CRISPR/Cas9 Mediated Gene Knockout Reveals The Involvement Of CYP304F1 In Β-cypermethrin And Chlorpyrifos Resistance In Spodoptera Litura

Spodoptera litura is a globally distributed omnivorous agricultural pest damaging 290 species ofthe host plant,which causes serious losses to agricultural production in China.At present,chemical control of S.litura is still the main way in agriculture.Pyrethroids and organophosphates have the advantages of high efficiency,broad-spectrum,and short residual period,which are widely used for the control of S.litura.However,the resistance of S.litura to pyrethroids and organophosphates is becoming more and more serious in some areas of China.The insecticide resistance of insects is closely related to the enhanced detoxification ability of metabolic enzymes.Cytochrome P450,as one of the most important detoxification enzymes in insects,has been widely reported to be involved in the metabolic resistance of insecticides.The P450 genes in insects are classified into six clans,namely CYP2,CYP3,CYP4,CYP16,CYP20,and mitochondrial（mito）clans.Among them,CYP16 and CYP20 clans only exist in some species of Apterygota and Paleoptera,and the specific functions remain enigmatic.CYP3 and CYP4 clans play a dominant role in insect resistance and have been widely reported to participate in the metabolism of xenobiotic compounds such as insecticides and plant allelochemicals through overexpression or induction.The functions of CYP2 and mito clans are generally considered to be relatively conserved in insects,mostly related to the biosynthesis and metabolism of endogenous compounds.Recent studies have proven that some genes in the CYP2 clan were also involved in the metabolism of insecticides and plant allelochemicals.Our previous study has found that CYP304F1 of the CYP2 clan as well as CYP6B47 and CYP6AE43 of the CYP3 clan were significantly overexpressed in the QJ population of S.litura resistant to pyrethroids and organophosphates.In this study,to further explore their role in insecticide metabolic resistance,the three candidates P450 genes in the QJ population were edited using CRISPR/Cas9technology,and they were successfully knockout and further confirmed by sequencing.However,only CYP304F1 knockout homozygotes strain was obtained in the subsequent screening.The susceptibility of CYP304F1 knockout homozygous strain to five insecticides,namelyβ-cypermethrin,fenvalerate,cyhalothrin,chlorpyrifos and phoxim,were detected by bioassay,revealing the role of CYP304F1 gene in the S.litura resistance to pyrethroids and organophosphates.The specific results are as follows:1.Bioinformatics analysis and the spatiotemporal expression of candidate P450 genes in S.lituraThe c DNA full-length of CYP304F1,CYP6B47 and CYP6AE43 are 1536 bp,1512 bp,and 1578bp,encoding 511,503,and 525 amino acids,respectively.The above three P450 genes all contain four typical conserved regions of P450 enzymes,including the C helix motif WXXXR,the K helix motif EXXR,the“meander”-binding sequences PXXF and the heme-binding motif FXXGXXXCXG.The phylogenetic tree analysis showed that CYP304F1 was categorized into the CYP2 clan,while CYP6B47 and CYP6AE43genes were categorized into the CYP3 clan.Sequence homology analysis showed that the CYP304F1 in S.litura had the highest sequence homology with CYP304F1 in Spodoptera exigua（ASO98028）and CYP304A1 in Spodoptera frugiperda（XP＿05056229）,with sequence similarity of 93.35%and 93.15%,respectively.CYP6B47 in S.litura showed the highest sequence homology with CYP6B48 in Spodoptera littoralis（AFP20587）and CYP6B68 in S.exigua（ASO98004）,with sequence similarity of 93.24%and86.48%,respectively.CYP6AE43 in S.litura had the highest sequence homology with CYP6AE43 in S.frugiperda（AID55427）and CYP6AE97 in S.exigua（ASO98010）,with sequence similarity of 88.38%and80.38%,respectively.To identify the expression characterizations of CYP304F1,CYP6B47 and CYP6AE43,the relative expression level of the above three genes in the RNA samples from the first,second,third,fourth,fifth and sixth instar larvae in QJ population,as well as the head,cuticle,foregut,midgut,hindgut,malpighian tubules and fat body of the fifth instar larvae,were analyzed by q RT-PCR.The results showed that CYP304F1 presented the highest expression level in the fifth instar larvae,and was highly expressed in the fat body and malpighian tubules,while the expression was relatively low in the midgut and hindgut.The relative expression of CYP6B47 was the highest in the first and fourth instar larvae,and it was highly expressed in the foregut and midgut,while the relative expression was the lowest in the fat body,cuticle and malpighian tubules.CYP6AE43 showed the highest expression level in the first and sixth instar larvae and was highly expressed in the foregut and midgut,with the lowest expression in the cuticle.2.CRISPR/Cas9 mediated gene knockout reveals the involvement of CYP304F1 inβ-cypermethrin and chlorpyrifos resistance in S.lituraPrimer pairs of CYP304F1,CYP6B47 and CYP6AE43 genes were designed online by Guide design resources-Zhang Lab.The concentrations of Cas9 protein and sg RNA were 300 ng/μL and 150ng/μL,respectively,which were mixed well and microinjected into the newly laid eggs within 2 h in the QJ population.The injected eggs were incubated carefully and the larvae developed into adults were named G0.Adults from G0 produced the G1 generation by mass-crossed.The knockout heterozygote of CYP304F1,CYP6B47 and CYP6AE43 was validated by sequencing.The heterozygote individuals were bred into adults,and their offspring were mass-crossed to obtain G2 generation.However,only CYP304F1 knockout homozygotes with a base-G deletion was detected in the G2 generation,and CYP6B47 and CYP6AE43knockout homozygotes were not detected.The CYP304F1 knockout homozygote larvae were carefully raised,and the G3 generation was obtained by mass-crossed.The G3 generation larvae were verified by mutation detection,and they were the base-G deficient CYP304F1 gene knockout homozygote population,which was named the QJ-CYP304F1 population.The susceptibility of S.litura to pyrethroids and organophosphates in QJ and QJ-CYP304F1 populations was measured by topical application.The results showed that the LD₅₀ of the QJ and QJ-CYP304F1 population toβ-cypermethrin were 38.61μg/larva and 21.36μg/larva,as well as 7.2μg/larva and 3.97μg/larva to chlorpyrifos,respectively.The LD₅₀ values toβ-cypermethrin and chlorpyrifos in QJ-CYP304F1 population were both 1.81-times than that in QJ population,while the LD₅₀values to fenvalerate,cyhalothrin and phoxim were not significantly changed,indicating that CYP304F1was involved in the metabolism resistance of S.litura toβ-cypermethrin and chlorpyrifos.3.Binding modes of CYP304F1 to pyrethroids and organophosphatesThe protein sequence of CYP304F1 was modeled by using the Swiss-model server and the most appropriate modeling template was selected from the Protein Data Bank.Autodock 4.2 was used for molecular docking.Molecular docking results showed thatβ-cypermethrin,cyhalothrin,fenvalerate,phoxim and chlorpyrifos were docked into the active site pockets of CYP304F1,and the binding free energies were-9.43,-5.64,-5.86,-6.38 and-7.20 Kcal/mol,respectively.Among them,the binding free energy ofβ-cypermethrin and chlorpyrifos with CYP304F1 was obviously lower than that of the other three insecticides,indicating the higher binding affinity between CYP304F1 toβ-cypermethrin and chlorpyrifos.These results were consistent with the conclusion that CYP304F1 was involved inβ-cypermethrin and chlorpyrifos resistance in S.litura.