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Bacillus thuringiensis gene combination and engineering bacteria and preparation method thereof

A technology of Bacillus thuringiensis and engineering bacteria, which is applied in botany equipment and methods, microbe-based methods, biochemical equipment and methods, etc., and can solve problems affecting peanut kernel quality, yield reduction, and mixed grubs, etc.

Inactive Publication Date: 2012-10-10
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 gene combination and engineering bacteria and preparation method thereof
  • Bacillus thuringiensis gene combination and engineering bacteria and preparation method thereof
  • Bacillus thuringiensis gene combination and engineering bacteria and preparation method thereof

Examples

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preparation example Construction

[0059] 2. Preparation of electroshock-competent Escherichia coli

[0060] 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.

[0061] 3. Preparation of Bt electric shock competent

[0062] 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

[0093] Example 1 Preparation of Escherichia coli SCS110 Electrocompetent

[0094] 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

[0095] Example 2 Preparation of Bt Electric Shock Competent

[0096] 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 the cells, aliquot, and store at -70°C for later use.

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Abstract

The invention relates to bacillus thuringiensis gene combination and engineering bacteria and a preparation method thereof and belongs to the field of biological prevention and control. The gene combination of cry8Eal, cry8Ca2 and cry3Aa7 has a synergistic effect between every two or among the three genes, particularly a good effect for the synergy between cry3Aa7 and cry8Eal as well as the synergy among the genes cry8Eal, cry8Ca2 and cry3Aa7. Engineering bacteria BIOT1853A thus constructed containing three genes are poisonous to holotrichia parallela, anomala corpulenta, potato beetles, colaphellus bowringi and other coleopter multiple pests at the same time, and in addition, maybe have higher poisonousness for several pests. The engineering bacteria BIOT1853A obtained through screening by allele homologous recombination are free of antibiotic genes, thereby enhancing the bio-safety of transgenic engineering bacteria.

Description

technical field [0001] The invention relates to the field of biological control, in particular to a gene combination of bacillus thuringiensis with broad-spectrum insecticidal activity. 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, 605 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 71 categories, belonging to 226 model genes (http: / / www.biols.susx.ac.uk / home / Neil_Crickmore / Bt / ). Among them, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N1/21C12N15/75C12N15/66C12Q1/68C12Q1/04A01N63/02A01P7/04C12R1/07
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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