Cloning, Expression And Insecticidal Activity Of Novel Cry9 Genes From Bacillus Thuringiensis

Bacillus thuringiensis is the most widely applied type of microbial pesticides because its high specificity and environmental safety. Primarily insecticidal protein was Insecticidal Crystal proteins (ICPs) which were encoded by cry or cyt genes. cry9 genes were promising tools for effectively controling many species of important lepidopteran pests, because of the limitations of identify methods, there were not so many cry9 genes been reported, especially with high activity. In order to identify more cry9 genes, we introduced a method combining template preparation, PCR-HRMA that can applied for rapid identification of cry9 genes. The nucleotide sequences of the known cry9 genes were aligned, and an appropriate 100-bp region was located, we designed a pair of universal primers in this region, and then used light scaner to analysis PCR productions.In our method, cry9 genes can be identified on the basis of the differences in melting-peak curve shapes, which result from differences in DNA sequences of PCR products. The method was able to identify both known and novel different cry9-type genes in B. thuringiensis isolates. The DNA templates, SC5-D2 containing known cry9Aa and SC5-E8 containing known cry9Ea gene (detected by PCR), used to test the applicability of the method. In the PCR-HRMA of the identification of the 72 standard strains, three of them were observed had the corresponding 100-bp PCR products. According to melting-peak curves and sequencing, T03C001 showed sequence identity with cry9Aa, T03B001 contained one cry9Da gene and two novel cry9 genes, T23001 contained one cry9Da gene. In the PCR-HRMA of the identification of the 2000 isolates,273 of them were observed had the corresponding 100-bp PCR products and the melting-peak curves to be divided into 20 types. The subsequent sequence analysis indicated that SC5-D4 contained cry9Ba gene. According to known cry9 gene sequences, we designed full-length primers and successfully cloned them, they were designated as cry9Aa3, cry9Ea8, cry9Aa4, cry9Da3, cry9Da4, cry9Eb2, cry9Ee1, cry9Ba3 by International Nomenclature Committee of Bt. They were successfully expressed in E.coli (Rosetta)(except cry9Ba3) by pEB expression vector, expressed products were 130 kDa. Cry9Aa3, Cry9Aa4, Cry9Eb2, Cry9Ee1 had insecticidal activity against Plutella xylostella, the LC50 were 1.83μg/ml,1.90μg/ml,1.03μg/ml and 1.70μg/ml; they had insecticidal activity against CrylAc-R strains of Plutella xylostella, the LC50 were 1.39μg/ml,1.74μg/ml,1.24μg/ml and 1.45μg/ml. The CrylAc-R strains of Plutella xylostella exhibited no cross-resistance to the four Cry9 proteins. Cry9Aa3, Cry9Aa4, Cry9Eb2, Cry9Ee1 had insecticidal activity against Ostrinia furnacalis, the LC50 were 6.93μg/ml,1.06μg/ml,5.93μg/ml and 2.20μg/ml, they had a better inhibitory effect on body weight of Helicoverpa armigera. Cry9Ea8, Cry9Da3 and Cry9Da4 had no insecticidal activity against P. xylostella and O. furnacalis. All of them had no insecticidal activity against Colaphellus bowringi and Agrotis ypsilon.