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Insecticidal crystalline gene cry7Bal of Bacillus thuringiensis

A technology of Bacillus thuringiensis, insecticidal crystal protein, applied in genetic engineering, plant genetic improvement, biocide and other directions

Inactive Publication Date: 2007-04-25
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, with the continuous promotion of the application of B.t insecticides and the continuous enhancement of the intensity of use, the resistance phenomenon of target pests has been continuously discovered by scientists

Method used

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  • Insecticidal crystalline gene cry7Bal of Bacillus thuringiensis
  • Insecticidal crystalline gene cry7Bal of Bacillus thuringiensis
  • Insecticidal crystalline gene cry7Bal of Bacillus thuringiensis

Examples

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Effect test

Embodiment 1

[0081] Cloning of the insecticidal crystal protein gene cry7Ba1 of Bacillus thuringiensis YBt-978 of embodiment 1

[0082] With reference to the method reported by Luo and Wing (2003) (see: Luo and Wing.2003.An improved method for plant BAClibrary construction, p.3-19.In Grotewold, Erich, Plant functional genomic: methods and protocols.Scientific and medical publishers, / Humana Press, Totowa, USA), utilize BAC library carrier pBeloBAC11 to construct Bacillus thuringiensis YBt-978 bacterial strain (the source of this bacterial strain sees literature: Dai J et al.1996.Bacillus thuringiensis subspecies huazhongnsis, serotypeH40.isolated from soils in the People ′s Republic of China. Letters in Applied Microbiology.22(1): 42-45, according to the relevant provisions of the Chinese Patent Law and Implementation Rules, the applicant has proposed to release the YBt to the public within 20 years from the date of application for this patent Proof of -978 strain) genomic BAC library. PC...

Embodiment 2

[0083] Example 2 Sequence analysis of the insecticidal crystal protein gene cry7Ba1 of Bacillus thuringiensis YBT-978

[0084] The nucleotide sequence of the 5kb XhoI fragment where the insecticidal crystal protein gene cry7Ba1 is located was analyzed to obtain a 5235bp sequence, including a 3465bp code. The code sequence is shown in SEQ ID NO:1.

[0085] The coding sequence of this section can encode a polypeptide consisting of 1154 amino acids, and its molecular weight is deduced to be 130558 Daltons (Da).

[0086] Comparing the amino acid sequences of this polypeptide with other known Cry and Cyt proteins, it was found that it was most similar to Cry7Ab2, Cry7Ab1, and Cry7Aa1, with similarities reaching 58.2%, 57.9%, and 57.1%, respectively. Among them, the general partial sequence of the N-terminal (1 to 658 amino acid sequence in SEQ ID NO: 1) has very low similarity, the similarity is 37.1%, 37.0%, and 36.4%, respectively. Due to the similarity with Cry7A insecticidal c...

Embodiment 3

[0087] Example 3 Expression of insecticidal crystal protein gene cry7Ba1

[0088] The 5kb XhoI fragment where cry7Ba1 is located was transferred to the Escherichia coli-Bacillus thuringiensis shuttle vector pHT304 (for the source of the vector, see: Arantes O and Lereclus D. 1991. Construction of cloning vectors for Bacillus thuringiensis. Gene 108: 115-119) to obtain recombinant Plasmid pBMB0495. Then it was transferred to the Bacillus thuringiensis amorphous CryB strain (see: Wu D and Federici BA.1993. A 20-kilodalton protein preserves cell viablility and promotes CytA crystal formation during sporulation in Bacillus thuringiensis. J. Bacteriol., 175:5276-5280 ), the obtained recombinant strain was named BMB0502.

[0089] After the above-mentioned recombinant bacteria named BMB0502 were activated overnight in LB medium (adding erythromycin at a final concentration of 20 μg / mL when used), they were transferred to ICPM medium (adding erythromycin at a final concentration of ...

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Abstract

The invention discloses the separation, clone and application of insecticidal crystal protein gene from Bacillus thuringiensis. In this invention a novel insecticidal crystal protein cry7Ba1 is separated from Bacillus thuringiensis Central China species YBt-978 strains. The genes code a novel insecticidal crystal protein Cry7Ba1 with insecticidal activity to Lepidoptera insects. Cry7Ba1 protein is consisted by 1,154 amino acid, and its homology with the existing published proteins is no more than 80% and the half of amino acid sequence near the protein N-terminal has no more than 50% homology with the existing published proteins. The invention also discloses the gene sequence of this novel insecticidal crystal protein and its coding protein Cry7Ba1 expressed by microorganism transformed by appropriate expression vector, qualifying it with lepidoptera insecticidal activity.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering. Specifically, it relates to the isolation and cloning of an insecticidal crystal protein gene from Bacillus thuringiensis, which is related to the genetic engineering of biological pesticides. Background technique [0002] Bacillus thuringiensis is a rod-shaped, Gram-positive bacteria that can form endogenous spores and widely exists in various ecological environments. In the budding stage, it can form parasporal crystals composed of insecticidal crystal proteins (Insecticidal Crystal Proteins, ICPs) that are specifically toxic to insects. More than 500 kinds of insects in 10 orders of Insecta and some harmful species in protozoa, nematodes and flatworms also have specific biological activities (Schnepf H E, Crickmore N, Rie J V, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean D H. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev, ...

Claims

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Application Information

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IPC IPC(8): C12N15/32C07K14/325A01N63/02A01N63/50
CPCC07K14/325A01N63/00A01N63/50A01N63/23
Inventor 孙明喻子牛陈守文张振宇阮丽芳郭素霞戴经元李林刘子铎
Owner HUAZHONG AGRI UNIV
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