The Cloning Of Cytochrome P450 Gene And Induction Of P450s By Phenobarbital From Ostrinia Furnacalis (Guenée)

This research mainly studies various effective preparation methods of Ostrinia furnacalis genome DNA as well as cloning and array analysis, and discusses the induction characteristic of Phenobarbital on Cytochrome P450 gene.The Asian corn borer, Ostrinia furnacalis (Guenee), as one of the key agricultural pests all over the world, has imperiled agricultural production seriously. Cytochrome P450, as one of the crucial enzyme department existing in organism extensively, has gained close attention for its ability to metabolize and plant more endogenous and other source materials. The results are as follows:1. Genome DNA extraction of Ostrinia furnacalis larvaeExtracting Genome DNA of Ostrinia furnacalis larvae through SDS-Pk, CTAB and gene box, the results show that high quality of DNA sample could be gained with the three methods for fresh larvae, while the quality of DNA sample different among the three methods for larvae kept in anhydrous ethanol.2. Cloning and analysis of fragments of Cytochrome P450 geneDesigning a pair of degenerate primers according to the conservative amino acid sequences of insect P450 from Genbank, with the genomic DNA of Ostrinia furnacalis field natural population as the template, a specific DNA fragment with 603bp, encoding 201 amino acid, is obtained. Homologous analysis on the deduced amino acid sequence with Blast and ClustalW EBI softwares demonstrates that the sequence is as high as 37~53% identity to members of CYP4 family, and suggests that the sequence belongs to CYP4 famliy.3. Induction of microsomal cytochrome P450 and b5 by Phenobarbital in Ostrinia furnacalisSrcibbling 2mg/g Phenobarbital solution in corn borer larval body well, then dissecting the check and sample and mensuating the quality of P450 and b5 in body well, midgut as well as fat body, the results indicate that Phenobarbital has complex induction mechanism for different induction function on different components of P450 enzyme department. Phenobarbital has no obvious induction functions on cytochrome b5. However, Phenobarbital induces various content of cytochrome P450 on different tissue as well as for different treated time. These different effects maybe result from the different time required during the running, accumulation of Phenobarbital and expression of P450 in larval body.