Investigation Of Novel Cry Genes Specific To Coleopteran Pests

Coleopteran pests caused heavy losses to agriculture and forestry every year. At present the main control measures being taken is the use of chemical pesticides. However, the obvious disadvantage of chemical pesticides is increasing: they brought out negative impact on the ecosystem; the residue of toxin is high; they were not safe to human and livestock and some of the pests have engendered resistance to them. In order to reduce pollution, increase the efficiency of control, scholars at home and abroad are exploring new technique and methods based on biological control. Bacillus thuringiensis (Bt) is one of the microbial pesticides that are widely used. The insecticidal activity of Bt is closely related to its insecticidal crystal protein (ICPs) coding by cry genes. Looking for new strains and cry genes with high toxicity to pests has been a hot spot in research. This work includes finding novel cry genes which are toxic to Coleopteran pests with two different methods:PCR-RFLP and Bioinformatics, at the same time the expression of these novel genes are also studied.1. 500 strains of Bacillus thuringiensis isolated in our laboratory were investigated. 43 strains which were insecticidal activity to larvae of cockchafers (Coleopteran, Scarabaeidae) were obtained. C9 was entomocidal to all the three species of cockchafers tested. Observation results of morphology indicated that all of these Bt strains contained crystals in spherical shape. SDS-PAGE analysis indicated that all these strains could express protein with 130 kD molecular mass. The study of cry gene-types of these 43 strains was carried out by PCR-RFLP method. Only cry8Ca type gene could be found in 21 strains, while both cry8Ca and cry8Ea type gene existed in 13 strains. Strain QCM may contain a novel cry8 gene for its unique PCR-RFLP patterns. Designed primers based on known cry8 genes, and cloned this gene cry8Xxx which named by International Nomenclature Committee of Bt.2. By Bioinformatics, three novel genes have been screened from strains NARC Bt17 and HD868 whose genome have been sequenced. These genes designated as cry61Xxx, cry7Xxx and cry8Ma3 are predicted to be toxic to coleopteran pests, and they are cloned successfully.3. These 4 genes above-mentioned are expressed in E. coli Rosetta (DE3) via expression vector pEB. SDS-PAGE analysis indicated that they were expressed protein of about 130kDa molecular mass successfully.4. cry8Xxx gene was transformed into a crystalliferous Bt strain HD73- by pSTK, a shuttle vector. Then a recombined strain designated as HD8X was obtained. SDS-PAGE analysis showed that the cry8Xxx gene was expressed in HD8X which can form spherical crystals.In conclusion, four cry genes were cloned via PCR-RFLP and bioinformatics methods in this work, and a new engineered strain HD8X was constructed. It enriches the gene resources of our country and provides a new approach for cry gene cloning, also it establishes a ground for application.