Cloning And Characterization Of Cytochrome P450 Genes From Mythimna Separata

Mythimna separata which has a long history about its harm,has always been focused on its prevention and control in our country,the most effective control means is still chemical treatment and the pyrethroid insecticide is the most commonly used.The pyrethroid insecticides are widely used because of their high activity and low toxicity.However,this insecticides which is used in a large area and unscientifically by farmers,has led to insect resistance.Cytochrome P450 is involved in the detoxification of insects and its detoxification is the main cause of insect resistance.In this paper,two cytochrome P450 genes were cloned,the specificity distribution in different tissues and different stages,the inhibitory effect of PBO and the induced effect of the two genes by the beta-cyfluthrin were studied.The results are as follows:We got CYP9A112 and CYP9A113,which is named by International P450 Nomenclature Committee,the Gen Bank accession number is respectively Ky436738 and Ky436739.The full-length c DNA sequence of CYP9A112 is 1785 bp,with an open reading frame 1593 bp,which could encode 531 amino acids with a Molecular weight of about 61.2 k Da and pI of 9.06.The full-length c DNA sequence of CYP9A113 is 1752 bp,with an open reading frame 1587 bp,which could encode 529 amino acids with a Molecular weight of about 60.4 k Da and pI of 8.81.The amino acid of CYP9A112 and CYP9A113 all have four conserved domain,which are noted in the fig1 and fig2.Compared with the amino acid sequence of CYP9 A from other noctuidae insects,the amino acid of CYP9A112 and CYP9A113 respectively showed above 72% and 67% sequence identities and respectively showed the highest identities up to 85% and 78% with Mamestra brassicae(Gen Bank accession no.: AAR26518).Detected the expression of these two genes in the fifth instar larvae in five different tissues by Real-Time PCR(All age of Mythimna separata used in the test were the first day after molting).The results showed CYP9A112 was highly expressed in fatbody,followed by midgut and foregut,and a small amount of expression in the hindgut and Malpighian tubules was detected.CYP9A113 was highly expressed in fatbody and midgut,and a small amount of expression in the foregut and hindgut and Malpighian tubules was detected.At the same time,we detected the expression of these two genes in different stages by Real-Time PCR.We found the expression of these two genes all was increasing with age,but the expression of CYP9A112 reached the highest level in sixth instar,while the expression of CYP9A113 reached the maximum in fifth instar.Different concentration of beta-cyfluthrin were treated on the third instar larvae,and the results showed that CYP9A112 showed the induced trend,and the highest expression under thedose of LD10 after 12 hours,was 2.7 times higher than the control.CYP9A113 was expressed highest under the dose of LD30 after 24 hours,was 2.8 times higher than the control,and the gene was not obviously induced under the dose of LD50..The same per weight of beta-cyfluthrin was treated on different stages of Mythimna separata,after 12 hours we found that the expression of CYP9A112 was the highest in the sixth instar larvae and the expression of CYP9A113 reached the maximum in the fifth instar larvae.The result was the same with the control,and the two genes were all generally induced by the low dose(LD10、LD30).The Mythimna separata was inhibited by the highest non-lethal dose of Piperonyl butoxide(PBO).The expression of CYP9A112 reached lowest level after 12 hours,which was 0.36 times of the control.CYP9A113 was also inhibited and reached the lowest level after 24 hours,which was0.27 times of the control.