Cloning Tissue Expression And Function Research Of Cytochrome P450 Genes From Bombyx Mandarina

Cytochrome P450-dependent monooxygenase is a very important enzymatic system,which has been found in all living organisms systems examined,and these monooxygenases are able to metabolize a phenomenal number of endogenous and exogenous compounds.A lot of evidences indicated that insect cytochrome P450 monooxygenase mediated resistance is the main mechanism of the development of insecticide resistance and adaptability to host plants.Bombyx mandarina has the same source with Bombyx mori,and was prevented as a mulberry orchard pet for a long time.The adaptability to infaust environment and resistance to pesticide of Bombyx mandarina is far stronger than that of Bombyx mori.Studying the resistance of Bombyx mandarina can provide a fundament for elevating the resistance of Bombyx.mori,and also provide a evidence for curing pets of Lepidoptera.For researching the resistance to insecticide of Bombyx.mandarina.we studied Cytochrome P450 genes,and the main results were as follow:1 Constuetion of cDNA Library of Midgut Tissue from Bombyx mandarinaA cDNA library of midgut tissue from Bombyx mandarina M was constructed.The library qualification evaluation showed that the titer of primary cDNA library was 6.2×10~5 pfu/ml,and the average size of insert DNA was 1.2 kb..We can screen new cytochrome P450 genes and other genes relating to resistance from this library later.2 Cloning and tissue expression of cytochrome P450 genes from Bombyx mandarinaAccording to the predicted P450 sequences from the genome of Bombyx mori,seven pairs of primers were designed and seven cytochrome P450 genes were successfully cloned from the midgut cDNA of Bombyx mandarina by RT-PCR,and were respectively named as: CYP6AE8,CYP6AE9,CYP6AE21,CYP6AB5,CYP6AE22,CYP4M5,CYP 6A U1.The tissue expression researches showed that:these genes' expression level were different in tissues tested,and have higher expression level in midgut and fatbody.And the expression level of cypermethrin(5ng) induced larva in midgut and fatbody showed that:the express level of CYP4M5 of cypermethrin induced larva in midgut and fatbody were higher than that of control larva as 1.1 fold and 4.4 fold,respectively;the express level of CYP6AE22 of cypermethrin induced larva in midgut and fatbody were higher than that of control larva as 1.5 fold and 2.5 fold,respectively.After these experiments,we get two genes whose express level were higher after induced,and inferred that these two genes may be involved in resistance to insecticide;this can help us study the possible function of cytochrome P450 in resistance to insecticide to follow.3 Expression Bombyx.mandarina CYP6AE22 in Baculovirus-insect cell expression systemUsing the Bac-to-Bac baculovirus expression system,we set up the BmNPV-CYP6AE22 expression system in insect cell.And express CYP6AE22 enzyme successfully.This can be foundation for constructing in vitro recombinant enzymatic systems to study CYP6AE22-mediated xenobiotics metabolism and finally confirm their functions.