| The small brown planthopper(SBPH),Laodelphax striatellus(Fallen)belonging to Delphacidae of Hemiptera,is a major pest on rice.Because of the unreasonable use of chemical pesticides,the SBPH has developed resistance to a variety of insecticides.Understanding the resistance mechanism is the basis of resistance management and pest control.In the previous work,the resistance strains of SBPH to deltamethrin,chlorpyrifos and imidacloprid were established by insecticide selection,respectively.And several detoxification enzymes were identified by comprarison the expression levels between the resistance and susceptible strains.Among the highly expressed genes,both CYP6AY2v2 and CYP353D1v2 were significantly up-regulated in three SBPH resisitance strains.Therefore,CYP6AY2v2 and CYP353D1v2 were transferred into the Drosophila and overexpressed by insect transgenesis.The genes functions were analyzed,and to understand the mechanism of metabolic resistance.The results of the study were summarized as follows:1.Construction the transgenic Drosophila and target gene expression analysisConstruction of transgenic vectors of pUAST-attB-CYP6A Y3v2 and pUAST-attB-CYP355D1v2.The vectors were introduced into Drosophila embryos by micro injection respectively.Two pure transgenic strains of UAS-CYP6A Y3v2 and UAS-CYP353D1v2 were successfully obtained by screening and purification.The tub-gal4 virgin flies with strong ?-tubulin promoter were crossed with the UAS-CYP6AY3v2 and UAS-CYP353D1v2 respectively.The offsprings were the transgenic flies of tub>CYP6AY3v2 and tub>CYP353D1v2,which overexpressed SBPH genes of CYP6AY3v2 and CYP353D1v2 in the whole body.The expression levels of target genes in transgenic flies of tub>CYP6AY3v2 and tub>CYP353D1v2 were determined by real time PCR.The results showed that the expression levels of target genes in strains of tub>CYP6AY3v2 and tub>CYP353D1v2 were 65.42 and 60.24 times higher than expression levels in the control strains of UAS-CYP6AY3v2 and UAS-CYP353D1v2,respectively.At the same time,the expression levels of endogenous CYP genes in Drosophlia with highly similaries with target genes were also detected.The results showed that the endogenous CYP genes expression levels were significantly lower than that of the target genes,and there was no significant expression differences(P>0.05)between the overexpression target genes flies and control flies.Therefore,the strains which are overexpression target genes of SBPH CYP6AY3v2 and CYP353D1v2 were successfully established.And the endogenous CYP genes with higly similarities to target genes won't interfere with the function analysis of target genes.2.Determination of the susceptibilities of transgenetic flies to different insecticidesIn order to explore the function of genes CYP6AY3v2 and CYP353D1v2,the susceptibilities of transgenic flies(tub>CYP6AY3v2 and tub>CYP353D1v2)and the control flies(UAS-CYP6AY3v2,UAS-CYP353D1v2)to four insecticides(chlorpyrifos,deltamethrin,imidacloprid and abamectin)were determined by bioassay.Bioassay results showed that the transgenic flies(tub>CYP6AY3v2)was more resistant to different concentrations of imidacloprid than that of control flies,and the resistance ratio was 1.54.However,the transgenic flies(tub>CYP6AY3v2)showed no senstivity differences to chlorpyrifos and deltamethrin.The results aslo showed the resistances of transgenic flies(tub>CYP353D1v2)to imidacloprid and abamectin were significantly increased compared to control flies,and the resistance ratios were 10.35 and 14.13 respcetively.The resistances of transgenic flies(tub>CYP353D1v2)to deltamethrin and chlorpyrifos were also improved,the resistance ratios were 2.02 and 1.56,respectively.Therefore,the up-regulation of expression levels of CYP6AY3v2 gene was one of the mechanisms of imidacloprid resisitance of SBPH.The up-regulation of expression levels of CYP353D1v2 gene was one of the mechanisms of insecticide resisitance(imidacloprid,abamectin,deltamethrin and chlorpyrifos)to SBPH,and also was the mechanisms of cross-resistance.In conclution,correlation between the overexpression of CYP6AY3v2 and CYP353D1v2 in SBPH and the insecticide detoxification metabolism was verified,and transgenenic Drosophila technical platform were established. |
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