| Nowadays, Bacillus thuringiensis (Bt), as one critical biopesticide, is widely and effectively utilized for insect pests biocontrol. It is very significant and necessary to screen Bt isolate with high toxicity and clone its novel insecticidal toxin genes for construction of genetically engineered bacteria and transgenic plants.In this study, cry-type genes of Bt isolates screened from China were identified using PCR-RFLP method. The research results showed that among 133 isolates, 61 contained cry] type gene, 59 contained cry2 type gene, and 15 contained cry9 type gene. 13 of them contained three type genes: cry], cry2, cry9. Two strains contained both cry] and cry9 type gene. None of them contained cry3, cty4, cry5, cry8 or cry]] type gene.On the basis of identification results, two isolates, B-Hm-16 and B-Pr-88, highly toxic to Lepidopteran pests, were chosen and studied on biological and molecular characterization systematically. Crystal shape of B-Hm-16 was bipyramidal, but in B-Pr-88 bipyramidal, spherical and irregular shapes were observed.Results of esterase pattern identification showed that both two strains belong to the galleriae type the same as standard strain of Bt subsp. galleriae and kurstaki. Biochemical reaction indicated that B-Hm- 16 was identical with Bt subsp. aizawai, while B-Pr-88 was the same as Bt subsp. galleriae.Results of cry-type gene identification showed that B-Hm- 16 contained crylAb and cry9Ea genes, while B-Pr-88 contained crylCb, crylDb, crylFb, cty]Gb, cry2Ab and cry9Ba genes. I3OkDa ICP was encoded in B-Hm-16, but l3OkDa and 6OkDa ICPs were encoded in B-Pr-88. Seven and three plasmids were detected from B-Hm-16 and B-Pr-88 respectively, and all genes of these two strains were located on its plasmids seperately.Southern hybridization and PCR amplification were used for cloning of novel cry genes. Five cry genes were isolated from B-Pr-88 successively. Four of them had been registered in GenBank, and designated by Bt ~ -endotoxin gene Nomenclature Committee as novel cry genes:cry]Gb2(Accession number is AF288683), cry]Fb5(AF3361 14), cry]Db2(AF358862) and cry2Ab4(AF336115). CrylGb2 protein of 133.OkDa, containing 1169 amino acids was deduced from cry]Gb2 (35 10bps) gene. CrylFb5 protein of 133.4kDa, containing 1174 amino acids was deduced from cry]Fb gene (3525bps). cry]Db2 (3483bps) and cry2Ab4 (1902bps) encoded 131.OkDa and 70.7kDa protein separately. cty9Ba (3354bps), encoding 126.4kDa protein of 1117amino acids, was obtained by PCR amplification.Plasmid library was constructed from B-Hm-l6, and two cry genes, obtained by usingPCR-RFLP method, had been registered in GenBank and named cIy1Ab]J (AF35886l) andcry9Ea2 (AF358863) respectively as novel genes by Bt 6 -endotoxin gene NomenclatureCommittee. CrylAbl5, l30.6kDa protein, was composed of l l55 amino acids deduced from3468bps nucletide sequence. Cry9Ea2, l29.9toa was composed of ll50 amino acids deducedfrom 3453bps DNA sequences. The latter gene, cryme, was the first cry9 gene cloned in ChinaFurthermore, another open reading frame was found on the upstream of CIy1Ab1J gene, and theamino acid sequences deduced from its DNA fragment as 70% homologous withN-acety l muramoy l -L-alanine am idas e in BaciHus s ubhas.Six pairs of specific primer were designed fOr amplification of cry1FbJ, cry1Db2, cWb4,cIy9Ba cry1Ab15 and cryM genes. The amplified productS of these genes were inserted intoexpression plasmid pET-2lb respectively and transfOrmed into E coli str8in BL2l(DE3). Afterbeing induced by IPTG, expression productS were performed by SDS-PAGE analysis. The resultSindicated cry genes could be expressed normally in E coli. Meanwhile, Cry1Ab1J and cryhagene, after transforming into Bt acrystalliferous mllbot stran BE20 resPectiveIy, the bipyramidalcrystal and l30soa protein band were observed and detected.Bioassay of expression products of these genes showed that Cry2Ab was highly tOxic to thelarvae of Plut6lla xylost6lla, ChilO suPPressalis and He... |
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