Secreted Insecticidal Protein and Gene Compositions from Bacillus Thuringiensis and Uses Therefor

a technology of insecticidal protein and bacillus thuringiensis, which is applied in the field of new family of genes encoding lepidopterantoxic proteins and insecticidal fragments, can solve the problems of high toxicity of crystal proteins to specific insects, all field crops, plants, commercial farming areas are susceptible to attack, etc., and achieve effective control of a particular insect pest species, reduce the likelihood of resistance to the insecticidal composition, and improve the effect of insect resistance managemen

Inactive Publication Date: 2008-02-14
DONOVAN JUDITH +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0024]A particular advantage of the present invention comprises an improvement in insect resistance management (RM). The ability to combine two or more insecticidal agents, each toxic to the same insect pest species, into a single composition, and each agent exhibiting a mode of action different from the other insecticidal agents with which it is combined, present a means for more effectively controlling a particular insect pest species by substantially reducing the likelihood that resistance to the insecticidal composition will develop in a population. The TIC900 protein an insecticidal fragment thereof, or any homolog thereof, of the present invention can be combined with any number of known insecticidal agents to achieve the level of resistance management in a particular composition, preferably by expression of the combination of insecticidal agents in plants. In particular TIC900 or related insecticidal protein compositions can be combined with a Cry1 or Cry2 amino acid sequence or a variant thereof to achieve control of various lepidopteran plant pest species, or with other appropriate Cry proteins, and with various insecticidal compositions derived from Xenorhabdus and Photorhabdus bacterium species that have been shown to exhibit insecticidal bioactivity directed to lepidopteran plant pest species. Preferably the in planta use of these compositions would be directed to enhanced expression of the proteins in the parts of the plant that exhibit the greatest vulnerability to lepidopteran insect predation. For protection of maize species against European corn borer (ECB), it would be preferable to achieve the highest levels of expression in the leaves and stems of the plant. For tobacco species susceptible to budworm, it would be preferable to achieve the highest levels of expression in the sprouting parts of the plant, i.e., within the bud systems of the plant. For protection of a cruciferous vegetable species against diamondback moth (DBM), it would be preferable to achieve the highest levels of expression in the leaves and stems of the plant.
[0025]The insecticidal proteins of the present invention can also be combined with insecticidal and/or fungicidal toxins expressed in planta to achieve a recombinant plant that exhibits multiple levels of resistance to infestation by pests that are not beneficial to plants. For example, a protein of the present invention can be expressed along with a protein that exhibits coleopteran insect control, and/or along with a protein or other agent that exhibits antifungal activity, to achieve a recombinant transgenic plant that exhibits improved resistance to lepidopteran insect pests, coleopteran insect pests, and fungal pests. Other permutations of levels of resistance are known to those of skill in the art, such as means for resistance to piercing and sucking insect infestation, and nematode infestation, etc. The insecticidal proteins of the present invention can also be combined with one or more nucleotide sequences expressed as one or more dsRNA's for use in suppression of one or more genes (1) in the target pest as a means for achieving a plant that exhibits multiple layers of resistance to infestation by a particular pest, (2) in the plant as a means for achieving desired plant traits, or (3) in various combinations to achieve the desired properties of (1) or (2) collectively.
[0026]Chimeric proteins consisting of all or a part of one or more proteins of the present invention fused to other proteins that are useful in plant protection from infestation or otherwise are contemplated herein. For example, domains of the proteins of the present invention have been found to exhibit a low level of similarity to other Bt toxins, such as Cry3Aa toxin domain I, Cry1Ca toxin domain II, and Cry1Ja toxin domain III (in particular, Domains I, II, and III of the toxin portion of the TIC900 protein, respectively). The proteins of the present inv...

Problems solved by technology

Almost all field crops, plants, and commercial farming areas are susceptible to attack by one or more insect pests.
Particularly problematic are Coleopteran and Lepidoptern pests.
Likewise, pests such as armyworm, sod webworm, and tropical sod webworm often attack turf grasses.
These B. thuringiensis crystal proteins are often highly toxic to specific insects.
Commercial B. thuringiensis i...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation and Bioassay of B. thuringiensis Strain EG5438 Culture Supernatant

[0171]B. thuringiensis strain EG5438 was grown in 60 ml of PYG culture medium with shaking overnight at 30° C. PYG medium contained the following: 11.8 g peptone, 23.6 g yeast extract, 4 ml glycerol, 19.4 g K2HPO4 anhydrous, and 2.2 g KH2PO4 anhydrous. Deionized water was added to 1 liter, and the medium was autoclaved for 15 min. The B. thuringiensis culture was centrifuged at 11,000×g for 30 min and the supematant was transferred to a clean flask. The supernatant was chilled to 4° C., and 34 grams of ammonium sulfate plus 1 ml of 1 M NaOH were slowly added to the supernatant while stirring. The mixture was centrifuged and the resulting pellet was dissolved in 2 ml of 20 mM Tris-HCl pH 7.5. The solution was transferred to dialysis tubing (6000 MWCO) and was dialyzed at 4° C. against 20 mM Tris-HCl pH 7.5. This is referred to as the dialyzed supernatant

[0172]The dialyzed supernatant was tested for toxicity...

example 2

Fractionation of Proteins in the Dialyzed Supernatant and Bioassay of Protein Fractions

[0173]Proteins in the dialyzed supernatant were initially fractionated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Thirty μl of the dialyzed supernatant was mixed with 15 μl of protein solubilization buffer, the mixture was heated to 100° C. for 5 min, and 25 μl of the mixture was applied to a polyacrylamide gel. An electric current was applied to the gel to size-separate proteins into the gel. The proteins were visualized after electrophoresis by staining with Coomassie dye. The dialyzed supernatant contained approximately twenty proteins ranging in size from approximately 20 kDa to about 100 kDa.

[0174]Proteins in the dialyzed supernatant were fractionated by DEAE ion-exchange chromatography. Two ml of the dialyzed supernatant was applied to a 1 ml DEAE column. The column was washed with 10 ml of 20 mM Tris-HCl, pH 7.5, followed by washing with 20 ml of a 0 to 1 M NaC...

example 3

Determination of the N-Terminal Sequence of a Fragment of a TIC900 Protein

[0176]mTIC900 protein was purified from the supernatant of strain EG5438 by DEAE and CM ion exchange chromatography. Attempts to determine the N-terminal sequence of the purified mTIC900 protein by standard methods were not successful. To overcome this difficulty, mTIC900 protein was fragmented by cyanogen bromide treatment (Cordoba et al., J. Biochem. Biophys. Methods 35: 1, 1997). The cyanogen bromide-generated TIC900 fragments were size-separated by SDS-PAGE without Coomassie staining. Separated TIC900 fragments were transferred from the SDS-PAGE to a polyvinylidene difluoride (PVDF) membrane by an electro transfer. The PVDF membrane was stained briefly with Coomassie dye and a portion of the membrane containing an approximately 14 kDa fragment of TIC900 protein was excised with a razor blade. The excised PVDF membrane containing the 14 kDa fragment was subjected to automated Edmund sequencing, revealing th...

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Abstract

The present invention relates to the isolation and characterization of nucleotide sequences encoding novel insecticidal proteins secreted into the extracellular space from Bacillus thuringiensis and related strains. The proteins are isolated from culture supernatants of Bacillus thuringiensis and related strains and display insecticidal activity against lepidopteran insects including European corn borer (ECB), tobacco budworm (TBW) and diamondback moth (DBM). Insecticidal proteins encoded by nucleotide sequences that hybridize under stringent conditions to the isolated and characterized nucleotide sequences are disclosed. Methods are disclosed for making and using transgenic cells and plants comprising the novel nucleotide sequence of the invention.

Description

BACKGROUND OF INVENTION[0001]The present invention relates to a new family of genes encoding lepidopteran-toxic proteins and insecticidal fragments thereof. In particular, the present invention is directed to exemplary proteins designated herein as TIC900, TIC402, TIC403, TIC404, TIC961, TIC962, TIC963, TIC965 and TIC966, and insecticidal fragments thereof, each encoded by exemplary nucleotide coding sequences designated herein respectively as tic900, tic402, tic403, tic404, tic434, tic961, tic962, tic963, tic965, and tic966, as well as to nucleotide sequence homologs that (1) encode insecticidal proteins and (2) hybridize to the tic900, tic402, tic403, tic404, tic434, tic961, tic962, tic963, tic965, and tic966 coding sequences under stringent hybridization conditions. The present invention also relates to host cells transformed with one or more nucleotide sequences of the present invention or transformed with variants of the nucleotide sequences set forth herein, genes related by i...

Claims

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Application Information

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IPC IPC(8): A01H5/00A01H1/00A61K38/00C07H21/04C07K14/325C12N15/00C12N15/82C12Q1/68
CPCC07K14/325C12N15/8286C07K14/32Y02A40/146
Inventor DONOVAN, JUDITHDONOVAN, WILLIAMENGLEMAN, JAMESMALVAR, THOMASPITKIN, JOHN
Owner DONOVAN JUDITH
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