| Diamondback moth(DBM),Plutella xylostella(L),is a worldwide major pest of cruciferous crops.So far,DBM had developed different levels of resistance to various insecticides.Chlorantraniliprole,owned a completely novel mode of action,was widely used to control the DBM.While high levels of resistance to this insecticide in DBM were reported just after 2-3 years extensive and repetitive application.The main mechanism of resistance was the point mutations on the insecticide target gene,Rynodine receptor(RyR).However,to date,there was a lack of in-depth research on the metabolic resistance mechanisms.This article mainly studied the role of P450 and UGTs in the metabolic resistance mechanisms of DBM against chlorantraniliprole and the results were briefly described as following:In this paper,the activities of three detoxification enzymes(carboxylesterase,P450 and glutathione S-transferase)in a susceptible population CHS,a laboratory selected resistant CHR and a field resistant population TH were detected.It was found that only the activity of cytochrome P450 was significantly higher than that in susceptible population in both resistant populations.They were 2.1-and 3.2-fold in CHR and TH population compared with that in CHS population,respectively.Then,the expression levels of 10 P450 genes were detected.CYP6BG1 was overexpressed in 5 tested resistant populations,especially in the TH population,which was 82-fold than that in the susceptible population.RNA interference technology could effectively silence CYP6BG1 gene.And the O-deethylation activity of 7-ethoxycoumarin of P450 was significantly reduced by 45.5%after interfered the gene.The mortality was significantly increased by 26.4%when the DBM larvae were treated with LC50 doses of chloramphenicol after RNAi.Moreover,the CYP6BG1 protein was expressed in Sf9 cell lines.The CYP6BG1 protein reduced the toxicity of chlorantraniliprole on Sf9 cells and P.xylostella larvae.Then,CYP6BG1 was specifically expressed in Drosophila melanogaster using the Gal4/UAS system.Overexpression of CYP6BG1 in D.melanogaster significantly increased the tolerance of fruit flies against chlorantraniliprole.For studying the mechanism of CYP6BG1 expression,about 1300bp 5’-flanking region of CYP6BG1 was obtained by chromosome walking method.Then a series of progressive 5’ deletion fragments of the CYP6BG1 promoter were used for constructing plasmids by subcloning into pGL4-basic vector and their promoter activity were detected using a luminescent reporter assay.The results showed that promoter constructs p(-152/+49),p(-340/+49)and p(-562/+49)had a higher luciferase activity than that of the promoterless control vector pGL4.Then a transcriptional factor FTZ-F1 was predicated and it was confirmed by RNAi technology that FTZ-F1 could regulate the expression of CYP6BG1.The inhibitors of UGTs,5-nitrouracil and sulfinpyrazone could significantly increase the toxicity of chlorantraniliprole to the third instars of P.xylostella.Then,the expression levels of 21 UGT genes in one susceptible and four resistant populations were compared by quantitative RT-PCR.The expression level of UGT33AA4 was significantly higher(from 30.7-to 77.4-fold)than that of susceptible population in all resistant populations.In addition,knockdown of the UGT33AA4 by RNAi,the mortality of DBM larvae caused by the LC50 chlorantraniliprole increased by 27.3%and 29.8%in CHS and CHR popupations,respectively.Previous study showed that overexpression of PxRyR was connected with chlorantraniliprole resistance in DBM.After knockdown of Dicer-1,a key gene in the process of mature miRNA formation,the expression of PxRyR was up-regulated,indicated that PxRyR might be regulated by miRNA.Then miR-7a and miR-8519 were predicted to target PxRyR by bioinformation software.In addition,the 3’-UTR of PxRyR was inserted into a pmirGLO vector to yield a recombined vector,pmirGLO-RyR.The firefly luciferase activity normalized to Renilla was significantly reduced after pmirGLO-RyR was co-transfected with agomir of miR-7a or miR-8519.Moreover,the expression of PxRyR decreased and the mortality of larvae caused by chlorantraniliprole increased when the larvae injected with the agomir of miR-7a or miR-8519.In contrast,the expression of PxRyR increased and the mortality of larvae caused by chlorantraniliprole decreased when the larvae injected with the antagomir of miR-7a or miR-8519.These results indicate that miR-8519 and miR-7a could be involved in chlorantraniliprole resistance by regulating the expression of PxRyR.In conclude,these fndings provide theoretical and practical significance for further revealing the molecular mechanism of resistance to diamide insecticides in lepidopteran pests and sustainable management of these insect pest. |
