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Bacillus thuringiensis engineering bacteria for killing coleopteran pests, preparation method and application thereof

The technology of Bacillus aureus and thuringiensis spore is applied to Bacillus thuringiensis engineering bacteria for killing coleopteran pests. Low toxicity, poor insecticidal specificity, etc., to achieve the effects of unique insecticidal characteristics, high protein yield, and high virulence

Active Publication Date: 2014-10-15
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the control of coleopteran pests can only rely on chemical pesticides. Chemical pesticides have poor insecticidal specificity, and at the same time cause environmental pollution and are harmful to humans and animals. are getting more and more attention
Bacillus thuringiensis is the most successful and extensive biological control model (Microbiology and Molecular Biology Reviews, 1998, 62 (3): 775-) to the control of Lepidoptera pests, but the application of Bt insecticides to kill Coleoptera pests is less This is related to the lack of Bt strains and gene resources for killing coleopteran insects, and the low toxicity of insecticidal crystal proteins, etc.

Method used

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  • Bacillus thuringiensis engineering bacteria for killing coleopteran pests, preparation method and application thereof
  • Bacillus thuringiensis engineering bacteria for killing coleopteran pests, preparation method and application thereof
  • Bacillus thuringiensis engineering bacteria for killing coleopteran pests, preparation method and application thereof

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Effect test

Embodiment 1

[0037] A preparation method of bacillus thuringiensis engineering bacteria, the steps are:

[0038] 1. Construction of the dissociation vector: The replication region ori1030 of the Bt-E.coil shuttle vector pHT304 was replaced with the replication region ori44 of the vector pBMB1205R to construct the dissociation vector pHT304 / res. The specific operation is: after pHT304 is digested and recovered by Hind III, it is incompletely digested with Sca I and the 3657bp fragment is recovered. At the same time, pBMB1205R was digested with Nco I, and the sticky ends were blunted with Klenow enzyme and recovered, and then the 3688bp fragment was recovered by Hind III digestion. The 3657bp fragment was connected with the 3688bp fragment to obtain the novel dissociation vector pHT304 / res, with a size of 7345bp (see figure 1 ).

[0039] 2. Using cry3A-F (5'GGA TCC TTT GAA AAT ATA ACT ACC3') and cry3A-R (5'GGA TCC AAG CTT ACA GAG AAA TAC) as primers, using PCR technology (polymerase chain ...

Embodiment 2

[0046] A kind of application of Bacillus thuringiensis engineered bacterium in the preparation of Bt insecticide, its steps are:

[0047] A. Shake flask fermentation:

[0048] The purpose of shake flask fermentation is to select the best fermentation formula, optimize the fermentation conditions, and conduct orthogonal experiments with different solid contents and different C / N ratios. Scraped cultures from plates cultured for more than 3 days were dissolved in sterile water to make spore suspension, treated at 80°C for 30 minutes for inoculation. 20 / 250ml filling volume, 30°C, 200 rpm shake flask culture for 72 hours, the optimal fermentation medium formula for strain Ace-38 is shown in Table 1.

[0049] B. Using the best fermentation medium formula screened out, put 20 ml of medium in a 250 ml Erlenmeyer flask for fermentation. The pH of the medium before elimination is 8.1, and the pH after elimination is 7.0. Inoculate the slope treated at 80°C for 30 minutes Strains, 30...

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Abstract

The invention discloses a bacillus thuringiensis engineering bacterium for killing coleopteran pests as well as a preparation method and application thereof. The preservation number is CCTCCM2013259. The preparation method comprises the following steps of: A, cloning insecticidal crystal protein gene cry3Aa8 from a Bt bacterial strain YC-03 for killing the coleopteran pests, inserting the gene into a disaggregation carrier pHT304 / res and obtaining a recombined plasmid pHT3Aa8 / res, converting the DNA (deoxyribonucleic acid) of the pHT3Aa8 / res into a Bt aphakic mutant strain BMB171 through electric shock, screening so as to obtain transformants, shifting an auxiliary plasmid pBMB1200 into the transformant, selecting a positive transformant not growing on an Amp / Erm flat panel but growing on an LB flat panel, breeding in an LB fluid nutrient medium, and selecting a single colony growing on the LB flat panel but not growing on a tetracycline flat panel; B, taking plagiodera versicolora and the larva of phaedon brassicae baly as the test pest, carrying out toxicity bioassay, comparing the toxicity lethal dose LC50, and screening the high-toxicity engineering bacterium strain Ace-38. The method is practicable, and is simple and convenient to operate, the bacillus thuringiensis engineering bacterium has high effect on killing a plurality of coleopteran pests such as plagiodera versicolora, potato beetles, vegetable phaedon brassicae baly, aulacophora indica and the like, and the bacterium has good fermenting property, and has development and application prospects.

Description

technical field [0001] The present invention relates to the technical field of microbial pesticides, in particular to a strain of Bacillus thuringiensis, more specifically to a method for preparing a Bacillus thuringiensis engineering bacterium that kills coleopteran pests, and also relates to a Bacillus thuringiensis engineering bacterium that kills coleopteran pests Use in pesticides. Background technique [0002] Coleoptera is the largest order in Insecta, and the harm of Coleoptera pests to agricultural and forestry production is second only to Lepidoptera pests. Coleoptera pests are widely distributed all over the world (Biodiversity of Coleoptera / Insect biodiversity: science and society, West Sussex: Blackwell Publishing, 2009, 265-301). Common ones such as potato beetle (Leptinotarsa ​​decemlineata), corn root beetle (Diabrotica virgifera), ape leaf beetle (Phaedon brassicae Baly), aeruginosa scarab (Anomala corpulenta), willow blue leaf beetle (Plagiodera versicolor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21A01N63/02A01P7/04C12R1/07
Inventor 高梅影余亚军刘朋明吴丹丹吴燕
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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