The Molecular Mechanism Of Cytochrome P450 Mediated Different Sensitivity Of Insecticides In B And Q Bemisia Tabaci

Bemisia tabaci(Gennadius)is a major agricultural invasive pest in the world.It causes crop damage by 30-50% annually and even destroys the crops totally.Based on the the monitoring results in our lab,it found that Q had replaced B B.tabaci in recent years,which was previously occupied the main niche.Previous studies had confirmed that the using pesticides were the key ecological mechanism leading to the replacement of B.B.tabaci by Q B.tabaci.At the same time,previous biochemical studies had shown that cytochrome P450 detoxifying enzyme activity was significantly higher in Q B.tabaci than in B B.tabaci.Thus,the study was fouced on the P450 genes.Many methods,including physiological,biochemical,cellular biology,molecular genetics and toxicology,were used and finally,the mechanism about the P450 family gene involing different sensitivity of insecticides in B and Q B.tabaci was revealed.The main conclusions were as follows:1.B and Q B.tabaci P450 gene family annotation: the numbers of B and Q B.tabaci P450 genes were 166 and 158,respectively,divided into 4 Clans.The phylogenetic tree showed that the Clan 3 and Clan 4 P450 genes were gene expansion,which may be related to the strong adaptive characteristics of B.tabaci.2.Different response of P450 gene in B and Q B.tabaci to imidacloprid: Quantitative PCR results showed that a total of 14 P450 genes expression levels were significantly higher in Q B.tabaci than in B.B.tabaci after treated with imidacloprid(CYP6JK1,CYP6DZ8,CYP6DZ9,CYP6DZ3,CYP6DW4,CYP6CM1,CYP4CS12,CYP4G128,CYP3133D4,CYP3133E1,CYP3133D3,CYP3133E8,CYP301A1,CYP18A1),which were the target genes for further functional analysis.3.Comparison of P450 expression levels in field B and Q B.tabaci: Quantitative PCR results showed that 9 P450 genes(CYP6CM1,CYP6EM1,CYP6DV5,CYP6CX3,CYP4CS5,CYP4CS12V2,CYP3133E4,CYP3133C1,CYP301B1)were overexpressed simultaneously in the 3 Q whitefly populations,only one P450 gene(CYP6CM1)was overexpressed in field B B.tabaci and the expression level of CYP6CM1 in B B.tabcai was lower than in Q B.tabaci.It indicated that the 9 P450 genes may plany an important role in the different sensitivity of insecticides in B and Q B.tabaci.4.Effect of RNAi on sensitivity to imidacloprid in B and Q B.tabaci: Based on the previous results and combined with the characteristics of the P450 gene family,9 P450 genes in CYP6 were choosed.The results showed that after feeding ds RNA for 48 h,the expression of 9 P450 genes was significantly reduced and the bioassay results showed that the mortality rate of B.tabaci Q increased significantly after knockdown the 9 P450 genes.However,the mortality rate was increased only after RNAi CYP6CM1 in B B.tabci.It indicated that the 8 P450 genes may play a role in the different sensitivity of insecticides in B and Q B.tabaci.5.NADPH cytochrome P450 reductase(CPR)study: the study used the PCR,RNAi,in vitro expression and enzyme activity to analyze the CPR function.The results showed that Bt CPR may affect the sensitivity of B.tabaci Q to imidacloprid.6.P450 protein expression and metabolism experiments in vitro: The results of carbon monoxide difference spectroscopy showed that 8 P450 genes were successfully expressed and further the results of metabolism experiments showed that only CYP6CM1 can metabolize imidacloprid,suggesting that the CYP6CM1 gene plays a key role in the resistance of B.tabaci to imidacloprid and it may involve in different sensitivity of insecticides in B and Q B.tabaci.7.CYP6CM1 polymorphism detection in B and Q B.tabcai: The results showed that the CYP6CM1 gene had 42 variants in the field Q B.tabaci population,while in the B B.tabaci,CYP6CM1 gene only had 12 variants.This indicated that the polymorphism of CYP6CM1 gene may affect the difference in insecticid sensitivity between B and Q B.tabaci.