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Bacillus thuringiensis toxins and genes for controlling coleopteran pests

A pest and amino acid technology, applied in genetic engineering, plant genetic improvement, bacteria, etc., can solve unpredictable problems

Inactive Publication Date: 2010-03-03
MYCOGEN CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the discovery of new Bacillus isolates, toxins and genes, and new uses for known B.t. isolates remains an entirely empirical and unpredictable art

Method used

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  • Bacillus thuringiensis toxins and genes for controlling coleopteran pests
  • Bacillus thuringiensis toxins and genes for controlling coleopteran pests

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Example 1 - Cultivation of B.t. isolates of the invention

[0110] Secondary cultures of B.t. isolates can be used to inoculate the following medium (peptone, glucose, salt medium, pH 7.2):

[0111] Bacto peptone 7.5g / l

[0112] Glucose 1.0g / l

[0113] K H 2 PO 4 3.4g / l

[0114] K 2 HPO 4 4.35g / l

[0115] Saline solution 5.0ml / l

[0116] CaCl 2 Solution 5.0ml / l

[0117] Saline solution (100ml)

[0118] MgSO 4 .7H 2 O 2.46g

[0119] MnSO 4 .H 2 O 0.04g

[0120] ZnSO 4 .7H 2 O 0.28g

[0121] FeSO 4 .7H 2 O 0.40g

[0122] CaCl 2 Solution (100ml)

[0123] CaCl 2 .2H 2 O 3.66g

[0124] Salt solution and CaCl 2 The solution was sterilized by filtration, and high-temperature sterilized culture medium was added at the time of inoculation. The shake flasks were incubated for 64 hours at 30°C on a shaker at 200 rpm.

[0125] The above process is readily scaled up to larger fermentors by procedures known in the art.

[0126] The B.t...

Embodiment 2

[0127] Example 2 - Molecular cloning, expression and sequencing of novel toxin genes from Bacillus thuringiensis strains PS52A1 and PS86A1

[0128] Total cellular DNA was prepared from cells of Bacillus thuringiensis (B.t.) PS52A1 and PS86A1 strains grown at 30°C to an optical density at 600 nm of 1.0. Cells were pelleted by centrifugation and resuspended in protoplast buffer (20 mg / ml lysozyme in 0.3 M sucrose, 25 mM Tris-Cl [pH 8.0], 25 mM EDTA). After incubation at 37°C for 1 hour, the protoplasts were lysed by two freeze-thaw cycles. 9 volumes of 0.1M NaCl, 0.1% SDS, 0.1M Tris-Cl [pH 8.0] solution were added to the complete lysate. The cleared lysate was extracted twice with phenol:chloroform (1:1). Nucleic acids were precipitated with two volumes of ethanol and pelleted by centrifugation. The pellet was resuspended in TE buffer (10 mM Tris-Cl [pH8.0], 1 mM EDTA), and RNase was added to a final concentration of 50 μg / ml. After incubation at 37°C for 1 hour, the solutio...

Embodiment 3

[0139] Example 3-MR509 / 86A1 (b) toxin-resistant Brassica frugata biological detection

[0140] Collect wild cruciferous flea beetles and store them in a rearing room at 25°C with a 16L:8D photoperiod. Sow 5 canola (Hyola 401) seeds in regular potting soil. Cotyledons were excised from seedlings and immersed in B.t.MR509 suspension (100 ug toxin / ml) made with 0.1% Bond (as adhesive). Individually treated cotyledons were dried and placed in plastic wells (NuTrend trays) filled with approximately 1 ml of 2% agar gel. The agar gel acts as a source of moisture to increase the lifespan of the excised cotyledons. One adult beetle was placed in each test well and kept at room temperature. Mortality and plant damage were tested 4 and 7 days after treatment. Cotyledon damage was rated on a scale of 1-10, with 10 corresponding to complete destruction of plant tissue.

[0141] Several treated samples showed reduced plant damage relative to untreated and CryB (amorphous B.t. strain)...

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PUM

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Abstract

The subject invention concerns materials and methods useful in the control of pests and, particularly, plant pests. More specifically, the subject invention concerns novel genes and pesticidal toxinsreferred to as 86A1(b) and 52A1(b). In preferred embodiments, the subject toxins are used for controlling flea beetles of the genus Phyllotreta. Using the genes described herein, the transformation ofplants can be accomplished using techniques known to those skilled in the art. In addition, the subject invention provides toxin genes optimized for expression in plants.

Description

[0001] Cross-references to related applications [0002] This application is a continuation-in-part of application serial number 09 / 076193, filed May 12,1998. Background of the invention [0003] The loss of crop yields caused by insects and other pests and the cost of controlling these pests cost farmers billions of dollars each year. Loss of agricultural habitat due to insects includes reduced crop yield, reduced quality, and increased harvest costs. [0004] Coleoptera insects are a group of important agricultural pests, causing huge crop losses every year. There are several species of beetles that can cause significant economic losses; examples include leaf beetles (eg, flea beetle and corn rootworm) and weevils (eg, clover weevil). [0005] Leap beetles include many genera (e.g., Altica, Apphthona, Argopistes, Disonycha, Epitrix, Longitarsus, Prodagricomela, Minor flea (Systena), Psylliodes and Phyllotreta). Flea beetles include striped flea beetles. Cruciferous jump...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/32C07K14/325A01N63/00C12N1/21
CPCC07K14/325Y02A40/146
Inventor G·A·布拉德费什马勒-科恩K·E·纳瓦J·M·富M·汤姆森
Owner MYCOGEN CORP