Identification, Cloning And Expression Of Insecticidal Genes From Bacillus Thuringiensis

Bacillus thuringiensis(Bt) has been widely used because of harmless to humans andanimals. It has important practical significance that screening of strains with high insecticidalactivity. The identification of insecticidal genes can provide a good genetic resources, and canprovide a way to deal with pest resistance.In this study,250 Bt strains which had preserved inthe laborory were identifided by use of PCR-RFLP,and the cloning,expression and the activityof the novel cry genes were studied.1. In this paper, 250 Bt strains were identifided by use of PCR-RFLP.The result show thatthose strains contain cry1,cry2,cry3,cry4/10,cry8,cry11,cry30 and cry34/35. Thirty-eightisolates harbored cry1 genes,35 isolates harbored cry2 genes,11 isolates harbored cry3 genes,8isolates harbored cry4/10 genes,68 isolates harbored cry8 genes,4 isolates harbored cry11genes,6 isolates harbored cry30 genes,5 isolates harbored cry34/35 genes. In the genecombination,Thirty isolates which harbored both cry1 and cry2 accounts for the biggestproportion,5 isolates which harbored both cry3 and cry8,1 isolates both cry8 and cry34/35,1isolates both cry4/10 and cry30,1 isolates both cry8 and cry2.134 stains which were notidentified genetypes,possibly include the unknown genetypes, and need to draw supports fromother methods to more research.2. All the strains carried on bioassay to Plutella xylostella,Heliothis armigera and Holotrichiaobeita.Forty isolates had high toxicity towards Plutella xylostella,and the 100% mortality wasgained after 4 days. 21 isolates had high toxicity towards Heliothis armigera,and the mortalityrate was past 70% after 4 days.The genes idenfication show that these stains contain cry1 orcry2 gene.Two isolates had high toxicity towards Holotrichia obeita, he mortality rate waspast 80% after 4 days.The genes idenfication show that these stains contain cry8G gene.Thebioassay of the strain HYW8 to Plutella xylostella and Heliothis armigera show that the100% mortality was gained after 2 days. The LC50 was 0.74 g/ml to (Hyphantriacunea(Drury),and the insect disinfestation effect is higher than standard strain HD-1。3. The cry gene which contained in the strain SCC4 and the strain JPL4 has not been determined by the PCR-RFLP.The fragment(897bp) of cry gene cloned from the strain SCC4had 91% sequence high-comparability with the Bt gene cry8B delta-toxin-likegene(GenBank:FJ770571.1).The fragment(1362bp) of cry gene cloned from the strain JPL4shared no comparablity with Bt gene which were known in GenBank.So one unknown genemight exist and further study.4. SDS-PAGE of the strain HYW8 show that it expressed 130 kDa protein.The identificationof cry gene indicate that the strain HYW8 contains cry1Ab,cry1E and a novel cry2.So weconjectures that 130kDa protein was expressed by cry1 gene,and cry2 gene that did notexpress in the strain was silent gene.5. Full-length of cry2A gene were cloned from the strain HYW8. The code area of this genewas 1896bp ,and had 98% sequence high-comparability with the Bt gene cry2Ah2 (GenBank:FJ550343), and had been nominated as cry2Ah3 by International Nomenclature Committee6. The cry32 Full-length gene were cloned from the strain FBG1. The code area of this genewas 3711bp ,and had 99% sequence high-comparability with the Bt gene cry32Aa1(GenBank:AAG36711),and had been nominated as cry32Aa2 by International NomenclatureCommittee of as GenBank accession numbers GU063849. Expression of cry32Ah2 had beenperformed by inserted it into E. coli expression vector pEB , and a 130 kDa protein bandcould be easily detected by SDS-PAGE. Results of bioassays proved that the toxicity towardsPlutella xylostella,that the 40% mortality was gained after 2 days ansd the 60% mortality wasgained after 4 days was higher than the toxicity towards Heliothis armigera,that the 20%mortality was gained after 2 days ansd the 34% mortality was gained after 4 days...