Characterization Cry-Type Genes In A B.Thuringiensis Collection From Soil Of Tropic Primitive Rain Forest In Hainan Province

In this study,one hundred seventy-six B.thuringiensis and 3,152 bacillus isolates were screened from 1,643 soil samples collected systematically according to the different altitudes from the tropic primeval rain-forest. So the average and isolating rate were 10.71% and 5.58% respectively .For rate Bt-harbour soil sample,the Wuzhishan Mountain is the highest(14.54%),while the Bawangling Primitive Tropic Rain Forest is the lowest(5.58%) among the all rain-forest. Several points attribute to this, firstly the ecosystem of the Wuzhishan Mountain area is very well preserved and the mountain area has been rated as a national primeval forest reserve. In addition the high percentage of forest cover,thick the humus horizon and all kinds of insects are related to the cause too. The regulars of distribution of B.thuringiensis and altitude were summarize,that soil samples are rich in strains of B.thuringiensis between 800 and 1400 metres above sea level. In general, the frequent farming and forestry activity of human destroy vegetation and effect on structure of soil microbial community below 800 metres,at the same time it exhibited a decreasing tendency along with altitude ascension for loosening soil structure and litter humic substances.Part of strains have been identified by 16S rDNA sequence analyse .Several shapes of crystals including bipyramidal, square, round and oval-shap etc, were observed in the B.thuringiensis isolates,among them round-shape is very abundant. Forty-serven pairs of universal primer have designed for characterization of insecticidal crystal protein genes. The cry-type genes of 176 B.thuringiensis isolates were identified based on the PCR-RFLP methods. The results showed that 137 B.thuringiensis isolates harbored cry1 genes and produced 130kDa crystal proteins formed bipyramidal crystals;38 isolates with 60kDa proteins formed bipyramidal crystals too, harbored cry2 genes;6 isolates with 70kDa protein formed square crystals harbored cry3 genes;8 isolates with 130kDa or 80kDa proteins constructed bipyramidal crystals as well, contained cry4 genes or cry11 genes. The cry1Ac genes of strain 4b-1 with high-yielding the crystal proteins have been cloned partly, suggested that strain 4b-1 contains cry1 Ac gene with high toxicity to Lepidoptera insects.