U.S. patent application number 12/171822 was filed with the patent office on 2009-01-08 for orally active androctonus amoreuxi pesticidal biopeptides.
This patent application is currently assigned to E.I.du Pont de Nemours and Company. Invention is credited to Rafael Herrmann, Albert L. Lu, Billy F. McCutchen, James K. Presnail, James F.H. Wong.
Application Number | 20090011999 12/171822 |
Document ID | / |
Family ID | 39687240 |
Filed Date | 2009-01-08 |
United States Patent
Application |
20090011999 |
Kind Code |
A1 |
Herrmann; Rafael ; et
al. |
January 8, 2009 |
ORALLY ACTIVE ANDROCTONUS AMOREUXI PESTICIDAL BIOPEPTIDES
Abstract
The present invention provides compositions and methods for
orally active Androctonus amoreuxi pesticidal polypeptides.
Compositions include novel Androctonus amoreuxi pesticidal
polypeptides and biologically active variants thereof. Further
provided are methods for modulating the pesticide resistance of
plants by expressing the sequences disclosed herein. One method
comprises stably transforming into the genome of a plant cell a
nucleotide sequence of the present invention operably linked to a
heterologous promoter and regenerating a stably transformed plant
that expresses the nucleotide sequence. An additional method
comprises incorporating a nucleotide sequence of the present
invention operably linked to a heterologous promoter into a
microorganism and applying said microorganism to the environment of
a plant.
Inventors: |
Herrmann; Rafael;
(Wilmington, DE) ; Lu; Albert L.; (Newark, DE)
; McCutchen; Billy F.; (College Station, TX) ;
Presnail; James K.; (Avondale, PA) ; Wong; James
F.H.; (Johnston, IA) |
Correspondence
Address: |
ALSTON & BIRD LLP;PIONEER HI-BRED INTERNATIONAL, INC.
BANK OF AMERICA PLAZA, 101 SOUTH TRYON STREET, SUITE 4000
CHARLOTTE
NC
28280-4000
US
|
Assignee: |
E.I.du Pont de Nemours and
Company
Wilmington
DE
|
Family ID: |
39687240 |
Appl. No.: |
12/171822 |
Filed: |
July 11, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10617978 |
Jul 11, 2003 |
7414173 |
|
|
12171822 |
|
|
|
|
60395428 |
Jul 12, 2002 |
|
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|
Current U.S.
Class: |
514/2.1 ;
530/350 |
Current CPC
Class: |
C07K 14/43522 20130101;
Y02A 40/146 20180101; Y02A 40/162 20180101; C12N 15/8286
20130101 |
Class at
Publication: |
514/12 ;
530/350 |
International
Class: |
A01N 63/00 20060101
A01N063/00; C07K 14/00 20060101 C07K014/00; A01P 15/00 20060101
A01P015/00 |
Claims
1. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: (a) an amino acid sequence
encoded by a nucleotide sequence set forth in SEQ ID NO:1, 3, 6, 8,
9, 11, 21, or 23; (b) an amino acid sequence set forth in SEQ ID
NO:2, 4, 7, 10, 20, 22, 24, or 27; (c) an amino acid sequence
having at least 80% sequence identity to the amino acid sequence
set forth in SEQ ID NO:2, 4, 7, 10, 20, 22, 24, or 27, or a
fragment thereof, wherein said polypeptide retains pesticidal
activity; and (d) an amino acid sequence consisting of at least 10
contiguous amino acids of the amino acid sequence set forth in SEQ
ID NO:2, 4, 7, 10, 20, 22, 24, or 27.
2. The isolated polypeptide of claim 1, wherein the polypeptide is
orally active.
3. A composition comprising the isolated polypeptide of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 10/617,978, filed Jul. 11, 2003, which claims the benefit of
U.S. Provisional Application No. 60/395,428, filed Jul. 12, 2002,
each of which is hereby incorporated in its entirety by reference
herein.
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA
EFS-WEB
[0002] The official copy of the sequence listing is submitted
concurrently with the specification as a text file via EFS-Web, in
compliance with the American Standard Code for Information
Interchange (ASCII), with a file name of 346403SequenceListing.txt,
a creation date of Jul. 11, 2008, and a size of 28,403 bytes. The
sequence listing filed via EFS-Web is part of the specification and
is hereby incorporated in its entirety by reference herein.
FIELD OF THE INVENTION
[0003] The present invention relates to naturally occurring
pesticides, in particular, to polypeptides isolated from
arthropods. The invention further relates to methods of impacting
pests, particularly insect pests, involving the pesticidal
polypeptides and the corresponding nucleic acid molecules that
encode the pesticidal polypeptides.
BACKGROUND OF THE INVENTION
[0004] Chemical insecticides are an integral component of modern
agriculture, and are an effective means for reducing crop damage by
controlling insect pests. However, chemical agents are under
continuous scrutiny due to the potential for environmental
contamination, selection of resistant populations of agronomic
pests, and toxicity to non-target organisms such as beneficial
insects, aquatic organisms, animals and humans. As a result,
alternative strategies for insect control are being sought that are
effective and yet benign to non-target populations and the
environment. One of these strategies is to utilize the mechanisms
of naturally occurring pathogens of target pest populations.
[0005] Examples of this type of strategy include the use of various
forms of the 6-endotoxin produced by the soil dwelling
microorganism Bacillus thuringiensis (Bt) as pesticidal agents.
These polypeptides have been found to be specifically toxic to
particular insects, and microbial formulations have been used
commercially for many years as foliarly applied insecticides. It
has also recently been found that various forms of the Bt toxin can
be toxic to insect pests when expressed inside the tissues of
plants on which the insects feed.
[0006] Many arthropods express polypeptides capable of killing or
incapacitating various pests. Therefore arthropods have been
identified as a group of organisms producing polypeptides
possessing pesticidal properties. In fact, scorpion venom contains
insect-selective toxins affecting ion channels (Zlotkin et al.
(1985) Arch. Biochem. Biophys. 240:877-87). The toxicity of the
different polypeptides operates through multiple pharmacological
and biochemical pathways (Zlotkin et al. (1971) Biochimie (Paris),
53:1073-1078).
[0007] Transgenic dicot plants expressing a toxin obtained from
Androctonus australis scorpions have been created (U.S. Pat. No.
5,177,308). The AaIT expressing plants have been crossbred to
transgenic plants carrying Bt .delta.-endotoxin yielding plants
with two independent insect-specific toxin traits (U.S. Pat. No.
5,177,308).
[0008] Insects predominate as rice pests throughout Asia and are
most serious in tropical regions where over 60 species are pests.
The most serious rice insect pests are commonly categorized as
Homoptera (sucking pests such as leafhoppers and planthoppers) and
Lepidoptera (stem borers and defoliators).
[0009] The identification of new pesticidal polypeptides is
desirable for use in pest-management strategies. It is of
particular importance to identify pesticidal toxins that are active
against insect pests from the orders Homoptera and Lepidoptera, and
against insects that have developed resistance to Bt toxins.
SUMMARY OF THE INVENTION
[0010] Compositions and methods for enhancing plant pest
resistance, particularly insect pests are provided. Compositions of
the invention include isolated polypeptides, peptides, and amino
acid sequences and nucleic acid molecules having a nucleotide
sequence encoding the polypeptides, peptides, and amino acid
sequences of the invention. The polypeptides of the invention were
isolated from arthropod venom and telsons and possess pesticidal
activity. In an embodiment, the polypeptides of the invention are
orally active and affect pests upon ingestion. The invention
provides expression cassettes, host cells, baculovirus expression
vectors, transformed plant cells, and plants comprising nucleic
acid molecules encoding the polypeptides of the invention. In an
embodiment, a nucleotide sequence encoding a pesticidal polypeptide
of the invention is inserted into the genome of a baculovirus to
produce a pesticidal recombinant baculovirus. In this embodiment
the presence of the pesticidal polypeptide in the baculovirus
increases the efficiency with which the virus acts to kill or
incapacitate the pest, thus, enhancing the effectiveness of the
baculovirus as an adjunct or replacement for chemical pest control
agents.
[0011] In an embodiment of the invention, the promoter to which a
nucleotide sequence of the invention or a nucleotide sequence
encoding a polypeptide of the invention is operably linked is
constitutive, inducible, or tissue-preferred. An embodiment of the
invention comprises a vascular tissue preferred promoter operably
linked to a nucleotide sequence encoding a polypeptide of the
invention. The operably linked promoter is an insect-inducible
promoter in one embodiment of the invention.
[0012] Methods for altering the pest resistance of a plant are
provided. The plant's resistance to pests such as, but not limited
to, insects and the pathogens transmitted by insects, may be
altered by the methods of the invention. A method of the invention
comprises stably transforming into a plant cell a nucleic acid
molecule encoding a polypeptide of the invention. The nucleic acid
molecule is operably linked to a promoter capable of driving
transcription in the plant cell. The transformed plant cell is used
to generate a transformed plant. The transformed plant cell and
transformed plant are capable of expressing a polypeptide of the
invention in the plant cell or plant cells. The polypeptide of the
invention possesses pesticidal activity. In an embodiment the
pesticidal activity of the polypeptide of the invention is orally
active upon ingestion by an insect such as, but not limited to, an
insect of the Homopteran, Lepidopteran, and Hymenopteran orders. In
an embodiment, the insect infected by the pesticidal activity of
the invention exhibits resistance to a Bt toxin.
[0013] Methods for identifying polypeptides possessing oral
pesticidal activity are provided. In an embodiment, polypeptides
are isolated from arthropod venom. The venom is obtained from
isolated venom glands. The isolated venom glands have been
surgically removed from the organism. In another embodiment, the
venom is obtained by harvesting venom from the organism through a
process such as, but not limited to, milking the organism. The
polypeptides are combined with at least one nutrient to generate a
polypeptide solution. The polypeptide solution is fed to insects.
Insecticidal activity is assayed. Insecticidal activity includes,
but is not limited to, mortality, weight loss, attraction, and
repellency.
[0014] Some pesticidal polypeptides modulate sodium channels and
are sodium channel neurotoxins. In another embodiment of the
invention, nucleic acid sequences encoding potential sodium channel
neurotoxins are identified from amino acid and nucleotide sequence
data derived from a cDNA library of arthropod telsons. The open
reading frames encoding potential sodium channels neurotoxins are
amplified from the cDNA and cloned into appropriate expression
cassettes. The potentially pesticidal amino acids are tested for
oral pesticidal activity.
[0015] Compositions of the invention provide antibodies that
selectively bind to a polypeptide of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 presents a chromatogram of the purification of Aam1
(SEQ ID NO:20) from a fraction of crude Androctonus amoreuxi venom
enriched for pesticidal activity. The enriched fraction was loaded
onto a Microbore LC C4 column, and the column was developed with an
acetonitrile gradient according to Method 5 as described elsewhere
herein. The fraction enriched for pesticidal activity was further
purified.
[0017] FIG. 2 presents a chromatogram of the further purification
of Aam1 (SEQ ID NO:20) from a Microbore LC fraction (see FIG. 1).
The enriched fraction was loaded onto a Microbore LC C18 column,
and the column was developed with an acetonitrile gradient
according to Method 9 as described elsewhere herein.
[0018] FIG. 3 depicts the results of mass spectroscopy analysis of
Aam1 (SEQ ID NO:20).
[0019] FIG. 4 presents a chromatogram of the purification of Aam2
(SEQ ID NO:27) from a fraction of crude Androctonus amoreuxi venom
enriched for pesticidal activity. The enriched fraction was loaded
onto a Microbore LC C4 column, and the column was developed with an
acetonitrile gradient according to Method 5 as described elsewhere
herein. The fraction enriched for pesticidal activity was further
purified.
[0020] FIG. 5 presents a chromatogram of the further purification
of Aam2 (SEQ ID NO:27) from a Microbore LC fraction (see FIG. 4).
The enriched fraction was loaded onto a Microbore LC C18 column,
and the column was developed with an acetonitrile gradient
according to Method 6 as described elsewhere herein.
[0021] FIG. 6 depicts the results of mass spectroscopy analysis of
Aam2 (SEQ ID NO:27).
[0022] FIG. 7 presents a chromatogram of the purification of CV1
(SEQ ID NO:2 and 4) from a fraction of crude Centruroides vittatus
venom enriched for pesticidal activity. The enriched fraction was
loaded onto a Microbore LC C18 column, and the column was developed
with an acetonitrile gradient according to Method 8 as described
elsewhere herein.
[0023] FIG. 8 presents a chromatogram of the purification of LqhIV
(SEQ ID NO:7) from a fraction of crude venom enriched for
pesticidal activity. The enriched fraction was loaded onto a
Microbore LC C18 column, and the column was developed with an
acetonitrile gradient according to Method 6 as described elsewhere
herein.
[0024] FIG. 9 depicts the results of mass spectroscopy analysis of
LqhIV (SEQ ID NO:7).
[0025] FIG. 10 presents a chromatogram of the purification of VC1
(SEQ ID NO:10) from a fraction of crude Vaejovis carolinanus venom
enriched for pesticidal activity. The enriched fraction was loaded
onto a Microbore LC C4 column, and the column was developed with an
acetonitrile gradient according to Method 4 as described elsewhere
herein. The fraction enriched for pesticidal activity was further
purified.
[0026] FIG. 11 presents a chromatogram of the further purification
of VC1 (SEQ ID NO:10) from a Microbore LC fraction (see FIG. 10).
The enriched fraction was loaded onto a Microbore LC C18 column,
and the column was developed with an acetonitrile gradient
according to Method 6 as described elsewhere herein.
[0027] FIG. 12 depicts the results of mass spectroscopy analysis of
VC1 (SEQ ID NO:10).
[0028] FIG. 13 depicts the results of RT-PCR analysis of transgenic
rice plants expressing SP-Aam1 (SEQ ID NOS:15 and 16). Lane 1
contains 100 bp size markers. Lane 2 contains a sample from a
vector alone control. Lane 3 contains a sample from a no vector
control. Lane 4 contains a sample from wild-type rice. Lanes 5 and
6 contain samples from 2 different transformants, while lanes 7 and
8 contain purified mRNA from the 2 transformants, respectively.
[0029] FIG. 14 depicts the results of a dose response assay of
Myzus persicae to Aam1 (SEQ ID NO:20). The percent mortality of M.
persicae 48 hours after exposure to the indicated doses of Aam1 is
presented. The Aam1 dose is in %, thus 1%=1 gm/100 ml, and 0.2%=2
mg/ml, 0.1%=1 mg/ml, 0.05%=0.5 mg/ml, 0.025%=0.25 mg/ml,
0.012%=0.12 mg/ml.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Compositions and methods for impacting pests, particularly
insect pests are provided. The polypeptides of the invention were
identified in the venom of various arthropods and possess
pesticidal properties. These polypeptides are orally active and are
toxic to pests upon ingestion by an insect such as, but not limited
to, an insect of the Homopteran, Lepidopteran, and Hymenopteran
orders. Insect pests of particular importance are those of the
Homopteran and Lepidopteran orders. Compositions of the invention
include polypeptides and nucleic acid molecules encoding the
polypeptides of the invention, expression cassettes comprising the
nucleic acid molecules, transformed microorganisms comprising the
nucleic acid molecules, vector sequences and host cells for the
expression of such polypeptides, and antibodies to the
polypeptides. The compositions of the invention further provide
plants, and seed thereof, transformed with the nucleic acid
molecules of the invention. The transgenic plants of the present
invention impact control of Bt toxin-resistant insect species.
[0031] The methods of the present invention include methods for
altering plant resistance to pests, including insects. Methods for
identifying and screening arthropod venom peptides and polypeptides
for pesticidal activity are also provided. The methods include HPLC
assays directed to the separation and purification of polypeptide
toxins, and small-scale, high throughput bioassays to measure the
oral pesticidal activity of polypeptides against various species of
plant pests.
[0032] Compositions of the invention include polypeptides and
nucleotide sequences that are involved in altering plant pest
resistance. In particular, the present invention provides for
isolated nucleic acid molecules comprising nucleotide sequences
encoding the amino acid sequences shown in SEQ ID NOS:2, 4, 7, 10,
20, 22, 24, or 27. Further provided are polypeptides having an
amino acid sequence encoded by a nucleic acid molecule described
herein, for example those set forth in SEQ ID NOS:1, 3, 6, 8, 9,
11, 21, or 23.
[0033] "Impacting insect pests" refers to effecting changes in
insect feeding, growth and/or behavior, at any stage of
development, including, but not limited to, killing the insect,
retarding growth, and preventing reproductive capability.
[0034] "Pesticidal property" or "pesticidal activity" are used
interchangeably herein and are defined as a property or activity of
an organism or a substance, such as, for example, a polypeptide,
that results in, but is not limited to, pest mortality, pest weight
loss, pest attraction, pest repellency, and other behavioral and
physical changes of a pest. "Pesticidal polypeptides," "pesticidal
peptides," or "pesticidal proteins" are polypeptides, peptides or
proteins that display pesticidal activity alone or in combination
with other polypeptides. Similarly, an "insecticidal property" or
"insecticidal activity" may be used to refer to a "pesticidal
activity" when the pest is an insect pest.
[0035] "Pesticidally effective amount" is intended as a quantity of
a substance or organism having pesticidal properties when present
in the environment of a pest. For each substance or organism, the
pesticidally effective amount is determined empirically for each
pest affected in a specific environment. Similarly an
"insecticidally effective amount" may be used to refer to a
"pesticidally effective amount" when the pest is an insect
pest.
[0036] By "orally active" it is intended that the polypeptide
retains activity after ingestion of the polypeptide. The orally
active polypeptides of the invention are not fully degraded by the
digestive system and retain activity after ingestion. After
ingestion an orally active polypeptide may be transferred from any
component of the digestive system including, but not limited to,
the foregut, midgut, hindgut, esophagus, salivary glands, crop,
proventriculus, gastric cecae, pyloric valve, Malpighian tubules,
ileum, colon, cuticle, nasal passages, pharynx, stomach, small
intestine, and large intestine. The polypeptide of the invention
may impact cells in any system including, but not limited to, the
nervous system, the circulatory system, the digestive system, the
musculoskeletal system, the reproductive system, and the excretory
system. Insect digestive systems degrade and inactivate most
peptides and polypeptides that enter it; the polypeptides of the
invention are not inactivated during digestion. The orally active
polypeptide may enter an insect target's hemolymph from the
digestive system. The orally active polypeptide may enter a
mammal's blood from the digestive system.
[0037] By "test unit" any structure or container is intended that
could contain a solution of the polypeptide to be screened and
insects. Such containers are known to one of skill in the art
including, but not limited to, 96 well plates, petri plates, 384
well plates, thermocycling plates, microplates, 8 well plates, 12
well plates, microfuge tubes, multi-well tissue culture plates,
thermocycling tubes, multiwell assay plates, ELISA plates, petri
dishes, dialysis tubing, polystyrene tubing, plastic tubing, and
microtiter plates.
[0038] By "membrane solution" a composition is intended that is
capable of forming a barrier on which an insect can stand and
through which the insect can feed. One of skill in the art will
recognize that multiple formulations of membrane solutions are
available including, but not limited to, 1% Fluoropolymer in PF
5080 solvent.
[0039] By "cold-immobilized" exposure to low temperatures is
intended that is sufficient to slow the metabolism of an organism
but not permanently damage the organism. One of skill in the art
will recognize that the conditions that result in
cold-immobilization vary depending on the organism in question;
such conditions include, but are not limited to, incubating the
organism on ice for 1 to 2 minutes.
[0040] By "disrupting" stimulation is intended by such means as
forceful movement, rapid movement, repeated movement, or incubation
at elevated temperatures. One of skill in the art will recognize
that disrupting may be accomplished by a variety of methods,
including but not limited to, tapping, shaking, rocking, or warming
the test unit at 42.degree. C. for 1 to 3 minutes.
[0041] The present invention provides new approaches for impacting
pests that do not depend on the use of traditional chemical
insecticides. The invention involves the isolation of polypeptides
and the genes that encode them. Such polypeptides comprise
pesticidal polypeptides with pesticidal activity against pests,
particularly insect pests of the Homopteran, Lepidopteran and
Hymenopteran orders, and more particularly insect pests of the
Homopteran and Lepidopteran orders. The pesticidal polypeptides of
the invention are isolated from the venom of arthropods such as
spiders, scorpions, centipedes and wasps; predators that rely on
insecticidal chemicals likely to have unique toxicity to
insects.
[0042] It has been demonstrated, notably with the toxins produced
by the soil dwelling microorganism Bacillus thuringiensis, that
pesticidal toxins can successfully be produced in plant cells so as
to render the transgenic plants toxic to pests that ingest them.
The Bacillus thuringiensis pesticidal toxins are selectively toxic
to insect pests and do not demonstrate toxicity to mammals. The
present invention is directed to pesticidal polypeptides that are
toxic to plant pests. In one embodiment, the peptides of the
present invention are not toxic to livestock or humans upon
consumption of plants expressing the claimed peptide or
polypeptide. In another embodiment, the peptides of the invention
are expressed in plants not intended for consumption by livestock
or humans, wherein mammalian toxicity is not an issue.
[0043] The invention provides compositions and methods for
producing transgenic plants that express the pesticidal
polypeptides of the invention, transgenic microorganisms that
express the pesticidal polypeptides of the invention, and
pesticidal polypeptide compositions that enhance the resistance of
plants to pests, particularly insect pests of the Homopteran,
Lepidopteran and Hymenopteran orders, and more particularly insect
pests of the Homopteran and Lepidopteran orders.
[0044] One embodiment of the invention provides transgenic rice
plants that express one or more pesticidal polypeptides of the
invention and possess enhanced resistance to insects. The
polypeptides of the invention exhibit pesticidal activity against
insects including, but not limited to, Homopteran, Lepidopteran and
Hymenopteran species. Insects of particular interest include the
green leafhopper, Nephotettix virescens; the brown planthopper,
Nilaparvata lugens; and Scirpophaga incertulas. In an embodiment of
the invention, transgenic rice plants control insects that vector
viruses causing diseases such as rice tungro and rice stunt,
thereby, significantly curtailing virus epizootics and adding
substantially to plant vigor and rice yield. An embodiment of the
invention is transgenic plants such as, but not limited to, rice,
that exhibit multiple pesticidal activities. The multiple
pesticidal activities result from expression of one or more of the
polypeptides of the invention in the transgenic plant. In an
embodiment, the transgenic plants possess resistance to multiple
species of insects. The polypeptides of the invention expressed by
the transgenic plant inhibit insect activity through one or more
pathways. The multiplicity of pesticidal activities exhibited by
the transgenic plant decreases the evolution of insects resistant
to the polypeptides of the invention. Provided in the invention is
a transgenic rice plant possessing resistance to both Homopteran
and Lepidopteran species of pests, and retaining resistance to
Lepidopteran pests resistant to Bt toxin.
[0045] Compositions of the invention include amino acid sequences
corresponding to pesticidal toxins isolated from various arthropod
venoms. In particular, the present invention provides isolated
polypeptides comprising the amino acid sequences shown in SEQ ID
NOS:2, 4, 7, 10, 13, 15, 16, 18, 19, 20, 22, 24, 26, and 27, and
the nucleotide sequences that encode these polypeptides. Also
included in the compositions of the invention are fragments and
variants of these polypeptides. Of particular interest are
optimized nucleotide sequences encoding the pesticidal polypeptides
of the invention. By "optimized nucleotide sequences," sequences
are intended that are optimized for expression in a particular
organism. Such optimized nucleotide sequences may be prepared for
any organism of interest using methods known in the art, described
elsewhere herein. SEQ ID NOS:5, 12, and 14 disclose nucleotide
sequences operably linked to signal sequences, optimized for
expression in rice, and encoding the polypeptides set forth in SEQ
ID NOS:4, 13, and 15 and 16. SEQ ID NO:17 discloses a nucleotide
sequence operably linked to a BAA signal sequence, optimized for
expression in plants, and encoding the polypeptides set forth in
SEQ ID NOS:18 and 19. SEQ ID NO:25 discloses a nucleotide sequence
operably linked to a BAA signal peptide, optimized for expression
in plants, and encoding the polypeptide set forth in SEQ ID NO:26.
Nucleotide sequences of the invention can be similarly optimized
for expression in any organism. Nucleotide sequences optimized for
expression in crop plants such as rice, wheat, corn, soybeans, rye,
barley, and alfalfa are of particular interest.
[0046] The polypeptides of the invention were isolated from the
venom of various species of arthropods. These polypeptides possess
pesticidal activity against pests including, but not limited to,
the southern corn rootworm and European corn borer.
[0047] The polypeptide set forth in SEQ ID NO:2 and SEQ ID NO:4,
was isolated from Centruroides vittatus venom and is also known as
CV1. Nucleotides 49 to 303 of SEQ ID NO:1 and SEQ ID NO:3 encode
CV1. SEQ ID NO:5 is an artificial sequence that encodes BAA-CV1
utilizing codons optimized for expression in Oryza sativa. SEQ ID
NO:5 contains a nucleotide sequence encoding the barley alpha
amylase (BAA) signal peptide (Rahmatullah, et al. (1989) Plant Mol.
Biol. 12:119-121, herein incorporated by reference) operably linked
to a nucleotide sequence encoding CV1.
[0048] The polypeptide set forth in SEQ ID NO:7 was isolated from
Leiurus quinquestriatus venom and is also known as LqhIV. The
nucleotide sequence set forth in SEQ ID NO:8 and nucleotides 38 to
289 of SEQ ID NO:6 encode the LqhIV polypeptide.
[0049] The polypeptide set forth in SEQ ID NO:10 was isolated from
Vaejovis carolinanus venom and is also known as VC1. The nucleotide
sequence set forth in SEQ ID NO:11 and nucleotides 65 to 358 of SEQ
ID NO:9 encode the VC1 polypeptide. SEQ ID NO:12 is an artificial
sequence that encodes PR1-VC1 utilizing codons optimized for
expression in Oryza sativa. SEQ ID NO:12 contains a nucleotide
sequence encoding the PR1 signal peptide (Cornelissen, et al.
(1986) Nature 321:531-532, herein incorporated by reference)
operably linked to a nucleotide sequence encoding VC1; the
polypeptide encoded by the nucleotide sequence set forth in SEQ ID
NO:12 is set forth in SEQ ID NO:13.
[0050] The polypeptide set forth in SEQ ID NO:20 was isolated from
Androctonus amoreuxi venom and is also known as Aam1. SEQ ID NO:14
is an artificial sequence that encodes SP-Aam1 utilizing codons
optimized for expression in Oryza sativa. SEQ ID NO:14 contains a
nucleotide sequence encoding the sweet potato sporamin (SP) signal
sequence (Hattori, et al (1985) Plant Mol. Biol. 5:313-320, herein
incorporated by reference) operably linked to a nucleotide sequence
encoding Aam1; the polypeptide encoded by the nucleotide sequence
set forth in SEQ ID NO:14 is set forth in SEQ ID NO:15 and SEQ ID
NO:16. SEQ ID NO:17 is an artificial sequence that encodes BAA-Aam1
utilizing codons optimized for expression in Streptomyces
coelicolor. SEQ ID NO: 17 contains a nucleotide sequence encoding
the BAA signal peptide operably linked to a nucleotide sequence
encoding Aam1; the polypeptide encoded by the nucleotide sequence
set forth in SEQ ID NO:17 is set forth in SEQ ID NO:18 and SEQ ID
NO:19.
[0051] The nucleotide sequence encoding the polypeptide set forth
in SEQ ID NO:22 and SEQ ID NO:24 was identified from a Centruroides
vittatus telsons cDNA library. SEQ ID NO:23 and nucleotides 117 to
359 of SEQ ID NO:21 encode Ts7, the polypeptide set forth in SEQ ID
NO:22 and 24. SEQ ID NO:25 is an artificial sequence that encodes
BAA-Ts7 utilizing codons optimized for expression in Streptomyces
coelicolor; the polypeptide encoded by the nucleotide sequence set
forth in SEQ ID NO:25 is set forth in SEQ ID NO:26. The polypeptide
set forth in SEQ ID NO:27 was isolated from Androctonus amoreuxi
venom and is also known as Aam2.
[0052] The invention discloses isolated polypeptides possessing
pesticidal properties and the nucleotide sequences that encode
them. These molecules find use in methods for impacting pests,
particularly insect pests. The nucleotide sequences of the
invention may be used to transform any organism to produce the
encoded pesticidal polypeptides. Methods are provided that involve
the use of such transformed organisms to impact or control plant
pests.
[0053] The invention encompasses a plant transformed with at least
one nucleotide sequence of the invention. The plant is stably
transformed with a nucleotide construct comprising at least one
nucleotide sequence of the invention operably linked to a promoter
that drives expression in a plant cell. While the invention does
not depend on a particular biological mechanism for increasing the
resistance of a plant to a plant pest, expression of the nucleotide
sequences of the invention in a plant can result in the production
of the pesticidal polypeptides of the invention and in an increase
in the resistance of the plant to a plant pest. The plants of the
invention find use in agriculture in methods for impacting insect
pests. Certain embodiments of the invention provide rice plants
that find use in methods for impacting both Homopteran and
Lepidopteran species of insect pests.
[0054] In one such embodiment, ingestion of transgenic rice plant
tissue expressing the Aam1 polypeptide by Homoptera and Lepidoptera
species of insect results in toxicity to both species of insect
pests. Aam1 (SEQ ID NO:20) is an orally active insecticidal
polypeptide active against both Homopteran and Lepidopteran species
of insect. Furthermore, transgenic rice plant tissue expressing
Aam1 is also toxic to insect populations resistant to the
Lepidopteran specific .delta.-endotoxin from Bacillus thuringiensis
(Bt). Thus, in an embodiment, the transgenic plants of the present
invention protect field crops and facilitate control of Bt
toxin-resistant insect species.
[0055] In another embodiment of the invention a nucleotide sequence
encoding an insecticidal polypeptide of the invention is inserted
into the genome of a baculovirus to yield an insecticidal
recombinant baculovirus. The recombinant baculovirus expresses the
insecticidal polypeptide. The presence of the insecticidal
polypeptide in the baculovirus increases the efficiency with which
the virus acts to kill or incapacitate the insect, thus enhancing
the effectiveness of the baculovirus in pest management techniques.
Compositions of the invention include such recombinant
baculoviruses.
[0056] The invention further encompasses microorganisms transformed
with at least one nucleic acid molecule of the invention, with an
expression cassette comprising the nucleotide molecule, or with a
vector molecule comprising the expression cassette. Microorganisms
that multiply on plants are of particular interest.
[0057] The invention provides pesticidal compositions. The
invention encompasses pesticidal compositions comprising an
isolated polypeptide of the invention, alone or in combination with
a transformed organism of the invention and a suitable carrier. In
an embodiment, the pesticidal composition comprises a transformed
organism of the invention. Such a pesticidal composition contains a
transformed microorganism at pesticidally effective levels and a
suitable carrier.
[0058] It is recognized that the pesticidal polypeptides may vary
in molecular characteristics and activity against particular pests.
However, by the methods set forth herein, polypeptides active
against a variety of pests may be isolated and characterized.
[0059] The pesticidal polypeptides of the invention can be used in
combination with Bt endotoxins or other insecticidal polypeptides
to increase insect target range. Furthermore, the use of the
pesticidal polypeptides of the present invention in combination
with Bt endotoxins or other types of insecticides of a distinct
nature has utility in the prevention and/or management of insect
resistance. Insecticides include, but are not limited to, protease
inhibitors (both serine and cysteine types), lectins,
.alpha.-amylases, and peroxidases.
[0060] The invention encompasses isolated or substantially purified
nucleic acid or protein compositions. An "isolated" or "purified"
nucleic acid molecule or protein, or biologically active portion
thereof, is substantially or essentially free from components that
normally accompany or interact with the nucleic acid molecule or
protein as found in its naturally occurring environment. Thus, an
isolated or purified nucleic acid molecule or protein 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 example, in various embodiments, the
isolated 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 protein that is
substantially free of cellular material includes preparations of
protein having less than about 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%,
4%, 3%, 2%, or 1% (by dry weight) of contaminating protein. When
the protein of the invention or biologically active portion thereof
is recombinantly produced, preferably culture medium represents
less than about 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or
1% (by dry weight) of chemical precursors or
non-protein-of-interest chemicals.
[0061] Fragments and variants of the disclosed nucleotide sequences
and polypeptides encoded thereby are also encompassed by the
present invention. By "fragment" a portion of the nucleotide
sequence and hence polypeptide encoded thereby, or a portion of the
amino acid sequence is intended. Fragments of a nucleotide sequence
may encode polypeptide fragments that retain the biological
activity of the native polypeptide and hence possess pesticidal
activity. Alternatively, fragments of a nucleotide sequence that
are useful as hybridization probes generally do not encode fragment
proteins retaining biological activity. Thus, fragments of a
nucleotide sequence may range from at least about 20 nucleotides,
about 50 nucleotides, about 100 nucleotides, and up to the
full-length nucleotide sequence encoding the proteins of the
invention.
[0062] A fragment of a nucleotide sequence of the invention that
encodes a biologically active portion of a pesticidal polypeptide
of the invention will encode at least 10, 15, 20, 25, 30, 35, 40,
45, 50, 55, 60, or 64 contiguous amino acids of SEQ ID NOS:2 and 4;
at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, or 87 contiguous amino acids of SEQ ID NO:7; at least 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
or 97 contiguous amino acids of SEQ ID NO:10; at least 10, 15, 20,
25, 30, 35, 40, 45, 50, 55, or 58 contiguous amino acids of SEQ ID
NO:20; at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 57
contiguous amino acids of SEQ ID NOS:22 and 24; and at least 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 64 contiguous amino
acids of SEQ ID NO:27 or up to the total number of amino acids
present in a polypeptide of the invention. Fragments of a
nucleotide sequence of the invention that are useful as
hybridization probes or PCR primers generally need not encode a
biologically active portion of a polypeptide of the invention.
[0063] Thus, a fragment of a nucleotide sequence of the invention
may encode a biologically active portion of a pesticidal
polypeptide, or it may be a fragment that can be used as a
hybridization probe or PCR primer using methods disclosed below. A
biologically active portion of a pesticidal polypeptide can be
prepared by isolating a portion of one of the nucleotide sequences
of the invention, expressing the encoded portion of the pesticidal
polypeptide (e.g., by recombinant expression in vitro), and
assessing the activity of the encoded portion of the pesticidal
polypeptide. Nucleic acid molecules that are fragments of a
nucleotide sequence of the invention comprise at least 15, 20, 30,
40, 50, 60, 70, 75, 80, 90, 100, 110, 120, 125, 130, 140, 150, 160,
170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,
290, 300, 310, 320, 330, 340, 350, 355, or up to the number of
nucleotides present in SEQ ID NO:1; at least 15, 20, 30, 40, 50,
60, 70, 75, 80, 90, 100, 110, 120, 125, 130, 140, 150, 160, 170,
175, 180, 190, 200, 210, 220, 230, 240, 250, 254, or up to the
number of nucleotides present in SEQ ID NO:3; at least 15, 20, 30,
40, 50, 60, 70, 75, 80, 90, 100, 110, 120, 125, 130, 140, 150, 160,
170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,
290, 300, 310, 320, 330, 340, 350, 355, 360, 365, 370, 375, or up
to the number of nucleotides present in SEQ ID NO:6; at least 15,
20, 30, 40, 50, 60, 70, 75, 80, 90, 100, 110, 120, 125, 130, 140,
150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 260,
261, or up to the number of nucleotides present in SEQ ID NO:8; at
least 15, 20, 30, 40, 50, 60, 70, 75, 80, 90, 100, 110, 120, 125,
130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240,
250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 355, 360,
365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 430, 440, 450,
460, 470, 480, 483, or up to the number of nucleotides present in
SEQ ID NO:9; at least 15, 20, 30, 40, 50, 60, 70, 75, 80, 90, 100,
110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210,
220, 230, 240, 250, 260, 270, 280, 290, 294, or up to the number of
nucleotides present in SEQ ID NO:11; at least 15, 20, 30, 40, 50,
60, 70, 75, 80, 90, 100, 110, 120, 125, 130, 140, 150, 160, 170,
175, or 177 nucleotides encoding a portion of the amino acid
sequence set forth in SEQ ID NO:20; at least 15, 20, 30, 40, 50,
60, 70, 75, 80, 90, 100, 110, 120, 125, 130, 140, 150, 160, 170,
175, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,
300, 310, 320, 330, 340, 350, 355, 360, 365, 370, 375, 380, 385,
390, 395, 400, 410, 420, 430, 440, 450, 460, 470, 479, or up to the
number of nucleotides present in SEQ ID NO:21; at least 15, 20, 30,
40, 50, 60, 70, 75, 80, 90, 100, 110, 120, 125, 130, 140, 150, 160,
170, 175, 180, 190, 200, 210, 220, 230, 240, 243, or up to the
number of nucleotides present in SEQ ID NO:23; and at least 15, 20,
30, 40, 50, 60, 70, 75, 80, 90, 100, 110, 120, 125, 130, 140, 150,
160, 170, 175, 180, 185, 190, or 195 nucleotides encoding a portion
of the amino acid sequence set forth in SEQ ID NO:27.
[0064] By "variants" substantially similar sequences are intended.
For nucleotide sequences, conservative variants include those
sequences that, because of the degeneracy of the genetic code,
encode the amino acid sequence of one of the pesticidal
polypeptides of the invention. Naturally occurring allelic variants
such as these can be identified with the use of well-known
molecular biology techniques, such as, for example, polymerase
chain reaction (PCR) and hybridization techniques as outlined
below. Variant nucleotide sequences also include synthetically
derived nucleotide sequences, such as those generated, for example,
by using site-directed mutagenesis but which still encode a
pesticidal polypeptide of the invention. Generally, variants of a
particular nucleotide sequence of the invention will have at least
about 40%, 45%, 50%, 55%, 60%, 65%, 70%, generally at least about
75%, 80%, 85%, preferably at least about 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, and more preferably at least about 98%, 99% or more
sequence identity to that particular nucleotide sequence as
determined by sequence alignment programs described elsewhere
herein using default parameters. Thus, isolated sequences that
encode a pesticidal polypeptide and which hybridize under stringent
conditions to the sequences disclosed herein, or to fragments
thereof, are encompassed by the present invention.
[0065] Variants of a particular nucleotide sequence of the
invention (i.e., the reference nucleotide sequence) can also be
evaluated by comparison of the percent sequence identity between
the polypeptide encoded by a variant nucleotide sequence and the
polypeptide encoded by the reference nucleotide sequence. Thus, for
example, isolated nucleic acids that encode a polypeptide with a
given percent sequence identity to the polypeptides of the
invention (for example, SEQ ID NO: 2, 4, 7, 10, 20, 22, 24, or 27)
are disclosed. Percent sequence identity between any two
polypeptides can be calculated using sequence alignment programs
described elsewhere herein using default parameters. Where any
given pair of polynucleotides of the invention is evaluated by
comparison of the percent sequence identity shared by the two
polypeptides they encode, the percent sequence identity between the
two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%,
65%, 70%, generally at least about 75%, 80%, 85%, preferably at
least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and more
preferably at least about 98%, 99% or more sequence identity.
[0066] By "variant protein" a protein is intended that is derived
from the native polypeptide by deletion (so-called truncation) or
addition of one or more amino acids to the N-terminal and/or
C-terminal end of the native polypeptide; deletion or addition of
one or more amino acids at one or more sites in the native
polypeptide; or substitution of one or more amino acids at one or
more sites in the native polypeptide. Variant proteins encompassed
by the present invention are biologically active, that is they
continue to possess the desired biological activity of the native
polypeptide, that is, pesticidal activity as described herein. Such
variants may result from, for example, genetic polymorphism or from
human manipulation. Biologically active variants of a native
pesticidal protein of the invention will have at least about 40%,
50%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%,
preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
and more preferably at least about 98%, 99% or more sequence
identity to the amino acid sequence for the native protein as
determined by sequence alignment programs described elsewhere
herein using default parameters. A biologically active variant of a
protein of the invention may differ from that protein by as few as
1-15 amino acid residues, as few as 1-10, such as 6-10, as few as
5, as few as 4, 3, 2, or even 1 amino acid residue.
[0067] The proteins of the invention 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
pesticidal polypeptides can be prepared by mutations in the DNA.
Methods for mutagenesis and nucleotide sequence alterations are
well known in the art. See, for example, Kunkel (1985) Proc. Natl.
Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol.
154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.
(1983) Techniques in Molecular Biology (MacMillan Publishing
Company, New York) and the references cited therein. Guidance as to
appropriate amino acid substitutions that do not affect biological
activity of the polypeptide of interest may be found in the model
of Dayhoff et al. (1978) Atlas of Polypeptide Sequence and
Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein
incorporated by reference. Conservative substitutions, such as
exchanging one amino acid with another having similar properties,
may be preferable.
[0068] Thus, the genes and nucleotide sequences of the invention
include both the naturally occurring sequences as well as mutant
forms. Likewise, the proteins of the invention encompass naturally
occurring proteins as well as variations and modified forms
thereof. Such variants will continue to possess the desired
pesticidal activity. Obviously, the mutations that will be made in
the DNA encoding the variant must not place the sequence out of
reading frame and preferably will not create complementary regions
that could produce secondary mRNA structure. See EP Patent
Application Publication No. 75,444, herein incorporated by
reference.
[0069] The deletions, insertions, and substitutions of the
polypeptide sequences encompassed herein are not expected to
produce radical changes in the characteristics of the polypeptide.
However, when it is difficult to predict the exact effect of the
substitution, deletion, or insertion in advance of doing so, one
skilled in the art will appreciate that the effect will be
evaluated by routine screening assays such as, but not limited to,
insect feeding assays. See, for example, Marrone et al. (1985) J.
Econ. Entomol. 78:290-293 and Czapla and Lang (1990) J. Econ.
Entomol. 83:2480-2485, herein incorporated by reference, or also in
Example 2, herein.
[0070] Variant nucleotide sequences and proteins also encompass
sequences and proteins derived from a mutagenic and recombinogenic
procedure such as DNA shuffling. With such a procedure, one or more
different orally active pesticidal coding sequences can be
manipulated to create new orally active pesticidal polypeptides
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,
full-length coding sequences, sequence motifs encoding a domain of
interest or any fragment of a nucleotide sequence of the invention
may be shuffled between a nucleotide sequence of the invention and
corresponding portions of other known pesticides to obtain a new
gene coding for a polypeptide with an improved property of
interest. Properties of interest include, but are not limited to,
pesticidal activity per unit of pesticidal polypeptide, polypeptide
stability, toxicity to non-target species particularly humans,
livestock, plants, baculovirus and other organisms that express the
pesticidal polypeptide of the invention, and an altered K.sub.m.
The invention is not bound by a particular shuffling strategy only
that at least one nucleotide sequence of the invention, or part
thereof, is involved in such a shuffling strategy. Shuffling may
involve only nucleotide sequences disclosed herein or may
additionally involve shuffling any other nucleotide sequences known
in the art including, but not limited to, GenBank Accession Nos.
U04364, U04365, and U04366. Strategies for 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.
[0071] The amino acid sequences of the invention can be used to
isolate corresponding sequences from other organisms, particularly
other arthropods. In this manner, methods such as PCR,
hybridization, and the like can be used to identify nucleotide
sequences encoding such amino acid sequences based on their
sequence homology to the sequences set forth herein. Sequences
isolated based on their sequence identity to the entire sequences
set forth herein or to fragments thereof are encompassed by the
present invention. Such sequences include sequences that are
orthologs of the disclosed sequences. By "orthologs" genes are
intended that are derived from a common ancestral gene and which
are found in different species as a result of speciation. Genes
found in different species are considered orthologs when their
nucleotide sequences and/or their encoded polypeptide sequences
share substantial identity as defined elsewhere herein. Functions
of orthologs are often highly conserved among species. Thus,
isolated sequences that encode for an orally active, pesticidal
protein and which hybridize under stringent conditions to the
pesticidal sequences disclosed herein, or to fragments thereof, are
encompassed by the present invention.
[0072] In a PCR approach, oligonucleotide primers can be designed
for use in PCR reactions to amplify corresponding DNA sequences
from cDNA or genomic DNA extracted from any organism of interest.
Methods for designing PCR primers and PCR cloning are generally
known in the art and are disclosed in Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor
Laboratory Press, Plainview, N.Y.). See also Innis et al., eds.
(1990) PCR Protocols: A Guide to Methods and Applications (Academic
Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies
(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR
Methods Manual (Academic Press, New York). Known methods of PCR
include, but are not limited to, methods using paired primers,
nested primers, single specific primers, degenerate primers,
gene-specific primers, vector-specific primers,
partially-mismatched primers, and the like.
[0073] In hybridization techniques, all or part of a known
nucleotide sequence is used as a probe that selectively hybridizes
to other corresponding nucleotide sequences present in a population
of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or
cDNA libraries) from a chosen organism. The hybridization probes
may be genomic DNA fragments, cDNA fragments, RNA fragments, or
other oligonucleotides, and may be labeled with a detectable group
such as .sup.32P, or any other detectable marker. Thus, for
example, probes for hybridization can be made by labeling synthetic
oligonucleotides based on the sequences of the invention. Methods
for preparation of probes for hybridization and for construction of
cDNA and genomic libraries are generally known in the art and are
disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory
Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview,
N.Y.).
[0074] For example, an entire sequence disclosed herein, or one or
more portions thereof, may be used as a probe capable of
specifically hybridizing to corresponding sequences and messenger
RNAs. To achieve specific hybridization under a variety of
conditions, such probes include sequences that are unique among the
sequences of the invention and are at least about 10 nucleotides in
length, and preferably at least about 20 nucleotides in length.
Such probes may be used to amplify corresponding 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 a
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.).
[0075] Hybridization of such sequences may be carried out under
stringent conditions. By "stringent conditions" or "stringent
hybridization conditions" conditions are intended 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 about 500 nucleotides in length.
[0076] 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. The duration of hybridization is generally less than
about 24 hours, usually about 4 to about 12 hours.
[0077] 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 .gtoreq.90% identity are sought, the T.sub.m can be
decreased 10.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.).
[0078] Thus, isolated sequences that encode a polypeptide of the
invention and which hybridize under stringent conditions to the
sequences disclosed herein, or to fragments thereof, are
encompassed by the present invention.
[0079] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides:
(a) "reference sequence", (b) "comparison window", (c) "sequence
identity", (d) "percentage of sequence identity", and (e)
"substantial identity".
[0080] (a) As used herein, "reference sequence" is a defined
sequence used as a basis for sequence comparison. A reference
sequence may be a subset or the entirety of a specified sequence;
for example, as a segment of a full-length cDNA or gene sequence,
or the complete cDNA or gene sequence.
[0081] (b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. Generally, the
comparison window is at least 20 contiguous nucleotides in length,
and optionally can be 30, 40, 50, 100, or longer. Those of skill in
the art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide sequence a
gap penalty is typically introduced and is subtracted from the
number of matches.
[0082] Methods of alignment of sequences for comparison are well
known in the art. Thus, the determination of percent identity
between any two sequences can be accomplished using a mathematical
algorithm. Non-limiting examples of such mathematical algorithms
are the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the
local alignment algorithm of Smith et al. (1981) Adv. Appl. Math.
2:482; the global alignment algorithm of Needleman and Wunsch
(1970) J. Mol. Biol. 48:443-453; the search-for-local alignment
method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.
85:2444-2448; the algorithm of Karlin and Altschul (1990) Proc.
Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul
(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
[0083] Computer implementations of these mathematical algorithms
can be utilized for comparison of sequences to determine sequence
identity. Such implementations include, but are not limited to:
CLUSTAL in the PC/Gene program (available from Intelligenetics,
Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP,
BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics
Software Package, Version 8 (available from Genetics Computer Group
(GCG), 575 Science Drive, Madison, Wis., USA). Alignments using
these programs can be performed using the default parameters. The
CLUSTAL program is well described by Higgins et al. (1988) Gene
73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet
et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)
CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol.
24:307-331. The ALIGN program is based on the algorithm of Myers
and Miller (1988) supra. A PAM120 weight residue table, a gap
length penalty of 12, and a gap penalty of 4 can be used with the
ALIGN program when comparing amino acid sequences. The BLAST
programs of Altschul et al (1990) J. Mol. Biol. 215:403 are based
on the algorithm of Karlin and Altschul (1990) supra. BLAST
nucleotide searches can be performed with the BLASTN program,
score=100, wordlength=12, to obtain nucleotide sequences homologous
to a nucleotide sequence encoding a polypeptide of the invention.
BLAST polypeptide searches can be performed with the BLASTX
program, score=50, wordlength=3, to obtain amino acid sequences
homologous to a polypeptide or polypeptides 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 (in BLAST 2.0)
can be used to perform an iterated search that detects distant
relationships between molecules. See Altschul et al. (1997) supra.
When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default
parameters of the respective programs (e.g., BLASTN for nucleotide
sequences, BLASTX for polypeptides) can be used. See
http://www.ncbi.hlm.nih.gov. Alignment may also be performed
manually by inspection.
[0084] Unless otherwise stated, nucleotide sequence
identity/similarity values provided herein refer to the value
obtained using GAP Version 10 using the following parameters: %
identity using GAP Weight of 50 and Length Weight of 3; %
similarity using Gap Weight of 12 and Length Weight of 4, or any
equivalent program. For amino acid sequences, amino acid sequence
identity values provided herein refer to the value obtained using
GAP Version 10 using the following parameters: % identity using GAP
Weight of 8 and Length Weight of 2, or any equivalent program. By
"equivalent program" any sequence comparison program is intended
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 the preferred program.
[0085] GAP uses the algorithm of Needleman and Wunsch (1970) J.
Mol. Biol. 48:443-453, to find the alignment of two complete
sequences that maximizes the number of matches and minimizes the
number of gaps. GAP considers all possible alignments and gap
positions and creates the alignment with the largest number of
matched bases and the fewest gaps. It allows for the provision of a
gap creation penalty and a gap extension penalty in units of
matched bases. GAP must make a profit of gap creation penalty
number of matches for each gap it inserts. If a gap extension
penalty greater than zero is chosen, GAP must, in addition, make a
profit for each gap inserted of the length of the gap times the gap
extension penalty. Default gap creation penalty values and gap
extension penalty values in Version 10 of the Wisconsin Genetics
Software Package for polypeptide sequences are 8 and 2,
respectively. For nucleotide sequences the default gap creation
penalty is 50 while the default gap extension penalty is 3. The gap
creation and gap extension penalties can be expressed as an integer
selected from the group of integers consisting of from 0 to 200.
Thus, for example, the gap creation and gap extension penalties can
be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65 or greater.
[0086] GAP presents one member of the family of best alignments.
There may be many members of this family, but no other member has a
better quality. GAP displays four figures of merit for alignments:
Quality, Ratio, Identity, and Similarity. The Quality is the metric
maximized in order to align the sequences. Ratio is the quality
divided by the number of bases in the shorter segment. Percent
Identity is the percent of the symbols that actually match. Percent
Similarity is the percent of the symbols that are similar. Symbols
that are across from gaps are ignored. A similarity is scored when
the scoring matrix value for a pair of symbols is greater than or
equal to 0.50, the similarity threshold. The scoring matrix used in
Version 10 of the Wisconsin Genetics Software Package is BLOSUM62
(see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA
89:10915).
[0087] For purposes of the present invention, comparison of
nucleotide or polypeptide sequences for determination of percent
sequence identity to the sequences disclosed herein is preferably
made using the GAP program in the Wisconsin Genetics Software
Package (Version 8 or later) or any equivalent program. For GAP
analyses of nucleotide sequences, a GAP Weight of 50 and a Length
of 3 was used.
[0088] (c) As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences makes
reference to the residues in the two sequences that are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to polypeptides it is recognized that residue positions which are
not identical often differ by conservative amino acid
substitutions, where amino acid residues are substituted for other
amino acid residues with similar chemical properties (e.g., charge
or hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity". Means for making
this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif.).
[0089] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0090] (e)(i) The term "substantial identity" of polynucleotide
sequences means that a polynucleotide comprises a sequence that has
at least 70% sequence identity, preferably at least 80%, more
preferably at least 90%, and most preferably at least 95%, compared
to a reference sequence using one of the alignment programs
described using standard parameters. One of skill in the art will
recognize that these values can be appropriately adjusted to
determine corresponding identity of polypeptides encoded by two
nucleotide sequences by taking into account codon degeneracy, amino
acid similarity, reading frame positioning, and the like.
Substantial identity of amino acid sequences for these purposes
normally means sequence identity of at least 60%, more preferably
at least 70%, 80%, 90%, and most preferably at least 95%.
[0091] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each other
under stringent conditions. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. However, stringent conditions encompass
temperatures in the range of about 1.degree. C. to about 20.degree.
C., depending upon the desired degree of stringency as otherwise
qualified herein. Nucleic acids that do not hybridize to each other
under stringent conditions are still substantially identical if the
polypeptides they encode are substantially identical. This may
occur, e.g., when a copy of a nucleic acid is created using the
maximum codon degeneracy permitted by the genetic code. One
indication that two nucleic acid sequences are substantially
identical is when the polypeptide encoded by the first nucleic acid
is immunologically cross reactive with the polypeptide encoded by
the second nucleic acid.
[0092] (e)(ii) The term "substantial identity" in the context of a
peptide indicates that a peptide comprises a sequence with at least
70% sequence identity to a reference sequence, preferably 80%, more
preferably 85%, most preferably at least 90% or 95% sequence
identity to the reference sequence over a specified comparison
window. Preferably, optimal alignment is conducted using the
homology alignment algorithm of Needleman and Wunsch (1970) J. Mol.
Biol. 48:443-453. An indication that two peptide sequences are
substantially identical is that one peptide is immunologically
reactive with antibodies raised against the second peptide. Thus, a
peptide is substantially identical to a second peptide, for
example, where the two peptides differ only by a conservative
substitution. Peptides that are "substantially similar" share
sequences as noted above except that residue positions that are not
identical may differ by conservative amino acid changes.
[0093] The use of the term "nucleotide constructs" herein is not
intended to limit the present invention to nucleotide constructs
comprising DNA. Those of ordinary skill in the art will recognize
that nucleotide constructs, particularly polynucleotides and
oligonucleotides, comprised of ribonucleotides and combinations of
ribonucleotides and deoxyribonucleotides may also be employed in
the methods disclosed herein. The nucleotide constructs, nucleotide
molecules and nucleotide sequences of the invention additionally
encompass all complementary forms of such constructs, molecules and
sequences. Further, the nucleotide constructs, nucleotide molecules
and nucleotide sequences of the present invention encompass all
nucleotide constructs, molecules and sequences which can be
employed in the methods of the present invention for transforming
plants including, but not limited to, those comprised of
deoxyribonucleotides, ribonucleotides and combinations thereof.
Such deoxyribonucleotides and ribonucleotides include both
naturally occurring molecules and synthetic analogues. The
nucleotide constructs, nucleotide molecules and nucleotide
sequences of the invention also encompass all forms of nucleotide
constructs including, but not limited to, single-stranded forms,
double-stranded forms, hairpins, stem-and-loop structures and the
like.
[0094] A further embodiment of the invention relates to a
transformed organism, preferably a transformed organism selected
from the group consisting of plant and insect cells, fungi, and
baculoviruses, comprising a DNA molecule of the invention, an
expression cassette comprising said DNA molecule or a vector
molecule comprising said expression cassette, preferably stably
incorporated into the genome of the transformed organism.
[0095] The sequences of the invention are provided in expression
cassettes for expression in the organism of interest, for example a
plant of interest. The cassette will include 5' and 3' regulatory
sequences operably linked to a sequence of the invention. By
"operably linked" a functional linkage is intended between a first
sequence, such as 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 polypeptide 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.
[0096] Such an expression cassette is provided with a plurality of
restriction sites for insertion of the sequence to be under the
transcriptional regulation of the regulatory regions. The
expression cassette may additionally contain selectable marker
genes.
[0097] The expression cassette will include in the 5'-3' direction
of transcription, a transcriptional and translational initiation
region (i.e., promoter), a DNA sequence of the invention, and a
transcriptional and translational termination region (i.e.,
termination region) functional in the host organism, for example, a
plant. The promoter may be native or analogous, or foreign or
heterologous, to the 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
"foreign" or "heterologous" to the host, it is intended that the
promoter is not found in the native host organism 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. While it may
be preferable to express the DNA sequences of the invention using
heterologous promoters, the native promoter sequences may be used
where functional in the host. As used herein, a chimeric gene
comprises a coding sequence operably linked to a transcription
initiation region that is heterologous to the coding sequence.
[0098] 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 host
organism, or may be derived from another source (i.e., foreign or
heterologous to the promoter, the DNA sequence of interest, the
host organism, or any combination thereof). Convenient
transcriptional and translational 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.
[0099] Where appropriate, the gene(s) may be optimized for
increased expression in the transformed host. For example, the
genes can be synthesized using plant-preferred codons for improved
expression in plants. 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.
[0100] Additional sequence modifications are known to enhance gene
expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon-intron
splice site signals, transposon-like repeats, and other such
well-characterized sequences that may be deleterious to gene
expression. The G-C content of the sequence may be adjusted to
levels average for a given cellular host, as calculated by
reference to known genes expressed in the host cell. When possible,
the sequence is modified to avoid predicted hairpin secondary mRNA
structures.
[0101] The expression cassettes may additionally contain 5' leader
sequences in the expression cassette construct. Such leader
sequences can act to enhance translation. Translation leaders are
known in the art and include: picornavirus leaders, for example,
EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein
et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus
leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et
al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic
Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain
binding polypeptide (BiP) (Macejak et al. (1991) Nature 353:90-94);
untranslated leader from the coat polypeptide mRNA of alfalfa
mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature
325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al.
(1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp.
237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et
al. (1991) Virology 81:382-385). See also, Della-Cioppa et al.
(1987) Plant Physiol. 84:965-968. Other methods known to enhance
translation can also be utilized, for example, introns, and the
like.
[0102] The expression cassettes may also contain sequences that act
to enhance proper folding or secretion. Proper folding or secretion
may be achieved by targeting operably linked polypeptides to
organelles where such modifications occur. Modifying organelles
include but are not limited to the endoplasmic reticulum and the
Golgi Apparatus. Sequences known to target operably linked
polypeptides to an appropriate organelle include, but are not
limited to, the sporamin signal sequence, the barley alpha amylase
sequence, and the vacuolar retention signal.
[0103] In preparing the expression cassette, the various DNA
fragments may be manipulated, so as to provide for the DNA
sequences in the proper orientation and, as appropriate, in the
proper reading frame. Toward this end, adapters or linkers may be
employed to join the DNA fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For
this purpose, in vitro mutagenesis, primer repair, restriction,
annealing, resubstitutions, e.g., transitions and transversions,
may be involved.
[0104] A number of promoters can be used in the practice of the
invention. The promoters can be selected based on the desired
outcome. The nucleic acids can be combined with constitutive,
inducible, tissue-preferred, or other promoters for expression in
plants.
[0105] Constitutive promoters include, for example, the core
promoter of the Rsyn7 promoter and other constitutive promoters
disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV
35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin
(McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin
(Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and
Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last
et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al.
(1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No.
5,659,026), and the like. Other constitutive promoters include, for
example, those disclosed in U.S. Pat. Nos. 5,608,149; 5,608,144;
5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142;
and 6,177,611, which are incorporated herein by reference.
[0106] Depending on the desired outcome, it may be beneficial to
express the gene from an inducible promoter. Of particular interest
for regulating the expression of the nucleotide sequences of the
present invention in plants are wound-inducible promoters. Such
wound-inducible promoters may respond to damage caused by insect
feeding, and include potato polypeptidase inhibitor (pin II) gene
(Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996)
Nature Biotechnology 14:494-498); wun1 and wun2, U.S. Pat. No.
5,428,148; win1 and win2 (Stanford et al. (1989) Mol. Gen. Genet.
215:200-208); systemin (McGurl et al. (1992) Science
225:1570-1573); WIPI (Rohmeier et al. (1993) Plant Mol. Biol.
22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76); MPI
gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like,
herein incorporated by reference.
[0107] Additionally, pathogen-inducible promoters may be employed
in the methods and nucleotide constructs of the present invention.
Such pathogen-inducible promoters include those from
pathogenesis-related polypeptides (PR polypeptides), which are
induced following infection by a pathogen; e.g., PR polypeptides,
SAR polypeptides, beta-1,3-glucanase, chitinase, etc. See, for
example, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254;
Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant
Mol. Virol. 4:111-116. See also WO 99/43819, herein incorporated by
reference.
[0108] Of interest are promoters that are expressed locally at or
near the site of pathogen infection. See, for example, Marineau et
al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989)
Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al.
(1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al.
(1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad.
Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J.
10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA
91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz et
al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386
(nematode-inducible); and the references cited therein. Of
particular interest is the inducible promoter for the maize PRms
gene, whose expression is induced by the pathogen Fusarium
moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol.
Plant. Path. 41:189-200).
[0109] Chemical-regulated promoters can be used to modulate the
expression of a gene in a plant through the application of an
exogenous chemical regulator. Depending upon the objective, the
promoter may be a chemical-inducible promoter, where application of
the chemical induces gene expression, or a chemical-repressible
promoter, where application of the chemical represses gene
expression. Chemical-inducible promoters are known in the art and
include, but are not limited to, the maize In2-2 promoter, which is
activated by benzenesulfonamide herbicide safeners, the maize GST
promoter, which is activated by hydrophobic electrophilic compounds
that are used as pre-emergent herbicides, and the tobacco PR-1a
promoter, which is activated by salicylic acid. Other
chemical-regulated promoters of interest include steroid-responsive
promoters (see, for example, the glucocorticoid-inducible promoter
in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425
and McNellis et al. (1998) Plant J. 14(2):247-257) and
tetracycline-inducible and tetracycline-repressible promoters (see,
for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and
U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by
reference.
[0110] Tissue-preferred promoters can be utilized to target
enhanced pesticidal polypeptide expression within a particular
plant tissue. See, for example, Yamamoto et al. (1997) Plant J.
12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol.
38(7):792-803; Hansen et al. (1997) Mol Gen Genet. 254(3):337-343;
Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al.
(1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996)
Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant
Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.
35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196;
Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; Matsuoka et
al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and
Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters
can be modified, if necessary, by methods known in the art, for
weak expression.
[0111] Vascular tissue-preferred promoters are known in the art.
See for example copending Application No. 60/305,362, filed Jul.
13, 2001, herein incorporated by reference in its entirety.
Vascular tissue preferred promoters allow tissue-preferred
expression of nucleotide sequences of interest in the vasculature
of plants. Vascular tissue preferred expression of pesticidal
agents allows targeting of sucking insect pests in particular.
[0112] Leaf-preferred promoters are known in the art. See, for
example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al.
(1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell
Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18;
Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka
et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
[0113] "Seed-preferred" promoters include both "seed-specific"
promoters (those promoters active during seed development such as
promoters of seed storage polypeptides) as well as
"seed-germinating" promoters (those promoters active during seed
germination). See Thompson et al. (1989) BioEssays 10:108, herein
incorporated by reference. Such seed-preferred promoters include,
but are not limited to, Cim1 (cytokinin-induced message); cZ19B1
(maize 19 kDa zein); milps (myo-inositol-1-phosphate synthase); and
celA (cellulose synthase) (see WO 00/11177 and U.S. Pat. No.
6,225,529; herein incorporated by reference). Gama-zein is a
preferred endosperm-preferred promoter. Glob-1 is a preferred
embryo-preferred promoter. For dicots, seed-preferred promoters
include, but are not limited to, bean .beta.-phaseolin, napin,
.beta.-conglycinin, soybean lectin, cruciferin, and the like. For
monocots, seed-preferred promoters include, but are not limited to,
maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken
1, shrunken 2, globulin 1, etc. See also WO 00/12733, where
seed-preferred promoters from end1 and end2 genes are disclosed and
herein incorporated by reference.
[0114] Where low level expression is desired, weak promoters will
be used. Low level expression is particularly desirable when
expression of a polypeptide of the invention in a plant has a
deleterious effect on the plant. Generally, by "weak promoter" is
intended a promoter that drives expression of a coding sequence at
a low level. By low level is intended at levels of about 1/1000
transcripts to about 1/100,000 transcripts to about 1/500,000
transcripts. Alternatively, it is recognized that weak promoters
also encompass promoters that are expressed in only a few cells and
not in others to give a total low level of expression. Where a
promoter drives expression at unacceptably high levels, portions of
the promoter sequence can be deleted or modified to decrease
expression levels.
[0115] Weak constitutive promoters include, for example the core
promoter of the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No.
6,072,050), the core 35S CaMV promoter, and the like.
[0116] Generally, the expression cassette will comprise a
selectable marker gene for the selection of transformed cells.
Selectable marker genes are utilized for the selection of
transformed cells or tissues. Marker genes include genes encoding
antibiotic resistance, such as those encoding neomycin
phosphotransferase II (NEO) and hygromycin phosphotransferase
(HPT), as well as genes conferring resistance to herbicidal
compounds, such as glufosinate ammonium, bromoxynil,
imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See
generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;
Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA
89:6314-6318; Yao et al (1992) Cell 71:63-72; Reznikoff (1992) Mol.
Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp.
177-220; Hu et al (1987) Cell 48:555-566; Brown et al (1987) Cell
49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al.
(1989) Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst et al (1989)
Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990)
Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of
Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA
90:1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356;
Zambretti et al (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956;
Baim et al (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski
et al. (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman
(1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb et al. (1991)
Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt et al
(1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,
University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad.
Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents
Chemother. 36:913-919; Hlavka et al. (1985) Handbook of
Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill
et al (1988) Nature 334:721-724. Such disclosures are herein
incorporated by reference.
[0117] The above list of selectable marker genes is not meant to be
limiting. Any selectable marker gene can be used in the present
invention.
[0118] 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
(Townsend et al., U.S. Pat. No. 5,563,055; Zhao et al., 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, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al.,
U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No. 5,886,244;
Bidney et al., 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); and Lec1 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 (onion); Christou et al. (1988) Plant Physiol.
87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926
(soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:
175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.
96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740
(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309
(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize);
Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos.
5,322,783 and 5,324,646; 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
(Springer-Verlag, Berlin) (maize); Klein et al. (1988) Plant
Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology
8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature
(London) 311:763-764; Bowen et al., U.S. Pat. No. 5,736,369
(cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA
84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental
Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New
York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell
Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.
84:560-566 (whisker-mediated transformation); D'Halluin et al.
(1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993)
Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals
of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature
Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all
of which are herein incorporated by reference. Protocols that are
useful for transformation of rice plants in particular include
biolistic methods (see Nayak et al. (1997) Proc. Natl. Acad. Sci.
94:2111-2116 and Christou, P. (1997) Plant Mol. Biol. 35:197-203)
and Agrobacterium mediated methods (see Hiei et al. (1994) Plant J.
6:271-282 and Ishida et al. (1996) Nat. Biotechnol.
14:745-750).
[0119] By "stable transformation" is intended that the nucleotide
construct introduced into a plant integrates into the genome of the
plant and is capable of being inherited by progeny thereof. By
"transient transformation" is intended that a nucleotide construct
introduced into a plant does not integrate into the genome of the
plant.
[0120] The nucleotide constructs of the invention may be introduced
into plants by contacting plants with a virus or viral nucleic
acids. Generally, such methods involve incorporating a nucleotide
construct of the invention within a viral DNA or RNA molecule. It
is recognized that the protein of interest of the invention may be
initially synthesized as part of a viral polyprotein, which later
may be processed by proteolysis in vivo or in vitro to produce the
desired recombinant protein. Further, it is recognized that
promoters of the invention also encompass promoters utilized for
transcription by viral RNA polymerases. Methods for introducing
nucleotide constructs into plants and expressing a protein encoded
therein, involving viral DNA or RNA molecules, are known in the
art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190,
5,866,785, 5,589,367 and 5,316,931; herein incorporated by
reference.
[0121] 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 that
expression of the desired phenotypic characteristic has been
achieved. In addition, the desired genetically altered trait can be
bred into other plant lines possessing other desirable
characteristics using conventional breeding methods and/or
top-cross technology.
[0122] Methods for cross pollinating plants are well known to those
skilled in the art, and are generally accomplished by allowing the
pollen of one plant, the pollen donor, to pollinate a flower of a
second plant, the pollen recipient, and then allowing the
fertilized eggs in the pollinated flower to mature into seeds.
Progeny containing the heterologous coding sequences of the two
parental plants can be selected from all of the progeny by standard
methods available in the art as described infra for selecting
transformed plants. If necessary, the selected progeny can be used
as either the pollen donor or pollen recipient in a subsequent
cross-pollination.
[0123] Parts of transgenic plants within the scope of the invention
comprise, for example, plant cells, protoplasts, tissues, callus,
embryos as well as flowers, stems, fruits, leaves, roots
originating in transgenic plants, and their progeny previously
transformed with a DNA molecule of the invention and therefore
consisting at least in part of transgenic cells. The invention
further relates to plant propagating material of a transformed
plant of the invention including, but not limited to, seeds,
tubers, corms, bulbs, leaves and cuttings of roots and shoots.
[0124] 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,
rice (Oryza sativa), corn (Zea mays), Brassica sp. (e.g., B. napus,
B. rapa, B. juncea), particularly those Brassica species useful as
sources of seed oil, alfalfa (Medicago sativa), rye (Secale
cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,
pearl millet (Pennisetum glaucum), proso millet (Panicum
miliaceum), foxtail millet (Setaria italica), finger millet
(Eleusine coracana)), sunflower (Helianthus annuus), safflower
(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine
max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum),
peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium
hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot
esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), flax
(Linum usitatissimum), castor (Ricinus communis), palm
(Cycadophyta), pineapple (Ananas comosus), citrus trees (Citrus
spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana
(Musa spp.), avocado (Persea americana), fig (Ficus casica), guava
(Psidium guajava), mango (Mangifera indica), olive (Olea europaea),
papaya (Carica papaya), cashew (Anacardium occidentale), macadamia
(Macadamia integrifolia), almond (Prunus amygdalus), sugar beets
(Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,
vegetables, ornamentals, and conifers.
[0125] Vegetables of interest include tomatoes (Lycopersicon
esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus
vulgaris), garden beans, lima beans (Phaseolus limensis), peas
(Lathyrus spp.), and members of the genus Cucumis such as cucumber
(C. sativus), cantaloupe (C. cantalupensis), and musk melon (C.
melo). Ornamentals of interest include azalea (Rhododendron spp.),
hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus
rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils
(Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus
caryophyllus), poinsettia (Euphorbia pulcherrima), and
chrysanthemum. Conifers that may be employed in practicing the
present invention include, for example, pines such as loblolly pine
(Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus
ponderosa), lodgepole pine (Pinus contorta), and Monterey pine
(Pinus radiata); Douglas fir (Pseudotsuga menziesii); Western
hemlock (Tsuga canadenensis); Sitka spruce (Picea glauca); redwood
(Sequoia sempervirens); true firs such as silver fir (Abies
amabilis) and balsam fir (Abies balsamea); and cedars such as
Western red cedar (Thuja plicata) and Alaska yellow-cedar
(Chamaecyparis nootkatensis). Preferably, plants of the present
invention are crop plants including grain plants that provide seeds
of interest, oil-seed plants, and leguminous plants (for example,
rice, corn, alfalfa, sunflower, Brassica, soybean, rye, barley,
cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.).
Leguminous plants include beans and peas. Beans include guar,
locust bean, fenugreek, cowpea, mung bean, fava bean, lentils,
chickpea, etc. More preferably, plants of the present invention are
corn, rice, and soybean plants, yet more preferably rice
plants.
[0126] In an embodiment, a transformed plant of the invention may
be treated with a protectant coating. Components of the protectant
coating include, but are not limited to, anti-desiccants,
herbicides, insecticides, fungicides, bactericides, nematocides,
and molluscicides. The protectant coating may include carriers,
surfactants or application-promoting adjuvants. The protectant
coating may be applied to seeds either by impregnating tubers or
grains with a liquid formulation or by coating them with a combined
wet or dry formulation. A transformed seed of the invention may be
treated with a seed protectant coating comprising a seed treatment
compound including, but not limited to, captan, carboxin, thiram,
methalaxyl, and pirimiphos-methyl. An embodiment of the invention
is a seed protectant coating comprising a pesticidal composition of
the invention. In addition, in special cases, other methods of
application to plants are possible, e.g. treatment directed at the
buds or the fruit.
[0127] In an embodiment, host cells expressing the polypeptides of
the invention are applied to the environment of the target pest or
pests. The host cells may be treated to prolong the activity of the
polypeptide of the invention. Host cells may be obtained from
organisms such as, but not limited to, fungi, yeast, plants,
mammals, and insects. Host organisms include, but are not limited
to, bacteria, fungi, and eukaryotic organisms. Hosts of particular
interest are the lower eukaryotes, such as fungi including
Phycomycetes and Ascomycetes, and yeast, such as Saccharomyces and
Schizosaccharromyces; and Basidiomycetes yeast, such as
Rhodotorula, Aureobasidium, Sporobolomyces.
[0128] Hosts that natively produce detrimental substances and
express a polypeptide of the invention are used at application
levels below the threshold for undesirable effects.
[0129] Numerous ways for introducing a gene expressing a pesticidal
polypeptide of the invention into a microorganism host under
conditions allowing stable maintenance and expression of the gene
exist. For example, expression cassettes can be constructed that
include the nucleotide constructs of interest operably linked with
the transcriptional and translational regulatory signals for
expression of the nucleotide constructs, and a nucleotide sequence
homologous with a sequence in the host organism, whereby
integration will occur, and/or a replication system which is
functional in the host, whereby integration or stable maintenance
will occur.
[0130] Alternatively, polypeptides of the invention are 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 a polypeptide of the
invention. The cells are treated under conditions that prolong the
activity of the polypeptide of the invention produced in the cell.
The transgenic host cell expresses a polypeptide of the invention
that retains pesticidal activity. These naturally encapsulated
pesticidal polypeptides 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.
[0131] Transcriptional and translational regulatory signals
necessary for the expression of the heterologous gene include, but
are not limited to, promoters, transcriptional initiation start
sites, operators, activators, enhancers, other regulatory elements,
ribosomal binding sites, an initiation codon, termination signals,
and the like. See, for example, U.S. Pat. No. 5,039,523; U.S. Pat.
No. 4,853,331; EPO 0480762A2; Sambrook et al. supra; Molecular
Cloning, a Laboratory Manual, Maniatis et al. (eds) Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y. (1982); Advanced
Bacterial Genetics, Davis et al. (eds.) Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1980); and the references
cited therein.
[0132] It is recognized that with these nucleotide sequences,
antisense constructions, which are complementary to at least a
portion of the messenger RNA (mRNA) for the orally active
pesticidal sequences, can be constructed. Antisense nucleotides are
constructed to hybridize with the corresponding mRNA. Modifications
of the antisense sequences may be made as long as the sequences
hybridize to and interfere with expression of the corresponding
mRNA. In this manner, antisense constructions having 70%,
preferably 80%, more preferably 85% sequence identity to the
corresponding antisensed sequences may be used. Furthermore,
portions of the antisense nucleotides may be used to disrupt the
expression of the target gene. Generally, sequences of at least 50
nucleotides, 100 nucleotides, 200 nucleotides, or greater may be
used.
[0133] It is further recognized that the genes encoding the
pesticidal polypeptides of the present invention can be used to
transform insect pathogenic organisms such as baculoviruses. Of
particular interest are recombinant baculoviruses expressing the
nucleic acid molecules of the invention. Such recombinant
baculovirus expression vectors may be prepared by protocols known
to those skilled in the art (e.g., Tomalski et al., U.S. Pat. No.
5,266,317; McCutchen et al. (1991) Bio/Technology 9:848-852; Maeda
et al. (1991) Virology 184:777-780; also see O'Reilly et al. (1992)
Baculovirus Expression Vectors: A Laboratory Manual, W. H. Freeman
and Company, New York; King and Possee (1992) The Baculovirus
Expression System, Chapman and Hall, London; U.S. Pat. No.
4,745,051; Emst et al. (1994) Nuc. Acid Res. 22:2855-2856; and WO
94/28114; herein incorporated by reference). Also of particular
interest are the phytosphere yeast species such as Rhodotorula
rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus,
C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis,
S. cerevisiae, Sporobolomyces rosues, S. odorus, Kluyveromyces
veronae, and Aureobasidium pollulans expressing the nucleic acid
molecules of the invention.
[0134] One of skill in the art can design and prepare recombinant
baculoviruses wherein a polypeptide of the invention is produced at
appropriate times during infection, expressed in toxic quantities,
and available for binding to target cells within the insect host.
Expression of the polypeptide of the invention can be confirmed
using a bioassay, LCMS, antibodies, or other methods known to one
of skill in the art. The pesticidal effect of the polypeptides of
the invention can be monitored in vivo. In vivo assays compare
biological activity of recombinant viruses to wild-type viruses.
Insect larvae can be infected orally by consumption of diet that
contains test or control viruses and the larvae monitored for
behavioral changes and mortality.
[0135] Characteristics of particular interest in selecting a host
cell or host organism for purposes of production of a polypeptide
of the invention include ease of introducing the pesticidal
polypeptide gene into the host, availability of expression systems,
efficiency of expression, stability of the polypeptide in the host,
and the presence of auxiliary genetic capabilities. Characteristics
of interest for use of a polypeptide of the invention as a
pesticide microcapsule include protective qualities for the
pesticide, such as thick cell walls, pigmentation, and
intracellular packaging or formation of inclusion bodies; leaf
affinity; lack of mammalian toxicity; attractiveness to pests for
ingestion; ease of killing and fixing without damage to the toxin;
and the like. Other considerations include ease of formulation and
handling, economics, storage stability, and the like.
[0136] The transformed microorganisms of the invention include
whole organisms, tissues, cells, spore(s), living or dead cells and
cell components including mixtures of living and dead cells and
cell components, including disrupted cells and cell components, or
an isolated pesticidal polypeptide. The living or dead cells and
cell components, mixtures of living and dead cells and cell
components, including disrupted cells and cell components, or an
isolated polypeptide can be formulated with an acceptable carrier
into a composition that is, for example, a suspension, a solution,
an emulsion, a dusting powder, a dispersible granule, a wettable
powder, and an emulsifiable concentrate, an aerosol, an impregnated
granule, an adjuvant, a coatable paste, and encapsulations in, for
example, polymer substances.
[0137] Such compositions may be obtained by the addition of a
surface-active agent, an inert carrier, a preservative, a
humectant, a feeding stimulant, an attractant, an encapsulating
agent, a binder, an emulsifier, a dye, a UV protectant, a buffer, a
flow agent or fertilizers, micronutrient donors or other
preparations that influence plant growth. One or more agrochemicals
including, but not limited to, herbicides, insecticides,
fungicides, bactericides, nematocides, molluscicides, acaracides,
plant growth regulators, harvest aids and fertilizers, can be
combined with carriers, surfactants or adjuvants employed in the
art of formulation or other components to facilitate product
handling and application for particular target pests. Suitable
carriers and adjuvants useful in the present invention can be solid
or liquid and correspond to the substances employed in formulation
technology including, but not limited to, natural or regenerated
mineral substances, solvents, dispersants, wetting agents,
tackifiers, binders or fertilizers. Compositions containing the
active ingredients of the present invention can be applied to the
crop area or plant to be treated alone, simultaneously, or in
succession with other compounds. Methods of applying an active
ingredient of the present invention or an agrochemical composition
of the present invention which contains at least one of the
pesticidal polypeptides of the present invention include, but are
not limited to, foliar application, seed coating and soil
application.
[0138] Suitable surface-active agents useful in the present
invention include, but are not limited to, anionic compounds such
as a carboxylate of, for example, a metal; a carboxylate of a long
chain fatty acid; an N-acylsarcosinate; mono or di-esters of
phosphoric acid with fatty alcohol ethoxylates or salts of such
esters; fatty alcohol sulfates such as sodium dodecyl sulfate,
sodium octadecyl sulfate or sodium cetyl sulfate; ethoxylated fatty
alcohol sulfates; ethoxylated alkylphenol sulfates; lignin
sulfonates; petroleum sulfonates; alkyl aryl sulfonates such as
alkyl-benzene sulfonates or lower alkylnaphtalene sulfonates, e.g.
butyl-naphthalene sulfonate; salts of sulfonated
naphthalene-formaldehyde condensates; salts of sulfonated
phenol-formaldehyde condensates; more complex sulfonates such as
the amide sulfonates, e.g. the sulfonated condensation product of
oleic acid and N-methyl taurine; or the dialkyl sulfosuccinates,
e.g., sodium sulfonate or dioctyl succinate. Non-ionic agents
useful in the present invention include condensation products of
fatty acid esters, fatty alcohols, fatty acid amides or
fatty-alkyl- or alkenyl-substituted phenols with ethylene oxide,
fatty esters of polyhydric alcohol ethers, e.g., sorbitan fatty
acid esters, condensation products of such esters with ethylene
oxide, e.g., polyoxyethylene sorbitar fatty acid esters, block
copolymers of ethylene oxide and propylene oxide, acetylenic
glycols such as 2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated
acetylenic glycols. Examples of cationic surface-active agents
useful in the present invention include, for instance, an aliphatic
mono-, di, or polyamine such as an acetate, naphthenate or oleate;
or oxygen-containing amine such as an amine oxide of
polyoxyethylene alkylamine; an amide-linked amine prepared by the
condensation of a carboxylic acid with a di- or polyamine; or a
quaternary ammonium salt.
[0139] Examples of inert materials useful in the present invention
include but are not limited to inorganic minerals such as kaolin,
phyllosilicates, carbonates, sulfates, phosphates or botanical
materials such as cork, powdered corncobs, peanut hulls, rice
hulls, and walnut shells.
[0140] The compositions of the present invention can be in a form
suitable for direct application or as a concentrate of a primary
composition that requires dilution with a suitable quantity of
water or other diluent before application. The pesticidal
concentration will vary depending upon the nature of the particular
formulation, specifically, whether it is a concentrate or it is to
be used directly. The composition contains 1 to 98% of a solid or
liquid inert carrier, and 0 to 50%, preferably 0.1 to 50% of a
surfactant. These compositions will be administered at the labeled
rate for the commercial product, preferably about 0.01 lb-5.0 lb.
per acre when in dry form and at about 0.01 pts.-10 pts. per acre
when in liquid form.
[0141] In a further embodiment, the compositions, as well as the
transformed microorganisms and pesticidal polypeptides, of the
invention can be treated prior to formulation to prolong the
pesticidal activity when applied to the environment of a target
pest. Such treatment can be by chemical and/or physical means.
Examples of chemical reagents include but are not limited to
halogenating agents; aldehydes such a formaldehyde and
glutaraldehyde; anti-infectives, such as zephiran chloride;
alcohols, such as isopropanol and ethanol; and histological
fixatives, such as Bouin's fixative and Helly's fixative (see, for
example, Humason, Animal Tissue Techniques, W.H. Freeman and Co.,
1967).
[0142] The compositions, including the transformed microorganisms
and pesticidal polypeptides, of the invention can be applied to the
environment of an insect pest by, for example, spraying, atomizing,
dusting, scattering, coating or pouring, introducing into or on the
soil, introducing into irrigation water, by seed treatment or
general application. In an embodiment, a polypeptide of the
invention is applied prophylactically, at times such as, but not
limited to, prior to the appearance of a pest, in early stages of
infestation, and in late stages of infestation. The compositions of
the invention may be applied at any stage of plant development
including, but not limited to, pre-germination, post-germination,
budding, flowering, ripening, pre-harvest, concomitant with
harvest, and post-harvest. The compositions of the invention may
contain additional insecticides if desired. One embodiment of the
invention is a granular form of a composition comprising an
agrochemical such as, for example, herbicides, insecticides,
fertilizers, inert carriers, and dead cells of a transformed
microorganism of the invention.
[0143] The invention is drawn to compositions and methods for
inducing resistance in a plant to plant pests. Accordingly, the
compositions and methods are also useful in protecting plants
against fungal pathogens, viruses, nematodes, insects and the
like.
[0144] By "disease resistance" it is intended that the plants avoid
the disease symptoms that are the outcome of plant-pathogen
interactions. That is, pathogens are prevented from causing plant
diseases and the associated disease symptoms, or alternatively, the
disease symptoms caused by the pathogen is minimized or lessened.
By "altered pest resistance" it is intended that the organism with
the altered resistance differs from wild type or untreated
organisms in its susceptibility to damage from pests. By "altered
insect resistance" it is intended that the organism differs from
wild type or untreated organisms in its susceptibility to damage
from insect pests. The substance or organism with an altered pest
resistance will differ from a wild-type or untreated organism in it
susceptibility to damage from pests by at least about 5% to about
50%, at least about 10% to about 60%, at least about 30% to about
70%, at least about 40% to about 80%, or at least about 50% to
about 90% or greater.
[0145] By "antipathogenic compositions" is intended that the
compositions of the invention have antipathogenic activity and thus
are capable of suppressing, controlling, and/or killing the
invading pathogenic organism. An antipathogenic composition of the
invention will reduce the disease symptoms resulting from pathogen
challenge by at least about 5% to about 50%, at least about 10% to
about 60%, at least about 30% to about 70%, at least about 40% to
about 80%, or at least about 50% to about 90% or greater. Hence,
the methods of the invention can be utilized to protect plants from
disease, particularly those diseases that are caused by plant
pathogens.
[0146] Assays that measure antipathogenic activity are commonly
known in the art, as are methods to quantitate disease resistance
in plants following pathogen infection. See, for example, U.S. Pat.
No. 5,614,395, herein incorporated by reference. Such techniques
include, measuring over time, the average lesion diameter, the
pathogen biomass, and the overall percentage of decayed plant
tissues. For example, a plant either expressing an antipathogenic
polypeptide or having an antipathogenic composition applied to its
surface shows a decrease in tissue necrosis (i.e., lesion diameter)
or a decrease in plant death following pathogen challenge when
compared to a control plant that was not exposed to the
antipathogenic composition. Alternatively, antipathogenic activity
can be measured by a decrease in pathogen biomass. For example, a
plant expressing an antipathogenic polypeptide or exposed to an
antipathogenic composition is challenged with a pathogen of
interest. Over time, tissue samples from the pathogen-inoculated
tissues are obtained and RNA is extracted. The percent of a
specific pathogen RNA transcript relative to the level of a plant
specific transcript allows the level of pathogen biomass to be
determined. See, for example, Thomma et al. (1998) Plant Biology
95:15107-15111, herein incorporated by reference.
[0147] Furthermore, in vitro antipathogenic assays include, for
example, the addition of varying concentrations of the
antipathogenic composition to paper disks and placing the disks on
agar containing a suspension of the pathogen of interest. Following
incubation, clear inhibition zones develop around the discs that
contain an effective concentration of the antipathogenic
polypeptide (Liu et al. (1994) Plant Biology 91:1888-1892, herein
incorporated by reference). Additionally, microspectrophotometrical
analysis can be used to measure the in vitro antipathogenic
properties of a composition (Hu et al. (1997) Plant Mol. Biol.
34:949-959 and Cammue et al. (1992) J. Biol. Chem. 267:2228-2233,
both of which are herein incorporated by reference).
[0148] Methods of rearing insect larvae and performing bioassays
are well known to one of ordinary skill in the art. General
procedures include addition of the experimental compound or
organism to the diet source in an enclosed container. Bioassays may
be performed as described in Czapla and Lang (1990) J. Econ.
Entomol. 83(6): 2480-2485, herein incorporated by reference, and as
described elsewhere herein. Pesticidal activity targeting insects
is tested on larval, immature, or adult stage insect pests. The
insects may be reared in total darkness at from about 20.degree. C.
to about 30.degree. C. and from about 30% to about 70% relative
humidity. Pesticidal activity can be measured by factors such as,
but not limited to, mortality, weight loss, attraction, repellency
and other behavioral and/or physical changes after feeding and
exposure to the pesticidal composition of the present
invention.
[0149] Pathogens of the invention include, but are not limited to,
viruses or viroids, bacteria, insects, nematodes, fungi, acarids,
protozoan pathogens and animal-parasitic liver flukes and the like.
Viruses include any plant virus, for example, tobacco or cucumber
mosaic virus, ringspot virus, necrosis virus, maize dwarf mosaic
virus, etc. Specific fungal and viral pathogens for the major crops
include: Soybeans: Phytophthora megasperma fsp. glycinea,
Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia
sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae
(Phomopsis sojae), Diaporthe phaseolorum var. caulivora, Sclerotium
rolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora
manshurica, Colletotrichum dematium (Colletotrichum truncatum),
Corynespora cassiicola, Septoria glycines, Phyllosticta solicola,
Alternaria alternata, Pseudomonas syringae p.v. glycinea,
Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa,
Fusarium semitectum, Phialophora gregata, Soybean mosaic virus,
Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus,
Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum,
Pythium debaryanum, Tomato spotted wilt virus, Heterodera glycines
Fusarium solani; Canola: Albugo candida, Alternaria brassicae,
Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia
sclerotiorum, Mycosphaerella brassiccola, Pythium ultimum,
Peronospora parasitica, Fusarium roseum, Alternaria alternata;
Alfalfa: Clavibacter michiganese subsp. insidiosum, Pythium
ultimum, Pythium irregulare, Pythium splendens, Pythium debaryanum,
Pythium aphanidermatum, Phytophthora megasperma, Peronospora
trifoliorum, Phoma medicaginis var. medicaginis, Cercospora
medicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis,
Fusarium oxysporum, Verticillium albo-atrum, Xanthomonas campestris
p.v. alfalfae, Aphanomyces euteiches, Stemphylium herbarum,
Stemphylium alfalfae, Colletotrichum trifolii, Leptosphaerulina
briosiana, Uromyces striatus, Sclerotinia trifoliorum, Stagnospora
meliloti, Stemphylium botryosum, Leptotrochila medicaginis; Wheat:
Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri,
Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v.
syringae, Alternaria alternata, Cladosporium herbarum, Fusarium
graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago
tritici, Ascochyta tritici, Cephalosporium gramineum,
Collotetrichum graminicola, Erysiphe graminis f.sp. tritici,
Puccinia graminis f.sp. tritici, Puccinia recondita f.sp. tritici,
Puccinia striifor is, Pyrenophora tritici-repentis, Septoria
nodorum, Septoria tritici, Septoria avenae, Pseudocercosporella
herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis,
Gaeumannomyces graminis var. tritici, Pythium aphanidermatum,
Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana,
Barley Yellow Dwarf Virus, Brome Mosaic Virus, Soil Borne Wheat
Mosaic Virus, Wheat Streak Mosaic Virus, Wheat Spindle Streak
Virus, American Wheat Striate Virus, Claviceps purpurea, Tilletia
tritici, Tilletia laevis, Tilletia indica, Pythium graminicola,
High Plains Virus, European wheat striate virus; Sunflower:
Plasmophora halstedii, Sclerotinia sclerotiorum, Aster Yellows,
Septoria helianthi, Phomopsis helianthi, Alternaria helianthi,
Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii,
Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae,
Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi,
Verticillium dahliae, Erwinia carotovorum p.v. carotovora,
Cephalosporium acremonium, Phytophthora cryptogea, Albugo
tragopogonis; Corn: Fusarium moniliforme var. subglutinans, Erwinia
stewartii, Fusarium moniliforme, Gibberella zeae (Fusarium
graminearum), Stenocarpella maydi (Diplodia maydis), Pythium
irregulare, Pythium debaryanum, Pythium graminicola, Pythium
splendens, Pythium ultimum, Pythium aphanidermatum, Aspergillus
flavus, Bipolaris maydis O, T (Cochliobolus heterostrophus),
Helminthosporium carbonum I, II & III (Cochliobolus carbonum),
Exserohilum turcicum I, II & III, Helminthosporium
pedicellatum, Physoderma maydis, Phyllosticta maydis, Kabatiella
maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi,
Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum,
Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata,
Curvularia inaequalis, Curvularia pallescens, Clavibacter
michiganense subsp. nebraskense, Trichoderma viride, Maize Dwarf
Mosaic Virus A & B, Wheat Streak Mosaic Virus, Maize Chlorotic
Dwarf Virus, Claviceps sorghi, Pseudomonas avenae, Erwinia
chrysanthemi p.v. zea, Erwinia carotovora, Corn stunt spiroplasma,
Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora
sorghi, Peronosclerospora philippinensis, Peronosclerospora maydis,
Peronosclerospora saccharin, Sphacelotheca reiliana, Physopella
zeae, Cephalosporium maydis, Cephalosporium acremonium, Maize
Chlorotic Mottle Virus, High Plains Virus, Maize Mosaic Virus,
Maize Rayado Fino Virus, Maize Streak Virus, Maize Stripe Virus,
Maize Rough Dwarf Virus; Sorghum: Exserohilum turcicum,
Colletotrichum graminicola (Glomerella graminicola), Cercospora
sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas
syringae p.v. syringae, Xanthomonas campestris p.v. holcicola,
Pseudomonas andropogonis, Puccinia purpurea, Macrophomina
phaseolina, Periconia circinata, Fusarium moniliforme, Alternaria
alternata, Bipolaris sorghicola, Helminthosporium sorghicola,
Curvularia lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonas
alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola,
Phyllachara saccharin, Sporisorium reilianum (Sphacelotheca
reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Sugarcane
mosaic H, Maize Dwarf Mosaic Virus A & B, Claviceps sorghi,
Rhizoctonia solani, Acremonium strictum, Sclerophthora macrospora,
Peronosclerospora sorghi, Peronosclerospora philippinensis,
Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum,
Pythium arrhenomanes, Pythium graminicola, etc.
[0150] 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.
[0151] The embodiments of the present invention may be effective
against a variety of pests. Target pests include, but are not
limited to, insect pests. "Insect pests" is intended to include
insects and other similar pests such as, for example, those of the
order Acari including, but not limited to, mites and ticks. Insect
pests of the present invention include, but are not limited to,
insects of the order Lepidoptera, e.g. Achoroia grisella, Acleris
gloverana, Acleris variana, Adoxophyes orana, Agrotis ipsilon,
Alabama argillacea, Alsophila pometaria, Amyelois transitella,
Anagasta kuehniella, Anarsia lineatella, Anisota senatoria,
Antheraea pernyi, Anticarsia gemmatalis, Archips sp., Argyrotaenia
sp., Athetis mindara, Bombyx mori, Bucculatrix thurberiella, Cadra
cautella, Choristoneura sp., Cochylls hospes, Colias eurytheme,
Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella, Datana
integerrima, Dendrolimus sibericus, Desmia feneralis, Diaphania
hyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraea
saccharalis, Ennomos subsignaria, Eoreuma loftini, Esphestia
elutella, Erannis tilaria, Estigmene acrea, Eulia salubricola,
Eupocoellia ambiguella, Eupoecilia ambiguella, Euproctis
chrysorrhoea, Euxoa messoria, Galleria mellonella, Grapholita
molesta, Harrisina americana, Helicoverpa subflexa, Helicoverpa
zea, Heliothis virescens, Hemileuca oliviae, Homoeosoma electellum,
Hyphantia cunea, Keiferia lycopersicella, Lambdina fiscellaria
fiscellaria, Lambdina fiscellaria lugubrosa, Leucoma salicis,
Lobesia botrana, Loxostege sticticalis, Lymantria dispar, Macalla
thyrisalis, Malacosoma sp., Mamestra brassicae, Mamestra
configurata, Manduca quinquemaculata, Manduca sexta, Maruca
testulalis, Melanchra picta, Operophtera brumata, Orgyia sp.,
Ostrinia nubilalis, Paleacrita vernata, Papilio cresphontes,
Pectinophora gossypiella, Phryganidia californica, Phyllonorycter
blancardella, Pieris napi, Pieris rapae, Plathypena scabra,
Platynotaflouendana, Platynota stultana, Platyptilia carduidactyla,
Plodia interpunctella, Plutella xylostella, Pontia protodice,
Pseudaletia unipuncta, Pseudoplasia includens, Sabulodes aegrotata,
Schizura concinna, Sitotroga cerealella, Spilonta ocellana,
Spodoptera sp., Thaurnstopoea pityocampa, Tinsola bisselliella,
Trichoplusia hi, Udea rubigalis, Xylomyges curiails, and Yponomeuta
padella.
[0152] Embodiments of the present invention may be effective
against Hemiptera such as, but not limited to, Lygus hesperus,
Lygus lineolaris, Lygus pratensis, Lygus rugulipennis Popp, Lygus
pabulinus, Calocoris norvegicus, Orthops compestris, Plesiocoris
rugicollis, Cyrtopeltis modestus, Cyrtopeltis notatus, Spanagonicus
albofasciatus, Diaphnocoris chlorinonis, Labopidicola allii,
Pseudatomoscelis seriatus, Adelphocoris rapidus, Poecilocapsus
lineatus, Blissus leucopterus, Nysius ericae, Nysius raphanus,
Euschistus servus, Nezara viridula, Eurygaster, Coreidae,
Pyrrhocoridae, Tinidae, Blostomatidae, Reduviidae, and
Cimicidae.
[0153] Insect pests include insects selected from the orders
Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga,
Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera,
Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly
Homoptera 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, sugarcane 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, 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; Siphaflava, 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; Frankliniella 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; Myzus persicae, green
peach aphid; Empoasca fabae, potato leafhopper; Acrosternum hilare,
green stink bug; Melanoplus femurrubrum, 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 Rape: Brevicoryne brassicae, cabbage aphid;
Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Bertha
armyworm; Plutella xylostella, Diamond-back moth; Delia spp., Root
maggots.
[0154] Arthropod venom may be obtained through a variety of methods
known in the art, including but not limited to, dissection and
isolation of the venom gland and milking of the venom
(Gopalakrishnakone et al. (1995) Lab Anim. 29:456-458, herein
incorporated by reference).
[0155] Compositions of the invention include antibodies that
selectively bind a polypeptide of the invention. Antibodies
specific to the polypeptides of the invention are prepared by
standard immunological techniques known to those skilled in the
art. See Harlow et al. (1988) Antibodies: A Laboratory Manual, Cold
Spring Harbor, N.Y.; Sambrook et al. (1989) Molecular Cloning: A
Laboratory Manual (2.sup.nd. ed., Cold Spring Harbor Laboratory
Press, Plain View, N.Y. and Ausubel et al., eds (1995) Current
Protocols in Molecular Biology, Wiley Interscience, New York,
herein incorporated by reference in their entirety.
[0156] Many peptides isolated from venoms modulate ion-channels.
Ion channels comprise a family of proteins classified according to
their biophysical and pharmacological characteristics. Modulation
of ion channel activity is known to affect the central nervous
system of mammals and other organisms. An embodiment of the
invention comprises the use of a composition of the invention to
modulate ion channel activity in mammals. An orally active
polypeptide of the invention may cross gut or nasal passages of
mammals. The compositions of the invention may be used to treat
heart and neurological diseases (e.g. U.S. Pat. No. 4,925,664; U.S.
Application No. 60/105,404; U.S. Application No. 60/140,227; and
U.S. Application No. 60/110,590).
[0157] Another embodiment involves the use of the compositions of
the invention in the treatment and preservation of textiles. Insect
pests devalue and destroy textiles and fabrics including, but not
limited to, carpets, draperies, clothing, blankets, and bandages.
The compositions of the invention may be applied to finished
textile products or may be expressed in plants yielding fibers that
are incorporated into fabrics. Insect pests that attack textiles
include, but are not limited to, webbing clothes moths and carpet
beetles.
[0158] The following examples are presented by way of illustration,
not by way of limitation.
EXPERIMENTAL
Example 1
Fractionation of Polypeptides From Arthropod Venom
[0159] Polypeptides were enriched from arthropod venom using a
variety of HPLC chromatography conditions. Crude arthropod venom
was applied to either HP1100 or Microbore LC columns as indicated.
The peptides were resolved using a method set forth herein. The
fractions were assayed for pesticidal activity as described
elsewhere herein.
[0160] For all HPLC chromatography methods Solvent A consists of
95% Water and Solvent B consists of 95% acetonitrile with either
0.1% TFA or HFBA as noted.
[0161] For gradients over HP1100 columns a 0.6 mL/min flow rate was
used unless otherwise noted.
TABLE-US-00001 METHOD 1: 60% Solvent B in 70 minutes TFA; C4 column
METHOD 2: 60% Solvent B in 70 minutes TFA; C4 column 0.325
mL/minute flow rate METHOD 3: 15-50% Solvent B in 70 minutes HFBA;
C4 column
[0162] For gradients over Microbore LC columns a 50 .mu.L/minute
flow rate was used unless otherwise noted.
TABLE-US-00002 METHOD 4: 60% Solvent B in 70 minutes TFA; C4 column
METHOD 5: 30% Solvent B in 60 minutes TFA; C4 column METHOD 6:
15-45% Solvent B in 65 minutes HFBA; C18 column METHOD 7: 50%
Solvent B in 70 minutes TFA; C4 column METHOD 8: 10-75% Solvent B
in 70 minutes HFBA; C18 column METHOD 9: 40% Solvent B in 70
minutes HFBA; C18 column METHOD 10: 15-15% Solvent B in 70 minutes
HFBA; C18 column
Example 2
Protein Sequencing
[0163] Purified peptides were reduced with 10 mM dithiothreitol and
alkylated with 4-vinylpyridine. The reduced and alkylated peptide
was then isolated from the chemicals on a Magic 2002 Microbore LC
using reversed phase chromatography. Protein sequencing was
performed on an Applied Biosystems Procise 494 protein sequencer
utilizing the Edman degradation reaction. Isolated peptides were
pipetted onto a pre-filtered glass fiber filter from Applied
Biosystems containing 100 mM Biobrene. The filter was dried and the
cartridge was re-assembled and placed onto the Procise 494.
N-terminal sequencing was determined with the pulsed-liquid method
standard with the Procise 494. Data were collected and analyzed on
the Model 610A data analysis program from Applied Biosystems.
Example 3
Full Length Peptide Sequencing
[0164] Purified peptides were reduced and alkylated as mentioned
previously. The peptides were then digested with GluC and LysC
endoproteinases from Boehringer-Mannheim. These endoproteinases
cleave on the C-terminal side of glutamic acid and lysine
respectively in the peptide. Peptide fragments were collected on
the Magic 2002 Microbore LC using reversed phase chromatography.
Each individual peptide was sequenced as described previously until
a full-length sequence map was obtained.
Example 4
Mass Spectroscopy Analysis
[0165] Mass spectroscopy analysis was performed on a Micromass
Platform LCZ electrospray instrument. The peptide was either
analyzed by LC/MS utilizing the Magic 2002 Microbore LC and
reversed phase chromatography or the purified peptide was directly
injected into the mass spec. probe. One of skill in the art will
recognize that a variety of conditions may be utilized for LC/MS.
Mass spectroscopy analysis of the present peptides was performed
under the following conditions: Capillary voltage=4.3, Cone
voltage=60, Extractor voltage=4, Source Block temperature=100,
Desolvation Temperature=200, Low Mass Resolution=12.5, High Mass
Resolution=12.5, Ion Energy=0.9, and Multiplier=650. Data was
analyzed on Micromass MassLynx software. The protein was
deconvoluted using MaxEnt and the accurate mass obtained.
Example 5
Southern Corn Rootworm Droplet Feeding Bioassay
[0166] This method assays the effects of polypeptides from crude
and fractionated venoms on first instar Diabrotica undecimpunctata
howardi (southern corn rootworm).
[0167] Southern corn rootworm (SCR), Diabrotica undecimpunctata
howardii, eggs were collected in the rearing chamber and placed in
a zip lock bag at 28.degree. C. in the dark. After hatching, the
larvae were incubated for 48 hours prior to testing.
[0168] On the day of testing, fifteen circles of parafilm that
approximately fit the inner diameter of a 15.times.60 mm glass
petri dish bottom were cut. Ten to fifteen circles of James River
Verigood.TM. blotter were prepared. A damp blotter circle was
placed in the inner lid of a polystyrene petri dish. The blotter
served as the mat or base of the test unit.
[0169] 2 mg of the freeze-dried polypeptides were suspended in 100
.mu.l of 5% sucrose/0.04% blue dye FD&C #1. The resuspended
polypeptide was stored on ice and used in a timely manner.
[0170] Three or more replicates of at least 10 insects per
replicate were performed on each test compound. A no compound
control containing only 5% sucrose/0.04% FD&C#1 blue dye was
also performed.
[0171] The parafilm circles were placed in the bottom of the Pyrex
glass petri dish and 3-5 .mu.l aliquots of the polypeptide solution
were placed in a ring-like fashion on the parafilm. Sample
desiccation was prevented with the top of the Pyrex dish.
[0172] Ten insects were placed in the center of the circle of
polypeptide aliquots. Larvae were handled one at a time to insure
injury-free larvae. Once the larvae were in the circle of droplets,
the larvae were forced to feed by pushing the heads into the
solution. When the entire gut of the larvae was blue, the larvae
were removed and placed in the polystyrene dish with the damp
blotter. Ten larvae were transferred for each replicate. The larvae
were rinsed with a droplet of dH.sub.2O to prevent any sucrose or
polypeptides on the insect from influencing the activity of the
larvae.
[0173] Insects were closely observed for the first minutes. The
accurate time of the appearance of symptoms was determined with a
stopwatch. Paralysis was detected by rolling larvae on their backs.
Inability to right themselves, contracted movements, and sluggish
behavior were also signs of intoxication. After 15 minutes, the
test unit was capped. Larvae were observed again at 30, 60, and 120
minutes. Insects were scored on the characteristics of paralysis,
lethargy, and contractedness at various intervals after treatment.
Insects fed crude and fractionated venoms exhibited a significantly
greater degree of paralysis, lethargy and contractedness than
controls. In some cases mortality was observed in the insects fed
the crude and fractionated venoms.
Example 6
Homopteran Membrane Feeding Bioassay For Screening Polypeptides
[0174] This assay can be used for a variety of homopterans
including, but not limited to, Myzus persicae and Perigrinus
maidis. The assay involves trapping the sample between two layers
of maximally stretched parafilm which act as a sachet on top of a
small vessel containing the insect of choice.
[0175] The assay was prepared as follows: 1 cm diameter polystyrene
tubing was cut into 15 mm lengths. One end of the tube was then
capped with a fine mesh screen. Five insects were then added to the
chamber after which the first layer of parafilm was stretched over
the remaining open end. 25 .mu.l of sample (polypeptide in a 5%
sucrose solution containing McCormick green food coloring) was then
placed on top of the stretched parafilm. A second layer of parafilm
was then stretched by hand and placed over the sample. The sample
was spread between the two layers of parafilm to make a continuous
sachet on which the insects fed. The sachet was then covered
tightly with saran wrap to prevent evaporation and produce a
slightly pressurized sample. The assay tubes were monitored for
insect reproduction and death on a 24 hour basis and compared to a
5% sucrose control. Insects fed crude and fractionated venoms
exhibited a significantly greater degree of mortality and
significantly less reproduction than controls.
Example 7
Construction of Recombinant Baculovirus Expressing Pesticidal
Polypeptides
[0176] The cDNAs encoding the instant polypeptides were introduced
into the baculovirus genome itself. For this purpose the cDNAs were
placed under the control of the polyhedrin promoter. The IE1
promoter or any other one of the baculovirus promoters may be
suitable also. The cDNA, together with appropriate leader sequences
was then inserted into a baculovirus transfer vector using standard
molecular cloning techniques. Following transformation of E. coli
DH5.alpha., isolated colonies were chosen and plasmid DNA was
prepared and analyzed by restriction enzyme analysis. Colonies
containing the appropriate fragment were isolated, propagated, and
plasmid DNA was prepared for cotransfection.
Example 8
Expression of Pesticidal Polypeptides in Insect Cells
[0177] Spodoptera frugiperda cells (Sf-9) were propagated in
ExCell.RTM. 401 media (JRH Biosciences, Lenexa, Kans.) supplemented
with 3.0% fetal bovine serum. Lipofectin.RTM. (50 .mu.L at 0.1
mg/mL, Gibco/BRL) was added to a 50 L aliquot of the transfer
vector containing the toxin gene (500 ng) and linearized
polyhedron-negative AcNPV (2.5 .mu.g, Baculogold.RTM. viral DNA,
Pharmigen, San Diego, Calif.). Sf-9 cells (approximate 50%
monolayer) were co-transfected with the viral DNA/transfer vector
solution. The supernatant fluid from the co-transfection experiment
was collected at 5 days post-transfection and recombinant viruses
were isolated employing standard plaque purification protocols,
wherein only polyhedron-positive plaques were selected (O'Reilly et
al. (1992), Baculovirus Expression Vectors: A Laboratory Manual, W.
H. Freeman and Company, New York.). Sf-9 cells in 35 mM petri
dishes (50% monolayer) were inoculated with 100 .mu.L of a serial
dilution of the viral suspension, and supernatant fluids were
collected at 5 days post infection. In order to prepare larger
quantities of virus for characterization, these supernatant fluids
are used to inoculate larger tissue cultures for large scale
propagation of recombinant viruses.
[0178] Expression of the encoded toxin gene by the recombinant
baculovirus was confirmed using a bioassay, LCMS, or antibodies.
The presence of toxin activity in the recombinant viruses was
monitored in vivo. These assays involve comparison of biological
activity of recombinant viruses to wild-type viruses. Third instar
larvae of H. virescens were infected orally by consumption of diet
that contains test and control viruses and the larvae were
monitored for behavioral changes and speed of kill. Larvae fed test
viruses exhibited a significantly faster speed of kill than larvae
fed control viruses.
[0179] Isolated plugs of a standard insect diet were inoculated
with approximately 5000 PIBs of each virus. Individual larvae that
had not fed for 12 h prior to beginning of the bioassay were
allowed to consume the diet for 24 h. The larvae were transferred
to individual wells in a diet tray and monitored for symptoms and
mortality on a daily basis (Zlotkin et al. (1991) Biochimie (Paris)
53:1073-1078). Again, larvae fed test viruses exhibited a
significantly faster speed of kill than larvae fed control
viruses.
Example 9
Construction of CV1 Expression Vectors
[0180] A synthetic version of CV1 with a barley alpha amylase
signal peptide (SEQ ID NO:5) was constructed with a codon bias
representative of Oryza sativa using standard molecular biology
methods. See Ausubel et al., eds. (1995) Current Protocols in
Molecular Biology, (Greene Publishing and Wiley-Interscience, New
York) and Sambrook et al. (1989) Molecular Cloning: A Laboratory
Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview,
N.Y.). The barley alpha amylase (BAA) signal sequence was added to
the 5' end of a nucleotide sequence corresponding to the mature
amino terminus of the CV1 peptide. The BAA signal peptide targets
operably linked polypeptides to the endoplasmic reticulum (ER),
where the polypeptide undergoes proper folding and subsequent
secretion out of the cell (Rahmatullah, et al (1989) Plant Mol.
Biol. The codon usage database used to determine a codon bias
representative of Oryza sativa is found in Table 1.
TABLE-US-00003 TABLE 1 Streptomyces coelicolor A3(2) [gbbct]: 6257
CDS's (2043281 codons) fields: [triplet] [frequency: per thousand]
([number]) UUU 0.4 (863) UUC 26.0 (53065) UUA 0.1 (128) UUG 2.4
(4935) CUU 1.5 (3129) CUC 36.6 (74736) CUA 0.3 (657) CUG 61.3
(125241) AUU 0.6 (1228) AUC 27.6 (56340) AUA 0.7 (1367) AUG 15.8
(32271) GUU 1.4 (2905) GUC 47.2 (96460) GUA 2.7 (5416) GUG 35.3
(72144) UCU 0.6 (1266) UCC 20.2 (41262) UCA 1.0 (2137) UCG 13.8
(28229) CCU 1.5 (2995) CCC 25.4 (51951) CCA 1.3 (2633) CCG 33.6
(68652) ACU 1.1 (2347) ACC 39.6 (80826) ACA 1.6 (3194) ACG 18.9
(38697) GCU 2.9 (5908) GCC 78.6 (160548) GCA 5.3 (10890) GCG 49.8
(101831) UAU 1.0 (1962) UAC 19.5 (39789) UAA 0.1 (290) UAG 0.5
(1089) CAU 1.6 (3366) CAC 21.5 (44018) CAA 1.3 (2593) CAG 25.1
(51248) AAU 0.7 (1436) AAC 16.2 (33191) AAA 1.0 (2041) AAG 19.7
(40293) GAU 2.9 (6024) GAC 58.0 (118595) GAA 8.5 (17445) GAG 48.5
(99056) UGU 0.7 (1448) UGC 7.0 (14341) UGA 2.4 (4878) UGG 15.1
(30770) CGU 5.5 (11183) CGC 39.1 (79956) CGA 2.5 (5124) CGG 32.0
(65332) AGU 1.5 (3030) AGC 12.3 (25187) AGA 0.8 (1574) AGG 3.7
(7488) GGU 9.3 (18920) GGC 61.4 (125467) GGA 7.1 (14608) GGG 18.2
(37288) Coding GC 72.38% 1st letter GC 72.74% 2nd letter GC 51.39%
3rd letter GC 93.00%
[0181] The synthetic gene was constructed using a series of
overlapping complementary oligonucleotides. The complementary
oligonucleotides were annealed, and the Klenow enzyme was used to
fill in the gaps. The full length gene was PCR amplified using
primers corresponding to the 5' and 3' ends of the gene sequence.
Restriction sites were incorporated into the 5' ends of the PCR
primers. The amplified product was TOPO cloned into pCR2.1
(Invitrogen), and the BAA-CV1 sequence was confirmed.
[0182] The BAA-CV1 fragment was subcloned into an expression vector
containing an appropriate promoter, such as the root-preferred IFS1
(isloflavone synthase 1) promoter (copending U.S. Application Ser.
Nos. 60/278,379, filed Mar. 23, 2001, and 60/311,461, filed Aug.
10, 2001, herein incorporated by reference) and the UCP1 promoter
(U.S. application Ser. No. 09/556,163 and U.S. Pat. No. 6,072,050,
herein incorporated by reference). The restriction sites introduced
during PCR amplification facilitated cloning of the nucleic acid
molecule behind the promoter of interest. The soybean-derived,
root-preferred IFS promoter directs expression of the CV1 peptide
in root tissue, the natural site of soybean cyst nematode feeding.
The UCP1 promoter is a constitutive, root-preferred promoter.
Additionally, the IFS vector contains the NOS termination
sequence.
Example 10
Construction of Aam1 Expression Vector
[0183] A synthetic version of Aam1 with a sweet potato sporamin
signal peptide (SEQ ID NOS:14, 15, and 16) was constructed using
overlapping oligonucleotides as described above herein. The codon
bias of the synthetic sequence was representative of Oryza sativa.
Selection of codons was based on the Kazusa codon usage database.
The sweet potato sporamin (SP) signal sequence was added to the 5'
end of a nucleotide sequence corresponding to the mature amino
terminus of the Aam1 peptide. The SP signal peptide (Hattori, et al
(1985) Plant Molecular Biology 5:313-320) targets operably linked
peptides to the ER and for export from the cell. Restriction sites
were added during the cloning process to facilitate cloning behind
the UCP1 promoter.
Example 11
Construction of VC1 Expression Vector
[0184] A synthetic version of VC1 with a PR1 signal peptide (SEQ ID
NOS:12 and 13) was constructed using overlapping oligonucleotides
as described above herein. The codon bias of the synthetic sequence
was representative of Oryza sativa. Selection of codons was based
on the Kazusa codon usage database. The PR1 signal sequence was
added to the 5' end of a nucleotide sequence corresponding to the
mature amino terminus of the VC1 peptide. The tobacco PR1 signal
peptide (Cornellison, et al (1986) Nature 321:531-532) targets
operably linked polypeptides to the ER for the purpose of proper
folding and disulfide bridge formation and for subsequent export
from the cell. Restriction sites were added during the cloning
process to facilitate cloning behind the IFS1 promoter.
Additionally, the IFS vector contains the NOS termination
sequence.
Example 12
Construction of Aam1 Expression Vector for Maize Expression
[0185] A synthetic version of Aam1 with a BAA signal peptide (SEQ
ID NOS:17, 18, and 19) was constructed using overlapping
oligonucleotides as described above herein. The codon bias of the
synthetic sequence was representative of Streptomyces coelicolor.
The codon bias of Streptomyces coelicolor was chosen because of its
overall similarity to codon usage in plants. Selection of codons
was based on the Kazusa codon usage database. See Table 1. The BAA
signal sequence was added to the 5' end of a nucleotide sequence
corresponding to the mature amino terminus of the Aam1 peptide. The
BAA signal peptide targets operably linked polypeptides to the
endoplasmic reticulum (ER), where the polypeptide undergoes proper
folding and subsequent secretion out of the cell (Rahmatullah, et
al (1989) Plant Mol. Biol., herein incorporated by reference).
[0186] The restriction sites introduced during PCR amplification
facilitated cloning of the nucleic acid molecule behind a promoter
of interest. The BAA-Aam1 fragment was subcloned into an expression
vector containing an appropriate promoter, such as the maize
ubiquitin promoter and the maize h2B promoter (U.S. Pat. No.
6,177,611, herein incorporated by reference). The ubiquitin
promoter and the h2B promoter are strong constitutive promoters.
Constitutive promoters were selected to allow testing of the
effects of Aam1 expression in a multiplicity of tissues including,
but not limited to, leaf, whorl, and roots. The expression vector
further contains the pinII terminator sequence 3' to the nucleotide
sequence of interest. Restriction sites flank the
promoter:Aam1:pinII cassette and facilitate cloning of the
expression cassette into binary plasmids for transformation.
Example 13
Identification of Ts7 and Assessment of Oral Activity
[0187] cDNAs encoding potential sodium channel neurotoxins were
identified from sequence data derived from a cDNA library of
Centruroides vittatus telsons. The nucleotide sequences, including
Ts7 (SEQ ID NO:21 and 23), exhibited homology to known toxins. Full
length genes were PCR amplified with oligonucleotide primers
introducing restriction enzyme sites at the 5' and 3' ends of the
gene. The PCR products were TOPO-cloned into pCR2.1 (Invitrogen
Co.) and the sequence was confirmed. The resulting constructs were
then cloned into a binary vector between the Cauliflower mosaic
virus 35S promoter and the NOS termination signal. The binary
vector also contained a selection marker cassette including the
Cauliflower mosaic 35S promoter, the BAR gene, and NOS
terminator.
[0188] Binary constructs were electroporated into Agrobacterium
tumefaciens strain C58. Transformed Agrobacterium cultures were
grown in YM media for 2 days at 28.degree. C. to an OD.sub.600 of
approximately 1.0. Cells were pelleted by centrifugation and
resuspended at the original culture volume in 5% sucrose with 0.05%
Silwet L-77. The Agrobacterium slurry was sprinkled onto the
inflorescences of flowering Arabidopsis thaliana (Landberg) plants
using a 10-25 ml pipette. The inflorescences were thoroughly
soaked. Plants were covered with a humidity dome in the dark for 24
hours and were grown under normal conditions thereafter. Seeds were
harvested six weeks later and were generously sown in flats filled
with soil. Transgenic events were selected by herbicide treatment
using BASTA and PCR confirmed. Those 3 week old events surviving
BASTA treatments were tested in bioassays using Myzus persicae
(Green Peach Aphid).
[0189] Bioassays were performed on individual transformation events
enclosed within plastic tubes with a fine mesh screen at the top to
confine the aphids. Adult aphids were applied to the base of each
plant. Plants were scored for total number of adult aphids and
nymphs at various intervals post infestation. Those events
expressing Ts7 cDNA were found to exhibit a greater degree of aphid
resistance than controls.
Example 14
Construction of Ts7 Expression Vector
[0190] A synthetic version of Ts7 with a BAA signal peptide (SEQ ID
NO:25) was constructed using overlapping oligonucleotides as
described above herein. The codon bias of the synthetic sequence
was representative of Streptomyces coelicolor as described
elsewhere herein. The BAA signal sequence was added to the 5' end
of a nucleotide sequence corresponding to the mature amino terminus
of the Ts7 peptide. The BAA signal peptide targets operably linked
polypeptides to the endoplasmic reticulum (ER), where the
polypeptide undergoes proper folding and subsequent secretion out
of the cell (Rahmatullah, et al (1989) Plant Mol. Biol., herein
incorporated by reference).
[0191] The BAA-Ts7 fragment was subcloned into an expression vector
containing an appropriate promoter, such as the maize ubiquitin
promoter and the maize h2B promoter (U.S. Pat. No. 6,177,611,
herein incorporated by reference). The restriction sites introduced
during PCR amplification facilitated cloning of the nucleic acid
molecule behind the promoter of interest. The ubiquitin promoter
and the h2B promoter are strong constitutive promoters.
Constitutive promoters were selected to allow testing of the
effects of Ts7 expression in a multiplicity of tissues including,
but not limited to, leaf, whorl, and roots. The expression vector
further contains the pinII terminator sequence 3' to the nucleotide
sequence of interest. Restriction sites flank the promoter:
Ts7:pinII cassette and facilitate cloning of the expression
cassette into binary plasmids for transformation.
Example 15
Transformation of Rice and Regeneration of Transgenic Rice
Plants
[0192] Embryogenic (E) callus was obtained from the scutellum of
immature and mature seed. Mature seeds were physically dehulled by
applying pressure with a plastic microcentrifuge tube rack.
Dehulled seeds were placed in a 50 ml tube. Seeds were sterilized
by agitating them in 40 mls of 70% ethanol for 1.5 minutes. The
seeds were rinsed twice with 40 mls of 20% bleach. 40 mls of 20%
bleach were added to the seeds. The bleach and seed mixture was
agitated at 25-50 rpm for 20 minutes. The bleach solution was
poured away and the seeds were rinsed three times with 40 mls of
sterile water. Two additional sterile water rinses were performed,
each under agitation, and for a duration of 10 minutes then 15
minutes. Water was removed and the seeds were transferred into a
sterile petri dish. 12-16 seeds were placed on each petri plate
containing callus initiation media. Callus initiation media
comprises MS salts, Nitsch and Nitsch vitamins, 1.0 mg/L 2,4-D and
10 .mu.M AgNO.sub.3. The plates were wrapped with fibertape and
incubated in the dark at 27-28.degree. C. for 18 days.
[0193] After 18 days a heterogeneous population of somatic
proembryos and embryos proliferated from the scutellum. The callus
initiated from the scutellum appears as a light-yellow bumpy
outgrowth. The callus was subcultured every 2 weeks on callus
maintenance media (CM) at a plating density of 100-200 mg/plate in
a size distribution pattern of 0.5-1.0 mm pieces, 1 mM apart. CM
comprises N6 salts, Nitsch and Nitsch vitamins, 1 mg/L 2,4-D (Chu,
et al (1985) Sci. Sinica 18:659-668, herein incorporated by
reference). During the culture period the phenotype of the callus
changed. The callus lines were maintained as clonal populations and
subcultured to perpetuate the desired E phenotype. A desired E
phenotype is typified by callus clumps ranging in size between 0.1
and 0.5 mm. E callus is smooth and regular as opposed to
non-embryogenic callus which displays a rough or jagged surface. E
phenotype callus tensile quality may be described as spongy and is
light yellow to white in color. E callus has a F.W. doubling time
of 3-4 days. Generally, the E callus used in transformation was
obtained from one subculture onto CM. Typically the E callus
cultures were transformed within 3-10 weeks of initiation.
[0194] Good E callus was prepared for transformation as described
below. Petri plates were prepared with CM medium in a circular
diameter of about 4 cm and a #541 Whatman paper was placed on the
CM medium. 0.5 ml of top agar were layered onto the Whatman paper.
0.5 to 1 mm pieces of E callus was transferred onto the top agar.
The plates were sealed with fibertape and incubated in the dark at
27'-28.degree. C. for 3-5 days in a 40-60% humidity environment.
The filters with the callus pieces were transferred to CM
supplemented with 0.5M osmoticum (0.25M mannitol plus 0.25M
sorbitol) for 3 hours in the dark. The plates were placed in a
flow-hood and the lids were left ajar for 20-45 minutes prior to
bombardment.
[0195] Gold particles were weighed so that the final concentration
was 60 mg/l ml or 0.6 .mu.M and placed in a sterile 1.5 ml
siliconized microcentrifuge tube. The gold particles were washed
with 1 ml of 100% ethanol, gently agitated for 3 minutes and
sonicated for 10 seconds. The gold particles were pelleted by
centrifugation. The supernatant was removed and the gold particles
were washed twice with 1 ml of sterile double distilled water. The
gold particles were resuspended in 1 ml of sterile double-distilled
water. The gold suspension was sonicated for ten seconds and 50
.mu.l aliquots were placed in 1.5 ml siliconized microcentrifuge
tubes.
[0196] Plasmid DNA was precipitated onto the prepared gold
particles in the following manner. The gold suspension was
sonicated twice briefly. Approximately 8 .mu.g of the trait gene
and 2 .mu.g of the hygromycin resistance (hpt) gene (a 2:1 molar
ratio), 50 .mu.l 2.5 M CaCl.sub.2, and 20 .mu.l 0.1 M spermidine
were added to the gold suspension. The solution was mixed gently
for 3 minutes, sonicated briefly, mixed for 30 seconds, and
centrifuged. The supernatant was removed and the particles were
washed twice with 1 ml 100% ethanol. The particles were resuspended
in 50 .mu.l 100% ethanol. The solution was finger vortexed several
times and sonicated briefly to relieve clumps and disperse
particles. The tubes were incubated at -70.degree. C. for at least
30 minutes. The mixture was sonicated briefly. 6 .mu.l of the
DNA/Gold solution was dispensed and evaporated onto a mylar
macrocarrier.
[0197] The callus-containing plates were bombarded with a
PDS-1000/He Gun with the following parameters: 5-10 .mu.g of total
DNA precipitated onto 3 mg of 0.6 .mu.M gold particles (from 50
.mu.l prep), 8 cm from the stopping screen, 27-29 inches Hg vacuum,
and 1050-1100 PSI He pressure. Each plate was bombarded twice.
[0198] Within an hour of bombardment, the filter paper supporting
the callus was placed on CM plates. The plates were wrapped with
fibertape and incubated in the dark at 27-28.degree. C. for 3-5
days. After 3-5 days, the callus was transferred to a sterile 50 ml
conical tube. The callus was weighed and cooled top agar was added
at a concentration of 40 mg/ml. Clumps were disrupted with a 10 ml
pipette. Three ml aliquots of the suspended callus were dispensed
onto SM50 media. SM50 media is comprised of CM media containing 50
PPM hygromycin B. The plates were wrapped with fibertape and
incubated in the dark at 27-28.degree. C. for 4 weeks. After 4
weeks as transformation events appeared, they were transferred to
SM50 and grown an additional two weeks. After two weeks, 8-10 mm
clumps of the transformants were transferred to RM1 media. RM1
comprises MS salts, Nitsch and Nitsch vitamins, 2% sucrose, 3%
sorbitol, 0.4% gelrite, and 50 PPM hygromycin B. The plates were
wrapped in fibertape and incubated at 25.degree. C. in the dark for
approximately 3 weeks. The transformants were transferred to RM2
media. RM2 media is comprised of MS salts, Nitsch and Nitsch
vitamins, 3% sucrose, 0.4% gelrite, and 50 PPM hygromycin B. The
RM2 plates were incubated in cool white light (.about.40
.mu.E.sup.-1) with a 12 hour photoperiod at 25.degree. C. and
30-40% humidity.
[0199] Plantlets emerged from the callus after 4-8 weeks on RM2
media. When the plantlets reached 2-3 cm they were gently
transferred to RM3 media in phytatrays (Sigma Chemical Co., St.
Louis, Mo.). RM3 media is comprised of 1/2.times. MS salts, Nitsch
and Nitsch vitamins, 1% sucrose, and 50 PPM hygromycin B. 2-6
plantlets were transferred per phytatray. Clumps of the smaller
plantlets were transferred to RM2 media for an additional 3 weeks
before transfer to RM3 media.
[0200] After sufficient root and shoot growth occurred, the plants
were transferred to a 24-spot tray containing Metromix. 3 plants
per clone were potted. The plants were grown in a 12/12 light cycle
at approximately 350 .mu.E.sup.-1 and 30.degree. C.
Example 16
Assay of Transgenic Plants for Enhanced Pesticidal Resistance
[0201] For Homopteran whole plant bioassays, regenerated transgenic
plants were assayed individually in single chamber containers
composed of the 4 inch pot containing the plant and a flexible
polystyrene sleeve capped with fine plastic mesh. An exact number
of insects was added to the chamber and the chamber was sealed to
prevent insect escape. At least three and preferably five
regenerated control plants of equivalent age to the transformants
were included in the experiment. Host plant resistance was assessed
based on insect mortality and population increase. Resistance was
defined as insect reproduction one standard deviation below the
average reproduction within the control plants or insect mortality
one standard deviation above the control plant average insect
mortality. Transgenic plants were insect resistant.
[0202] For lepidopteran assays, 3 1 cm segments from a mature leaf
of the regenerated plant were placed in three separate chambers of
a 12 well microtiter plate. 3 neonate larvae were added to each
chamber, and the chamber was sealed to prevent escape of the
insects. As with the Homopteran assay, 3 to 5 control plants were
assayed in a similar way for each experiment. Plant resistance was
determined based on mortality and leaf damage in comparison to the
control plant performance. Again, the transgenic plants were
resistant.
[0203] In some assays the radish leaves used to rear Myzus persicae
were placed at the base of the transgenic plant. The transgenic
plant was enclosed in a plastic tube covered with a fine mesh top.
As the radish leaf dried, the green peach aphids (M. persicae)
moved from the radish leaf to the transgenic plants. The transgenic
plants were resistant to Myzus persicae infestation and/or
damage.
Example 17
Leaf Segment Assay for Lepidopteran Activity
Identification of Aam1
A Homopteran and Lepidopteran Orally Active Peptide
[0204] Initial screening of five minute fractions of the venom from
Androctonus amoreuxi indicated activity against the corn plant
hopper Perigrinus maidis. Initial activity was observed in Fraction
8. Based on this activity, Fraction 8 was further fractionated by
HPLC (see Example 1 for conditions). The Homopteran activity was
then found to be associated with Fraction 8-6. Sufficient
quantities of this purified protein were collected to allow
confirmation of activity and to initiate protein sequencing by
Edmund degradation as described in Example 2. The mature sequence
was determined to be that indicated in SEQ ID NO:20. Molecular
weight of the mature peptide was confirmed by Mass spectroscopy to
be 6534.5 Daltons. This information was used to create a synthetic
gene codon biased for expression in rice. This synthetic gene was
then cloned into the appropriate vector for transformation of rice
as described in Example 15. A synthetic gene was also generated for
expression in the Pichia yeast and baculovirus systems. Activity of
expressed toxins from both systems was confirmed in Homopteran
feeding assays. A dose response assay was performed on the aphid
Myzus persicae. The results are found in FIG. 14.
[0205] Transformed rice was bioassayed for activity against Aphis
fabeae by a combination choice, reproduction assay. Two
transformant rice plants containing the 35s:Aam1 transgene were
combined with 2 control plants in a large insect cage. Each plant
was infested twice with 50 aphids and the aphids were allowed to
move to their preferred plant. A second infestation was
subsequently performed to increase infestation levels. 5 days later
the plants were removed from the common cage and placed on an open
shelf in water moats to prevent movement between plants. The plants
were then monitored for insect population growth. Aphid populations
on the control plants increased rapidly while the aphid populations
on the transgenic plants grew relatively little and the aphids
continued to disperse widely rather than settle near the mater and
form foci of infestation as seen in the control plants (data not
shown).
[0206] Lepidopteran activity was assessed in a leaf segment assay
against ECB as described here. Substantial leaf protection was
demonstrated in the 35s:Aam1 leaves. Microscopic observation also
disclosed classic symptoms of paralysis among the ECB larvae.
Example 18
Transformation of Maize by Particle Bombardment and Regeneration of
Transgenic Plants
[0207] Immature maize embryos from greenhouse donor plants are
bombarded with a plasmid containing a nucleotide sequence encoding
a polypeptide of the invention operably linked to a ubiquitin
promoter and the selectable marker gene PAT (Wohlleben et al.
(1988) Gene 70:25-37), which confers resistance to the herbicide
Bialaphos. Alternatively, the selectable marker gene is provided on
a separate plasmid. Transformation is performed as follows. Media
recipes follow below.
Preparation of Target Tissue
[0208] The ears are husked and surface sterilized in 30% Clorox
bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two
times with sterile water. The immature embryos are excised and
placed embryo axis side down (scutellum side up), 25 embryos per
plate, on 560Y medium for 4 hours and then aligned within the
2.5-cm target zone in preparation for bombardment.
Preparation of DNA
[0209] A plasmid vector comprising a nucleotide sequence encoding a
polypeptide of the invention operably linked to a ubiquitin
promoter is made. This plasmid DNA plus plasmid DNA containing a
PAT selectable marker is precipitated onto 1.1 .mu.m (average
diameter) tungsten pellets using a CaCl.sub.2 precipitation
procedure as follows: [0210] 100 .mu.l prepared tungsten particles
in water [0211] 10 .mu.l (1 .mu.g) DNA in Tris EDTA buffer (1 .mu.g
total DNA) [0212] 100 .mu.l 2.5 M CaCl.sub.2 [0213] 10 .mu.l 0.1 M
spermidine
[0214] Each reagent is added sequentially to the tungsten particle
suspension, while maintained on the multitube vortexer. The final
mixture is sonicated briefly and allowed to incubate under constant
vortexing for 10 minutes. After the precipitation period, the tubes
are centrifuged briefly, liquid removed, washed with 500 .mu.l 100%
ethanol, and centrifuged for 30 seconds. Again the liquid is
removed, and 105 .mu.l 100% ethanol is added to the final tungsten
particle pellet. For particle gun bombardment, the tungsten/DNA
particles are briefly sonicated and 10 .mu.l spotted onto the
center of each macrocarrier and allowed to dry about 2 minutes
before bombardment.
Particle Gun Treatment
[0215] The sample plates are bombarded at level #4 in particle gun
#HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI,
with a total of ten aliquots taken from each tube of prepared
particles/DNA.
Subsequent Treatment
[0216] Following bombardment, the embryos are kept on 560Y medium
for 2 days, then transferred to 560R selection medium containing 3
mg/liter Bialaphos, and subcultured every 2 weeks. After
approximately 10 weeks of selection, selection-resistant callus
clones are transferred to 288J medium to initiate plant
regeneration. Following somatic embryo maturation (2-4 weeks),
well-developed somatic embryos are transferred to medium for
germination and transferred to the lighted culture room.
Approximately 7-10 days later, developing plantlets are transferred
to 272V hormone-free medium in tubes for 7-10 days until plantlets
are well established. Plants are then transferred to inserts in
flats (equivalent to 2.5'' pot) containing potting soil and grown
for 1 week in a growth chamber, subsequently grown an additional
1-2 weeks in the greenhouse, then transferred to classic 600 pots
(1.6 gallon) and grown to maturity. Plants are monitored and scored
for expression of the polypeptide of the invention by assays known
in the art, such as, for example, insect feeding assays.
Bombardment and Culture Media
[0217] Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts
(SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix
(1000.times.SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose,
1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with
D-1H.sub.2O following adjustment to pH 5.8 with KOH); 2.0 g/l
Gelrite (added after bringing to volume with D-1H.sub.2O); and 8.5
mg/l silver nitrate (added after sterilizing the medium and cooling
to room temperature). Selection medium (560R) comprises 4.0 g/l N6
basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix
(1000.times.SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose,
and 2.0 mg/l 2,4-D (brought to volume with D-1H.sub.2O following
adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after
bringing to volume with D-1H.sub.2O); and 0.85 mg/l silver nitrate
and 3.0 mg/l bialaphos (both added after sterilizing the medium and
cooling to room temperature).
[0218] Plant regeneration medium (288J) comprises 4.3 g/l MS salts
(GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g
nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and
0.40 g/l glycine brought to volume with polished D-I H.sub.2O)
(Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/l
myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1
mM abscisic acid (brought to volume with polished D-I H.sub.2O
after adjusting to pH 5.6); 3.0 g/l Gelrite (added after bringing
to volume with D-I H.sub.2O); and 1.0 mg/l indoleacetic acid and
3.0 mg/l bialaphos (added after sterilizing the medium and cooling
to 60.degree. C.). Hormone-free medium (272V) comprises 4.3 g/l MS
salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100
g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL,
and 0.40 g/l glycine brought to volume with polished D-I H.sub.2O),
0.1 g/1 myo-inositol, and 40.0 g/l sucrose (brought to volume with
polished D-I H.sub.2O after adjusting pH to 5.6); and 6 g/l
bacto-agar (added after bringing to volume with polished D-I
H.sub.2O), sterilized and cooled to 60.degree. C.
Example 19
Agrobacterium-Mediated Transformation of Maize and Regeneration of
Transgenic Plants
[0219] For Agrobacterium-mediated transformation of maize with a
nucleotide sequence encoding a polypeptide of the invention,
preferably the method of Zhao is employed (U.S. Pat. No. 5,981,840,
and PCT patent publication WO98/32326; the contents of which are
hereby incorporated by reference). Briefly, immature embryos are
isolated from maize and the embryos contacted with a suspension of
Agrobacterium, where the bacteria are capable of transferring the
nucleotide sequence encoding a polypeptide of the invention to at
least one cell of at least one of the immature embryos (step 1: the
infection step). In this step the immature embryos are preferably
immersed in an Agrobacterium suspension for the initiation of
inoculation. The embryos are co-cultured for a time with the
Agrobacterium (step 2: the co-cultivation step). Preferably the
immature embryos are cultured on solid medium following the
infection step. Following this co-cultivation period an optional
"resting" step is contemplated. In this resting step, the embryos
are incubated in the presence of at least one antibiotic known to
inhibit the growth of Agrobacterium without the addition of a
selective agent for plant transformants (step 3: resting step).
Preferably the immature embryos are cultured on solid medium with
antibiotic, but without a selecting agent, for elimination of
Agrobacterium and for a resting phase for the infected cells. Next,
inoculated embryos are cultured on medium containing a selective
agent and growing transformed callus is recovered (step 4: the
selection step). Preferably, the immature embryos are cultured on
solid medium with a selective agent resulting in the selective
growth of transformed cells. The callus is then regenerated into
plants (step 5: the regeneration step), and preferably calli grown
on selective medium are cultured on solid medium to regenerate the
plants.
Example 20
Soybean Embryo Transformation
[0220] Soybean embryos are bombarded with a plasmid containing a
nucleotide sequence of the invention operably linked to a SCP1
promoter as follows. To induce somatic embryos, cotyledons, 3-5 mm
in length dissected from surface-sterilized, immature seeds of the
soybean cultivar A2872, are cultured in the light or dark at
26.degree. C. on an appropriate agar medium for six to ten weeks.
Somatic embryos producing secondary embryos are then excised and
placed into a suitable liquid medium. After repeated selection for
clusters of somatic embryos that multiplied as early,
globular-staged embryos, the suspensions are maintained as
described below.
[0221] Soybean embryogenic suspension cultures are maintained in 35
ml liquid media on a rotary shaker, 150 rpm, at 26.degree. C. with
florescent lights on a 16:8 hour day/night schedule. Cultures are
subcultured every two weeks by inoculating approximately 35 mg of
tissue into 35 ml of liquid medium.
[0222] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Klein et al.
(1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A Du
Pont Biolistic PDS1000/HE instrument (helium retrofit) can be used
for these transformations.
[0223] A selectable marker gene that can be used to facilitate
soybean transformation is a transgene composed of the 35S promoter
from Cauliflower Mosaic Virus (Odell et al. (1985) Nature
313:810-812), the hygromycin phosphotransferase gene from plasmid
pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188), and the
3' region of the nopaline synthase gene from the T-DNA of the Ti
plasmid of Agrobacterium tumefaciens. The expression cassette
comprising a nucleotide sequence of the invention operably linked
to the SCP1 promoter can be isolated as a restriction fragment.
This fragment can then be inserted into a unique restriction site
of the vector carrying the marker gene.
[0224] To 50 .mu.l of a 60 mg/ml 1 .mu.m gold particle suspension
is added (in order): 5 .mu.l DNA (1 .mu.g/.mu.l), 20 .mu.l
spermidine (0.1 M), and 50 .mu.l CaCl.sub.2 (2.5 M). The particle
preparation is then agitated for three minutes, spun in a microfuge
for 10 seconds and the supernatant removed. The DNA-coated
particles are then washed once in 400 .mu.l 70% ethanol and
resuspended in 40 .mu.l of anhydrous ethanol. The DNA/particle
suspension can be sonicated three times for one second each. Five
microliters of the DNA-coated gold particles are then loaded on
each macro carrier disk.
[0225] Approximately 300-400 mg of a two-week-old suspension
culture is placed in an empty 60.times.15 mm petri dish and the
residual liquid removed from the tissue with a pipette. For each
transformation experiment, approximately 5-10 plates of tissue are
normally bombarded. Membrane rupture pressure is set at 1100 psi,
and the chamber is evacuated to a vacuum of 28 inches mercury. The
tissue is placed approximately 3.5 inches away from the retaining
screen and bombarded three times. Following bombardment, the tissue
can be divided in half and placed back into liquid and cultured as
described above.
[0226] Five to seven days post bombardment, the liquid media may be
exchanged with fresh media, and eleven to twelve days
post-bombardment with fresh media containing 50 mg/ml hygromycin.
This selective media can be refreshed weekly. Seven to eight weeks
post-bombardment, green, transformed tissue may be observed growing
from untransformed, necrotic embryogenic clusters. Isolated green
tissue is removed and inoculated into individual flasks to generate
new, clonally propagated, transformed embryogenic suspension
cultures. Each new line may be treated as an independent
transformation event. These suspensions can then be subcultured and
maintained as clusters of immature embryos or regenerated into
whole plants by maturation and germination of individual somatic
embryos.
Example 21
Fluorescence Imaging Ratiometric Assay
[0227] Embryonic neuronal cultures of the American cockroach
Periplaneta americana were established following the method of
Beadle and Lees (See Beadle D. J. and Lees G., Chapter 5 in Cell
Culture Approaches to Invertebrate Neuroscience, Beadle, D. J.,
Lees, G. and Kater, S. B. (eds.) Academic Press, New York 1988)
with minor modification. Briefly, 24-day old oothecae were
collected, surface-sterilized and cut open one third below the
dorsal midline. The heads were then removed and placed in
Schneider's Drosophila medium. Using fine forceps 32 brains were
individually removed and placed in a small glass vial containing
790 .mu.l of `5+4` medium. The brains were dissociated using a
Pasteur pipette with tip flamed to slightly less than half its
original diameter. A 5 .mu.l volume of the suspension was placed at
the center of each well of a poly-L-lysine coated 96-well plate.
After cells were allowed to attach, wells were filled with a 1:1
mixture of Leibovitz's L-15 medium and Yunker's modified Grace's
medium in which 20-hydroxyecdysone was added. Cells were maintained
in a high humidity incubator at 29.degree. C. until testing.
[0228] Cells were rinsed in standard physiological saline having
the following composition (mM): NaCl 190; CaCl.sub.2 9; KCl 3.1;
probenicid 1; Tris buffer 10; pH 7.2. Cells were then bathed in
saline containing the calcium sensitive fluoroprobe Fluo-4 AM (2
.mu.M) and Pluronic F127 (0.002%) for 45 minutes then rinsed in
saline for at least 15 minutes prior to testing. (One could also
use Fluo-3 AM, Fura-2 AM or other calcium-sensitive fluoroprobes).
The 96-well plate was placed on a computer-controlled stage mounted
onto a Nikon Diaphot microscope and imaged using a 20.times. Fluor
objective (NA 0.75). Individual wells from the 96-well plate were
sequentially excited with light at 495 nm and the emitted
fluorescence at 530 nm detected using a Hamamatsu ORCA ER digital
camera.
[0229] Acquisition control, image processing and image analysis
were conducted using Universal Imaging Corporation's MetaMorph.TM.
imaging software. A series of "control" images were obtained for
each well. Test and control compounds were added directly to each
well via a multi-channel pipettor (one could use an automated
fluidic system) and subsequent "treated" images were obtained.
Subsequently, post-acquisition image processing was performed
consisting of the following operations. A threshold with respect to
minimum gray level was set for all images in order to facilitate
image morphometry. Individual cells were separated and identified
based on meeting designated morphometric criteria for total size
and shape as previously determined experimentally for this cell
type. Regions of interest were generated from the periphery of each
identified cell and the mean pixel value for each region was
generated for each control and treated image. Compound-stimulated
increase in intracellular free calcium concentration resulted in an
increase of Fluo-4 fluorescence emission. The application of Aam1
to the neuronal cells resulted in changes in fluorescence
characteristic of a sodium channel agonist. This confirms that Aam1
likely acts at the level of the sodium channel.
[0230] All publications, patents and patent applications mentioned
in the specification are indicative of the level of those skilled
in the art to which this invention pertains. All publications,
patents and patent applications are herein incorporated by
reference to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated by reference.
[0231] 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
271355DNACentruroides
vittatusCDS(49)...(303)misc_feature(0)...(0)CV1 1ggatcccccg
ggctgcagga gaatttatac gttatcagaa aactcaaa atg aat tat 57Met Asn
Tyr1ttt ata ttg att ttg gtt gca gct cta tta ata ttg gat gca aat tgt
105Phe Ile Leu Ile Leu Val Ala Ala Leu Leu Ile Leu Asp Ala Asn Cys5
10 15aag aaa gac gga tat cca gtt gat gcg gag gaa tgt aga tat aat
tgt 153Lys Lys Asp Gly Tyr Pro Val Asp Ala Glu Glu Cys Arg Tyr Asn
Cys20 25 30 35tgg aaa aac gaa tac tgc gac aaa atc tgc aaa gag aag
aaa ggt gaa 201Trp Lys Asn Glu Tyr Cys Asp Lys Ile Cys Lys Glu Lys
Lys Gly Glu40 45 50agt gga tat tgt tac gga tgg aat ctg tcg tgt tgg
tgt ata ggt ctt 249Ser Gly Tyr Cys Tyr Gly Trp Asn Leu Ser Cys Trp
Cys Ile Gly Leu55 60 65cct gat gat aca aat aca aaa atg aat ccc ttt
tgt cag ggt ttg gat 297Pro Asp Asp Thr Asn Thr Lys Met Asn Pro Phe
Cys Gln Gly Leu Asp70 75 80ggg taa acgaaattta accaataaaa aaaaaaaaaa
ggggaaatct gcttttacta 353Gly *at 355284PRTCentruroides vittatus
2Met Asn Tyr Phe Ile Leu Ile Leu Val Ala Ala Leu Leu Ile Leu Asp1 5
10 15Ala Asn Cys Lys Lys Asp Gly Tyr Pro Val Asp Ala Glu Glu Cys
Arg20 25 30Tyr Asn Cys Trp Lys Asn Glu Tyr Cys Asp Lys Ile Cys Lys
Glu Lys35 40 45Lys Gly Glu Ser Gly Tyr Cys Tyr Gly Trp Asn Leu Ser
Cys Trp Cys50 55 60Ile Gly Leu Pro Asp Asp Thr Asn Thr Lys Met Asn
Pro Phe Cys Gln65 70 75 80Gly Leu Asp Gly3255DNACentruroides
vittatusCDS(1)...(255) 3atg aat tat ttt ata ttg att ttg gtt gca gct
cta tta ata ttg gat 48Met Asn Tyr Phe Ile Leu Ile Leu Val Ala Ala
Leu Leu Ile Leu Asp1 5 10 15gca aat tgt aag aaa gac gga tat cca gtt
gat gcg gag gaa tgt aga 96Ala Asn Cys Lys Lys Asp Gly Tyr Pro Val
Asp Ala Glu Glu Cys Arg20 25 30tat aat tgt tgg aaa aac gaa tac tgc
gac aaa atc tgc aaa gag aag 144Tyr Asn Cys Trp Lys Asn Glu Tyr Cys
Asp Lys Ile Cys Lys Glu Lys35 40 45aaa ggt gaa agt gga tat tgt tac
gga tgg aat ctg tcg tgt tgg tgt 192Lys Gly Glu Ser Gly Tyr Cys Tyr
Gly Trp Asn Leu Ser Cys Trp Cys50 55 60ata ggt ctt cct gat gat aca
aat aca aaa atg aat ccc ttt tgt cag 240Ile Gly Leu Pro Asp Asp Thr
Asn Thr Lys Met Asn Pro Phe Cys Gln65 70 75 80ggt ttg gat ggg taa
255Gly Leu Asp Gly *464PRTCentruroides vittatus 4Lys Lys Asp Gly
Tyr Pro Val Asp Ala Glu Glu Cys Arg Tyr Asn Cys1 5 10 15Trp Lys Asn
Glu Tyr Cys Asp Lys Ile Cys Lys Glu Lys Lys Gly Glu20 25 30Ser Gly
Tyr Cys Tyr Gly Trp Asn Leu Ser Cys Trp Cys Ile Gly Leu35 40 45Pro
Asp Asp Thr Asn Thr Lys Met Asn Pro Phe Cys Gln Gly Leu Asp50 55
605267DNAArtificial SequenceCodon biased nucleotide sequence
encoding CV1. Codon biased for rice. 5atg gcc aac aag cac ctc tcc
ctg agc ctt ttc ttg gtg ctc cta ggc 48Met Ala Asn Lys His Leu Ser
Leu Ser Leu Phe Leu Val Leu Leu Gly-20 -15 -10ctg tcg gcg tct tta
gct tca ggg aag aaa gac ggc tac ccg gtg gat 96Leu Ser Ala Ser Leu
Ala Ser Gly Lys Lys Asp Gly Tyr Pro Val Asp-5 1 5gcc gag gaa tgc
cgc tat aac tgt tgg aag aat gag tac tgc gac aag 144Ala Glu Glu Cys
Arg Tyr Asn Cys Trp Lys Asn Glu Tyr Cys Asp Lys10 15 20atc tgc aag
gag aaa aag ggg gaa tcc gga tac tgt tat ggc tgg aac 192Ile Cys Lys
Glu Lys Lys Gly Glu Ser Gly Tyr Cys Tyr Gly Trp Asn25 30 35 40ctc
agc tgc tgg tgc att ggc ctg ccc gat gac acc aat acg aag atg 240Leu
Ser Cys Trp Cys Ile Gly Leu Pro Asp Asp Thr Asn Thr Lys Met45 50
55aac cca ttc tgc cag ggg ctt gat tga 267Asn Pro Phe Cys Gln Gly
Leu Asp *606375DNALeiurus
quinquestriatusCDS(38)...(298)misc_feature(0)...(0)LghIV
6gaattcggca cctcgtgnaa tttcggcaca gtncaaa atg aat tac ttg atg ata
55att agt ttg gct ctt ctt cta atg aca ggt gtg gag agc ggt gta cgt
103gat gct tat att gcc gac gat aaa aac tgt gtg tac act tgt ggt gca
151aat tca tat tgc aac aca gaa tgt acc aag aac ggt gct gag agt ggc
199tat tgt caa tgg ttt ggt aaa tat gga aat gcc tgc tgg tgc ata aag
247ttg ccc gat aaa gta cct att aga ata cca gga aag tgc cgt ggc cga
295taa atttaagatg gaatataacc taaatataac tgttaaataa atataattta
348aaaatttaaa aaaaaaaaaa aaaaanc 375786PRTLeiurus quinquestriatus
7Met Asn Tyr Leu Met Ile Ile Ser Leu Ala Leu Leu Leu Met Thr Gly1 5
10 15Val Glu Ser Gly Val Arg Asp Ala Tyr Ile Ala Asp Asp Lys Asn
Cys20 25 30Val Tyr Thr Cys Gly Ala Asn Ser Tyr Cys Asn Thr Glu Cys
Thr Lys35 40 45Asn Gly Ala Glu Ser Gly Tyr Cys Gln Trp Phe Gly Lys
Tyr Gly Asn50 55 60Ala Cys Trp Cys Ile Lys Leu Pro Asp Lys Val Pro
Ile Arg Ile Pro65 70 75 80Gly Lys Cys Arg Gly Arg858261DNALeiurus
quinquestriatusCDS(1)...(261)misc_feature(0)...(0)LghIV 8atg aat
tac ttg atg ata att agt ttg gct ctt ctt cta atg aca ggt 48Met Asn
Tyr Leu Met Ile Ile Ser Leu Ala Leu Leu Leu Met Thr Gly1 5 10 15gtg
gag agc ggt gta cgt gat gct tat att gcc gac gat aaa aac tgt 96Val
Glu Ser Gly Val Arg Asp Ala Tyr Ile Ala Asp Asp Lys Asn Cys20 25
30gtg tac act tgt ggt gca aat tca tat tgc aac aca gaa tgt acc aag
144Val Tyr Thr Cys Gly Ala Asn Ser Tyr Cys Asn Thr Glu Cys Thr
Lys35 40 45aac ggt gct gag agt ggc tat tgt caa tgg ttt ggt aaa tat
gga aat 192Asn Gly Ala Glu Ser Gly Tyr Cys Gln Trp Phe Gly Lys Tyr
Gly Asn50 55 60gcc tgc tgg tgc ata aag ttg ccc gat aaa gta cct att
aga ata cca 240Ala Cys Trp Cys Ile Lys Leu Pro Asp Lys Val Pro Ile
Arg Ile Pro65 70 75 80gga aag tgc cgt ggc cga taa 261Gly Lys Cys
Arg Gly Arg *859483DNAVaejovis
carolinanusCDS(65)...(358)misc_feature(0)...(0)VC1 9gccgctctag
aactagtgga tcccccgggc tgcaggtttc tccgtttgga taatcgtcta 60gaaa atg
aac gct aaa ata act gtt cta ttt ttc ctc gta gcc att aca 109Met Asn
Ala Lys Ile Thr Val Leu Phe Phe Leu Val Ala Ile Thr1 5 10 15att gct
tct tgt gcc tgg ata agt gag aaa aaa gtt caa gat gtc att 157Ile Ala
Ser Cys Ala Trp Ile Ser Glu Lys Lys Val Gln Asp Val Ile20 25 30gat
aaa aaa ttg cca aac gga atg gtg aag aat gca atc aaa gcc gta 205Asp
Lys Lys Leu Pro Asn Gly Met Val Lys Asn Ala Ile Lys Ala Val35 40
45gta cac aaa gca gcg aag aat aag cac ggc tgt ttt gct gat ttt gat
253Val His Lys Ala Ala Lys Asn Lys His Gly Cys Phe Ala Asp Phe
Asp50 55 60gta gga gga gga tgc gaa cag cac tgc cag aaa acg gaa agt
aaa gca 301Val Gly Gly Gly Cys Glu Gln His Cys Gln Lys Thr Glu Ser
Lys Ala65 70 75gga atc tgt cac gga acc aaa tgc aaa tgc ggt att ccc
cgt gcc tat 349Gly Ile Cys His Gly Thr Lys Cys Lys Cys Gly Ile Pro
Arg Ala Tyr80 85 90 95aaa aaa taa atcactgatt aatgctaacg gtgaatacat
ataatatttc 398Lys Lys *tatccaagct ttagtcaaaa ataataaaat gaattatttg
cacacttaca ttctatgtaa 458tatacacaaa ataaatcgaa tttgg
4831097PRTVaejovis carolinanus 10Met Asn Ala Lys Ile Thr Val Leu
Phe Phe Leu Val Ala Ile Thr Ile1 5 10 15Ala Ser Cys Ala Trp Ile Ser
Glu Lys Lys Val Gln Asp Val Ile Asp20 25 30Lys Lys Leu Pro Asn Gly
Met Val Lys Asn Ala Ile Lys Ala Val Val35 40 45His Lys Ala Ala Lys
Asn Lys His Gly Cys Phe Ala Asp Phe Asp Val50 55 60Gly Gly Gly Cys
Glu Gln His Cys Gln Lys Thr Glu Ser Lys Ala Gly65 70 75 80Ile Cys
His Gly Thr Lys Cys Lys Cys Gly Ile Pro Arg Ala Tyr Lys85 90
95Lys11294DNAVaejovis
carolinanusCDS(1)...(294)misc_feature(0)...(0)VC1 11atg aac gct aaa
ata act gtt cta ttt ttc ctc gta gcc att aca att 48Met Asn Ala Lys
Ile Thr Val Leu Phe Phe Leu Val Ala Ile Thr Ile1 5 10 15gct tct tgt
gcc tgg ata agt gag aaa aaa gtt caa gat gtc att gat 96Ala Ser Cys
Ala Trp Ile Ser Glu Lys Lys Val Gln Asp Val Ile Asp20 25 30aaa aaa
ttg cca aac gga atg gtg aag aat gca atc aaa gcc gta gta 144Lys Lys
Leu Pro Asn Gly Met Val Lys Asn Ala Ile Lys Ala Val Val35 40 45cac
aaa gca gcg aag aat aag cac ggc tgt ttt gct gat ttt gat gta 192His
Lys Ala Ala Lys Asn Lys His Gly Cys Phe Ala Asp Phe Asp Val50 55
60gga gga gga tgc gaa cag cac tgc cag aaa acg gaa agt aaa gca gga
240Gly Gly Gly Cys Glu Gln His Cys Gln Lys Thr Glu Ser Lys Ala
Gly65 70 75 80atc tgt cac gga acc aaa tgc aaa tgc ggt att ccc cgt
gcc tat aaa 288Ile Cys His Gly Thr Lys Cys Lys Cys Gly Ile Pro Arg
Ala Tyr Lys85 90 95aaa taa 294Lys *12312DNAArtificial
SequenceCDS(76)...(312)sig_peptide(1)...(75)PR1 signal peptide
12atg aac ttc ctc aag tcc ttt ccg ttc tac gcc ttc ctg tgc ttt ggc
48Met Asn Phe Leu Lys Ser Phe Pro Phe Tyr Ala Phe Leu Cys Phe
Gly-25 -20 -15 -10cag tat ttc gtg gcg gtc acc cac gct gcc tgg atc
tcc gag aag aaa 96Gln Tyr Phe Val Ala Val Thr His Ala Ala Trp Ile
Ser Glu Lys Lys-5 1 5gtg cag gac gtc att gat aag aag ctc ccg aac
ggc atg gtt aag aat 144Val Gln Asp Val Ile Asp Lys Lys Leu Pro Asn
Gly Met Val Lys Asn10 15 20gcg ata aaa gct gta gtg cac aag gca gcc
aag aac aag cat ggg tgc 192Ala Ile Lys Ala Val Val His Lys Ala Ala
Lys Asn Lys His Gly Cys25 30 35ttc gcg gac ttt gat gtc gga ggc ggc
tgt gaa caa cac tgc cag aaa 240Phe Ala Asp Phe Asp Val Gly Gly Gly
Cys Glu Gln His Cys Gln Lys40 45 50 55acc gag agc aag gct ggg atc
tgc cat gga acg aag tgt aag tgc ggc 288Thr Glu Ser Lys Ala Gly Ile
Cys His Gly Thr Lys Cys Lys Cys Gly60 65 70atc ccc cgc gcc tac aaa
aag tag 312Ile Pro Arg Ala Tyr Lys Lys *7513103PRTArtificial
SequencePR1 signal peptide linked to VC1 13Met Asn Phe Leu Lys Ser
Phe Pro Phe Tyr Ala Phe Leu Cys Phe Gly1 5 10 15Gln Tyr Phe Val Ala
Val Thr His Ala Ala Trp Ile Ser Glu Lys Lys20 25 30Val Gln Asp Val
Ile Asp Lys Lys Leu Pro Asn Gly Met Val Lys Asn35 40 45Ala Ile Lys
Ala Val Val His Lys Ala Ala Lys Asn Lys His Gly Cys50 55 60Phe Ala
Asp Phe Asp Val Gly Gly Gly Cys Glu Gln His Cys Gln Lys65 70 75
80Thr Glu Ser Lys Ala Gly Ile Cys His Gly Thr Lys Cys Lys Cys Gly85
90 95Ile Pro Arg Ala Tyr Lys Lys10014240DNAArtificial SequenceCodon
biased nucleotide sequence encoding Aaml. Codon biased rice. 14atg
aag gcc ttc acc ctc gcg ctg ttt ctc gct ctc tcc ttg tat ctt 48Met
Lys Ala Phe Thr Leu Ala Leu Phe Leu Ala Leu Ser Leu Tyr Leu-20 -15
-10ctc ccc aac cca gcg gct gac gtc ccg gga aac tac cca ctt gat tct
96Leu Pro Asn Pro Ala Ala Asp Val Pro Gly Asn Tyr Pro Leu Asp Ser-5
1 5 10tcc gac aat acc tac ctg tgc gcc cct ttg gga gat aat ccg gac
tgc 144Ser Asp Asn Thr Tyr Leu Cys Ala Pro Leu Gly Asp Asn Pro Asp
Cys15 20 25att aag atc tgt cag aaa cac ggt gtg gat tac ggg tat tgc
tac gcc 192Ile Lys Ile Cys Gln Lys His Gly Val Asp Tyr Gly Tyr Cys
Tyr Ala30 35 40ttc caa tgc tgg tgt gaa ttt ctg aag gat gag aac gtg
aag gtc tga 240Phe Gln Cys Trp Cys Glu Phe Leu Lys Asp Glu Asn Val
Lys Val *45 50 551579PRTArtificial SequenceSIGNAL(1)...(21)Codon
biased nucleotide sequence encoding Aam1. Codon biased to rice.
15Met Lys Ala Phe Thr Leu Ala Leu Phe Leu Ala Leu Ser Leu Tyr
Leu-20 -15 -10Leu Pro Asn Pro Ala Ala Asp Val Pro Gly Asn Tyr Pro
Leu Asp Ser-5 1 5 10Ser Asp Asn Thr Tyr Leu Cys Ala Pro Leu Gly Asp
Asn Pro Asp Cys15 20 25Ile Lys Ile Cys Gln Lys His Gly Val Asp Tyr
Gly Tyr Cys Tyr Ala30 35 40Phe Gln Cys Trp Cys Glu Phe Leu Lys Asp
Glu Asn Val Lys Val45 50 551679PRTArtificial SequenceSPAam1
sporamin signal and Aam1 16Met Lys Ala Phe Thr Leu Ala Leu Phe Leu
Ala Leu Ser Leu Tyr Leu-20 -15 -10Leu Pro Asn Pro Ala Ala Asp Val
Pro Gly Asn Tyr Pro Leu Asp Ser-5 1 5 10Ser Asp Asn Thr Tyr Leu Cys
Ala Pro Leu Gly Asp Asn Pro Asp Cys15 20 25Ile Lys Ile Cys Gln Lys
His Gly Val Asp Tyr Gly Tyr Cys Tyr Ala30 35 40Phe Gln Cys Trp Cys
Glu Phe Leu Lys Asp Glu Asn Val Lys Val45 50 5517249DNAArtificial
SequenceCodon biased nucleotide sequence encoding Aam1. Codon
biased to Streptomyces coelicolor. 17atg gcc aac aag cac ctg tcc
ctg tcg tta ttc ctg gtc ctc ctc ggc 48Met Ala Asn Lys His Leu Ser
Leu Ser Leu Phe Leu Val Leu Leu Gly-20 -15 -10ctc tcc gcc tcc ctc
gcg agc ggt gcc gac gtg cca ggg aac tac ccg 96Leu Ser Ala Ser Leu
Ala Ser Gly Ala Asp Val Pro Gly Asn Tyr Pro-5 1 5ctg gac agc tcg
gac aac acc tac ctg tgc gca ccc ctg ggc gac aac 144Leu Asp Ser Ser
Asp Asn Thr Tyr Leu Cys Ala Pro Leu Gly Asp Asn10 15 20ccg gac tgc
atc aag atc tgc cag aag cac ggc gtc gac tac ggc tac 192Pro Asp Cys
Ile Lys Ile Cys Gln Lys His Gly Val Asp Tyr Gly Tyr25 30 35 40tgc
tac gcg ttc cag tgt tgg tgc gag ttc ctg aag gac gag aac gtc 240Cys
Tyr Ala Phe Gln Cys Trp Cys Glu Phe Leu Lys Asp Glu Asn Val45 50
55aag gtg tga 249Lys Val *1882PRTArtificial
SequenceSIGNAL(1)...(24)Codon biased nucleotide sequence encoding
Aam1. Codon biased to Streptomyces coelicolor. 18Met Ala Asn Lys
His Leu Ser Leu Ser Leu Phe Leu Val Leu Leu Gly-20 -15 -10Leu Ser
Ala Ser Leu Ala Ser Gly Ala Asp Val Pro Gly Asn Tyr Pro-5 1 5Leu
Asp Ser Ser Asp Asn Thr Tyr Leu Cys Ala Pro Leu Gly Asp Asn10 15
20Pro Asp Cys Ile Lys Ile Cys Gln Lys His Gly Val Asp Tyr Gly Tyr25
30 35 40Cys Tyr Ala Phe Gln Cys Trp Cys Glu Phe Leu Lys Asp Glu Asn
Val45 50 55Lys Val1982PRTArtificial SequenceBAAAam1 BAA signal and
Aam1 19Met Ala Asn Lys His Leu Ser Leu Ser Leu Phe Leu Val Leu Leu
Gly-20 -15 -10Leu Ser Ala Ser Leu Ala Ser Gly Ala Asp Val Pro Gly
Asn Tyr Pro-5 1 5Leu Asp Ser Ser Asp Asn Thr Tyr Leu Cys Ala Pro
Leu Gly Asp Asn10 15 20Pro Asp Cys Ile Lys Ile Cys Gln Lys His Gly
Val Asp Tyr Gly Tyr25 30 35 40Cys Tyr Ala Phe Gln Cys Trp Cys Glu
Phe Leu Lys Asp Glu Asn Val45 50 55Lys Val2058PRTAndroctonus
amoreuxi 20Ala Asp Val Pro Gly Asn Tyr Pro Leu Asp Ser Ser Asp Asn
Thr Tyr1 5 10 15Leu Cys Ala Pro Leu Gly Asp Asn Pro Asp Cys Ile Lys
Ile Cys Gln20 25 30Lys His Gly Val Asp Tyr Gly Tyr Cys Tyr Ala Phe
Gln Cys Trp Cys35 40 45Glu Phe Leu Lys Asp Glu Asn Val Lys Val50
5521479DNACentruroides
vittatusCDS(117)...(359)misc_feature(0)...(0)Ts7 21ggccgctcta
gaactagtgg atcccccggg ctgcaggaat tcggcacgag acattttacc 60ataacggtaa
aaacgtttct attaatactt tctttagtga aaaaaaactt gaaagt atg 119Met1aaa
ttc ttc cta att gtg tca ttg gca ata atg tcg tgt ttc atg gaa 167Lys
Phe Phe Leu Ile Val Ser Leu Ala Ile Met Ser Cys Phe Met Glu5 10
15atg aaa gaa gta tac gca ggt acg aaa gga aat ttt ccc gtc gat ttt
215Met Lys Glu Val Tyr Ala Gly Thr Lys Gly Asn Phe Pro Val Asp
Phe20 25 30caa gga ata ttt tac gaa tgc atc gta tac aat aga tgt gaa
cgc gac 263Gln Gly Ile Phe Tyr Glu Cys Ile Val Tyr Asn Arg Cys Glu
Arg Asp35 40
45tgc aag tta cat gga tcg agt tat ggc tat tgc tac gct gga gtt tgc
311Cys Lys Leu His Gly Ser Ser Tyr Gly Tyr Cys Tyr Ala Gly Val
Cys50 55 60 65tac tgc gaa ggt tta gct gac gaa gat aaa tat ttc ctg
gga atg taa 359Tyr Cys Glu Gly Leu Ala Asp Glu Asp Lys Tyr Phe Leu
Gly Met *70 75 80tgaaaaaaca atgccgatta aatgtaaaat caatatcgtt
attgccctac aataagcgat 419taatcntttt gngagattaa ccttgggaat
aatggttacc taaaaaactn gggaataaaa 4792280PRTCentruroides vittatus
22Met Lys Phe Phe Leu Ile Val Ser Leu Ala Ile Met Ser Cys Phe Met1
5 10 15Glu Met Lys Glu Val Tyr Ala Gly Thr Lys Gly Asn Phe Pro Val
Asp20 25 30Phe Gln Gly Ile Phe Tyr Glu Cys Ile Val Tyr Asn Arg Cys
Glu Arg35 40 45Asp Cys Lys Leu His Gly Ser Ser Tyr Gly Tyr Cys Tyr
Ala Gly Val50 55 60Cys Tyr Cys Glu Gly Leu Ala Asp Glu Asp Lys Tyr
Phe Leu Gly Met65 70 75 8023243DNACentruriodes vittatus
23atgaaattct tcctaattgt gtcattggca ataatgtcgt gtttcatgga aatgaaagaa
60gtatacgcag gtacgaaagg aaattttccc gtcgattttc aaggaatatt ttacgaatgc
120atcgtataca atagatgtga acgcgactgc aagttacatg gatcgagtta
tggctattgc 180tacgctggag tttgctactg cgaaggttta gctgacgaag
ataaatattt cctgggaatg 240taa 2432457PRTCentruroides vittatus 24Gly
Thr Lys Gly Asn Phe Pro Val Asp Phe Gln Gly Ile Phe Tyr Glu1 5 10
15Cys Ile Val Tyr Asn Arg Cys Glu Arg Asp Cys Lys Leu His Gly Ser20
25 30Ser Tyr Gly Tyr Cys Tyr Ala Gly Val Cys Tyr Cys Glu Gly Leu
Ala35 40 45Asp Glu Asp Lys Tyr Phe Leu Gly Met50
5525292DNAArtificial SequenceCodon biased nucleotide sequence
encoding Ts7. Codon biased to Streptomyces coelicolor. 25atg gcg
aac aag cac ctc tcc ctg tcg ctg ttc ctc gtc ctg ctg ggc 48Met Ala
Asn Lys His Leu Ser Leu Ser Leu Phe Leu Val Leu Leu Gly-20 -15
-10ctg tcg gcg agc ctc gcc tcc ggc ggg acc aag ggc aac ttc ccg gtc
96Leu Ser Ala Ser Leu Ala Ser Gly Gly Thr Lys Gly Asn Phe Pro Val-5
1 5gac ttc cag ggt atc ttc tac gag tgc atc gtg tac aac cgc tgc gag
144Asp Phe Gln Gly Ile Phe Tyr Glu Cys Ile Val Tyr Asn Arg Cys
Glu10 15 20cgg gac tgt aag ctg cac ggc agc tcc tac ggc tac tgc tac
gcc ggc 192Arg Asp Cys Lys Leu His Gly Ser Ser Tyr Gly Tyr Cys Tyr
Ala Gly25 30 35 40gtg tgc tac tgc gag ggg ctc gcc gac gaa gac aag
tac ttc ctg gga 240Val Cys Tyr Cys Glu Gly Leu Ala Asp Glu Asp Lys
Tyr Phe Leu Gly45 50 55atg taa gac gct ccc cga gcg gct gct tct gtt
cat gaa gga ccc tta 288Met * Asp Ala Pro Arg Ala Ala Ala Ser Val
His Glu Gly Pro Leu60 65 70cat t 292His2681PRTArtificial
SequenceBAATs7 BAA signal and Ts7 26Met Ala Asn Lys His Leu Ser Leu
Ser Leu Phe Leu Val Leu Leu Gly-20 -15 -10Leu Ser Ala Ser Leu Ala
Ser Gly Gly Thr Lys Gly Asn Phe Pro Val-5 1 5Asp Phe Gln Gly Ile
Phe Tyr Glu Cys Ile Val Tyr Asn Arg Cys Glu10 15 20Arg Asp Cys Lys
Leu His Gly Ser Ser Tyr Gly Tyr Cys Tyr Ala Gly25 30 35 40Val Cys
Tyr Cys Glu Gly Leu Ala Asp Glu Asp Lys Tyr Phe Leu Gly45 50
55Met2764PRTAdroctonus amoreuxi 27Val Arg Asp Gly Tyr Ile Ala Asp
Ala Gly Asn Cys Gly Tyr Thr Cys1 5 10 15Val Ala Asn Asp Tyr Cys Asn
Thr Glu Cys Thr Lys Asn Gly Ala Glu20 25 30Ser Gly Tyr Cys Gln Trp
Phe Gly Arg Tyr Gly Asn Ala Cys Trp Cys35 40 45Ile Lys Leu Pro Asp
Lys Val Pro Ile Lys Val Pro Gly Lys Cys Asn50 55 60
* * * * *
References