CYP6CX4 Gene Mediated Metabolic Resistance Of Flupyradifurone In Bemisia Tabaci And Related Regulation Mechanisms

Bemisia tabaci is a major agricultural pest worldwide and in a variety of cash crops.It causes widespread damage by feeding directly on crops,degrading crop quality,secreting honeydew and spreading plant viruses.B.tabaci is known for its genetic complexity and diversity,and is thought to be a complex of biological types,or to use the now widely accepted definition: a complex of different cryptic species.B.tabaci is an extremely difficult pest to control due to its adaptability,rapid reproduction and voracious feeding.At present,the control of B.tabaci mainly relies on the use of insecticides,among which neonicotinoids are the most important insecticides for the control of B.tabaci due to their low toxicity and environmental friendliness.However,it has rapidly developed resistance to most insecticides,posing a serious challenge to integrated pest management.It is believed that B.tabaci Q is more resistant to neonicotinoid insecticides than Bemisi B,which is one of the reasons why B.tabaci Q is gradually replacing Bemisi B.Cytochrome P450-dependent monooxygenases(P450)are the most widely studied detoxification enzymes in the resistance mechanism of B.tabaci to neonicotinoid insecticides.Multiple P450 genes have been reported to be involved in metabolic resistance of insects to a variety of insecticides,especially to neonicotinoids.At present,P450 gene has been widely reported to be regulated at pre-transcriptional level,transcriptional level or post-transcriptional level.The regulation of post-transcriptional level of insect P450 gene mainly focuses on non-coding RNAs,especially micro RNA(miRNA).In a previous study,we found that CYP6CX4 gene may also be involved in the resistance of B.tabaci to flupyradifurone.On this basis,this study took CYP6CX4 gene of B.tabaci Q type as the research object,and finally revealed the CYP6CX4 gene mediated metabolic resistance of flupyradifurone in B.tabaci and related regulation mechanisms.The main research is as follows:1.CYP6CX4 gene was successfully cloned,and its sequence was analyzed and compared: the full length of CYP6CX4 gene(open reading frame)was 1599 bp,encoding 533 amino acids,with typical DNA binding motifs of P450.Through phylogenetic tree construction,it was found that CYP6CX3 and CYP6CX1v1 converged with CYP6 family CX subfamily of B.tabaci.In addition to CYP6 family genes of Bethinia tabaci,CYP6CX4 is also associated with genes associated with classic nicotinoid insecticide resistance in other hemiptera insects.2.Quantitative Real-time PCR(qRT-PCR)was used to analyze the temporal and spatial expression pattern of CYP6CX4 gene.In the different development,the high expression of CYP6CX4 gene in the adult stage of B.tabaci may be related to detoxification metabolism after feeding.In the tissue expression profile,it is highly expressed in the chest and abdomen,which may be related to tissues related to detoxification metabolism,such as the midgut and fat body.3.Studies on the metabolic resistance of flupyradifurone mediated by CYP6CX4 in transgenic Drosophila: Tub Gal4 > UAS-CYP6CX4 transgenic Drosophila were constructed,and their PCR sequences were verified.qRT-PCR was used to verify the relative expression level of significantly high expression.Finally,the bioassay of flupyradifurone confirmed that CYP6CX4 gene overexpression mediated resistance to flupyradifurone.4.The regulatory mechanism of CYP6CX4 gene will be studied to predict the miRNA targeting CYP6CX4.The stability of 11 miRNAs in B.tabaci samples after treatment with 11 insecticides(pymetrozine,chlorpyrifos,imidacloprid,flupyradifurone,sulfoxaflor,flonicamid,cyantraniliprole,afidopyropen,avermectin,deltamethrin,and β-cypermethrin)was screened.And other factors(different developmental stages,different tissues,and different genders).