Cloning And Expression Of Cry7Ab7 And Construction Of Fusion Gene And Engineering Strain From Bacillus Thuringiensis

Bacillus thuringiensis(Bt) is the most in-depth study of microbial pathogens, and its insecticidal protein gene is the most widely and potentially used against pest insect, Therefore, exploring new Bt strains and isolating novel insecticidal gene to control pest are of great significance. Using the temperature screening method, 9 Bacillus thuringiensis isolates were obtained from 182 soil samples collected from some locations of Baoding, Hebei provinces, identification of cry gene-type of Bt strains through PCR system and cloning, expression and insecticidal activity assay of new protein gene that is toxic to Coccinellidae, and subsequently a novel Bt fusion gene gene was cloned by PCR technique. In order to provide new genetic resources for constructing high Bt engineered strain and cultivating genetically modified plant. The main contents of this study are as follows:1. 9 Bt strains were isolated from 182 soil samples in Baoding, Hebei Province by temperature screening method. On an average, 4.95% Separate rate of Bt strain. Crystal shapes could be observed, such as bipyramidal, square and so on.2. The DNA template from 9 Wild-type Bt strains were amplified by using 40 pairs universal primers, then a pair of special primer with cry7 gene from GW6 strain only for the identification by PCR techniques. The results of sequencing and structural analysis showed that the gene, named Cry7Ab7 (GenBank accession number: FJ940776) by International Nomenclature Committee ofδ-endotoxin was the open reading frame encoded a 129.64kDa 1138 amino acids.3. The recombinant plasmids, pET21b-7Ab7 and pSXY422b-7Ab7 were constructed by insertion of the cry7Ab7 gene into E.coli expression vector pET21b and Bt-E.coli shuttle vector pSXY422b. These two plasmids were transformed into E.coli BL21 and Bt HD73(cry-) strains to acquire two engineering strains. E.coli EC7Ab7 and Bt HD73(cry-), respectively. The results of SDS-PAGE suggested that cry7Ab7 gene was expressed with a high level, and the expressed products from the two engineering strains were toxic to second instar larvae of Henosepilachna vigintioctomaculata with the LC50 values of 1.1664μg/μL and 0.779μg/μL, respectively.4. The DNA template from Bacillus thuringiensis (Bt) strain GW6 and Bt11 was used for PCR amplification with one pairs special primers designed on the basis of the sequence of CRY7AbF/CRY1IaR gene from Bt cry7Ab7 and cry1Ia14 gene to obtain fusion gene Bt7Ia, the full-length gene was designated Bt7Ia in GenBank (Accession No. GU462130). Bt7Ia gene was inserted into the BamHⅠ/SalⅠsites of E.coli expression vector pET21b and Bt-E.coli shuttle vector pSXY422b to obtain the recombinant plasmid pET21b-7Ia and pSXY422b-7Ia, While E.coli EC7Ia and the engineering strains Bt HD7Ia were acquired by constructed plasmid transformed into E.coli BL21 and Bt HD73(cry-) respectively. EC7Ia were high level expressed in E.coli, cry1I silence gene fragment containing the fusion Bt7Ia gene, which was not expressed in the acrystalliferous mutant (HD73cry-). The results of bioassay showed that the expressed product of the EC7Ia was highly toxic to the first-instar larvae of Trichoplusia ni and Plutella xylostella with the LC50 values of 2.0953μg/mL and 0.3927μg/mL, An expression plasmid with the fusion protein was also created for further research relating to transgenic crops and engineered microorganism to control pest.