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Baciallus thuringiensis engineering bacterium Go33A and preparing method thereof

A technology of engineering bacteria, pstk-3a, applied in the fields of botanical equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems of easy resistance of pests, single species, narrow insecticidal spectrum, etc.

Inactive Publication Date: 2007-05-30
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

[0005] At present, the Bt preparations used to control Lepidoptera and Coleoptera pests in the world have narrow insecticidal spectrum, single species, and pests are prone to develop resistance
Moreover, the safety problems of genetically modified organisms brought by the engineering bacteria of the prior art are more prominent

Method used

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  • Baciallus thuringiensis engineering bacterium Go33A and preparing method thereof
  • Baciallus thuringiensis engineering bacterium Go33A and preparing method thereof
  • Baciallus thuringiensis engineering bacterium Go33A and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Identification of insecticidal crystal protein gene and determination of insecticidal activity in G03

[0072] Utilize the CAPS identification system of cry gene (Kuo W-S, Chak K-F, 1996. Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA. Appl. Environ Microbiol., 62 (4) : 1369-1377; Song Fuping, Bacillus thuringiensis cry gene PCR-RFLP identification system establishment, Chinese Agricultural Sciences, 1998, 31 (3), 13-18.), identified the cry genotype of strain G03. The 1.6 kb cry1 gene 3' end and the 1.4 kb cry1 gene 5' end characteristic band were amplified using the Kun2 primer pair and Kun3 primer pair respectively, indicating that G03 contains cry1 genes; the 1.2 kb cry2 gene was amplified using the Sun2 primer pair The gene characteristic band indicates that the cry2 gene is contained. The results of PCR-RFLP analysis showed that the cry1 gene was cry1Ac, ...

Embodiment 2

[0073] The insecticidal activity assay of embodiment 2 G03

[0074] The insecticidal crystal protein of the G03 strain was extracted, and after quantitative analysis by SDS-PAGE, it was mixed into the artificial feed placed in the petri dish with different concentration gradients, and 20 newly hatched larvae were inserted into each petri dish, and cultured at room temperature. For beet armyworm, count the number of live insects every 24 hours until 120 hours; for cotton bollworms, count them every 24 hours. Then count. Table 1 shows the insecticidal activity of G03 protein against beet armyworm; Table 2 shows the insecticidal activity against cotton bollworm. The initial concentration of protein was 2.0 μg / μl. Indoor bioactivity tests showed that the strain was highly virulent to cotton bollworm and beet armyworm, and its LC for cotton bollworm and beet armyworm 50 They were 0.113 μg / g and 2.62859 μg / g, respectively.

[0075] Table 1 Insecticidal activity of G03 protein ag...

Embodiment 3

[0079] Example 3 PCR amplification of thymidylate synthase gene (thy) full-length gene

[0080] According to the sequence of the thymidylate synthase gene, a pair of primers for amplifying its full-length gene, the upstream primer M11 and the downstream primer M12, were designed, and an EcoRI restriction site was added to the primers at the same time. The primer sequence is shown in SEQ ID NO:1. Using the plasmid pUCM1 containing the full-length thymidylate synthase gene as a template (Yang Hongjie, Master’s Thesis of Peking University, 1998), combined with upstream primers and downstream primers M11 and M12, PCR amplification was performed to obtain the thymidylate synthase gene Full-length fragment (1.1 kb). The PCR reaction system is as follows: PCR 10 times buffer 10uL, 20mmol / L MgCl 2 12uL, 10mmol / LdNTP 2uL, 10umol / L upstream and downstream primers 2uL each, DNA template 0.1-2ng, 2U / uL pfu enzyme 0.5uL, make up to 100uL with ultrapure water, mix and centrifuge, add 50u...

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Abstract

The invention provides a method for constructing Suyunjin gemma of a fungus bacillus project fungus which has broad spectrum insect killing activity, it gets a kind of project fungus which has high toxicity to coleopteran and Lepidoptera insects. At the same time, in order to increases the safety of the project fungus; it constructs a kind of environment safe Bt-E.coli shuttle expressing carrier and the correspondent mutant.

Description

Technical field: [0001] The invention relates to a Bacillus thuringiensis engineering bacterium with broad-spectrum insecticidal activity and a preparation method thereof; the invention also relates to the construction of an environment-safe Bt-E.coli shuttle expression vector and corresponding mutants. The present invention also relates to the use of wild-type Bacillus thuringiensis strain G03. 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, 268 insecticidal crystal protein genes have been cloned from different Bt stra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N15/64C12N15/70C12N15/11C12N15/52C12N1/21C12N15/32A01N63/00A01P7/04C12R1/07
Inventor 王广君张杰宋福平黄大昉冯书亮王容燕
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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