U.S. patent application number 10/958008 was filed with the patent office on 2005-08-18 for axmi-010, a delta-endotoxin gene and methods for its use.
This patent application is currently assigned to Athenix Corporation. Invention is credited to Carozzi, Nadine, Carr, Brian, Duck, Nicholas B., Hargiss, Tracy, Koziel, Michael G..
Application Number | 20050183161 10/958008 |
Document ID | / |
Family ID | 34467980 |
Filed Date | 2005-08-18 |
United States Patent
Application |
20050183161 |
Kind Code |
A1 |
Carozzi, Nadine ; et
al. |
August 18, 2005 |
AXMI-010, a delta-endotoxin gene and methods for its use
Abstract
Compositions and methods for conferring pesticidal activity to
bacteria, plants, plant cells, tissues and seeds are provided.
Compositions comprising a coding sequence for a delta-endotoxin
polypeptide are provided. The coding sequences can be used in DNA
constructs or expression cassettes for transformation and
expression in plants and bacteria. Compositions also comprise
transformed bacteria, plants, plant cells, tissues, and seeds. In
particular, isolated delta-endotoxin nucleic acid molecules are
provided. Additionally, amino acid sequences corresponding to the
polynucleotides are encompassed. In particular, the present
invention provides for isolated nucleic acid molecules comprising
nucleotide sequences encoding the amino acid sequence shown in SEQ
ID NO:2, or the nucleotide sequence set forth in SEQ ID NO: 1, as
well as variants thereof.
Inventors: |
Carozzi, Nadine; (Raleigh,
NC) ; Hargiss, Tracy; (Cary, NC) ; Koziel,
Michael G.; (Raleigh, NC) ; Duck, Nicholas B.;
(Apex, NC) ; Carr, Brian; (Raleigh, NC) |
Correspondence
Address: |
ALSTON & BIRD LLP
BANK OF AMERICA PLAZA
101 SOUTH TRYON STREET, SUITE 4000
CHARLOTTE
NC
28280-4000
US
|
Assignee: |
Athenix Corporation
Durham
NC
|
Family ID: |
34467980 |
Appl. No.: |
10/958008 |
Filed: |
October 4, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60510982 |
Oct 14, 2003 |
|
|
|
Current U.S.
Class: |
800/279 ;
435/419; 435/468; 435/6.15; 514/2.1; 514/4.5; 530/370;
536/23.6 |
Current CPC
Class: |
Y02A 40/162 20180101;
C07K 14/325 20130101; Y02A 40/146 20180101; C12N 15/8286
20130101 |
Class at
Publication: |
800/279 ;
435/468; 435/419; 530/370; 514/012; 536/023.6; 435/006 |
International
Class: |
A01H 001/00; C12N
015/82; A01N 063/00; C12Q 001/68; C07H 021/04 |
Claims
That which is claimed:
1. An isolated nucleic acid molecule selected from the group
consisting of: a) a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:1, or a complement thereof; b) a nucleic acid
molecule comprising a nucleotide sequence having at least 90%
sequence identity to the nucleotide sequence of SEQ ID NO:1, or a
complement thereof; c) a nucleic acid molecule comprising a
nucleotide sequence having at least 95% sequence identity to the
nucleotide sequence of SEQ ID NO:1, or a complement thereof; d) a
nucleic acid molecule that encodes a polypeptide comprising the
amino acid sequence of SEQ ID NO:2; e) a nucleic acid molecule that
encodes a polypeptide having at least 90% sequence identity to the
amino acid sequence of SEQ ID NO:2; and, f) a nucleic acid molecule
that encodes a polypeptide having at least 95% sequence identity to
the amino acid sequence of SEQ ID NO:2.
2. A vector comprising the nucleic acid molecule of claim 1.
3. The vector of claim 2, further comprising a nucleic acid
molecule encoding a heterologous polypeptide.
4. A host cell that contains the vector of claim 2.
5. The host cell of claim 4 that is a bacterial host cell.
6. The host cell of claim 4 that is a plant cell.
7. A transgenic plant comprising the host cell of claim 6.
8. The transgenic plant of claim 7, wherein said plant is selected
from the group consisting of maize, sorghum, wheat, cabbage,
sunflower, tomato, crucifers, peppers, potato, cotton, rice,
soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed
rape.
9. Transformed seed of the plant of claim 8.
10. An isolated polypeptide with pesticidal activity, selected from
the group consisting of: a) a polypeptide comprising the amino acid
sequence of SEQ ID NO:2; b) a polypeptide comprising an amino acid
sequence having at least 90% sequence identity to the amino acid
sequence of SEQ ID NO:2; c) a polypeptide comprising an amino acid
sequence having at least 95% sequence identity to the amino acid
sequence of SEQ ID NO:2; d) a polypeptide that is encoded by the
nucleotide sequence of SEQ ID NO:1; e) a polypeptide that is
encoded by a nucleic acid molecule comprising a nucleotide sequence
having at least 90% sequence identity to the nucleotide sequence of
SEQ ID NO:1; and, f) a polypeptide that is encoded by a nucleic
acid molecule comprising a nucleotide sequence having at least 95%
sequence identity to the nucleotide sequence of SEQ ID NO:1.
11. The polypeptide of claim 10 further comprising heterologous
amino acid sequences.
12. A composition comprising the polypeptide of claim 10.
13. The composition of claim 12, wherein said composition is
selected from the group consisting of a powder, dust, pellet,
granule, spray, emulsion, colloid, and solution.
14. The composition of claim 12, wherein said composition is
prepared by desiccation, lyophilization, homogenization,
extraction, filtration, centrifugation, sedimentation, or
concentration of a culture of Bacillus thuringiensis cells.
15. The composition of claim 12, comprising from about 1% to about
99% by weight of said polypeptide.
16. A method for controlling a lepidopteran or coleopteran pest
population comprising contacting said population with a
pesticidally effective amount of a polypeptide of claim 10.
17. A method for killing a lepidopteran or coleopteran pest,
comprising contacting said pest with, or feeding to said pest, a
pesticidally effective amount of a polypeptide of claim 10.
18. A method for producing a polypeptide with pesticidal activity,
comprising culturing the host cell of claim 4 under conditions in
which a nucleic acid molecule comprising a nucleotide sequence
encoding the polypeptide is expressed, said polypeptide being
selected from the group consisting of: a) a polypeptide comprising
the amino acid sequence of SEQ ID NO:2; b) a polypeptide that is
encoded by the nucleotide sequence of SEQ ID NO:1; c) a polypeptide
comprising an amino acid sequence having at least 90% sequence
identity to the amino acid sequence of SEQ ID NO:2; d) a
polypeptide comprising an amino acid sequence having at least 95%
sequence identity to the amino acid sequence of SEQ ID NO:2; e) a
polypeptide that is encoded by a nucleic acid molecule comprising a
nucleotide sequence having at least 90% sequence identity to the
nucleotide sequence of SEQ ID NO:1; and, f) a polypeptide that is
encoded by a nucleic acid molecule comprising a nucleotide sequence
having at least 95% sequence identity to the nucleotide sequence of
SEQ ID NO:1.
19. A plant having stably incorporated into its genome a DNA
construct comprising a nucleotide sequence that encodes a protein
having pesticidal activity, wherein said nucleotide sequence is
selected from the group consisting of: a) the nucleotide sequence
of SEQ ID NO:1; b) a nucleotide sequence having at least 90%
sequence identity to the nucleotide sequence of SEQ ID NO:1; c) a
nucleotide sequence having at least 95% sequence identity to the
nucleotide sequence of SEQ ID NO:1; d) a nucleotide sequence
encoding a polypeptide comprising the amino acid sequence of SEQ ID
NO:2; e) a nucleotide sequence encoding a polypeptide having at
least 90% sequence identity to the amino acid sequence of SEQ ID
NO:2; and f) a nucleotide sequence encoding a polypeptide having at
least 95% sequence identity to the amino acid sequence of SEQ ID
NO:2; wherein said nucleotide sequence is operably linked to a
promoter that drives expression of a coding sequence in a plant
cell.
20. A plant cell having stably incorporated into its genome a DNA
construct comprising a nucleotide sequence that encodes a protein
having pesticidal activity, wherein said nucleotide sequence is
selected from the group consisting of: a) the nucleotide sequence
of SEQ ID NO:1; b) a nucleotide sequence having at least 90%
sequence identity to the nucleotide sequence of SEQ ID NO:1; c) a
nucleotide sequence having at least 95% sequence identity to the
nucleotide sequence of SEQ ID NO:1; d) a nucleotide sequence
encoding a polypeptide comprising the amino acid sequence of SEQ ID
NO:2; e) a nucleotide sequence encoding a polypeptide having at
least 90% sequence identity to the amino acid sequence of SEQ ID
NO:2; f) a nucleotide sequence encoding a polypeptide having at
least 95% sequence identity to the amino acid sequence of SEQ ID
NO:2; and, wherein said nucleotide sequence is operably linked to a
promoter that drives expression of a coding sequence in a plant
cell.
21. A method for protecting a plant from a pest, comprising
introducing into said plant or cell thereof at least one expression
vector comprising a nucleotide sequence that encodes a pesticidal
polypeptide, wherein said nucleotide sequence is selected from the
group consisting of: a) a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:1; b) a nucleic acid molecule
comprising a nucleotide sequence having at least 90% sequence
identity to the nucleotide sequence of SEQ ID NO:1; c) a nucleic
acid molecule comprising a nucleotide sequence having at least 95%
sequence identity to the nucleotide sequence of SEQ ID NO:1; d) a
nucleic acid molecule that encodes a polypeptide comprising the
amino acid sequence of SEQ ID NO:2; e) a nucleic acid molecule that
encodes a polypeptide having at least 90% sequence identity to the
amino acid sequence of SEQ ID NO:2; and, f) a nucleic acid molecule
that encodes a polypeptide having at least 95% sequence identity to
the amino acid sequence of SEQ ID NO:2.
22. The method of claim 21, wherein said plant produces a
pesticidal polypeptide having pesticidal activity against a
lepidopteran or coleopteran pest.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/510,982, filed Oct. 14, 2003, the contents
of which are hereby incorporated in their entirety by reference
herein.
FIELD OF THE INVENTION
[0002] This invention relates to the field of molecular biology.
Provided are novel genes that encode pesticidal proteins. These
proteins and the nucleic acid sequences that encode them are useful
in preparing pesticidal formulations and in the production of
transgenic pest-resistant plants.
BACKGROUND OF THE INVENTION
[0003] Bacillus thuringiensis is a Gram-positive spore forming soil
bacterium characterized by its ability to produce crystalline
inclusions that are specifically toxic to certain orders and
species of insects, but are harmless to plants and other
non-targeted organisms. For this reason, compositions including
Bacillus thuringiensis strains or their insecticidal proteins can
be used as environmentally-acceptable insecticides to control
agricultural insect pests or insect vectors for a variety of human
or animal diseases.
[0004] Crystal (Cry) proteins (delta-endotoxins) from Bacillus
thuringiensis have potent insecticidal activity against
predominantly lepidopteran, dipteran, and coleopteran larvae. These
proteins also have shown activity against Hymenoptera, Homoptera,
Phthiraptera, Mallophaga, and Acari pest orders, as well as other
invertebrate orders such as Nemathelminthes, Platyhelminthes, and
Sarcomastigorphora (Feitelson (1993) The Bacillus Thuringiensis
Family Tree. In Advanced Engineered Pesticides. Marcel Dekker,
Inc., New York, N.Y.) These proteins were originally classified as
CryI to CryV based primarily on their insecticidal activity. The
major classes were Lepidoptera-specific (I), Lepidoptera- and
Diptera-specific (II), Coleoptera-specific (III), Diptera-specific
(IV), and nematode-specific (V) and (VI). The proteins were further
classified into subfamilies; more highly related proteins within
each family were assigned divisional letters such as Cry1A, Cry1B,
Cry1C, etc. Even more closely related proteins within each division
were given names such as Cry1C1, Cry1C2, etc.
[0005] A new nomenclature was recently described for the Cry genes
based upon amino acid sequence homology rather than insect target
specificity (Crickmore et al. (1998) Microbiol. Mol. Biol. Rev.
62:807-813). In the new classification, each toxin is assigned a
unique name incorporating a primary rank (an Arabic number), a
secondary rank (an uppercase letter), a tertiary rank (a lowercase
letter), and a quarternary rank (another Arabic number). In the new
classification, Roman numerals have been exchanged for Arabic
numerals in the primary rank.
[0006] The crystal protein does not exhibit insecticidal activity
until it has been ingested and solubilized in the insect midgut.
The ingested protoxin is hydrolyzed by proteases in the insect
digestive tract to an active toxic molecule. (Hofte and Whiteley
(1989) Microbiol. Rev. 53:242-255). This toxin binds to apical
brush border receptors in the midgut of the target larvae and
inserts into the apical membrane creating ion channels or pores,
resulting in larval death.
[0007] Because of the devastation that insects can confer there is
a continual need to discover new forms of Bacillus thuringiensis
delta-endotoxins.
SUMMARY OF INVENTION
[0008] Compositions and methods for conferring pesticide resistance
to bacteria, plants, plant cells, tissues and seeds are provided.
Compositions include nucleic acid molecules encoding sequences for
delta-endotoxin polypeptides, vectors comprising those nucleic acid
molecules, and host cells comprising the vectors. Compositions also
include the polypeptide sequences of the endotoxin, and antibodies
to those polypeptides. The nucleotide sequences can be used in DNA
constructs or expression cassettes for transformation and
expression in organisms, including microorganisms and plants. The
nucleotide or amino acid sequences may be synthetic sequences that
have been designed for expression in an organism including, but not
limited to, a microorganism or a plant. Compositions also comprise
transformed bacteria, plants, plant cells, tissues, and seeds.
[0009] In particular, isolated nucleic acid molecules corresponding
to a delta-endotoxin nucleic acid sequence are provided.
Additionally, amino acid sequences corresponding to the
polynucleotide are encompassed. In particular, the present
invention provides for an isolated nucleic acid molecule comprising
the nucleotide sequences encoding the amino acid sequence shown in
SEQ ID NO:2, or the nucleotide sequence set forth in SEQ ID NO:1,
as well as variants and fragments thereof.
[0010] Methods are provided for producing the polypeptides of the
invention, and for using those polypeptides for controlling or
killing a lepidopteran or coleopteran pest. Methods and kits for
detecting the nucleic acids and polypeptides of the invention in a
sample are also included.
[0011] The compositions and methods of the invention are useful for
the production of organisms with pesticide resistance, specifically
bacteria and plants. These organisms and compositions derived from
them are desirable for agricultural purposes. The compositions of
the invention are also useful for generating altered or improved
delta-endotoxin proteins that have pesticidal activity, or for
detecting the presence of delta-endotoxin proteins or nucleic acids
in products or organisms.
DESCRIPTION OF FIGURES
[0012] FIG. 1 shows an alignment of AXMI-010 (SEQ ID NO:2) with
BinB4 (Accession No. CAA04290) (SEQ ID NO:3), cry36Aa1 (Accession
No. AAK64558) (SEQ ID NO:4), and cry35Ab (AAG41672) (SEQ ID NO:5).
The alignment shows the most highly conserved amino acid residues
highlighted in black, and highly conserved amino acid residues
highlighted in gray.
DETAILED DESCRIPTION
[0013] The present invention is drawn to compositions and methods
for regulating pest resistance in organisms, particularly plants or
plant cells. The methods involve transforming organisms with a
nucleotide sequence encoding a delta-endotoxin protein of the
invention. In particular, the nucleotide sequences of the invention
are useful for preparing plants and microorganisms that possess
pesticidal activity. Thus, transformed bacteria, plants, plant
cells, plant tissues and seeds are provided. Compositions are
delta-endotoxin nucleic acids and proteins of Bacillus
thuringiensis. The sequences find use in the construction of
expression vectors for subsequent transformation into organisms of
interest, as probes for the isolation of other delta-endotoxin
genes, and for the generation of altered pesticidal proteins by
methods known in the art, such as domain swapping or DNA shuffling.
The proteins find use in controlling or killing lepidopteran or
coleopteran pest populations and for producing compositions with
pesticidal activity.
[0014] Definitions
[0015] By "delta-endotoxin" is intended a toxin from Bacillus
thuringiensis that has toxic activity against one or more pests,
including, but not limited to, members of the Lepidoptera, Diptera,
and Coleoptera orders, or a protein that has homology to such a
protein. In some cases, delta-endotoxin proteins have been isolated
from other organisms, including Clostridium bifermentans and
Paenibacillus popilliae. Delta-endotoxin proteins include amino
acid sequences deduced from the full-length nucleotide sequences
disclosed herein, and amino acid sequences that are shorter than
the full-length sequences, either due to the use of an alternate
downstream start site, or due to processing that produces a shorter
protein having pesticidal activity. Processing may occur in the
organism the protein is expressed in, or in the pest after
ingestion of the protein. Delta-endotoxins include proteins
identified as cry1 through cry43, cyt1 and cyt2, and Cyt-like
toxin. There are currently over 250 known species of
delta-endotoxins with a wide range of specificities and toxicities.
For an expansive list see Crickmore et al. (1998), Microbiol. Mol.
Biol. Rev. 62:807-813, and for regular updates see Crickmore et al.
(2003) "Bacillus thuringiensis toxin nomenclature," at
www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index- .
[0016] Bacterial genes, such as the AXMI-010 gene of this
invention, quite often possess multiple methionine initiation
codons in proximity to the start of the open reading frame. Often,
translation initiation at one or more of these start codons will
lead to generation of a functional protein. These start codons can
include ATG codons. However, bacteria such as Bacillus sp. also
recognize the codon GTG as a start codon, and proteins that
initiate translation at GTG codons contain a methionine at the
first amino acid. Furthermore, it is not often determined a priori
which of these codons are used naturally in the bacterium. Thus, it
is understood that use of one of the alternate methionine codons
may also lead to generation of delta-endotoxin proteins that encode
pesticidal activity. These delta-endotoxin proteins are encompassed
in the present invention and may be used in the methods of the
present invention.
[0017] By "plant cell" is intended all known forms of plant,
including undifferentiated tissue (e.g. callus), suspension culture
cells, protoplasts, leaf cells, root cells, phloem cells, plant
seeds, pollen, propagules, embryos and the like. By "plant
expression cassette" is intended a DNA construct that is capable of
resulting in the expression of a protein from an open reading frame
in a plant cell. Typically these contain a promoter and a coding
sequence. Often, such constructs will also contain a 3'
untranslated region. Such constructs may contain a `signal
sequence` or `leader sequence` to facilitate co-translational or
post-translational transport of the peptide to certain
intracellular structures such as the chloroplast (or other
plastid), endoplasmic reticulum, or Golgi apparatus.
[0018] By "signal sequence" is intended a sequence that is known or
suspected to result in cotranslational or post-translational
peptide transport across the cell membrane. In eukaryotes, this
typically involves secretion into the Golgi apparatus, with some
resulting glycosylation. By "leader sequence" is intended any
sequence that when translated, results in an amino acid sequence
sufficient to trigger co-translational transport of the peptide
chain to a sub-cellular organelle. Thus, this includes leader
sequences targeting transport and/or glycosylation by passage into
the endoplasmic reticulum, passage to vacuoles, plastids including
chloroplasts, mitochondria, and the like.
[0019] By "plant transformation vector" is intended a DNA molecule
that is necessary for efficient transformation of a plant cell.
Such a molecule may consist of one or more plant expression
cassettes, and may be organized into more than one `vector` DNA
molecule. For example, binary vectors are plant transformation
vectors that utilize two non-contiguous DNA vectors to encode all
requisite cis- and trans-acting functions for transformation of
plant cells (Hellens and Mullineaux (2000) Trends in Plant Science
5:446-451). "Vector" refers to a nucleic acid construct designed
for transfer between different host cells. "Expression vector"
refers to a vector that has the ability to incorporate, integrate
and express heterologous DNA sequences or fragments in a foreign
cell.
[0020] "Transgenic plants" or "transformed plants" or "stably
transformed" plants or cells or tissues refers to plants that have
incorporated or integrated exogenous nucleic acid sequences or DNA
fragments into the plant cell. These nucleic acid sequences include
those that are exogenous, or not present in the untransformed plant
cell, as well as those that may be endogenous, or present in the
untransformed plant cell.
[0021] "Heterologous" generally refers to the nucleic acid
sequences that are not endogenous to the cell or part of the native
genome in which they are present, and have been added to the cell
by infection, transfection, microinjection, electroporation,
microprojection, or the like.
[0022] "Promoter" refers to a nucleic acid sequence that functions
to direct transcription of a downstream coding sequence. The
promoter together with other transcriptional and translational
regulatory nucleic acid sequences (also termed "control sequences")
are necessary for the expression of a DNA sequence of interest.
[0023] Provided herein are novel isolated nucleotide sequences that
confer pesticidal activity. Also provided are the amino acid
sequences of the delta-endotoxin proteins. The protein resulting
from translation of this gene allows cells to control or kill pests
that ingest it.
[0024] An "isolated" or "purified" nucleic acid molecule or
protein, or biologically active portion thereof, is substantially
free of other cellular material, or culture medium when produced by
recombinant techniques, or substantially free of chemical
precursors or other chemicals when chemically synthesized.
Preferably, an "isolated" nucleic acid is free of sequences
(preferably protein encoding sequences) that naturally flank the
nucleic acid (i.e., sequences located at the 5' and 3' ends of the
nucleic acid) in the genomic DNA of the organism from which the
nucleic acid is derived. For purposes of the invention, "isolated"
when used to refer to nucleic acid molecules excludes isolated
chromosomes. For example, in various embodiments, the isolated
delta-endotoxin encoding nucleic acid molecule can contain less
than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of
nucleotide sequences that naturally flank the nucleic acid molecule
in genomic DNA of the cell from which the nucleic acid is derived.
A delta-endotoxin protein that is substantially free of cellular
material includes preparations of protein having less than about
30%, 20%, 10%, or 5% (by dry weight) of non-delta-endotoxin protein
(also referred to herein as a "contaminating protein"). Various
aspects of the invention are described in further detail in the
following subsections.
[0025] Isolated Nucleic Acid Molecules, and Variants and Fragments
Thereof
[0026] One aspect of the invention pertains to isolated nucleic
acid molecules comprising nucleotide sequences encoding
delta-endotoxin proteins and polypeptides or biologically active
portions thereof, as well as nucleic acid molecules sufficient for
use as hybridization probes to identify delta-endotoxin encoding
nucleic acids. As used herein, the term "nucleic acid molecule" is
intended to include DNA molecules (e.g., cDNA or genomic DNA) and
RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated
using nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA.
[0027] Nucleotide sequences encoding the proteins of the present
invention include the sequence set forth in SEQ ID NO:1, and
complements thereof. By "complement" is intended a nucleotide
sequence that is sufficiently complementary to a given nucleotide
sequence such that it can hybridize to the given nucleotide
sequence to thereby form a stable duplex. The corresponding amino
acid sequence for the delta-endotoxin protein encoded by this
nucleotide sequence are set forth in SEQ ID NO:2.
[0028] Nucleic acid molecules that are fragments of these
delta-endotoxin encoding nucleotide sequences are also encompassed
by the present invention. By "fragment" is intended a portion of
the nucleotide sequence encoding a delta-endotoxin protein. A
fragment of a nucleotide sequence may encode a biologically active
portion of a delta-endotoxin protein, or it may be a fragment that
can be used as a hybridization probe or PCR primer using methods
disclosed below. Nucleic acid molecules that are fragments of a
delta-endotoxin nucleotide sequence comprise at least about 15, 20,
50, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,
800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,
1400, 1450 contiguous nucleotides, or up to the number of
nucleotides present in a full-length delta-endotoxin encoding
nucleotide sequence disclosed herein (for example, 1467 nucleotides
for SEQ ID NO:1) depending upon the intended use. By "contiguous"
nucleotides is intended nucleotide residues that are immediately
adjacent to one another. Fragments of the nucleotide sequences of
the present invention will encode protein fragments that retain the
biological activity of the delta-endotoxin protein and, hence,
retain pesticidal activity. By "retains activity" is intended that
the fragment will have at least about 30%, preferably at least
about 50%, more preferably at least about 70%, even more preferably
at least about 80% of the pesticidal activity of the
delta-endotoxin protein. Methods for measuring pesticidal activity
are well known in the art. See, for example, Czapla and Lang (1990)
J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J.
252:199-206; Marrone et al. (1985) J. of Economic Entomology
78:290-293; and U.S. Pat. No. 5,743,477, all of which are herein
incorporated by reference in their entirety.
[0029] A fragment of a delta-endotoxin encoding nucleotide sequence
that encodes a biologically active portion of a protein of the
invention will encode at least about 15, 25, 30, 50, 75, 100, 125,
150, 175, 200, 250, 300, 350, 400, and 450 contiguous amino acids,
or up to the total number of amino acids present in a full-length
delta-endotoxin protein of the invention (for example, 489 amino
acids for SEQ ID NO:2).
[0030] Preferred delta-endotoxin proteins of the present invention
are encoded by a nucleotide sequence sufficiently identical to the
nucleotide sequence of SEQ ID NO:1. By "sufficiently identical" is
intended an amino acid or nucleotide sequence that has at least
about 60% or 65% sequence identity, preferably about 70% or 75%
sequence identity, more preferably about 80% or 85% sequence
identity, most preferably about 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% sequence identity compared to a reference sequence
using one of the alignment programs described herein 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.
[0031] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes. The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences (i.e., percent identity=number of identical
positions/total number of positions (e.g., overlapping
positions).times.100). In one embodiment, the two sequences are the
same length. The percent identity between two sequences can be
determined using techniques similar to those described below, with
or without allowing gaps. In calculating percent identity,
typically exact matches are counted.
[0032] The determination of percent identity between two sequences
can be accomplished using a mathematical algorithm. A nonlimiting
example of a mathematical algorithm utilized for the comparison of
two sequences is the algorithm of Karlin and Altschul (1990) Proc.
Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul
(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm
is incorporated into the BLASTN and BLASTX programs of Altschul et
al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be
performed with the BLASTN program, score=100, wordlength=12, to
obtain nucleotide sequences homologous to delta-endotoxin-like
nucleic acid molecules of the invention. BLAST protein searches can
be performed with the BLASTX program, score=50, wordlength=3, to
obtain amino acid sequences homologous to delta-endotoxin protein
molecules of the invention. 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 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, and
PSI-Blast programs, the default parameters of the respective
programs (e.g., BLASTX and BLASTN) can be used. Alignment may also
be performed manually by inspection.
[0033] Another non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is the ClustalW algorithm
(Higgins et al. (1994) Nucleic Acids Res. 22:4673-4680). ClustalW
compares sequences and aligns the entirety of the amino acid or DNA
sequence, and thus can provide data about the sequence conservation
of the entire amino acid sequence. The ClustalW algorithm is used
in several commercially available DNA/amino acid analysis software
packages, such as the ALIGNX module of the vector NTi Program Suite
(Informax, Inc). After alignment of amino acid sequences with
ClustalW, the percent amino acid identity can be assessed. A
non-limiting example of a software program useful for analysis of
ClustalW alignments is GeneDoc.TM.. Genedoc.TM. (Karl Nicholas)
allows assessment of amino acid (or DNA) similarity and identity
between multiple proteins. Another non-limiting example of a
mathematical algorithm utilized for the comparison of sequences is
the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an
algorithm is incorporated into the ALIGN program (version 2.0),
which is part of the GCG Wisconsin Genetics Software Package,
Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San
Diego, Calif., USA). When utilizing the ALIGN program for comparing
amino acid sequences, a PAM120 weight residue table, a gap length
penalty of 12, and a gap penalty of 4 can be used.
[0034] A preferred program is GAP version 10, which uses the
algorithm of Needleman and Wunsch, 1970, supra. GAP Version 10 may
be used with 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. Equivalent
programs may also be used. By "equivalent program" is intended 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.
[0035] The invention also encompasses variant nucleic acid
molecules. "Variants" of the delta-endotoxin encoding nucleotide
sequences include those sequences that encode the delta-endotoxin
proteins disclosed herein but that differ conservatively because of
the degeneracy of the genetic code as well as those that are
sufficiently identical as discussed above. Naturally occurring
allelic variants can be identified with the use of well-known
molecular biology techniques, such as polymerase chain reaction
(PCR) and hybridization techniques as outlined below. Variant
nucleotide sequences also include synthetically derived nucleotide
sequences that have been generated, for example, by using
site-directed mutagenesis but which still encode the
delta-endotoxin proteins disclosed in the present invention as
discussed below. Variant proteins encompassed by the present
invention are biologically active, that is they continue to possess
the desired biological activity of the native protein, that is,
retaining pesticidal activity. By "retains activity" is intended
that the variant will have at least about 30%, preferably at least
about 50%, more preferably at least about 70%, even more preferably
at least about 80% of the pesticidal activity of the native
protein. Methods for measuring pesticidal activity are well known
in the art. See, for example, Czapla and Lang (1990) J Econ.
Entomol. 83: 2480-2485; Andrews et al. (1988) Biochem. J.
252:199-206; Marrone et al. (1985) J of Economic Entomology
78:290-293; and U.S. Pat. No. 5,743,477, all of which are herein
incorporated by reference in their entirety.
[0036] The skilled artisan will further appreciate that changes can
be introduced by mutation into the nucleotide sequences of the
invention thereby leading to changes in the amino acid sequence of
the encoded delta-endotoxin proteins, without altering the
biological activity of the proteins. Thus, variant isolated nucleic
acid molecules can be created by introducing one or more nucleotide
substitutions, additions, or deletions into the corresponding
nucleotide sequence disclosed herein, such that one or more amino
acid substitutions, additions or deletions are introduced into the
encoded protein. Mutations can be introduced by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Such variant nucleotide sequences are also encompassed
by the present invention.
[0037] For example, preferably, conservative amino acid
substitutions may be made at one or more predicted, preferably
nonessential amino acid residues. A "nonessential" amino acid
residue is a residue that can be altered from the wild-type
sequence of a delta-endotoxin protein without altering the
biological activity, whereas an "essential" amino acid residue is
required for biological activity. A "conservative amino acid
substitution" is one in which the amino acid residue is replaced
with an amino acid residue having a similar side chain. Families of
amino acid residues having similar side chains have been defined in
the art. These families include amino acids with basic side chains
(e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g.,
glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine).
[0038] Amino acid substitutions may be made in nonconserved regions
that retain function. In general, such substitutions would not be
made for conserved amino acid residues, or for amino acid residues
residing within a conserved motif, where such residues are
essential for protein activity. Examples of residues that are
conserved and that may be essential for protein activity include,
for example, residues that are identical between all proteins
contained in the alignment of FIG. 1. Examples of residues that are
conserved but that may allow conservative amino acid substitutions
and still retain activity include, for example, residues that have
only conservative substitutions between all proteins contained in
the alignment of FIG. 1. However, one of skill in the art would
understand that functional variants may have minor conserved or
nonconserved alterations in the conserved residues.
[0039] Alternatively, variant nucleotide sequences can be made by
introducing mutations randomly along all or part of the coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for ability to confer delta-endotoxin
activity to identify mutants that retain activity. Following
mutagenesis, the encoded protein can be expressed recombinantly,
and the activity of the protein can be determined using standard
assay techniques.
[0040] Using methods such as PCR, hybridization, and the like
corresponding delta-endotoxin sequences can be identified, such
sequences having substantial identity to the sequences of the
invention. See, for example, Sambrook J., and Russell, D. W. (2001)
Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.) and Innis, et al.
(1990) PCR Protocols: A Guide to Methods and Applications (Academic
Press, NY).
[0041] In a hybridization method, all or part of the
delta-endotoxin nucleotide sequence can be used to screen cDNA or
genomic libraries. Methods for construction of such cDNA and
genomic libraries are generally known in the art and are disclosed
in Sambrook and Russell, 2001. The so-called hybridization probes
may be genomic DNA fragments, cDNA fragments, RNA fragments, or
other oligonucleotides, and may be labeled with a detectable group
such as .sup.32P, or any other detectable marker, such as other
radioisotopes, a fluorescent compound, an enzyme, or an enzyme
co-factor. Probes for hybridization can be made by labeling
synthetic oligonucleotides based on the known
delta-endotoxin-encoding nucleotide sequence disclosed herein.
Degenerate primers designed on the basis of conserved nucleotides
or amino acid residues in the nucleotide sequence or encoded amino
acid sequence can additionally be used. The probe typically
comprises a region of nucleotide sequence that hybridizes under
stringent conditions to at least about 12, preferably about 25,
more preferably at least about 50, 75, 100, 125, 150, 175, 200,
250, 300, 350, or 400 consecutive nucleotides of delta-endotoxin
encoding nucleotide sequence of the invention or a fragment or
variant thereof. Preparation of probes for hybridization is
generally known in the art and is disclosed in Sambrook and
Russell, 2001, herein incorporated by reference.
[0042] For example, the entire delta-endotoxin sequence disclosed
herein, or one or more portions thereof, may be used as a probe
capable of specifically hybridizing to corresponding
delta-endotoxin-like sequences and messenger RNAs. To achieve
specific hybridization under a variety of conditions, such probes
include sequences that are unique and are preferably at least about
10 nucleotides in length, and most preferably at least about 20
nucleotides in length. Such probes may be used to amplify
corresponding delta-endotoxin 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 et al. (1989) Molecular Cloning: A Laboratory Manual (2d
ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
[0043] Hybridization of such sequences may be carried out under
stringent conditions. By "stringent conditions" or "stringent
hybridization conditions" is intended 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 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 nucleotides in
length, preferably less than 500 nucleotides in length.
[0044] 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 sulphate) 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 wash in 0.1.times.SSC at 60 to
65.degree. C. Optionally, wash buffers may comprise about 0.1% to
about 1% SDS. Duration of hybridization is generally less than
about 24 hours, usually about 4 to about 12 hours.
[0045] 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
T.sub.m can be approximated from the equation of Meinkoth and Wahl
(1984) Anal. Biochem. 138:267-284: T.sub.m=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 T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of a complementary target
sequence hybridizes to a perfectly matched probe. T.sub.m is
reduced by about 1.degree. C. for each 1% of mismatching; thus,
T.sub.m, 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 T.sub.m can be
decreased 110.degree. C. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) 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 thermal melting point (T.sub.m);
moderately stringent conditions can utilize a hybridization and/or
wash at 6, 7, 8, 9, or 10.degree. C. lower than the thermal melting
point (T.sub.m); low stringency conditions can utilize a
hybridization and/or wash at 11, 12, 13, 14, 15, or 20.degree. C.
lower than the thermal melting point (T.sub.m). Using the equation,
hybridization and wash compositions, and desired T.sub.m, 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 T.sub.m of less than
45.degree. C. (aqueous solution) or 32.degree. C. (formamide
solution), it is preferred to increase the SSC concentration 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 Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Plainview, N.Y.).
[0046] Isolated Proteins and Variants and Fragments Thereof
[0047] Delta-endotoxin proteins are also encompassed within the
present invention. By "delta-endotoxin protein" is intended a
protein having the amino acid sequence set forth in SEQ ID NO:2.
Fragments, biologically active portions, and variants thereof are
also provided, and may be used to practice the methods of the
present invention.
[0048] "Fragments" or "biologically active portions" include
polypeptide fragments comprising amino acid sequences sufficiently
identical to the amino acid sequence set forth in SEQ ID NO:2, and
exhibit delta-endotoxin activity. A biologically active portion of
a delta-endotoxin protein can be a polypeptide that is, for
example, 10, 25, 50, 100 or more amino acids in length. Such
biologically active portions can be prepared by recombinant
techniques and evaluated for delta-endotoxin activity. Methods for
measuring pesticidal activity are well known in the art. See, for
example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485;
Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al.
(1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No.
5,743,477, all of which are herein incorporated by reference in
their entirety. As used here, a fragment comprises at least 8
contiguous amino acids of SEQ ID NO:2. The invention encompasses
other fragments, however, such as any fragment in the protein
greater than about 10, 20, 30, 50, 100, 150, 200, 250, 300, 350,
400, 450, and 489 amino acids.
[0049] By "variants" is intended proteins or polypeptides having an
amino acid sequence that is at least about 60%, 65%, preferably
about 70%, 75%, more preferably 80%, 85%, most preferably 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino
acid sequence of SEQ ID NO:2. Variants also include polypeptides
encoded by a nucleic acid molecule that hybridizes to the nucleic
acid molecule of SEQ ID NO:1, or a complement thereof, under
stringent conditions. Such variants generally retain pesticidal
activity. Variants include polypeptides that differ in amino acid
sequence due to mutagenesis. Variant proteins encompassed by the
present invention are biologically active, that is they continue to
possess the desired biological activity of the native protein, that
is, retaining pesticidal activity. Methods for measuring pesticidal
activity are well known in the art. See, for example, Czapla and
Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988)
Biochem. J. 252:199-206; Marrone et al. (1985) J. of Economic
Entomology 78:290-293; and U.S. Pat. No. 5,743,477, all of which
are herein incorporated by reference in their entirety.
[0050] Antibodies to the polypeptides of the present invention, or
to variants or fragments thereof, are also encompassed. Methods for
producing antibodies are well known in the art (see, for example,
Harlow and Lane (1988) "Antibodies: A Laboratory Manual," Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; U.S. Pat. No.
4,196,265).
[0051] Altered or Improved Variants
[0052] It is recognized that DNA sequences of a delta-endotoxin may
be altered by various methods, and that these alterations may
result in DNA sequences encoding proteins with amino acid sequences
different than that encoded by the delta-endotoxin of the present
invention. This protein may be altered in various ways including
amino acid substitutions, deletions, truncations, and insertions.
Methods for such manipulations are generally known in the art. For
example, amino acid sequence variants of the delta-endotoxin
protein can be prepared by mutations in the DNA. This may also be
accomplished by one of several forms of mutagenesis and/or in
directed evolution. In some aspects, the changes encoded in the
amino acid sequence will not substantially affect the function of
the protein. Such variants will possess the desired pesticidal
activity. However, it is understood that the ability of
delta-endotoxin to confer pesticidal activity may be improved by
the use of such techniques upon the compositions of this invention.
For example, one may express delta-endotoxin in host cells that
exhibit high rates of base misincorporation during DNA replication,
such as XL-1 Red (Stratagene). After propagation in such strains,
one can isolate the delta-endotoxin DNA (for example by preparing
plasmid DNA, or by amplifying by PCR and cloning the resulting PCR
fragment into a vector), culture the delta-endotoxin mutations in a
non-mutagenic strain, and identify mutated delta-endotoxin genes
with pesticidal activity, for example by performing an assay to
test for pesticidal activity. Generally, the protein is mixed and
used in feeding assays. See, for example Marrone et al. (1985) J.
of Economic Entomology 78:290-293. Such assays can include
contacting plants with one or more pests and determining the
plant's ability to survive and/or cause the death of the pests.
Examples of mutations that result in increased toxicity are found
in Schnepf et al. (1998) Microbiol. Mol. Biol. Rev. 62:775-806.
[0053] Alternatively, alterations may be made to the protein
sequence of many proteins at the amino or carboxy terminus without
substantially affecting activity. This can include insertions,
deletions, or alterations introduced by modem molecular methods,
such as PCR, including PCR amplifications that alter or extend the
protein coding sequence by virtue of inclusion of amino acid
encoding sequences in the oligonucleotides utilized in the PCR
amplification. Alternatively, the protein sequences added can
include entire protein-coding sequences, such as those used
commonly in the art to generate protein fusions. Such fusion
proteins are often used to (1) increase expression of a protein of
interest (2) introduce a binding domain, enzymatic activity, or
epitope to facilitate either protein purification, protein
detection, or other experimental uses known in the art (3) target
secretion or translation of a protein to a subcellular organelle,
such as the periplasmic space of Gram-negative bacteria, or the
endoplasmic reticulum of eukaryotic cells, the latter of which
often results in glycosylation of the protein.
[0054] Variant nucleotide and amino acid sequences of the present
invention also encompass sequences derived from mutagenic and
recombinogenic procedures such as DNA shuffling. With such a
procedure, one or more different delta-endotoxin protein coding
regions can be used to create a new delta-endotoxin protein
possessing the desired properties. In this manner, libraries of
recombinant polynucleotides are generated from a population of
related sequence polynucleotides comprising sequence regions that
have substantial sequence identity and can be homologously
recombined in vitro or in vivo. For example, using this approach,
sequence motifs encoding a domain of interest may be shuffled
between the delta-endotoxin gene of the invention and other known
delta-endotoxin genes to obtain a new gene coding for a protein
with an improved property of interest, such as an increased
insecticidal activity. Strategies for such DNA shuffling are known
in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci.
USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et
al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol.
Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA
94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S.
Pat. Nos. 5,605,793 and 5,837,458.
[0055] Domain swapping or shuffling is another mechanism for
generating altered delta-endotoxin proteins. Domains II and III may
be swapped between delta-endotoxin proteins, resulting in hybrid or
chimeric toxins with improved pesticidal activity or target
spectrum. Methods for generating recombinant proteins and testing
them for pesticidal activity are well known in the art (see, for
example, Naimov et al. (2001) Appl. Environ. Microbiol.
67:5328-5330; de Maagd et al. (1996) Appl. Environ. Microbiol.
62:1537-1543; Ge et al. (1991) J. Biol. Chem. 266:17954-17958;
Schnepf et al. (1990) J. Biol. Chem. 265:20923-20930; Rang et al.
91999) Appl. Environ. Micriobiol. 65:2918-2925).
[0056] Plant Transformation
[0057] Transformation of plant cells can be accomplished by one of
several techniques known in the art. First, one engineers the
delta-endotoxin gene in a way that allows its expression in plant
cells. Typically a construct that expresses such a protein would
contain a promoter to drive transcription of the gene, as well as a
3' untranslated region to allow transcription termination and
polyadenylation. The organization of such constructs is well known
in the art. In some instances, it may be useful to engineer the
gene such that the resulting peptide is secreted, or otherwise
targeted within the plant cell. For example, the gene can be
engineered to contain a signal peptide to facilitate transfer of
the peptide to the endoplasmic reticulum. It may also be preferable
to engineer the plant expression cassette to contain an intron,
such that mRNA processing of the intron is required for
expression.
[0058] Typically this `plant expression cassette` will be inserted
into a `plant transformation vector`. This plant transformation
vector may be comprised of one or more DNA vectors needed for
achieving plant transformation. For example, it is a common
practice in the art to utilize plant transformation vectors that
are comprised of more than one contiguous DNA segment. These
vectors are often referred to in the art as `binary vectors`.
Binary vectors as well as vectors with helper plasmids are most
often used for Agrobacterium-mediated transformation, where the
size and complexity of DNA segments needed to achieve efficient
transformation is quite large, and it is advantageous to separate
functions onto separate DNA molecules. Binary vectors typically
contain a plasmid vector that contains the cis-acting sequences
required for T-DNA transfer (such as left border and right border),
a selectable marker that is engineered to be capable of expression
in a plant cell, and a `gene of interest` (a gene engineered to be
capable of expression in a plant cell for which generation of
transgenic plants is desired). Also present on this plasmid vector
are sequences required for bacterial replication. The cis-acting
sequences are arranged in a fashion to allow efficient transfer
into plant cells and expression therein. For example, the
selectable marker gene and the gene of interest are located between
the left and right borders. Often a second plasmid vector contains
the trans-acting factors that mediate T-DNA transfer from
Agrobacterium to plant cells. This plasmid often contains the
virulence functions (Vir genes) that allow infection of plant cells
by Agrobacterium, and transfer of DNA by cleavage at border
sequences and vir-mediated DNA transfer, as in understood in the
art (Hellens and Mullineaux (2000) Trends in Plant Science
5:446-451). Several types of Agrobacterium strains (e.g. LBA4404,
GV3101, EHA101, EHA105, etc.) can be used for plant transformation.
The second plasmid vector is not necessary for transforming the
plants by other methods such as microprojection, microinjection,
electroporation, polyethylene glycol, etc.
[0059] In general, plant transformation methods involve
transferring heterologous DNA into target plant cells (e.g.
immature or mature embryos, suspension cultures, undifferentiated
callus, protoplasts, etc.), followed by applying a maximum
threshold level of appropriate selection (depending on the
selectable marker gene) to recover the transformed plant cells from
a group of untransformed cell mass. Explants are typically
transferred to a fresh supply of the same medium and cultured
routinely. Subsequently, the transformed cells are differentiated
into shoots after placing on regeneration medium supplemented with
a maximum threshold level of selecting agent. The shoots are then
transferred to a selective rooting medium for recovering rooted
shoot or plantlet. The transgenic plantlet then grows into a mature
plant and produces fertile seeds (e.g. Hiei et al. (1994) The Plant
Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology
14:745-750). Explants are typically transferred to a fresh supply
of the same medium and cultured routinely. A general description of
the techniques and methods for generating transgenic plants are
found in Ayres and Park (1994) Critical Reviews in Plant Science
13:219-239 and Bommineni and Jauhar (1997) Maydica 42:107-120.
Since the transformed material contains many cells; both
transformed and non-transformed cells are present in any piece of
subjected target callus or tissue or group of cells. The ability to
kill non-transformed cells and allow transformed cells to
proliferate results in transformed plant cultures. Often, the
ability to remove non-transformed cells is a limitation to rapid
recovery of transformed plant cells and successful generation of
transgenic plants.
[0060] Generation of transgenic plants may be performed by one of
several methods, including but not limited to introduction of
heterologous DNA by Agrobacterium into plant cells
(Agrobacterium-mediated transformation), bombardment of plant cells
with heterologous foreign DNA adhered to particles, and various
other non-particle direct-mediated methods (e.g. Hiei et al. (1994)
The Plant Journal 6:271-282; Ishida et al. (1996) Nature
Biotechnology 14:745-750; Ayres and Park (1994) Critical Reviews in
Plant Science 13:219-239; Bommineni and Jauhar (1997) Maydica
42:107-120) to transfer DNA.
[0061] Transformation protocols as well as protocols for
introducing nucleotide sequences into plants may vary depending on
the type of plant or plant cell, i.e., monocot or dicot, targeted
for transformation. Suitable methods of introducing nucleotide
sequences into plant cells and subsequent insertion into the plant
genome include microinjection (Crossway et al. (1986) Biotechniques
4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad.
Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (U.S.
Pat. No. 5,563,055; U.S. Pat. No. 5,981,840), direct gene transfer
(Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic
particle acceleration (see, for example, U.S. Pat. No. 4,945,050;
U.S. Pat. No. 5,879,918; U.S. Pat. No. 5,886,244; U.S. Pat. No.
5,932,782; Tomes et al. (1995) "Direct DNA Transfer into Intact
Plant Cells via Microprojectile Bombardment," in Plant Cell,
Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and
Phillips (Springer-Verlag, Berlin); McCabe et al. (1988)
Biotechnology 6:923-926); aerosol beam transformation (U.S.
Published Application No. 20010026941; U.S. Pat. No. 4,945,050;
International Publication No. WO 91/00915; U.S. Published
Application No. 2002015066); and Lecl transformation (WO 00/28058).
Also see Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477;
Sanford et al. (1987) Particulate Science and Technology 5:27-37;
Christou et al. (1988) Plant Physiol. 87:671-674; McCabe et al.
(1988) Bio/Technology 6:923-926; Finer and McMullen (1991) In Vitro
Cell Dev. Biol. 27P:175-182; Singh et al. (1998) Theor. Appl.
Genet. 96:319-324; Datta et al. (1990) Biotechnology 8:736-740;
Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309; U.S.
Pat. No. 5,240,855; U.S. Pat. Nos. 5,322,783 and 5,324,646; Klein
et al. (1988) Plant Physiol. 91:440-444; Hooykaas-Van Slogteren et
al. (1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369;
Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349; De
Wet et al. (1985) in The Experimental Manipulation of Ovule
Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209;
Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et
al. (1992) Theor. Appl. Genet. 84:560-566; D'Halluin et al. (1992)
Plant Cell 4:1495-1505; Li et al. (1993) Plant Cell Reports
12:250-255 and Christou and Ford (1995) Annals of Botany
75:407-413; Osjoda et al. (1996) Nature Biotechnology 14:745-750;
all of which are herein incorporated by reference.
[0062] Following integration of heterologous foreign DNA into plant
cells, one then applies a maximum threshold level of appropriate
selection in the medium to kill the untransformed cells and
separate and proliferate the putatively transformed cells that
survive from this selection treatment by transferring regularly to
a fresh medium. By continuous passage and challenge with
appropriate selection, one identifies and proliferates the cells
that are transformed with the plasmid vector. Then molecular and
biochemical methods will be used for confirming the presence of the
integrated heterologous gene of interest in the genome of
transgenic plant.
[0063] The cells that have been transformed may be grown into
plants in accordance with conventional ways. See, for example,
McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants
may then be grown, and either pollinated with the same transformed
strain or different strains, and the resulting hybrid having
constitutive expression of the desired phenotypic characteristic
identified. Two or more generations may be grown to ensure that
expression of the desired phenotypic characteristic is stably
maintained and inherited and then seeds harvested to ensure
expression of the desired phenotypic characteristic has been
achieved. In this manner, the present invention provides
transformed seed (also referred to as "transgenic seed") having a
nucleotide construct of the invention, for example, an expression
cassette of the invention, stably incorporated into their
genome.
[0064] The delta-endotoxin sequences of the invention may be
provided in expression cassettes for expression in the plant of
interest. The cassette will include 5' and 3' regulatory sequences
operably linked to a sequence of the invention. By "operably
linked" is intended a functional linkage between a promoter and a
second sequence, wherein the promoter sequence initiates and
mediates transcription of the DNA sequence corresponding to the
second sequence. Generally, operably linked means that the nucleic
acid sequences being linked are contiguous and, where necessary to
join two protein coding regions, contiguous and in the same reading
frame. The cassette may additionally contain at least one
additional gene to be cotransformed into the organism.
Alternatively, the additional gene(s) can be provided on multiple
expression cassettes.
[0065] Such an expression cassette is provided with a plurality of
restriction sites for insertion of the delta-endotoxin sequence to
be under the transcriptional regulation of the regulatory
regions.
[0066] The expression cassette will include in the 5'-3' direction
of transcription, a transcriptional and translational initiation
region (i.e., a promoter), a DNA sequence of the invention, and a
transcriptional and translational termination region (i.e.,
termination region) functional in plants. The promoter may be
native or analogous, or foreign or heterologous, to the plant host
and/or to the DNA sequence of the invention. Additionally, the
promoter may be the natural sequence or alternatively a synthetic
sequence. Where the promoter is "native" or "homologous" to the
plant host, it is intended that the promoter is found in the native
plant into which the promoter is introduced. Where the promoter is
"foreign" or "heterologous" to the DNA sequence of the invention,
it is intended that the promoter is not the native or naturally
occurring promoter for the operably linked DNA sequence of the
invention.
[0067] The termination region may be native with the
transcriptional initiation region, may be native with the operably
linked DNA sequence of interest, may be native with the plant host,
or may be derived from another source (i.e., foreign or
heterologous to the promoter, the DNA sequence of interest, the
plant host, or any combination thereof). Convenient termination
regions are available from the Ti-plasmid of A. tumefaciens, such
as the octopine synthase and nopaline synthase termination regions.
See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144;
Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev.
5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et
al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.
17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res.
15:9627-9639.
[0068] Where appropriate, the gene(s) may be optimized for
increased expression in the transformed host cell. That is, the
genes can be synthesized using host cell-preferred codons for
improved expression, or may be synthesized using codons at a
host-preferred codon usage frequency. Generally, the GC content of
the gene will be increased. See, for example, Campbell and Gowri
(1990) Plant Physiol. 92: 1-11 for a discussion of host-preferred
codon usage. Methods are available in the art for synthesizing
plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831,
and 5,436,391, and Murray et al. (1989) Nucleic Acids Res.
17:477-498, herein incorporated by reference.
[0069] In one embodiment, the nucleic acids of interest are
targeted to the chloroplast for expression. In this manner, where
the nucleic acid of interest is not directly inserted into the
chloroplast, the expression cassette will additionally contain a
nucleic acid encoding a transit peptide to direct the gene product
of interest to the chloroplasts. Such transit peptides are known in
the art. See, for example, Von Heijne et al. (1991) Plant Mol.
Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem.
264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol.
84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun.
196:1414-1421; and Shah et al. (1986) Science 233:478-481.
[0070] Methods for transformation of chloroplasts are known in the
art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci.
USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA
90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method
relies on particle gun delivery of DNA containing a selectable
marker and targeting of the DNA to the plastid genome through
homologous recombination. Additionally, plastid transformation can
be accomplished by transactivation of a silent plastid-borne
transgene by tissue-preferred expression of a nuclear-encoded and
plastid-directed RNA polymerase. Such a system has been reported in
McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.
[0071] The nucleic acids of interest to be targeted to the
chloroplast may be optimized for expression in the chloroplast to
account for differences in codon usage between the plant nucleus
and this organelle. In this manner, the nucleic acids of interest
may be synthesized using chloroplast-preferred codons. See, for
example, U.S. Pat. No. 5,380,831, herein incorporated by
reference.
[0072] Evaluation of Plant Transformation
[0073] Following introduction of heterologous foreign DNA into
plant cells, the transformation or integration of heterologous gene
in the plant genome is confirmed by various methods such as
analysis of nucleic acids, proteins and metabolites associated with
the integrated gene.
[0074] PCR Analysis: PCR analysis is a rapid method to screen
transformed cells, tissue or shoots for the presence of
incorporated gene at the earlier stage before transplanting into
the soil (Sambrook and Russell (2001) Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.). PCR is carried out using oligonucleotide primers
specific to the gene of interest or Agrobacterium vector
background, etc.
[0075] Southern Analysis: Plant transformation is confirmed by
Southern blot analysis of genomic DNA (Sambrook and Russell, 2001,
supra). In general, total DNA is extracted from the transformant,
digested with appropriate restriction enzymes, fractionated in an
agarose gel and transferred to a nitrocellulose or nylon membrane.
The membrane or "blot" then is probed with, for example,
radiolabeled .sup.32P target DNA fragment to confirm the
integration of introduced gene in the plant genome according to
standard techniques (Sambrook and Russell, 2001, supra).
[0076] Northern Analysis: RNA is isolated from specific tissues of
transformant, fractionated in a formaldehyde agarose gel, blotted
onto a nylon filter according to standard procedures that are
routinely used in the art (Sambrook and Russell, 2001, supra).
Expression of RNA encoded by the delta-endotoxin is then tested by
hybridizing the filter to a radioactive probe derived from a
delta-endotoxin, by methods known in the art (Sambrook and Russell,
2001, supra).
[0077] Western blot and Biochemical assays: Western blot and
biochemical assays and the like may be carried out on the
transgenic plants to confirm the presence of protein encoded by the
delta-endotoxin gene by standard procedures (Sambrook and Russell,
2001, supra) using antibodies that bind to one or more epitopes
present on the delta-endotoxin protein.
[0078] Pesticidal Activity in Plants
[0079] In another aspect of the invention, one may generate
transgenic plants expressing delta-endotoxin that have pesticidal
activity. Methods described above by way of example may be utilized
to generate transgenic plants, but the manner in which the
transgenic plant cells are generated is not critical to this
invention. Methods known or described in the art such as
Agrobacterium-mediated transformation, biolistic transformation,
and non-particle-mediated methods may be used at the discretion of
the experimenter. Plants expressing delta-endotoxin may be isolated
by common methods described in the art, for example by
transformation of callus, selection of transformed callus, and
regeneration of fertile plants from such transgenic callus. In such
process, one may use any gene as a selectable marker so long as its
expression in plant cells confers ability to identify or select for
transformed cells.
[0080] A number of markers have been developed for use with plant
cells, such as resistance to chloramphenicol, the aminoglycoside
G418, hygromycin, or the like. Other genes that encode a product
involved in chloroplast metabolism may also be used as selectable
markers. For example, genes that provide resistance to plant
herbicides such as glyphosate, bromoxynil, or imidazolinone may
find particular use. Such genes have been reported (Stalker et al.
(1985) J. Biol. Chem. 263:6310-6314 (bromoxynil resistance
nitrilase gene); and Sathasivan et al. (1990) Nucl. Acids Res.
18:2188 (AHAS imidazolinone resistance gene).
[0081] Fertile plants expressing delta-endotoxin may be tested for
pesticidal activity, and the plants showing optimal activity
selected for further breeding. Methods are available in the art to
assay for pest activity. Generally, the protein is mixed and used
in feeding assays. See, for example Marrone et al. (1985) J of
Economic Entomology 78:290-293. The present invention may be used
for transformation of any plant species, including, but not limited
to, monocots and dicots. Examples of plants of interest include,
but are not limited to, corn (maize), sorghum, wheat, sunflower,
tomato, crucifers, peppers, potato, cotton, rice, soybean,
sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica
sp., alfalfa, rye, millet, safflower, peanuts, sweet potato,
cassaya, coffee, coconut, pineapple, citrus trees, cocoa, tea,
banana, avocado, fig, guava, mango, olive, papaya, cashew,
macadamia, almond, oats, vegetables, ornamentals, and conifers.
[0082] Vegetables include, but are not limited to, tomatoes,
lettuce, green beans, lima beans, peas, and members of the genus
Cucumis such as cucumber, cantaloupe, and musk melon. Ornamentals
include, but are not limited to, azalea, hydrangea, hibiscus,
roses, tulips, daffodils, petunias, carnation, poinsettia, and
chrysanthemum. Preferably, plants of the present invention are crop
plants (for example, maize, sorghum, wheat, sunflower, tomato,
crucifers, peppers, potato, cotton, rice, soybean, sugarbeet,
sugarcane, tobacco, barley, oilseed rape., etc.).
[0083] Use in Pesticidal Control
[0084] General methods for employing strains comprising the
nucleotide sequence of the present invention, or a variant thereof,
in pesticide control or in engineering other organisms as
pesticidal agents are known in the art. See, for example U.S. Pat.
No. 5,039,523 and EP 0480762A2.
[0085] The Bacillus strains containing the nucleotide sequence of
the present invention, or a variant thereof, or the microorganisms
that have been genetically altered to contain the pesticidal gene
and protein may be used for protecting agricultural crops and
products from pests. In one aspect of the invention, whole, i.e.,
unlysed, cells of a toxin (pesticide)-producing organism are
treated with reagents that prolong the activity of the toxin
produced in the cell when the cell is applied to the environment of
target pest(s).
[0086] Alternatively, the pesticide is produced by introducing a
heterologous gene into a cellular host. Expression of the
heterologous gene results, directly or indirectly, in the
intracellular production and maintenance of the pesticide. In one
aspect of this invention, these cells are then treated under
conditions that prolong the activity of the toxin produced in the
cell when the cell is applied to the environment of target pest(s).
The resulting product retains the toxicity of the toxin. These
naturally encapsulated pesticides may then be formulated in
accordance with conventional techniques for application to the
environment hosting a target pest, e.g., soil, water, and foliage
of plants. See, for example EPA 0192319, and the references cited
therein. Alternatively, one may formulate the cells expressing the
genes of this invention such as to allow application of the
resulting material as a pesticide.
[0087] The active ingredients of the present invention are normally
applied in the form of compositions and can be applied to the crop
area or plant to be treated, simultaneously or in succession, with
other compounds. These compounds can be fertilizers, weed killers,
cryoprotectants, surfactants, detergents, pesticidal soaps, dormant
oils, polymers, and/or time-release or biodegradable carrier
formulations that permit long-term dosing of a target area
following a single application of the formulation. They can also be
selective herbicides, chemical insecticides, virucides,
microbicides, amoebicides, pesticides, fungicides, bacteriocides,
nematocides, mollusocides or mixtures of several of these
preparations, if desired, together with further agriculturally
acceptable carriers, surfactants or application-promoting adjuvants
customarily employed in the art of formulation. Suitable carriers
and adjuvants can be solid or liquid and correspond to the
substances ordinarily employed in formulation technology, e.g.
natural or regenerated mineral substances, solvents, dispersants,
wetting agents, tackifiers, binders or fertilizers. Likewise the
formulations may be prepared into edible "baits" or fashioned into
pest "traps" to permit feeding or ingestion by a target pest of the
pesticidal formulation.
[0088] Preferred methods of applying an active ingredient of the
present invention or an agrochemical composition of the present
invention that contains at least one of the pesticidal proteins
produced by the bacterial strains of the present invention are leaf
application, seed coating and soil application. The number of
applications and the rate of application depend on the intensity of
infestation by the corresponding pest.
[0089] The composition may be formulated as a powder, dust, pellet,
granule, spray, emulsion, colloid, solution, or such like, and may
be preparable by such conventional means as desiccation,
lyophilization, homogenation, extraction, filtration,
centrifugation, sedimentation, or concentration of a culture of
cells comprising the polypeptide. In all such compositions that
contain at least one such pesticidal polypeptide, the polypeptide
may be present in a concentration of from about 1% to about 99% by
weight.
[0090] Lepidopteran or coleopteran pests may be killed or reduced
in numbers in a given area by the methods of the invention, or may
be prophylactically applied to an environmental area to prevent
infestation by a susceptible pest. Preferably the pest ingests, or
is contacted with, a pesticidally effective amount of the
polypeptide. By "pesticidally effective amount" is intended an
amount of the pesticide that is able to bring about death to at
least one pest, or to noticeably reduce pest growth, feeding, or
normal physiological development. This amount will vary depending
on such factors as, for example, the specific target pests to be
controlled, the specific environment, location, plant, crop, or
agricultural site to be treated, the environmental conditions, and
the method, rate, concentration, stability, and quantity of
application of the pesticidally effective polypeptide composition.
The formulations may also vary with respect to climatic conditions,
environmental considerations, and/or frequency of application
and/or severity of pest infestation.
[0091] The pesticide compositions described may be made by
formulating either the bacterial cell, crystal and/or spore
suspension, or isolated protein component with the desired
agriculturally-acceptable carrier. The compositions may be
formulated prior to administration in an appropriate means such as
lyophilized, freeze-dried, desiccated, or in an aqueous carrier,
medium or suitable diluent, such as saline or other buffer. The
formulated compositions may be in the form of a dust or granular
material, or a suspension in oil (vegetable or mineral), or water
or oil/water emulsions, or as a wettable powder, or in combination
with any other carrier material suitable for agricultural
application. Suitable agricultural carriers can be solid or liquid
and are well known in the art. The term "agriculturally-acceptable
carrier" covers all adjuvants, inert components, dispersants,
surfactants, tackifiers, binders, etc. that are ordinarily used in
pesticide formulation technology; these are well known to those
skilled in pesticide formulation. The formulations may be mixed
with one or more solid or liquid adjuvants and prepared by various
means, e.g., by homogeneously mixing, blending and/or grinding the
pesticidal composition with suitable adjuvants using conventional
formulation techniques. Suitable formulations and application
methods are described in U.S. Pat. No. 6,468,523, herein
incorporated by reference.
[0092] "Pest" includes but is not limited to, insects, fungi,
bacteria, nematodes, mites, ticks, and the like. Insect pests
include insects selected from the orders Coleoptera, Diptera,
Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera,
Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura,
Siphonaptera, Trichoptera, etc., particularly Coleoptera and
Lepidoptera. Insect pests of the invention for the major crops
include: Maize: Ostrinia nubilalis, European corn borer; Agrotis
ipsilon, black cutworm; Helicoverpa zea, corn earworm; Spodoptera
frugiperda, fall armyworm; Diatraea grandiosella, southwestern corn
borer; Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea
saccharalis, surgarcane borer; Diabrotica virgifera, western corn
rootworm; Diabrotica longicornis barberi, northern corn rootworm;
Diabrotica undecimpunctata howardi, southern corn rootworm;
Melanotus spp., wireworms; Cyclocephala borealis, northern masked
chafer (white grub); Cyclocephala immaculata, southern masked
chafer (white grub); Popillia japonica, Japanese beetle;
Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize
billbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis
maidiradicis, corn root aphid; Blissus leucopterus leucopterus,
chinch bug; Melanoplus femurrubrum, redlegged grasshopper;
Melanoplus sanguinipes, migratory grasshopper; Hylemya platura,
seedcorn maggot; Agromyza parvicornis, corn blot leafminer;
Anaphothrips obscrurus, grass thrips; Solenopsis milesta, thief
ant; Tetranychus urticae, twospotted spider mite; Sorghum: Chilo
partellus, sorghum borer; Spodoptera frugiperda, fall armyworm;
Helicoverpa zea, corn earworm; Elasmopalpus lignosellus, lesser
cornstalk borer; Feltia subterranea, granulate cutworm; Phyllophaga
crinita, white grub; Eleodes, Conoderus, and Aeolus spp.,
wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema
pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug;
Rhopalosiphum maidis; corn leaf aphid; Sipha flava, yellow
sugarcane aphid; Blissus leucopterus leucopterus, chinch bug;
Contarinia sorghicola, sorghum midge; Tetranychus cinnabarinus,
carmine spider mite; Tetranychus urticae, twospotted spider mite;
Wheat: Pseudaletia unipunctata, army worm; Spodoptera frugiperda,
fall armyworm; Elasmopalpus lignosellus, lesser cornstalk borer;
Agrotis orthogonia, western cutworm; Elasmopalpus lignosellus,
lesser cornstalk borer; Oulema melanopus, cereal leaf beetle;
Hypera punctata, clover leaf weevil; Diabrotica undecimpunctata
howardi, southern corn rootworm; Russian wheat aphid; Schizaphis
graminum, greenbug; Macrosiphum avenae, English grain aphid;
Melanoplus femurrubrum, redlegged grasshopper; Melanoplus
differentialis, differential grasshopper; Melanoplus sanguinipes,
migratory grasshopper; Mayetiola destructor, Hessian fly;
Sitodiplosis mosellana, wheat midge; Meromyza americana, wheat stem
maggot; Hylemya coarctata, wheat bulb fly; Frankliniella fusca,
tobacco thrips; Cephus cinctus, wheat stem sawfly; Aceria tulipae,
wheat curl mite; Sunflower: Suleima helianthana, sunflower bud
moth; Homoeosoma electellum, sunflower moth; zygogramma
exclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle;
Neolasioptera murtfeldtiana, sunflower seed midge; Cotton:
Heliothis virescens, cotton budworm; Helicoverpa zea, cotton
bollworm; Spodoptera exigua, beet armyworm; Pectinophora
gossypiella, pink bollworm; Anthonomus grandis, boll weevil; Aphis
gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton
fleahopper; Trialeurodes abutilonea, bandedwinged whitefly; Lygus
lineolaris, tarnished plant bug; Melanoplus femurrubrum, redlegged
grasshopper; Melanoplus differentialis, differential grasshopper;
Thrips tabaci, onion thrips; Franklinkiella fusca, tobacco thrips;
Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae,
twospotted spider mite; Rice: Diatraea saccharalis, sugarcane
borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn
earworm; Colaspis brunnea, grape colaspis; Lissorhoptrus
oryzophilus, rice water weevil; Sitophilus oryzae, rice weevil;
Nephotettix nigropictus, rice leafhopper; Blissus leucopterus
leucopterus, chinch bug; Acrosternum hilare, green stink bug;
Soybean: Pseudoplusia includens, soybean looper; Anticarsia
gemmatalis, velvetbean caterpillar; Plathypena scabra, green
cloverworm; Ostrinia nubilalis, European corn borer; Agrotis
ipsilon, black cutworm; Spodoptera exigua, beet armyworm; Heliothis
virescens, cotton budworm; Helicoverpa zea, cotton bollworm;
Epilachna varivestis, Mexican bean beetle; Myzuspersicae, green
peach aphid; Empoasca fabae, potato leafhopper; Acrosternum hilare,
green stink bug; Melanoplusfemurrubrum, redlegged grasshopper;
Melanoplus differentialis, differential grasshopper; Hylemya
platura, seedcorn maggot; Sericothrips variabilis, soybean thrips;
Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry
spider mite; Tetranychus urticae, twospotted spider mite; Barley:
Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black
cutworm; Schizaphis graminum, greenbug; Blissus leucopterus
leucopterus, chinch bug; Acrosternum hilare, green stink bug;
Euschistus servus, brown stink bug; Delia platura, seedcorn maggot;
Mayetiola destructor, Hessian fly; Petrobia latens, brown wheat
mite; Oil Seed Raipe: Brevicoryne brassicae, cabbage aphid;
Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Bertha
armyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Root
maggots.
[0093] Nematodes include parasitic nematodes such as root-knot,
cyst, and lesion nematodes, including Heterodera spp., Meloidogyne
spp., and Globodera spp.; particularly members of the cyst
nematodes, including, but not limited to, Heterodera glycines
(soybean cyst nematode); Heterodera schachtii (beet cyst nematode);
Heterodera avenae (cereal cyst nematode); and Globodera
rostochiensis and Globodera pailida (potato cyst nematodes). Lesion
nematodes include Pratylenchus spp.
[0094] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
Example 1
Extraction of Plasmid DNA
[0095] A pure culture of strain ATX13026 was grown in large
quantities of rich media. The culture was spun to harvest the cell
pellet. The cell pellet was then prepared by treatment with SDS by
methods known in the art, resulting in breakage of the cell wall
and release of DNA. Proteins and large genomic DNA was then
precipitated by a high salt concentration. The plasmid DNA was then
precipitated by standard ethanol precipitation. The plasmid DNA was
separated from any remaining chromosomal DNA by high-speed
centrifugation through a cesium chloride gradient. The DNA was
visualized in the gradient by UV light and the band of lower
density (i.e. the lower band) was extracted using a syringe. This
band contained the plasmid DNA from Strain ATX13026. The quality of
the DNA was checked by visualization on an agarose gel by methods
known in the art.
Example 2
Cloning of Genes
[0096] The purified plasmid DNA was sheared into 5-10 kb sized
fragments and the 5' and 3' single stranded overhangs repaired
using T4 DNA polymerase and Klenow fragment in the presence of all
four dNTPs, as known in the art. Phosphates were then attached to
the 5' ends by treatment with T4 polynucleotide kinase, as known in
the art. The repaired DNA fragments were then ligated overnight
into a standard high copy vector (i.e. pBluescript SK+), suitably
prepared to accept the inserts as known in the art (for example by
digestion with a restriction enzyme producing blunt ends).
[0097] The quality of the library was analyzed by digesting a
subset of clones with a restriction enzyme known to have a cleavage
site flanking the cloning site. A high percentage of clones were
determined to contain inserts, with an average insert size of 5-6
kb.
Example 3
High Throughput Sequencing of Library Plates
[0098] Once the shotgun library quality was checked and confirmed,
colonies were grown in a rich broth in 2 ml 96-well blocks
overnight at 37.degree. C. at a shaking speed of 350 rpm. The
blocks were spun to harvest the cells to the bottom of the block.
The blocks were then prepared by standard alkaline lysis prep in a
high throughput format.
[0099] The end sequences of clones from this library were then
determined for a large number of clones from each block in the
following way: The DNA sequence of each clone chosen for analysis
was determined using the fluorescent dye terminator sequencing
technique (Applied Biosystems) and standard primers flanking each
side of the cloning site. Once the reactions had been carried out
in the thermocycler, the DNA was precipitated using standard
ethanol precipitation. The DNA was resuspended in water and loaded
onto a capillary sequencing machine. Each library plate of DNA was
sequenced from either end of the cloning site, yielding two reads
per plate over each insert.
Example 4
Assembly and Screening of Sequencing Data
[0100] DNA sequences obtained were compiled into an assembly
project and aligned together to form contigs. This can be done
efficiently using a computer program, such as Vector NTi, or
alternatively by using the Pred/Phrap suite of DNA alignment and
analysis programs. These contigs, along with any individual read
that may not have been added to a contig, were compared to a
compiled database of all classes of known pesticidal genes. Contigs
or individual reads identified as having identity to a known
endotoxin or pesticidal gene were analyzed further. Among the
sequences obtained, clone pAX010 contained DNA identified as having
homology to known endotoxin genes. Therefore, pAX010 was selected
for further sequencing.
Example 5
Sequencing of pATX-010, and Identification of AXMI-010
[0101] Primers were designed to anneal to pAX-010, in a manner such
that DNA sequences generated from such primers will overlap
existing DNA sequence of the clone(s). This process, known as
"oligo walking", is well known in the art. This process was
utilized to determine the entire DNA sequence of the region
exhibiting homology to a known endotoxin gene. In the case of
pAX-010, this process was used to determine the DNA sequence of the
entire clone, resulting in a single nucleotide sequence. The
completed DNA sequence was then placed back into the original large
assembly for further validation. This allowed incorporation of more
DNA sequence reads into the contig, resulting in multiple reads of
coverage over the entire region.
[0102] Analysis of the DNA sequence of pAX-010 by methods known in
the art identified an open reading frame with homology to known
delta endotoxin genes. This open reading frame is designated as
AXMI-010. The DNA sequence of AXMI-010 is provided as SEQ ID NO:1,
and the amino acid sequence of the predicted AXMI-010 protein is
provided in SEQ ID NO:2.
Example 6
Homology of AXMI-010 to Known Endotoxin Genes
[0103] Searches of DNA and protein databases with the DNA sequence
and amino acid sequence of AXMI-010 reveal that AXMI-010 is
homologous to a set of known endotoxins.
[0104] Blast searches identify ET69 (cry36Aa1) as having the
strongest block of homology to AXMI-010. The overall amino acid
identity of AXMI-010 to cry36Aa1 is 35% (see Table 1). Alignment of
AXMI-010 protein (SEQ ID NO:2) to a set of related toxin proteins
(FIG. 1) shows that the most homologous protein is cry36Aa1.
Searches of the pFAM database identify AXMI-010 as having homology
to the Insecticidal Crystal Toxin, P42 family (`Toxin10` family) of
endotoxins (PFAM Accession No. PF05431). Strains of Bacillus that
have this insecticidal activity use a binary toxin comprised of two
proteins, P51 and P42 (this family). The P42 protein alone has been
shown to have activity against mosquitos (Baumann et al. (1985) J.
Bacteriol. 163:738-47). ET69 has been shown to have activity
against Western Corn Root Worm (WCRW) (International Publication
No. WO0066742). Members of this family are highly conserved between
strains of different serotypes and phage groups (Humphreys and
Berry (1998) J. Invert. Pathol. 71:184-185). These toxins differ
somewhat from the `typical` endotoxin in that they do not contain
the set of five conserved domains shared by, for example
Cry1Aa-like toxins. AXMI-010 contains conserved domains that are
present in this `B. Sphaericus-like` toxin subfamily. Inspection of
the amino acid sequence of AXMI-010 suggests that it does not
contain a C-terminal non-toxic domain as is present in several
endotoxin families.
1TABLE 1 Amino Acid Identity of AXMI-010 with Related Toxins Amino
Acid Identity to Toxin GenBank Accession No: AXMI-010 Cry36Aa1
(ET69) AAK64558.1 35% BinB4 CAA04920.1 24% Cry35Ab (149-B1)
AAG41672.1 14%
Example 7
Assay for Pesticidal Activity
[0105] The ability of a pesticidal protein to act as a pesticide
upon a pest is often assessed in a number of ways. One way well
known in the art is to perform a feeding assay. In such a feeding
assay, one exposes the pest to a sample containing either compounds
to be tested, or control samples. Often this is performed by
placing the material to be tested, or a suitable dilution of such
material, onto a material that the pest will ingest, such as an
artificial diet. The material to be tested may be composed of a
liquid, solid, or slurry. The material to be tested may be placed
upon the surface and then allowed to dry. Alternatively, the
material to be tested may be mixed with a molten artificial diet,
then dispensed into the assay chamber. The assay chamber may be,
for example, a cup, a dish, or a well of a microtiter plate.
[0106] Assays for sucking pests (for example aphids) may involve
separating the test material from the insect by a partition,
ideally a portion that can be pierced by the sucking mouth parts of
the sucking insect, to allow ingestion of the test material. Often
the test material is mixed with a feeding stimulant, such as
sucrose, to promote ingestion of the test compound.
[0107] Other types of assays can include microinjection of the test
material into the mouth, or gut of the pest, as well as development
of transgenic plants, followed by test of the ability of the pest
to feed upon the transgenic plant. Plant testing may involve
isolation of the plant parts normally consumed, for example, small
cages attached to a leaf, or isolation of entire plants in cages
containing insects.
[0108] Other methods and approaches to assay pests are known in the
art, and can be found, for example in Robertson, J. L. & H. K.
Preisler. 1992. Pesticide bioassays with arthropods. CRC, Boca
Raton, Fla. Alternatively, assays are commonly described in the
journals "Arthropod Management Tests" and "Journal of Economic
Entomology" or by discussion with members of the Entomological
Society of America (ESA).
Example 8
Expression of AXMI-0010 in Bacillus
[0109] The insecticidal AXMI-010 gene is amplified by PCR from
pATX-010, and cloned into the Bacillus Expression vector pAX916 by
methods well known in the art. The resulting clone is assayed for
expression of AXMI-010 protein after transformation into cells of a
cry(-) Bacillus thuringiensis strain. A Bacillus strain containing
the AXMI-010 clone and expressing the AXMI-010 insecticidal protein
is grown in CYS media (10 g/l Bacto-casitone; 3 g/l yeast extract;
6 g/l KH.sub.2PO.sub.4; 14 g/l K.sub.2HPO.sub.4; 0.5 mM MgSO.sub.4;
0.05 mM MnCl.sub.2; 0.05 mM FeSO.sub.4), until sporulation is
evident by microscopic examination. Samples are prepared, and
AXMI-010 is tested for insecticidal activity in bioassays against
important insect pests.
[0110] Methods
[0111] To prepare CYS media: 10 g/l Bacto-casitone; 3 g/l yeast
extract; 6 g/l KH.sub.2PO.sub.4; 14 g/l K.sub.2HPO 4; 0.5 mM
MgSO.sub.4; 0.05 mM MnCl.sub.2; 0.05 mM FeSO.sub.4. The CYS mix
should be pH 7, if adjustment is necessary. NaOH or HCl are
preferred. The media is then autoclaved and 100 ml of 10.times.
filtered glucose is added after autoclaving. If the resultant
solution is cloudy it can be stirred at room temperature to
clear.
Example 9
Vectoring of AXMI-010 for Plant Expression
[0112] The AXMI-010 coding region DNA is operably connected with
appropriate promoter and terminator sequences for expression in
plants. Such sequences are well known in the art and may include
the rice actin promoter or maize ubiquitin promoter for expression
in monocots, the Arabidopsis UBQ3 promoter or CaMV .sup.35S
promoter for expression in dicots, and the nos or PinII
terminators. Techniques for producing and confirming
promoter--gene--terminator constructs also are well known in the
art. The plant expression cassettes described above are combined
with an appropriate plant selectable marker to aid in the
selections of transformed cells and tissues, and ligated into plant
transformation vectors. These may include binary vectors from
Agrobacterium-mediated transformation or simple plasmid vectors for
aerosol or biolistic transformation.
Example 10
Transformation of Maize Cells with AXMI-010
[0113] Maize ears are collected 8-12 days after pollination.
Embryos are isolated from the ears, and those embryos 0.8-1.5 mm in
size are used for transformation. Embryos are plated scutellum
side-up on a suitable incubation media, such as DN62A5S media (3.98
g/L N6 Salts; 1 mL/L (of 1000.times. Stock) N6 Vitamins; 800 mg/L
L-Asparagine; 100 mg/L Myo-inositol; 1.4 g/L L-Proline; 100 mg/L
Casaminoacids; 50 g/L sucrose; 1 mL/L (of 1 mg/mL Stock) 2,4-D),
and incubated overnight at 25.degree. C. in the dark.
[0114] The resulting explants are transferred to mesh squares
(30-40 per plate), transferred onto osmotic media for 30-45
minutes, then transferred to a beaming plate (see, for example, PCT
Publication No. WO/0138514 and U.S. Pat. No. 5,240,842).
[0115] DNA constructs designed to express AXMI-010 in plant cells
are accelerated into plant tissue using an aerosol beam
accelerator, using conditions essentially as described in PCT
Publication No. WO/0138514. After beaming, embryos are incubated
for 30 min on osmotic media, then placed onto incubation media
overnight at 25.degree. C. in the dark. To avoid unduly damaging
beamed explants, they are incubated for at least 24 hours prior to
transfer to recovery media. Embryos are then spread onto recovery
period media, for 5 days, 25.degree. C. in the dark, then
transferred to a selection media. Explants are incubated in
selection media for up to eight weeks, depending on the nature and
characteristics of the particular selection utilized. After the
selection period, the resulting callus is transferred to embryo
maturation media, until the formation of mature somatic embryos is
observed. The resulting mature somatic embryos are then placed
under low light, and the process of regeneration is initiated by
methods known in the art. The resulting shoots are allowed to root
on rooting media, and the resulting plants are transferred to
nursery pots and propagated as transgenic plants.
[0116] Materials
2 DN62A5S Media Components Per Liter Source Chu'S N6 Basal 3.98 g/L
Phytotechnology Labs Salt Mixture (Prod. No. C 416) Chu's N6
Vitamin Solution 1 mL/L Phytotechnology Labs (Prod. No. C 149) (of
1000x Stock) L-Asparagine 800 mg/L Phytotechnology Labs
Myo-inositol 100 mg/L Sigma L-Proline 1.4 g/L Phytotechnology Labs
Casaminoacids 100 mg/L Fisher Scientific Sucrose 50 g/L
Phytotechnology Labs 2,4-D (Prod. No. D-7299) 1 mL/L Sigma (of 1
mg/mL Stock)
[0117] Adjust the pH of the solution to pH to 5.8 with 1N KOH/1N
KCl, add Gelrite (Sigma) to 3g/L, and autoclave. After cooling to
50.degree. C., add 2 ml/L of a 5 mg/ml stock solution of Silver
Nitrate (Phytotechnology Labs). Recipe yields about 20 plates.
Example 11
Transformation of AXMI-010 into Plant Cells by
Agrobacterium-Mediated Transformation
[0118] Ears are collected 8-12 days after pollination. Embryos are
isolated from the ears, and those embryos 0.8-1.5 mm in size are
used for transformation. Embryos are plated scutellum side-up on a
suitable incubation media, and incubated overnight at 25.degree. C.
in the dark. However, it is not necessary per se to incubate the
embryos overnight. Embryos are contacted with an Agrobacterium
strain containing the appropriate vectors for Ti plasmid mediated
transfer for 5-10 min, and then plated onto co-cultivation media
for 3 days (25.degree. C. in the dark). After co-cultivation,
explants are transferred to recovery period media for five days (at
25.degree. C. in the dark). Explants are incubated in selection
media for up to eight weeks, depending on the nature and
characteristics of the particular selection utilized. After the
selection period, the resulting callus is transferred to embryo
maturation media, until the formation of mature somatic embryos is
observed. The resulting mature somatic embryos are then placed
under low light, and the process of regeneration is initiated as
known in the art. The resulting shoots are allowed to root on
rooting media, and the resulting plants are transferred to nursery
pots and propagated as transgenic plants.
[0119] All publications and patent applications mentioned in the
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. 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 to be incorporated by
reference.
[0120] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
Sequence CWU 1
1
5 1 1467 DNA Bacillus thuringiensis CDS (1)...(1467) 1 atg aat gta
aat caa aga gat gat aga tat aat cag cag cat acg aca 48 Met Asn Val
Asn Gln Arg Asp Asp Arg Tyr Asn Gln Gln His Thr Thr 1 5 10 15 aat
gaa cag gtg cat gag aat ggg aat tct aat agt cga att cat gct 96 Asn
Glu Gln Val His Glu Asn Gly Asn Ser Asn Ser Arg Ile His Ala 20 25
30 gga gct tgt tct tgc ggt tgt cag caa gga ata tat gat aat tac tcc
144 Gly Ala Cys Ser Cys Gly Cys Gln Gln Gly Ile Tyr Asp Asn Tyr Ser
35 40 45 aca aaa aat aat aag ggg agt aat tat tct gta ata aaa ggt
tct tca 192 Thr Lys Asn Asn Lys Gly Ser Asn Tyr Ser Val Ile Lys Gly
Ser Ser 50 55 60 caa aat gat atg aac tat gaa aac aca aac tat aat
gga ttg aat agt 240 Gln Asn Asp Met Asn Tyr Glu Asn Thr Asn Tyr Asn
Gly Leu Asn Ser 65 70 75 80 tgt gtc cca cca gta tta aat tta cct att
gaa agt act caa ttt caa 288 Cys Val Pro Pro Val Leu Asn Leu Pro Ile
Glu Ser Thr Gln Phe Gln 85 90 95 acg ata agc gcc tca ggt gag tcg
act atg tgt tta gat tct tgg aat 336 Thr Ile Ser Ala Ser Gly Glu Ser
Thr Met Cys Leu Asp Ser Trp Asn 100 105 110 att agg aaa ggc act gat
ttg aat aat gga atg tcc gga gtg tgt cgg 384 Ile Arg Lys Gly Thr Asp
Leu Asn Asn Gly Met Ser Gly Val Cys Arg 115 120 125 aaa gtg cct aat
gat tat caa gtt act att tat cct ctt aat aca gcg 432 Lys Val Pro Asn
Asp Tyr Gln Val Thr Ile Tyr Pro Leu Asn Thr Ala 130 135 140 aat gat
tca caa tat ttt ata ttt tac cgg tta gat gat ggg aat ttt 480 Asn Asp
Ser Gln Tyr Phe Ile Phe Tyr Arg Leu Asp Asp Gly Asn Phe 145 150 155
160 ata ata gct agt cag aat cac gga cgt gtt ttt gat aag gga tta agc
528 Ile Ile Ala Ser Gln Asn His Gly Arg Val Phe Asp Lys Gly Leu Ser
165 170 175 gat cat agt att gtg gca agt tta tac act ggt aat aat gat
caa aga 576 Asp His Ser Ile Val Ala Ser Leu Tyr Thr Gly Asn Asn Asp
Gln Arg 180 185 190 ttt tcg aaa gtt act act tca agt aat aat ttt act
tta aga aga aat 624 Phe Ser Lys Val Thr Thr Ser Ser Asn Asn Phe Thr
Leu Arg Arg Asn 195 200 205 gga aga tgg gtg gat gct tgt gat cgt aat
atg gca aac gat cgc ctt 672 Gly Arg Trp Val Asp Ala Cys Asp Arg Asn
Met Ala Asn Asp Arg Leu 210 215 220 ctt gta gct gat act act act act
tct act gcg aca ttc cgt cat agt 720 Leu Val Ala Asp Thr Thr Thr Thr
Ser Thr Ala Thr Phe Arg His Ser 225 230 235 240 gat gta aga aat ata
gat aac tta aat tta tct tgt gta aca gca tta 768 Asp Val Arg Asn Ile
Asp Asn Leu Asn Leu Ser Cys Val Thr Ala Leu 245 250 255 ggt cca ctg
cca gat tta acg gga ttg aat gat tca gga cca tct cca 816 Gly Pro Leu
Pro Asp Leu Thr Gly Leu Asn Asp Ser Gly Pro Ser Pro 260 265 270 gaa
gca gca tca aga gca acc atg ggt agt tgg ctt atc cct tgt ata 864 Glu
Ala Ala Ser Arg Ala Thr Met Gly Ser Trp Leu Ile Pro Cys Ile 275 280
285 ttt ata aat gat gta atc cca tta gag aac aga atc aaa cag agt cct
912 Phe Ile Asn Asp Val Ile Pro Leu Glu Asn Arg Ile Lys Gln Ser Pro
290 295 300 tat tat tta tta gaa tat aga cag tat tgg cat aga tta tgg
tca gat 960 Tyr Tyr Leu Leu Glu Tyr Arg Gln Tyr Trp His Arg Leu Trp
Ser Asp 305 310 315 320 gtg att cct gct tca gat tca aga atc ttt gaa
gaa aca aca ggg ata 1008 Val Ile Pro Ala Ser Asp Ser Arg Ile Phe
Glu Glu Thr Thr Gly Ile 325 330 335 gaa cct gat agt caa tcg aat atg
agc cgt aca gta gat ata atg ata 1056 Glu Pro Asp Ser Gln Ser Asn
Met Ser Arg Thr Val Asp Ile Met Ile 340 345 350 ggg gca gat tgg aat
tta aga ttc gga agt ctt tca aca ccg ttt aga 1104 Gly Ala Asp Trp
Asn Leu Arg Phe Gly Ser Leu Ser Thr Pro Phe Arg 355 360 365 caa caa
att ttg tcg ggt tta aat acg cta agc tca tat tct aat atg 1152 Gln
Gln Ile Leu Ser Gly Leu Asn Thr Leu Ser Ser Tyr Ser Asn Met 370 375
380 aat tta gga ata aga aca aac ctt cca cgt tat aca aat ttc aat agt
1200 Asn Leu Gly Ile Arg Thr Asn Leu Pro Arg Tyr Thr Asn Phe Asn
Ser 385 390 395 400 cag gca gtt aga tat gcc aga ttt aca aga gcg tat
gag tat aga tta 1248 Gln Ala Val Arg Tyr Ala Arg Phe Thr Arg Ala
Tyr Glu Tyr Arg Leu 405 410 415 aca cgt att gat gga aca cgc gta gga
aca tgg gta gcc cta gat aat 1296 Thr Arg Ile Asp Gly Thr Arg Val
Gly Thr Trp Val Ala Leu Asp Asn 420 425 430 aga agc atg tat ctg aaa
aca ttc cct cat aat atg caa tta tct gta 1344 Arg Ser Met Tyr Leu
Lys Thr Phe Pro His Asn Met Gln Leu Ser Val 435 440 445 caa gat aac
aaa ata aaa aga agt gat aac agc tat gat cta tcc gta 1392 Gln Asp
Asn Lys Ile Lys Arg Ser Asp Asn Ser Tyr Asp Leu Ser Val 450 455 460
tgg aaa aca cca atg gta ata aaa gat ggt gaa atg aaa ata aaa aat
1440 Trp Lys Thr Pro Met Val Ile Lys Asp Gly Glu Met Lys Ile Lys
Asn 465 470 475 480 aaa cat aat tca aaa cca tac aat gag 1467 Lys
His Asn Ser Lys Pro Tyr Asn Glu 485 2 489 PRT Bacillus
thuringiensis 2 Met Asn Val Asn Gln Arg Asp Asp Arg Tyr Asn Gln Gln
His Thr Thr 1 5 10 15 Asn Glu Gln Val His Glu Asn Gly Asn Ser Asn
Ser Arg Ile His Ala 20 25 30 Gly Ala Cys Ser Cys Gly Cys Gln Gln
Gly Ile Tyr Asp Asn Tyr Ser 35 40 45 Thr Lys Asn Asn Lys Gly Ser
Asn Tyr Ser Val Ile Lys Gly Ser Ser 50 55 60 Gln Asn Asp Met Asn
Tyr Glu Asn Thr Asn Tyr Asn Gly Leu Asn Ser 65 70 75 80 Cys Val Pro
Pro Val Leu Asn Leu Pro Ile Glu Ser Thr Gln Phe Gln 85 90 95 Thr
Ile Ser Ala Ser Gly Glu Ser Thr Met Cys Leu Asp Ser Trp Asn 100 105
110 Ile Arg Lys Gly Thr Asp Leu Asn Asn Gly Met Ser Gly Val Cys Arg
115 120 125 Lys Val Pro Asn Asp Tyr Gln Val Thr Ile Tyr Pro Leu Asn
Thr Ala 130 135 140 Asn Asp Ser Gln Tyr Phe Ile Phe Tyr Arg Leu Asp
Asp Gly Asn Phe 145 150 155 160 Ile Ile Ala Ser Gln Asn His Gly Arg
Val Phe Asp Lys Gly Leu Ser 165 170 175 Asp His Ser Ile Val Ala Ser
Leu Tyr Thr Gly Asn Asn Asp Gln Arg 180 185 190 Phe Ser Lys Val Thr
Thr Ser Ser Asn Asn Phe Thr Leu Arg Arg Asn 195 200 205 Gly Arg Trp
Val Asp Ala Cys Asp Arg Asn Met Ala Asn Asp Arg Leu 210 215 220 Leu
Val Ala Asp Thr Thr Thr Thr Ser Thr Ala Thr Phe Arg His Ser 225 230
235 240 Asp Val Arg Asn Ile Asp Asn Leu Asn Leu Ser Cys Val Thr Ala
Leu 245 250 255 Gly Pro Leu Pro Asp Leu Thr Gly Leu Asn Asp Ser Gly
Pro Ser Pro 260 265 270 Glu Ala Ala Ser Arg Ala Thr Met Gly Ser Trp
Leu Ile Pro Cys Ile 275 280 285 Phe Ile Asn Asp Val Ile Pro Leu Glu
Asn Arg Ile Lys Gln Ser Pro 290 295 300 Tyr Tyr Leu Leu Glu Tyr Arg
Gln Tyr Trp His Arg Leu Trp Ser Asp 305 310 315 320 Val Ile Pro Ala
Ser Asp Ser Arg Ile Phe Glu Glu Thr Thr Gly Ile 325 330 335 Glu Pro
Asp Ser Gln Ser Asn Met Ser Arg Thr Val Asp Ile Met Ile 340 345 350
Gly Ala Asp Trp Asn Leu Arg Phe Gly Ser Leu Ser Thr Pro Phe Arg 355
360 365 Gln Gln Ile Leu Ser Gly Leu Asn Thr Leu Ser Ser Tyr Ser Asn
Met 370 375 380 Asn Leu Gly Ile Arg Thr Asn Leu Pro Arg Tyr Thr Asn
Phe Asn Ser 385 390 395 400 Gln Ala Val Arg Tyr Ala Arg Phe Thr Arg
Ala Tyr Glu Tyr Arg Leu 405 410 415 Thr Arg Ile Asp Gly Thr Arg Val
Gly Thr Trp Val Ala Leu Asp Asn 420 425 430 Arg Ser Met Tyr Leu Lys
Thr Phe Pro His Asn Met Gln Leu Ser Val 435 440 445 Gln Asp Asn Lys
Ile Lys Arg Ser Asp Asn Ser Tyr Asp Leu Ser Val 450 455 460 Trp Lys
Thr Pro Met Val Ile Lys Asp Gly Glu Met Lys Ile Lys Asn 465 470 475
480 Lys His Asn Ser Lys Pro Tyr Asn Glu 485 3 448 PRT Bacillus
sphaericus 3 Met Cys Asp Ser Lys Asp Asn Ser Gly Val Ser Glu Lys
Cys Gly Lys 1 5 10 15 Lys Phe Thr Asn Tyr Pro Leu Asn Thr Thr Pro
Thr Ser Leu Asn Tyr 20 25 30 Asn Leu Pro Glu Ile Ser Lys Lys Phe
Tyr Asn Leu Lys Asn Lys Tyr 35 40 45 Ser Arg Asn Gly Tyr Gly Leu
Ser Lys Thr Glu Phe Pro Ser Ser Ile 50 55 60 Glu Asn Cys Pro Ser
Asn Glu Tyr Ser Ile Met Tyr Asp Asn Lys Asp 65 70 75 80 Pro Arg Phe
Leu Ile Arg Phe Leu Leu Asp Asp Gly Arg Tyr Ile Ile 85 90 95 Ala
Asp Arg Asp Asp Gly Glu Val Phe Asp Glu Ala Pro Ile Tyr Leu 100 105
110 Asp Asn Asn Asn His Pro Ile Ile Ser Arg His Tyr Thr Gly Glu Glu
115 120 125 Arg Gln Lys Phe Glu Gln Val Gly Ser Gly Asp Tyr Ile Thr
Gly Glu 130 135 140 Gln Phe Phe Gln Phe Tyr Thr Gln Asn Lys Thr Arg
Val Leu Ser Asn 145 150 155 160 Cys Arg Ala Leu Asp Ser Arg Thr Ile
Leu Leu Ser Thr Ala Lys Ile 165 170 175 Phe Pro Ile Tyr Pro Pro Ala
Ser Glu Thr Gln Leu Thr Ala Phe Val 180 185 190 Asn Ser Ser Phe Tyr
Ala Ala Ala Ile Pro Gln Leu Pro Gln Thr Ser 195 200 205 Leu Leu Glu
Asn Ile Pro Glu Pro Thr Ser Leu Asp Asp Ser Gly Val 210 215 220 Leu
Pro Lys Asp Ala Val Arg Ala Val Lys Gly Ser Ala Leu Leu Pro 225 230
235 240 Cys Ile Ile Val His Asp Pro Asn Leu Asn Asn Ser Asp Lys Met
Lys 245 250 255 Phe Asn Thr Tyr Tyr Leu Leu Glu Tyr Lys Glu Tyr Trp
His Gln Leu 260 265 270 Trp Ser Gln Ile Ile Pro Ala His Gln Thr Val
Lys Ile Gln Glu Arg 275 280 285 Thr Gly Ile Ser Glu Val Val Gln Asn
Ser Met Ile Glu Asp Leu Asn 290 295 300 Met Tyr Ile Gly Ala Asp Phe
Gly Met His Phe Tyr Leu Arg Ser Ser 305 310 315 320 Gly Phe Lys Glu
Gln Ile Thr Arg Gly Leu Asn Arg Pro Leu Ser Gln 325 330 335 Thr Thr
Thr Gln Leu Gly Glu Arg Val Glu Glu Met Glu Tyr Tyr Asn 340 345 350
Ser Asn Asp Leu Asp Val Arg Tyr Val Lys Tyr Ala Leu Ala Arg Glu 355
360 365 Phe Thr Leu Lys Arg Val Asn Gly Glu Ile Val Lys Asn Trp Val
Ala 370 375 380 Val Asp Tyr Arg Met Ala Gly Ile Gln Ser Tyr Pro Asn
Ala Pro Ile 385 390 395 400 Thr Asn Pro Leu Thr Leu Thr Lys His Thr
Ile Ile Arg Cys Glu Asn 405 410 415 Ser Tyr Asp Gly His Ile Phe Lys
Thr Pro Leu Ile Phe Lys Asn Gly 420 425 430 Glu Val Ile Val Lys Thr
Asn Glu Glu Leu Ile Pro Lys Ile Asn Gln 435 440 445 4 520 PRT
Bacillus thuringiensis 4 Met Asn Val Asn His Gly Met Ser Cys Gly
Cys Gly Cys Gln Gln Gly 1 5 10 15 Lys Glu Glu Tyr Asn Asp Tyr His
Val Ser Asn Glu Tyr Arg Asp Glu 20 25 30 Asn Pro Ser Thr Thr Cys
Asn Ser Gln Gln Gly Asn Tyr Glu Tyr Glu 35 40 45 Gln Ser Lys Glu
Thr Tyr Asn Asn Asp Tyr Gln Ser Tyr Glu Tyr Asn 50 55 60 Gln Gln
Asn Tyr Asn Thr Cys Gly Arg Asn Gln Gly Thr Met Glu Gln 65 70 75 80
Glu Ser Met Gln Lys Asp Arg Asn Trp Glu Asn Ala Asn Tyr Ser Gly 85
90 95 Tyr Asp Gly Cys Ser Pro Asn Gln Leu Asn Ala Leu Asn Leu Pro
Asp 100 105 110 Glu Ser Thr Arg Phe Gln Lys Ile Thr Asn Val Asn Thr
Arg Asp Ser 115 120 125 His Arg Val Leu Asp Met Met Asp Val Pro Ser
Gly Thr Arg Leu Asp 130 135 140 Thr Arg Val Pro Pro Ile Cys Ser Gln
Thr Glu Phe Thr Asn Thr Val 145 150 155 160 Ser Asn Glu Leu Val Ser
Thr Asn His Asp Thr Gln Phe Leu Ile Phe 165 170 175 Tyr Gln Thr Asp
Asp Ser Ser Phe Ile Ile Gly Asn Arg Gly Asn Gly 180 185 190 Arg Val
Leu Asp Val Phe Pro Ser Asn Arg Asn Gly Tyr Thr Ile Val 195 200 205
Ser Asn Val Tyr Ser Gly Ser Arg Asn Asn Gln Arg Phe Arg Met Asn 210
215 220 Lys Ala Ser Asn Asn Gln Phe Ser Leu Gln Thr Ile Phe Lys Asp
Arg 225 230 235 240 Val Asn Ile Cys Gly His Ile His Asn Phe Asn Ala
Ile Ile Thr Ala 245 250 255 Thr Thr Leu Gly Glu Asn Asp Ser Asn Ala
Leu Phe Gln Val Gln Ser 260 265 270 Ser Thr Asn Ile Thr Leu Pro Thr
Leu Pro Pro Arg Thr Thr Leu Glu 275 280 285 Pro Pro Arg Ala Leu Thr
Asn Ile Asn Asp Thr Gly Asp Ser Pro Ala 290 295 300 Gln Ala Pro Arg
Ala Val Glu Gly Ser Val Leu Ile Pro Ala Ile Ala 305 310 315 320 Val
Asn Asp Val Ile Pro Val Ala Gln Arg Met Gln Glu Ser Pro Tyr 325 330
335 Tyr Val Leu Thr Tyr Asn Thr Tyr Trp His Arg Val Ile Ser Ala Ile
340 345 350 Leu Pro Gly Ser Gly Gln Thr Thr Arg Phe Asp Val Asn Leu
Pro Gly 355 360 365 Pro Asn Gln Ser Thr Met Val Asp Val Leu Asp Thr
Ala Ile Thr Ala 370 375 380 Asp Phe Arg Leu Gln Phe Val Gly Ser Gly
Arg Thr Asn Val Phe Gln 385 390 395 400 Gln Gln Ile Arg Asn Gly Leu
Asn Ile Leu Asn Ser Thr Thr Ser His 405 410 415 Arg Leu Gly Asp Glu
Thr Arg Asn Trp Asp Phe Thr Asn Arg Gly Ala 420 425 430 Gln Gly Arg
Leu Ala Phe Phe Val Lys Ala His Glu Phe Val Leu Thr 435 440 445 Arg
Ala Asn Gly Thr Arg Val Ser Asp Pro Trp Val Ala Leu Asp Pro 450 455
460 Asn Val Thr Ala Ala Gln Thr Phe Gly Gly Val Leu Leu Thr Leu Glu
465 470 475 480 Lys Glu Lys Ile Val Cys Ala Ser Asn Ser Tyr Asn Leu
Ser Val Trp 485 490 495 Lys Thr Pro Met Glu Ile Lys Asn Gly Lys Ile
Tyr Thr Lys Asn Glu 500 505 510 Trp Asn Thr Lys Pro Asn Tyr Lys 515
520 5 383 PRT Bacillus thuringiensis 5 Met Leu Asp Thr Asn Lys Val
Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10 15 Leu Tyr Ala Ala Thr
Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Asn Lys
Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 Leu
Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 55
60 Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser
65 70 75 80 Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys
Ala Asn 85 90 95 Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys
Val Leu Thr Ala 100 105 110 Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg
Leu Thr Asp Glu Ser Ser 115 120 125 Asn Asn Pro Asn Gln Gln Trp Asn
Leu Thr Ser Val Gln Thr Ile Gln 130 135 140 Leu Pro Gln Lys Pro Ile
Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160 Tyr Ser Pro
Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165
170 175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile
Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu
Lys Lys Tyr 195 200 205 Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val
Ala Leu Arg Pro His 210 215 220 Glu Lys Lys Ser Tyr Thr Tyr Glu Trp
Gly Thr Glu Ile Asp Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr
Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe Asp
Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys 260 265 270 Thr Gln
Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys 275 280 285
Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290
295 300 Gln Ser Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu
Leu 305 310 315 320 Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln
Ile Gln Thr Ser 325 330 335 Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr
Pro Asn His Gln Gln Ala 340 345 350 Leu Leu Leu Leu Thr Asn His Ser
Tyr Glu Glu Val Glu Glu Ile Thr 355 360 365 Asn Ile Pro Lys Ser Thr
Leu Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375 380
* * * * *
References