Cloning Of Aminopeptidase N And Cadherin-Receptor Proteins For Bacillus Thuringiensis Toxin In Chilo Suppressalis (Walker) And Expression In Trichoplusia Ni Cells Line

The rice stem borer Chilo suppressalis (Walker) is a serious pest damaging on rice production in China. Recently, C. suppressalis has disserved more seriously year after year, because of the effect of unreasonally change cropping system and farming pattern, extensive planting of high yield and high quality rice, and suitable weather conditions, etc. The resistance of C. suppressalis to chemical insecticide has obviously increased and let it more difficult to be controlled, the emergence of Bt transgenic rice offer a new effective method to control the rice stem borer. But from the biological angle, the resistance of insect pests to transgenic crop is a kind of intimidated evolution phenomenon, after Bt transgenic rice has been cultivated in the field, the resistant C. suppressalis to Bt rice will inevitably produced. Therefore, to research the mode of action of Bt to C. suppressalis and the resistance mechanism will offer the academic basic for extending the transgenic Bt rice in the future, constituting the resistance manangement strategy and prolonging the using longevity of Bt rice. In this dissertation, the aminopeptidase N (APN) and cadherin-receptor for Bt toxin in C. suppressalis were studied.The full length genes of APN and cadherin were cloned using PCR and RACE methods, and these two receptor proteins were successfully expressed in Insect Baculovirus Expression System. The results were as follows:The full length genes of APN and cadherin were cloned using PCR and RACE methods. The APN was a member of aminopeptidase family by comparing analysed in data-base. The APN gene were accessed in GENEBANK and the accession number was DQ342305. The full length of APN was 3292 bp, encoding 1075 amino acids, having a 25 amino acids segment in the C-Terminal which had lipophicity and transmembrane characters. The accession number of cadherin was DQ821523. The full length of cadherin gene was 5148 bp, encoding 1715 amino acids, having 16 cadherin repeats, the deduced protein sequence was highly similar to that of Ostrinia nubilalis with 73.8% identity and other lepidopteran cadherin.As one of four expression systems, baculovirus-insect expression system has been widely utilized in synthesis of recombinant proteins. This system has many advantages, such as high yield regulated by polyhedron promoter, proper post-translation modification, easy of purification from culture cells, and lake of endotoxin contamination. After sequencing the gene, the fragment encoding for APN and cadherin without signal peptide were cloned into the Bac-to-Bac baculovirus expression system transfer vector pFastBacHTb under the polyhedron gene promoter. The recombinant transposing plasmid pFastBacHTb/APN (or pFastBacHTb/CAD) was screened and then transformed into E scherichia coli DH10Bac. It was cultured in LB plate, which contained Te~+, Kan~+, Ge~+, X-gal and IPTG. The resulting recombinant bacmid was transfected into cells of the insect Trichoplusia ni (Tn) and recombinant baculoviruse was obtained. The lysate of cells infected with recombinant baculoviruse was analyzed by SDS-PAGE and blot. The resuLts showed that the recombinant baculoviruse was fully capable of expressing APN and cadgerin, and both of these two proteins were receptors for Cry1 Ab.