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Dipel molecular companion gene, carrier containing same and bacterial strain

A technology of Bacillus thuringiensis and molecular chaperone, applied in the field of genetic engineering of Bacillus thuringiensis

Inactive Publication Date: 2004-08-04
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above 20kDa protein can increase the expression level of ICP "expressed" genes, there is no report that it can stimulate ICP "silenced" genes

Method used

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  • Dipel molecular companion gene, carrier containing same and bacterial strain
  • Dipel molecular companion gene, carrier containing same and bacterial strain
  • Dipel molecular companion gene, carrier containing same and bacterial strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The preparation process of embodiment 1.p21zb gene:

[0041] The plasmid was extracted from the newly isolated strain Bt-ZB522 in our laboratory, and the plasmid DNA was digested with PstI, randomly cloned on the shuttle plasmid pHT3101, and directly transformed into the crystal-deficient strain BtK-BE20 by electric shock, and the transformed receptor was coated with They were cultured on LB plate medium containing 50ug / ml Em and 100ug / ml Ap, and observed under an optical microscope for the production of paraspora crystals to determine effective recombinants. JB001, one of the recombinant strains screened by this method, can produce large oval crystals. All the plasmids of JB001 were extracted, transformed into Escherichia coli JM101, and the recombinant shuttle plasmid pHZB001 was obtained, and the size of the exogenous fragment was 1.6kbp.

[0042] The gene p21zb can also be directly extracted from the Bt-YPF22 (CCTCC M98018) bacterial strain as described above, and ...

Embodiment 2

[0043] Embodiment 2. Construction of the recombinant plasmid pHZB1 containing p21zb gene:

[0044] a. Design 2 primers: Primer I: C[CTGCAG]GTCAACCCTGGGTCAA, the PstI site in brackets; PrimerII: G[ATGCAT]TCATTGATGTTCGG, the NsiI site in brackets; the template is plasmid pWF45, containing the gene cryIA(c) Promoter. A 195bp DNA fragment was obtained by PCR reaction, and sequence determination showed that the PCR product was expected;

[0045] b. The above fragment was ligated and transformed with pALTER-EX1 / PstI and NsiI to obtain the recombinant plasmid pAL-P. ;

[0046] c. Plasmid pAL-P was digested with PstI / HindIII double enzymes to obtain a 295bp promoter-containing DNA fragment, which was inserted into BluscriptM13- to obtain plasmid pBL-P;

[0047] d. The ORF, promoter regulatory region and transcription termination region of the p21zb gene were located in the 1.2kb fragment digested with NsiI / ClaI, and this fragment was inserted into the plasmid pBL-P to obtain the re...

Embodiment 3

[0051] Embodiment 3. Contain the construction of the engineering bacterium Bt-YPF22 of p21zb gene:

[0052] a. Select the Bt-F2 strain with higher insecticidal toxicity as the starting strain;

[0053] b. Prepare Bacillus thuringiensis competent cells according to the methods of Schurter (1989) and Brain & Luke (1988);

[0054] c. Add 0.01-0.05ug / ul plasmid pHZB1 to the prepared competent cells, mix well, place in ice bath for 10 minutes, transfer to a pre-cooled electric shock cup (0.2mm), voltage 1500v, resistance 400 ~800 for electric shock, after electric shock, quickly move to ice bath for 10 minutes, add 800ml SOC, 30~35℃ static culture for 1~2 hours, then take samples, spread on penicillin and erythromycin double antibody plate to screen positive colonies . Since pHZB1 is a shuttle plasmid of Bt-E.coli, the plasmids of positive colonies can be extracted, transformed into Escherichia coli JM101 or TG1, and the plasmids can be extracted again, and analyzed by enzyme dig...

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Abstract

The present invention relates to a molecular chaperone gene p21zb cloned from Bacillus thuringiensis (Bt) and its carrier containing said carrier and strain. The transfer of said gene into Bt strain not only can obviously raise the yield of inserticidal crystal protein (ICPs) of wild type Bacillus thuringiensis, but also can excite high-level expression of insecticidal crystal protein silent gene of Bacillus thuringiensis. Said invention creates the recombinant carrier and engineering strain containing p21zb gene, and the ICPs expression quantity of said engineering strain is raised by above once as compared with that of initial strain. The tests show that said engineering strain possesses high toxicity for lepidopterous pests and good genetic stability, and has high-yield and high-toxicity property.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering of Bacillus thuringiensis, and specifically relates to a molecular chaperone gene of Bacillus thuringiensis insecticidal crystal protein, a carrier containing the gene and a strain. Background technique [0002] Bacillus thuringiensis (Bt) insecticide is currently the largest production volume and the most widely used microbial insecticide at home and abroad. Advantages such as non-target biosafety. The pathogenic effect of Bacillus thuringiensis on insects mainly depends on insecticidal crystal proteins (Insecticidal crystal proteins, ICPs). Increasing the yield of ICPs can increase the total virulence of crystals produced and reduce production costs. In the past, we only started with conventional screening and breeding strains, but their yields were unstable or reduced as the strains degenerated. [0003] Ellis and Hemmingsen define chaperones as a class of unrelated cellular prote...

Claims

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Application Information

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IPC IPC(8): C12N15/32
Inventor 余健秀庞义邓日强
Owner SUN YAT SEN UNIV
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