Isolation And Genotyping Of Bacillus Thuringiensis Strains In

Bacillus thuringiensis (Bt) is a kind of bacteria which is studied in-depth, and used widely at present in the world. The gene of Bt are usually used in genetically engineered crops. The field trials that is used for a long time causes some insect pest of plant have the resistance. So separating abundant Bt resources and isolating the effective Bt can not only enrich the species resources in our country, but also has important theoretical and practical significance to the cultivation of transgenic insect resistant plants for the construction of genetically engineered bacteria for the basis for isolating new high foundation.In this paper, we investigated the Bt resources in different ecological zones of Tibet.74 strains of Bacillus thuringiensis were isolated from 54 soil samples. And the gene types were identified by PCR. Biological activity of high virulence strains of Lepidoptera pests was determined to find the optimal fermentation conditions of Bt strain B. XZ18-1 with the highest virulence.1. With using temperature screening method,74 wild type Bt strains were isolated from from 53 soil samples, and a preliminary identificated (morphological, physiological and biochemical identification)."Berger’s Bacterial Identification Manual", the result of identification shows:59 subspecies type strains:5 strains of Bt.spp.tochigiensis,6 strains of Bt.spp.kumamotoensis,5 strains of Bt.spp.wuhanensis. 15 strains of Bt.spp.tohokuensis,5 strains of Bt.spp.yuannanensis,7 strains of Bt.spp.monisoni,2 strains of Bt.spp.darmstadiensis,2 strains of Bt.spp.subtoxicus,2 strains of Bt.spp.shandongiensis 5 strains of Bt.spp.israelensis.3 strains of Bt.spp.entomocidus,1 strains of Bt.spp.kurstaki,1 strains of Bt.spp.alesti, respectively. The RFLP analysis of PCR products shows that 24 strains contain Cry1 gene,32.43% of the isolates:12 strains contain Cry2 gene.16.22%of the isolates; 4 strains contain Cry3 gene.5.4% of the isolated strains; 5 strains contain gene 4/10 of the isolates,6.76%; 11 strains contain Cry5 gene.14.86% of the isolates; 11 strains contain Cry6 gene,14.86% of the isolates; 15strains contain Cry9 gene 20.27% of the isolates; 1 strains contain Cry13/14 type,1.35% of the isolated strains; 2 strains contain Cry11 gene.2.7% of the isolates; 23 strains contain Cry18 gene,31.08%of the isolates; 4 strains contain Cry30 gene,5.4% of the isolates; 2 strains contain Cry39/40 gene,2.7% of the isolates. In conclusion, the Tibet area has abundant resources of Bt.2.74 strains of Bacillus thuringiensis isolated, the result of toxicity test of sensitive diamondback moth in Incubation Chamber indicates that all strains had virulence on diamondback moth, especially B.XZ18-1, the LC50 is 8.4mg/L; B.XZ9-2, B.XZ1-1, B.XZ4-6, B.XZ2-4,B.XZ5-2followed, the LC50 range from 64.lto 114.5 mg/L; LC50is 143.8mg/L for B.XZ6-4.The protein analysis of SDS-PAGE showed these strains mainly expressed 130kDa,90kDa,50 kDa protein. We identified the 16S rDNA of high virulent strains:7 strains of high virulent were identified as Bacillus thuringiensis sp.; the similarity between B.XZ1-1 B.XZ4-6, B.XZ5-2, B.XZ9-2, which is compared with Bacillus thuringiensis ATCC 10792(T) is 95%、 87%、80%.79% respectively; the similarity of B.XZ2-4 and Bacillus toyonensis BCT-7112(T) is 87%; the similarity between B.XZ6-4 and Bacillus safensis FO-036b(T) is 97%; the similarity between B.XZ18-1 and Bacillus thuringiensis HD-771 is 100%3.In the strains isolated which is sensitive to the diamond back moth,B.XZ18-1 is particularly prominent. With series of shake-flask fermentation for the B.XZ18-1, in the optimum culture conditions (10h of seed liquor age, fermentation temperature of 30℃,2% of inoculation quantity,200r/min of rotation speed, the initial pH value 6.5-7.5, shaking speed of 200r/min) fermented 48h, the viable count and living spores number respectively reached 2.71×109CFU/mL and 8.75×108CFU/mL, crystal protein content was 18.5mg/mL. To detemine the optimal culture medium components of strain B.XZ18-1, designing that 7 factors and 3 levels orthogonal experiment: glucose 0.3%, corn flour 1.5%, soybean meal 1.5%, fish meal 1%, yeast powder 1.5%, K2HPO40.3%, CaCl22.0%.