Surver Of Insecticidal Protein Genes Of High Toxicity Bacillus Thuringiensis Agaist Lymantria Dispar And Establishment Of PCR Assay For Detecting Of Ldmnpv

Asian type of Lymantria dispar is a kind of omnivorous insect pest, the area it damaged is developing and spreading, which brought immense economic loses to the forest of China. Bacillus thuringiensis (Bt) and Lymantria dispar multiple-embedded nucleopolyhedrovirus (LdMNPV) are toxic to many insect pests. In this paper, rhombic parasporal crystal of high toxicity Bt stains against L. Dispar has been analyzed, the genotype of high toxicity protein were speculated; on the basic of the sequence of LdMNPV, a pair of primers was designed, then the PCR method was found to detection LdMNPV in a low level.In this paper four high toxicity Bacillus thuringiensis stains against L. dispar are selected from seven stains, they are LSB-1、LSB-2、LSB-6、LSB-7, LC50of these stains were1.77×107cfu/ml,1.84×107cfu/ml,1.37×107cfu/ml and2.95×107cfu/ml. Respectively at96h, they all have rhombic parasporal crystal.130kDa and60kDa proteins were the major components of Bt by SDS-PAGE, that is the recombinant protein of cry1and cry2. After amplification, the products were verified by double-enzyme cleavage method. Results showed that cry1gene fragments of LSB-1、LSB-2were1117、103、518; cryl gene fragments of LSB-6、LSB-7werel117、518; the cry2gene fragments of the four stains were791、297. Moreover, both cry1Aa and cry2Ab genes were presented in all of the high toxicity stains by PCR-RFLP.In this paper, a technical architecture used for microdetermination of the LdMNPV was set up successfully. Based on egt gene of LdMNPV, a pair of primers was designed with the sensitivity of1fg·mL-1DNA. By using this pair of primers, the partial sequences of egt gene were amplified from the eggs, larvae and pupae of the infected L. dispar, which verified the feasibility of this method. The partial sequences of egt gene were also amplified from the polyhedron suspension and the minimum amount of detection could be as low as5OBs·mL-1of polyhedron. Morphology research indicated that the surface of a few of polyhedrons were rough pitted and released virions. It could be one reason that the target DNA fragment could be amplified from the virus suspension. So in the detection of LdMNPV, suspension after trituration, filtration and centrifugalism can be used as template.