Novel pesticidal toxins

A technology of toxins and insects, applied in biocides, viruses/bacteriophages, plant cells, etc.

Inactive Publication Date: 2004-09-08
SYNGENTA PARTICIPATIONS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, only a few specific insect pests can be controlled with current methods

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0161] Example 1: Construction of Bacillus thuringiensis strain AB1183 cosmid library

[0162] Total DNA was isolated from Bacillus thuringiensis named AB1183 by treating freshly grown cells resuspended in 100 mM Tris pH 8, 10 mM EDTA with 2 mg / ml lysozyme at 37°C for 30 minutes. Protease was added to 1% SDS, 50mM EDTA, 1M urea to a final concentration of 100μg / ml, and incubated at 55°C. Add an equal volume of phenol-chloroform-isoamyl alcohol. Gently mix the sample for 5 minutes and centrifuge at 3K. Repeat this step 2 times. Then mix the aqueous phase with 0.7 volume of isopropanol and centrifuge. Wash the DNA pellet 3 times with 70% ethanol, and gently resuspend in 0.5 X TE. At 37°C, 12 μg DNA was treated with 0.3 unit Sau3A / μg DNA in a volume of 100 μl. Sampling every 2 minutes interval for 10 minutes. Then add 1 / 10 volume of 10X TE and heat the sample at 65°C for 30 minutes to inactivate the enzyme. The sample was electrophoresed to determine the 40 kb range, and the sample w...

Embodiment 2

[0164] Example 2: Bioassay of cosmid cloning

[0165] The 1200 colonies of Example 1 were screened for their insecticidal activity against newborn larvae of Heliothis virescens. On artificial bait, the surface contamination method is used for bioassay. The bioassay is scored after 7 days. Eight clones were found to have insecticidal activity against Heliothis virescens.

Embodiment 3

[0166] Example 3: Analysis of Insecticidal Activity Cosmid Clones

[0167] In order to identify the homologous sequence of vip3, the method of Carozzi et al. (1991, Appl. Env. Microbiol. 57: 3057-3061) was used, using primers derived from the 5'priming region of the vip3A gene (SEQ ID NO: 4) in 8 Myzus persicae PCR analysis was performed on the positive clones of Heliothis virescens. The primers used for this analysis are

[0168] Forward: 5'-GTGATCTAACCCTAGACG-3' (SEQ ID NO: 8)

[0169] Reverse: 5’-GCTTTAGTTCCATTCACTCC-3’ (SEQ ID NO: 9).

[0170] One clone produced a DNA band of the expected size of vip3 related genes. The 3.8 kb EcoRI fragment from this clone was subcloned into pBluescript (Stratagene) and transformed into E. coli. PCR analysis confirmed that the E. coli clone contained the homologous coding sequence of vip3. This vip3 homologous coding sequence was named vip3B. The bioassay results of the E. coli clone containing the vip3B coding sequence proved that the Vip3B ...

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PUM

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Abstract

A novel pesticidal toxin that is highly active against a wide range of lepidopteran insect pests is disclosed. The DNA encoding the pesticidal toxin can be used to transform various prokaryotic and eukaryotic organisms to express the pesticidal toxin. These recombinant organisms can be used to control lepidopteran insects in various environment.

Description

Technical field [0001] The present invention relates to a new Vip3 toxin derived from Bacillus thuringiensis, which expresses a nucleic acid sequence that causes the toxin, a method for preparing the toxin, and a method for controlling insects by using the toxin and the corresponding nucleic acid sequence. Background technique [0002] Plant pests are the main factor in the loss of important crops in the world. In the United States alone, about 8 billion U.S. dollars is lost every year from infestation by non-mammalian pests, including insects. In addition to crop losses in farmland, insect pests are also a burden for vegetable and fruit growers, ornamental flower producers, and home gardeners. [0003] At present, insect pests are mainly controlled through the widespread application of chemical insecticides, which can actively inhibit the growth of insects, hinder the feeding or reproduction of insects, or cause death. So good insect control can be achieved, but these chemicals ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N37/46A01N63/50C07K14/32C12N7/01C12N15/32C12N15/82
CPCA01N63/00A01N37/46C12N15/8286A01N63/02C07K14/32Y02A40/146A01N63/50A01N63/23
Inventor P·迈尔斯V·克拉默沈志诚F·肖特科斯基G·W·沃伦
Owner SYNGENTA PARTICIPATIONS AG
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