar);
Datana integerrima Grote & Robinson (walnut caterpillar);
Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomos
subsignaria Hubner (elm spanworm); Erannis tiliaria Harris (linden
looper); Euproctis chrysorrhoea Linnaeus (browntail moth);
Harrisina americana Guerin-Meneville (grapeleaf skeletonizer);
Hemileuca oliviae Cockrell (range caterpillar); Hyphantria cunea
Drury (fall webworm); Keiferia lycopersicella Walsingham (tomato
pinworm); Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock
looper); L. fiscellaria lugubrosa Hulst (Western hemlock looper);
Leucoma salicis Linnaeus (satin moth); Lymantria dispar Linnaeus
(gypsy moth); Manduca quinquemaculata Haworth (five spotted hawk
moth, tomato hornworm); M. sexta Haworth (tomato hornworm, tobacco
hornworm); Operophtera brumata Linnaeus (winter moth); Paleacrita
vernata Peck (spring cankerworm); Papilio cresphontes Cramer (giant
swallowtail, orange dog); Phryganidia californica Packard
(California oakworm); Phyllocnistis citrella Stainton (citrus
leafminer); Phyllonorycter blancardella Fabricius (spotted
tentiform leafminer); Pieris brassicae Linnaeus (large white
butterfly); P. rapae Linnaeus (small white butterfly); P. napi
Linnaeus (green veined white butterfly); Platyptilia carduidactyla
Riley (artichoke plume moth); Plutella xylostella Linnaeus
(diamondback moth); Pectinophora gossypiella Saunders (pink
bollworm); Pontia protodice Boisduval & Leconte (Southern
cabbageworm); Sabulodes aegrotata Guenee (omnivorous looper);
Schizura concinna J. E. Smith (red humped caterpillar); Sitotroga
cerealella Olivier (Angoumois grain moth); Thaumetopoea pityocampa
Schiffermuller (pine processionary caterpillar); Tineola
bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick
(tomato leafminer); Yponomeuta padella Linnaeus (ermine moth);
Heliothis subflexa Guenee; Malacosoma spp. and Orgyia spp.
[0087] In particular embodiments, the insect pests are from the
order of Lepidopteran insects including European corn borer, e.g.,
Ostrinia nubilalis; corn earworm, e.g., Helicoverpa zea; common
stalk borer, e.g., Papiapema nebris; armyworm, e.g., Pseudaletia
unipuncta; Southwestern corn borer, e.g., Diatraea grandiosella;
black cutworm, e.g., Agrotis ipsilon; fall armyworm, e.g.,
Spodoptera frugiperda; beet armyworm, e.g., Spodoptera exigua; and
diamond-back moth, e.g., Plutella xylostella. In specific
embodiments, the insect pests are European corn borer, Ostrinia
nubilalis, and corn earworm Helicoverpa zea.
[0088] Determinations can be made using whole plants, tissues
thereof, or plant cell culture.
[0089] All publications and patent applications in this
specification are indicative of the level of ordinary skill in the
art to which embodiments of this invention pertain. All
publications and patent applications are herein incorporated by
reference to the same extent as if each individual publication or
patent application was specifically and individually indicated by
reference. The disclosure of each reference set forth herein is
incorporated herein by reference in its entirety.
EXAMPLES
[0090] Embodiments of this invention can be better understood by
reference to the following examples. The foregoing and following
description of embodiments of the present invention and the various
embodiments are not intended to limit the claims, but rather are
illustrative thereof. Therefore, it will be understood that the
claims are not limited to the specific details of these examples.
It will be appreciated by those skilled in the art that other
embodiments of the invention may be practiced without departing
from the spirit and the scope of the disclosure, the scope of which
is defined by the appended claims.
Example 1
[0091] The evaluation of potential synergism between Bt proteins
IP1-88 (a Cry1) and IP2-127 (a Cry2) using the bioassay method
according to Colby, S. R., Calculating synergistic and antagonistic
responses of herbicide combinations, Weeds, 15(1):20-22 (1967). Bt
proteins IP1-88 (a Cry1) and IP2-127 (a Cry2) were utilized as test
substances to qualitatively confirm the presence and resultant
effect of the Bt proteins. Evaluation of the interactive effects
between the two insecticidal compounds was required to understand
the effectiveness of the two toxins stacked in transgenic plants.
Resultant synergy was examined using neonate larvae of European
corn borer (ECB), Ostrinia nubilalis.
[0092] Standardized Lepidoptera LC50 diet incorporation bioassays
were utilized to evaluate the effects of insecticidal proteins Cry1
and Cry2 on Lepidoptera larvae (referring to the immature stage of
an insect between the egg and pupal stage of an insect with
complete metamorphosis). All synergism experiments were conducted
in completely randomized designs with 5 replications. Each
replicate consisted of eight wells in a 96-well bioassay plate.
There were two basic doses/treatments for each protein based on
preliminary data, one close to but <LC50s based on mortality (M)
data (as M1.times.), and the other close to but <IC50s based on
response (R) data (as R1.times., mortality+severe stunted or
<0.1 mg/larva). Apart from the basic 1.times.:1.times. ratio,
higher doses using 2.times. dose for one of the two proteins might
be also used (1.times.:2.times. or 2.times.:1.times.). The
insecticidal proteins were combined with a Lepidoptera specific
artificial diet to create the bioassay diet. Ingredients including
boiling water, agar (SeaPlaque), agar (NuSeive), cooling water and
Southland Premix were used for the diet. The diet was dispensed and
combined with the proteins in 96-well plates and one neonate larva
was placed in each well.
[0093] All plates in bioassays were placed in a growth chamber with
a target temperature of 27.+-.1.degree. C. and relative humidity of
>60%. Approximately 4 days after initiation of each bioassay,
mortality and severe stunted (ss, <0.1 mg/larva) counts were
scored. Data analysis was based on the following: X=Observed result
from Compound A at dose p; Y=Observed result from Compound B at
dose q; E=Expected result for mixture of A and B at dose (p+q) if
there is no synergy or antagonism (assuming responses range from 0
to 100), whereby E=X+(100.about.X)(Y/100)=X+Y-(X*Y)/100; if
observed value is greater than expected result (Obs>E):
synergism; if observed value is similar as expected result (Obs=E):
additive; and if observed value is less than expected result
(Obs<E): antagonism. Results provided in Table 1 below showed
synergism on ECB in the mixture of IP1-88 and IP2-127.
TABLE-US-00001 TABLE 1 Mortality of ECB caused by IP1-88 or IP2-127
alone and in mixture (n = 40, 4 d at 27.degree. C.) Protein in
Expected single or Observed % mortality, combination Dose %
mortality* Colby's equation IP 1-88 M1x 32.5 -- M2x 23.1 -- IP2-127
M1x 7.7 -- M2x 20.5 -- IP1-88: IP2-127 M1x: 1x 63.2 37.7 M1x: 2x
81.6 46.4 M2x: 1x 66.7 29.0 *Corrected data based on Abbott
equation with CK mortality < 2.5%.
Example 2
[0094] Bt proteins IP1-88 (a Cry1) and IP2-127 (a Cry2), as well as
additional proteins were utilized as test substances to further
confirm the presence and resultant effect of the Bt proteins,
according to the same methods of Example 1. Both European corn
borers (ECB) and corn earworms (CEW), Helicoverpa zea, were used in
the bioassays. The observed response (mortality+ss) of both ECB and
CEW in two of three mixture treatments was evidently higher than
expected mortality based on Colby's equation (results provided in
Table 2), also indicative of synergism between IP1-88 and IP2-127
for both ECB and CEW.
TABLE-US-00002 TABLE 2 Response (mortality + ss) of ECB and CEW
caused by IP1-88 or IP2- 127 alone and in mixture (n = 40, 4 d at
27.degree. C.) Protein in Observed/ single or expected combination
Dose % response ECB CEW IP 1-88 R1x Observed 31.6 19.4 R2x Observed
94.5 27.8 IP2-127 R1x Observed 7.7 21.5 R2x Observed 20.5 83.8
IP1-88: IP2-127 R1x: 1x Observed 94.7 86.8 Expected 36.9 36.7 R1x:
2x Observed 76.9 94.7 Expected 45.6 86.9 R2x: 1x Observed 66.7 68.4
Expected 94.9 36.7
Example 3
[0095] Bt proteins Cry1Ah and IP2-127 (a Cry2), as well as
additional proteins were utilized as test substances to further
confirm the presence and resultant effect of the Bt proteins,
according to the same methods of Example 1. Both European corn
borers (ECB) and corn earworms (CEW), Helicoverpa zea, were used in
the bioassays. The observed mortality and response (mortality+ss)
of both ECB and CEW in the mixture treatments was higher than
expected based on Colby's equation (results provided in Table 3),
also indicative of synergism between Cry1Ah and IP2-127.
TABLE-US-00003 TABLE 3 Mortality (mort) and response (resp;
mortality + ss) of ECB and CEW caused by Cry1Ah or IP2-127 alone
and in mixture. (Trial 1: n = 40, trial 2: n = 32; both 4 d at
27.degree. C.) Protein in single Observed/expected ECB - trial 1
CEW - trial 1 ECB - trial 2 CEW - trial 2 or combination Dose %
response % mort % resp % mort % resp % mort % resp % mort % resp
Cry1Ah R1x Observed 59.5 50.6 28.2 23.7 30.2 62.5 45.1 40.9 R2x
Observed 77.2 92.2 66.7 44.7 30.2 75.0 70.9 70.4 IP2-127 R1x
Observed 28.8 6.9 21.7 7.9 59.4 55.3 11.9 34.9 R2x Observed 72.6
33.3 26.3 34.2 67.7 73.3 42.4 62.7 Cry1Ah:1P2-127 R1x:1x Observed
74.0 65.4 69.9 69.5 77.0 92.9 62.8 72.9 Expected 71.2 54.0 43.8
29.7 71.6 83.2 51.6 61.5 R1x:2x Observed 92.2 82.0 74.0 65.8 93.4
100.0 79.7 96.6 Expected 88.9 67.1 47.1 49.8 77.5 90.0 68.4 78.0
R2x:1x Observed 92.2 75.4 81.4 62.2 54.1 89.7 86.9 90.2 Expected
83.8 92.7 73.9 49.1 71.6 88.8 74.4 80.8
Sequence CWU 1
1
411182PRTBacillus thuringiensis 1Met Gly His Asn Asn Pro Asn Ile
Asn Glu Cys Ile Pro Tyr Asn Cys 1 5 10 15 Leu Ser Asn Pro Glu Val
Glu Val Leu Gly Gly Glu Arg Ile Glu Thr 20 25 30 Gly Tyr Thr Pro
Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu 35 40 45 Ser Glu
Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Val 50 55 60
Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln 65
70 75 80 Ile Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg
Asn Gln 85 90 95 Ala Ile Ser Arg Val Glu Gly Leu Ser Asn Leu Tyr
Gln Ile Tyr Ala 100 105 110 Glu Ser Phe Arg Glu Trp Glu Ala Asp Pro
Thr Asn Pro Ala Leu Lys 115 120 125 Glu Glu Met Arg Thr Gln Phe Asn
Asp Met Asn Ser Ala Leu Thr Thr 130 135 140 Ala Ile Pro Leu Phe Ala
Val Gln Asn Tyr Gln Val Pro Leu Leu Ser 145 150 155 160 Val Tyr Val
Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val 165 170 175 Ser
Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser 180 185
190 Arg Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala
195 200 205 Val Arg Trp His Asn Thr Gly Leu Glu Arg Ile Trp Gly Pro
Asp Ser 210 215 220 Arg Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu
Leu Thr Leu Thr 225 230 235 240 Val Leu Asp Ile Val Ser Leu Phe Pro
Asn Tyr Asp Ser Arg Thr Tyr 245 250 255 Pro Ile Arg Thr Ala Ser Gln
Leu Thr Arg Glu Ile Tyr Thr Asn Pro 260 265 270 Val Leu Glu Asn Phe
Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile 275 280 285 Glu Gly Ser
Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile 290 295 300 Thr
Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His 305 310
315 320 Gln Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr
Phe 325 330 335 Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln
Arg Ile Val 340 345 350 Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu
Ser Ser Thr Leu Tyr 355 360 365 Arg Arg Pro Phe Asn Ile Gly Ile Asn
Asn Gln Gln Leu Ser Val Leu 370 375 380 Asp Gly Thr Glu Phe Ala Tyr
Gly Thr Ser Ser Asn Leu Pro Ser Ala 385 390 395 400 Val Tyr Arg Lys
Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro 405 410 415 Gln Asn
Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser 420 425 430
His Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile 435
440 445 Ile Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe
Asn 450 455 460 Asn Thr Ile Asp Pro Glu Arg Ile Asn Gln Ile Pro Leu
Thr Lys Ser 465 470 475 480 Thr Asn Leu Gly Ser Gly Thr Ser Val Val
Lys Gly Pro Gly Phe Thr 485 490 495 Gly Gly Asp Ile Leu Arg Arg Thr
Ser Pro Gly Gln Ile Ser Thr Leu 500 505 510 Arg Val Asn Ile Thr Ala
Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile 515 520 525 Arg Tyr Ala Ser
Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly 530 535 540 Arg Pro
Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser 545 550 555
560 Asn Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe
565 570 575 Asn Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His
Val Phe 580 585 590 Asn Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu
Phe Val Pro Ala 595 600 605 Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu
Glu Arg Ala Gln Lys Val 610 615 620 Val Asn Ala Leu Phe Thr Ser Ser
Asn Gln Ile Gly Leu Lys Thr Asp 625 630 635 640 Val Thr Asp Tyr His
Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu 645 650 655 Ser Asp Glu
Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val 660 665 670 Lys
His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro 675 680
685 Asn Phe Arg Gly Ile Asn Arg Gln Pro Asp Arg Gly Trp Arg Gly Ser
690 695 700 Thr Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu
Asn Tyr 705 710 715 720 Val Thr Leu Pro Gly Thr Val Asp Glu Cys Tyr
Pro Thr Tyr Leu Tyr 725 730 735 Gln Lys Ile Asp Glu Ser Lys Leu Lys
Ala Tyr Thr Arg Tyr Glu Leu 740 745 750 Arg Gly Tyr Ile Glu Asp Ser
Gln Asp Leu Glu Ile Tyr Leu Ile Arg 755 760 765 Tyr Asn Ala Lys His
Glu Ile Val Asn Val Pro Gly Thr Gly Ser Leu 770 775 780 Trp Pro Leu
Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn 785 790 795 800
Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys 805
810 815 Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Thr Leu
Asp 820 825 830 Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly
Val Trp Val 835 840 845 Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala
Arg Leu Gly Asn Leu 850 855 860 Glu Phe Leu Glu Glu Lys Pro Leu Leu
Gly Glu Ala Leu Ala Arg Val 865 870 875 880 Lys Arg Ala Glu Lys Lys
Trp Arg Asp Lys Arg Glu Lys Leu Gln Leu 885 890 895 Glu Thr Asn Ile
Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu 900 905 910 Phe Val
Asn Ser Gln Tyr Asp Arg Leu Gln Val Asp Thr Asn Ile Ala 915 920 925
Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu Ala Tyr 930
935 940 Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe
Glu 945 950 955 960 Glu Leu Glu Gly Arg Ile Phe Thr Ala Tyr Ser Leu
Tyr Asp Ala Arg 965 970 975 Asn Val Ile Lys Asn Gly Asp Phe Asn Asn
Gly Leu Leu Cys Trp Asn 980 985 990 Val Lys Gly His Val Asp Val Glu
Glu Gln Asn Asn His Arg Ser Val 995 1000 1005 Leu Val Ile Pro Glu
Trp Glu Ala Glu Val Ser Gln Glu Val Arg 1010 1015 1020 Val Cys Pro
Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys 1025 1030 1035 Glu
Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp 1040 1045
1050 Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Val
1055 1060 1065 Tyr Pro Asn Asn Thr Val Thr Cys Asn Asn Tyr Thr Gly
Thr Gln 1070 1075 1080 Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn
Gln Gly Tyr Asp 1085 1090 1095 Glu Ala Tyr Gly Asn Asn Pro Ser Val
Pro Ala Asp Tyr Ala Ser 1100 1105 1110 Val Tyr Glu Glu Lys Ser Tyr
Thr Asp Gly Arg Arg Glu Asn Pro 1115 1120 1125 Cys Glu Ser Asn Arg
Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala 1130 1135 1140 Gly Tyr Val
Thr Lys Asp Leu Glu Tyr Phe Pro Glu Thr Asp Lys 1145 1150 1155 Val
Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 1160 1165
1170 Ser Val Glu Leu Leu Leu Met Glu Glu 1175 1180 2634PRTBacillus
thuringiensis 2Met Gly Asn Ser Val Leu Asn Ser Gly Arg Thr Thr Ile
Cys Asp Ala 1 5 10 15 Tyr Asn Val Ala Ala His Asp Pro Phe Ser Phe
Gln His Lys Ser Leu 20 25 30 Asp Thr Val Gln Arg Glu Trp Thr Glu
Trp Lys Lys Asn Asn His Ser 35 40 45 Leu Tyr Leu Asp Pro Ile Val
Gly Thr Val Ala Ser Phe Leu Leu Lys 50 55 60 Lys Val Gly Ser Leu
Val Gly Lys Arg Ile Leu Ser Glu Leu Arg Asn 65 70 75 80 Leu Ile Phe
Pro Ser Gly Ser Thr Asn Leu Met Gln Asp Ile Leu Arg 85 90 95 Glu
Thr Glu Gln Phe Leu Asn Gln Arg Leu Asp Thr Asp Thr Leu Ala 100 105
110 Arg Val Asn Ala Glu Leu Thr Gly Leu Gln Ala Asn Val Glu Glu Phe
115 120 125 Asn Arg Gln Val Asp Asn Phe Leu Asn Pro Asn Arg Asn Ala
Val Pro 130 135 140 Leu Ser Ile Thr Ser Ser Val Asn Thr Met Gln Gln
Leu Phe Leu Asn 145 150 155 160 Arg Leu Pro Gln Phe Gln Met Gln Gly
Tyr Gln Leu Leu Leu Leu Pro 165 170 175 Leu Phe Ala Gln Ala Ala Asn
Leu His Leu Ser Phe Ile Arg Asp Val 180 185 190 Ile Leu Asn Ala Asp
Glu Trp Gly Ile Ser Ala Ala Thr Leu Arg Thr 195 200 205 Tyr Arg Asp
Tyr Leu Lys Asn Tyr Thr Arg Asp Tyr Ser Asn Tyr Cys 210 215 220 Ile
Asn Thr Tyr Gln Ser Ala Phe Lys Gly Leu Asn Thr Arg Leu His 225 230
235 240 Gly Thr Leu Glu Phe Arg Thr Tyr Met Phe Leu Asn Val Phe Glu
Tyr 245 250 255 Val Ser Ile Trp Ser Leu Phe Lys Tyr Gln Ser Leu Leu
Val Ser Ser 260 265 270 Gly Ala Asn Leu Tyr Ala Ser Gly Ser Gly Pro
Gln Gln Thr Gln Ser 275 280 285 Phe Thr Ser Gln Asp Trp Pro Phe Leu
Tyr Ser Leu Phe Gln Val Asn 290 295 300 Ser Asn Tyr Val Leu Asn Gly
Phe Ser Gly Ala Arg Leu Ser Asn Thr 305 310 315 320 Phe Pro Asn Ile
Gly Gly Leu Pro Gly Ser Thr Thr Thr His Ala Leu 325 330 335 Leu Ala
Ala Arg Val Asn Tyr Ser Gly Gly Ile Ser Ser Gly Asp Ile 340 345 350
Gly Ala Ser Pro Phe Asn Gln Asn Phe Asn Cys Ser Thr Phe Leu Pro 355
360 365 Pro Leu Leu Thr Pro Phe Val Arg Ser Trp Leu Asp Ser Gly Ser
Asp 370 375 380 Arg Glu Gly Val Ala Thr Val Thr Asn Trp Gln Thr Glu
Ser Phe Glu 385 390 395 400 Thr Thr Leu Gly Leu Arg Ser Gly Ala Phe
Thr Ala Arg Gly Asn Ser 405 410 415 Asn Tyr Phe Pro Asp Tyr Phe Ile
Arg Asn Ile Ser Gly Val Pro Leu 420 425 430 Val Val Arg Asn Glu Asp
Leu Arg Arg Pro Leu His Tyr Asn Glu Ile 435 440 445 Arg Asn Ile Ala
Ser Pro Ser Gly Thr Pro Gly Gly Ala Arg Ala Tyr 450 455 460 Met Val
Ser Val His Asn Arg Lys Asn Asn Ile His Ala Val His Glu 465 470 475
480 Asn Gly Ser Met Ile His Leu Ala Pro Asn Asp Tyr Thr Gly Phe Thr
485 490 495 Ile Ser Pro Ile His Ala Thr Gln Val Asn Asn Gln Thr Arg
Thr Phe 500 505 510 Ile Ser Glu Lys Phe Gly Asn Gln Gly Asp Ser Leu
Arg Phe Glu Gln 515 520 525 Asn Asn Thr Thr Ala Arg Tyr Thr Leu Arg
Gly Asn Gly Asn Ser Tyr 530 535 540 Asn Leu Tyr Leu Arg Val Ser Ser
Ile Gly Asn Ser Thr Ile Arg Val 545 550 555 560 Thr Ile Asn Gly Arg
Val Tyr Thr Ala Thr Asn Val Asn Thr Thr Thr 565 570 575 Asn Asn Asp
Gly Val Asn Asp Asn Gly Ala Arg Phe Ser Asp Ile Asn 580 585 590 Ile
Gly Asn Val Val Ala Ser Ser Asn Ser Asp Val Pro Leu Asp Ile 595 600
605 Asn Val Thr Phe Asn Ser Gly Thr Gln Phe Asp Leu Met Asn Thr Met
610 615 620 Leu Val Pro Thr Asn Ile Ser Pro Leu Tyr 625 630
32136DNABacillus thuringiensis 3atggcttata ataataatca aaatcaatgc
ataccttata attgtttgaa taatcccgaa 60atcgaaatat tagaaggcgg aagaatatca
gttggtaata ccccaattga tatttctctt 120tcgcttactc agtttctttt
gagtgaattt gtcccaggtg cggggtttgt attaggatta 180attgatttaa
tatggggatt tgtaggtcct tcccaatggg acgcatttct tgctcaagtg
240gaacagttaa ttaaccaaag aatagcagaa gctgtaagaa atacagcaat
tcaggaatta 300gagggaatgg cacgggttta tagaacctat gctactgctt
ttgctgagtg ggaaaaagct 360cctgatgacc cagagctaag agaagcacta
cgtacacaat ttacagcaac tgagacttat 420ataagtggaa gaatatccgt
tttaaaaatt caaacttttg aagtacagct gttatcagtg 480tttgcccaag
ctgcaaattt acatttatct ttattaagag acgttgtgtt ttttgggcaa
540agatggggtt tttcaacgac aaccgtaaat aattactaca atgatttaac
agaagggatt 600agtacctata cagattatgc tgtacgctgg tacaatacgg
gattagaacg tgtatgggga 660ccggattcta gagattgggt aaggtataat
caatttagaa gagaattaac actaactgta 720ttagatatcg ttgctctgtt
cccgaattat gatagtagaa gatatccaat tcgaacagtt 780tcccaattaa
caagagaaat ttatacaaac ccagtattag aaaattttga tggtagtttt
840cgaggctcgg ctcagggcat agaaagaagt attaggagtc cacatttgat
ggatatactt 900aacagtataa ccatctatac ggatgctcat aggggttatt
attattggtc agggcatcaa 960ataatggctt ctcctgtcgg tttttcgggg
ccagaattca cgtttccgct atatggaacc 1020atgggaaatg cagctccaca
acaacgtatt gttgcccaac taggtcaggg cgtgtataga 1080acattatcct
ctacttttta tagaagacct tttaatatag ggataaataa tcaacaacta
1140tctgttcttg acgggacaga atttgcttat ggaacctcct caaatttgcc
atccgctgta 1200tacagaaaaa gcggaacggt agattcgctg gatgaaatac
caccacagaa taacaacgtg 1260ccacctaggc aaggatttag tcatcgatta
agccatgttt caatgtttcg ttcaggctct 1320agtagtagtg taagtataat
aagagctcct atgttctctt ggatacatcg tagtgctgaa 1380tttaataata
taattgcatc ggatagtatt actcaaatcc ctgcagtgaa gggaaacttt
1440ctttttaatg gttctgtaat ttcaggacca ggatttactg gtggggactt
agttagatta 1500aatagtagtg gaaataacat tcagaataga gggtatattg
aagttccaat tcacttccca 1560tcgacatcta ccagatatcg agttcgtgta
cggtatgctt ctgtaacccc gattcacctc 1620aacgttaatt ggggtaattc
atccattttt tccaatacag taccagctac agctacgtca 1680ttagataatc
tacaatcaag tgattttggt tattttgaaa gtgccaatgc ttttacatct
1740tcattaggta atatagtagg tgttagaaat tttagtggga ctgcaggagt
gataatagac 1800agatttgaat ttattccagt tactgcaaca ctcgaggctg
aatataatct ggagagagcg 1860cagaaggcgg tggatgcgct gtttacgtct
acagaccaac tagggctaaa aacaaatgta 1920acggattatc atattgatca
agtgtccaat ttagttacgt gtttatcgga tgaatttggt 1980ctggatgaaa
agcgagaatt gtccgagaaa gtcaaacatg cgaagcgact cagtgatgaa
2040cgcaatttac tccaagattc aaatttcaaa gacattaata ggcaaccaga
acgtgggtgg 2100ggcggaatta ctccttatgg aggaattagc ggctag
21364711PRTBacillus thuringiensis 4Met Ala Tyr Asn Asn Asn Gln Asn
Gln Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Asn Asn Pro Glu Ile Glu
Ile Leu Glu Gly Gly Arg Ile Ser Val Gly 20 25 30 Asn Thr Pro Ile
Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 Glu Phe
Val Pro Gly Ala Gly Phe Val Leu Gly Leu Ile Asp Leu Ile 50 55 60
Trp Gly Phe Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Ala Gln Val 65
70 75 80 Glu Gln Leu Ile Asn Gln Arg Ile Ala Glu Ala Val Arg Asn
Thr Ala 85 90 95 Ile Gln Glu Leu Glu Gly Met Ala Arg Val Tyr Arg
Thr Tyr Ala Thr 100 105 110 Ala Phe Ala Glu Trp Glu Lys Ala Pro Asp
Asp Pro Glu Leu Arg Glu 115 120 125 Ala Leu Arg Thr Gln Phe Thr
Ala
Thr Glu Thr Tyr Ile Ser Gly Arg 130 135 140 Ile Ser Val Leu Lys Ile
Gln Thr Phe Glu Val Gln Leu Leu Ser Val 145 150 155 160 Phe Ala Gln
Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Val Val 165 170 175 Phe
Phe Gly Gln Arg Trp Gly Phe Ser Thr Thr Thr Val Asn Asn Tyr 180 185
190 Tyr Asn Asp Leu Thr Glu Gly Ile Ser Thr Tyr Thr Asp Tyr Ala Val
195 200 205 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp
Ser Arg 210 215 220 Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu
Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Val Ala Leu Phe Pro Asn
Tyr Asp Ser Arg Arg Tyr Pro 245 250 255 Ile Arg Thr Val Ser Gln Leu
Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 Leu Glu Asn Phe Asp
Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 Arg Ser Ile
Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 Ile
Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310
315 320 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe
Pro 325 330 335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg
Ile Val Ala 340 345 350 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser
Ser Thr Phe Tyr Arg 355 360 365 Arg Pro Phe Asn Ile Gly Ile Asn Asn
Gln Gln Leu Ser Val Leu Asp 370 375 380 Gly Thr Glu Phe Ala Tyr Gly
Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390 395 400 Tyr Arg Lys Ser
Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405 410 415 Asn Asn
Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His 420 425 430
Val Ser Met Phe Arg Ser Gly Ser Ser Ser Ser Val Ser Ile Ile Arg 435
440 445 Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn
Ile 450 455 460 Ile Ala Ser Asp Ser Ile Thr Gln Ile Pro Ala Val Lys
Gly Asn Phe 465 470 475 480 Leu Phe Asn Gly Ser Val Ile Ser Gly Pro
Gly Phe Thr Gly Gly Asp 485 490 495 Leu Val Arg Leu Asn Ser Ser Gly
Asn Asn Ile Gln Asn Arg Gly Tyr 500 505 510 Ile Glu Val Pro Ile His
Phe Pro Ser Thr Ser Thr Arg Tyr Arg Val 515 520 525 Arg Val Arg Tyr
Ala Ser Val Thr Pro Ile His Leu Asn Val Asn Trp 530 535 540 Gly Asn
Ser Ser Ile Phe Ser Asn Thr Val Pro Ala Thr Ala Thr Ser 545 550 555
560 Leu Asp Asn Leu Gln Ser Ser Asp Phe Gly Tyr Phe Glu Ser Ala Asn
565 570 575 Ala Phe Thr Ser Ser Leu Gly Asn Ile Val Gly Val Arg Asn
Phe Ser 580 585 590 Gly Thr Ala Gly Val Ile Ile Asp Arg Phe Glu Phe
Ile Pro Val Thr 595 600 605 Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu
Arg Ala Gln Lys Ala Val 610 615 620 Asp Ala Leu Phe Thr Ser Thr Asp
Gln Leu Gly Leu Lys Thr Asn Val 625 630 635 640 Thr Asp Tyr His Ile
Asp Gln Val Ser Asn Leu Val Thr Cys Leu Ser 645 650 655 Asp Glu Phe
Gly Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys 660 665 670 His
Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Ser Asn 675 680
685 Phe Lys Asp Ile Asn Arg Gln Pro Glu Arg Gly Trp Gly Gly Ile Thr
690 695 700 Pro Tyr Gly Gly Ile Ser Gly 705 710
* * * * *