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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: 2013-08-15
MONSANTO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a way to manage insect resistance in plants by combining two or more insecticidal agents with different modes of action. This helps to prevent the development of resistance in the pest population. The invention also includes the use of proteins from Bacillus thuringiensis (Bt) in plants to control lepidopteran pests. The Bt proteins can be fused with other Bt proteins or with other insecticidal proteins to improve their activity and host range. The invention also includes the use of recombinant DNA technology to combine Bt proteins with other genes to achieve desired traits in plants. Overall, the invention provides a more effective way to manage insect resistance in plants and protect them from harmful pests.

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 insecticide formulations typically contain dried sporulated B. thuringiensis fermentation cultures whose crystal proteins are toxic to various insect species.
However, three secreted non-crystal proteins of B. thuringiensis designated Vip1, Vip2 and Vip3 have been reported to be toxic to coleopteran or lepidopteran insects (Estruch et al., 1996; U.S. Pat. No. 5,866,326; WO94 / 21795; WO96 / 10083).

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 supernatant 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 toxicit...

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 NaCl...

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 the...

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PUM

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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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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/32C12N15/82C07K14/325
CPCC07K14/325C12N15/8286C07K14/32Y02A40/146
Inventor DONOVAN, JUDITH C.DONOVAN, WILLIAM P.ENGLEMAN, JAMES T.MALVAR, THOMAS M.PITKIN, JOHN W.
Owner MONSANTO TECH LLC
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