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Bacillus thuringiensis engineering bacteria and application thereof

A technology of Bacillus thuringiensis and Bacillus aureus, which is applied in the field of Bacillus thuringiensis engineering bacteria, can solve the problems of mixed hair grubs, production reduction, affecting the quality of peanut grains, etc.

Active Publication Date: 2014-12-10
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the biggest threat to peanut production is insect pests - grubs, which directly cause production reduction and affect the quality of peanut seeds. The damage is very serious, and there are many kinds of grubs mixed in the field.

Method used

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  • Bacillus thuringiensis engineering bacteria and application thereof
  • Bacillus thuringiensis engineering bacteria and application thereof
  • Bacillus thuringiensis engineering bacteria and application thereof

Examples

Experimental program
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preparation example Construction

[0027] 2. Preparation of electroshock-competent Escherichia coli

[0028] In order to improve the transformation efficiency of Escherichia coli SCS110, prepare electric shock transformation competent cells of SCS110 (Sambrook J, Fritsch, EF, and Maniatis T.1989. Molecular cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY) , SCS110 recipient cells were transformed by electric shock transformation to remove the effect of methylation in the exogenous gene and obtain more transformants containing the exogenous gene.

[0029] 3. Preparation of Bt electric shock competent

[0030] In order to improve the conversion efficiency of Bt, prepare Bt electric shock transformation competent cells (Sambrook J, Fritsch E F, and Maniatis T, 1989. Molecular cloning: A Laboratory Manual (Cold Spring Harbor Lab, Cold Spring Harbor, NY), using electric shock transformation The method of transforming Bt recipient bacterial cells to obtain more transformants containing ...

Embodiment 1

[0041] Example 1 Preparation of Escherichia coli SCS110 Electrocompetent

[0042] Pick a single colony and culture overnight at 37°C with shaking in 5ml LB. Inoculate in 100ml LB liquid medium according to 1% inoculum amount, culture at 37°C, 230rpm for 2hrs (OD 600 =0.5). Centrifuge at 4000rpm for 6min at 4°C. Discard the supernatant and rinse with pre-cooled ultrapure water twice. Add pre-cooled ultrapure water to suspend the cells. Centrifuge at 9000 rpm for 6 minutes at 4°C to recover the cells. Add pre-cooled ultrapure water to wash twice, add 1-2ml of 10% glycerol to resuspend the cells, aliquot, and store at -70°C for later use.

Embodiment 2

[0043] Example 2 Preparation of Bt Electric Shock Competent

[0044] Pick a single colony and culture overnight at 30°C with shaking in 5ml LB. Inoculate in 100ml BH liquid medium according to 1% inoculum amount, culture at 37°C, 230rpm for 3-4hr (OD 600 =2.0). Centrifuge at 9000 rpm for 6 min at 4°C. Discard the supernatant and add pre-cooled ultrapure water to wash twice. Add pre-cooled ultrapure water to suspend the cells, centrifuge at 9000 rpm for 6 min at 4°C, and recover the cells. Add pre-cooled ultrapure water to wash twice, add 40% PEG to resuspend cells, aliquot, and store at -70°C for later use.

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Abstract

The invention relates to bacillus thuringiensis engineering bacteria and application thereof, and belongs to the field of biological control. A shuttle vector pSTK-8G containing a cry8Gal gene is led to a bacterial strain BIOT185, so as to obtain a recombination strain BIOT1858G. The recombinant strain BIOT1858G has toxicity on coleopteran multiple pest holotrichia parallela, anomala corpulenta, holotrichia oblita and the like; and the toxicity on a plurality of insect pests is also improved.

Description

technical field [0001] The invention relates to the field of biological control, in particular to a Bacillus thuringiensis engineered bacterium with broad-spectrum insecticidal activity and its application. Background technique [0002] Bacillus thuringiensis (Bt) is a Gram-positive bacterium with a wide distribution. While forming spores, parasporal crystals composed of one or several insecticidal crystal proteins can be produced. This insecticidal crystal protein (Insecticidal Crystal Proteins, ICPs), also known as δ-endotoxin (delta-endotoxin), is harmless to humans and animals, and does not pollute the environment, so Bt has been the most widely used in the biological control of pests. [0003] So far, 746 insecticidal crystal protein genes have been cloned from different Bt strains. According to the homology of insecticidal crystal protein amino acids, they can be divided into 75 categories and belong to 292 model genes (http: / / www.biols.susx.ac.uk / home / Neil_Crickmor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/75A01N47/44A01P7/04C12R1/07
Inventor 贾艳华张杰束长龙王庆雷宋福平黄大昉彭琦
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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