Cloning And Functional Analysis Of Cytochrome P450 Genes From Mamestra Brassicae Linnaeus

In recent years, it is very serious on vegetables by Mamestra brassicae larvaes damaged. Now, pyrethroid insecticides is also the main control method of using chemical insecticides which is most widely used. The use of pyrethroid insecticide has led to the insecticide resistant. Cytochrome P450’s as one of the three detoxification enzymes in insect, At present, Gene families of. CYP4,CYP6 and CYP9 are the most important study which produce the insecticide resistance. Based on the M.brassicae as the research object and a full-length c DNAs of cytochrome P450 genes was cloned, according to two cytochrome P450 genes obtained by our laboratory to study the molecular characterization and the metabolism of deltamethrin resistance of three cytochrome P450 genes. The main results are as follows:1. The full length c DNA of CYP9A90 is 1828bp(Gen Bank accession No: KR676343), containing a 731 bp open reading frame, a 586 bp 5′ un-translate region, a 653 bp 3′un-translate region. Encoding 532 amino acid with a predicted molecular weight of 61.1 k Da and p I 8.84.2. Analysis the expression of M. brassicae in different organs in three cytochrome P450 genes. Our results displayed that CYP4M51 was observed in fatbodies, followed by the foregut and midgut; CYP6AB56 was mainly expressed in the midgut and fatbodies, CYP9A90 was relatively high in fatbodies. Analysis the expression of the three cytochrome P450 genes at different development stages of M.brassicae. Among the four stages from the 3rd to 6th instar larvas showed CYP4M51 and CYP6AB56 were relatively high in the 5th instar larvas, whereas CYP9A90 was relatively high in the 6th instar larvas.3. The different concentrations of deltamethrin were treated of M. brassicae, cytochrome P450 mRNA expression levels of genes showed the induced trend; CYP4M51 at the lower dose induced, at the concentration of LD10 in deltamethrin the expression in M. brassicae compared with the control, and with increase of concentration led to the induced lower; The induced more obvious with increase of concentration of CYP6AB56, the highest expression under the LD50 dose; CYP9A90 showed from inhibit to induce expressed highest of LD30 dose.After treatment at LD10 dose these P450 genes imparitied at different stress times. The expression of CYP6AB56 level from inhibition to induction, then to inhibition; CYP4M51 show from induction to inhibition which inhibition the expression in 48 h, the two genes both be seen that the highest induced expression appears in 12h; The expression of CYP9A90 level induced at the lower dose which expressed highest for 24 h.After the treated of different stages at the same dose of per weight at 12 h, CYP4M51 and CYP6AB56 were relatively high in the 5th instar larvas, whereas CYP9A90 was relatively high in the 6th instar larvas; It was same as the induced without deltamethrin at different stages.4. In order to study the metabolism of deltamethrin by silenced CYP4M51,CYP6AB56 and CYP9A90.After the treatment group were injected dsCYP4M51, dsCYP6AB56 and dsCYP9A90.The larva corrected mortalities treatment increased to 77%, while no significant rise in 48h; CYP4M51 were treated by deltamethrin which the mortalities increased 11.4%, CYP6AB56 were treated by deltamethrin which the mortalities increased 26.1%,while there was no significant difference after injection ds CYP9A90.