Functional Characterization Of Detoxification Genes Related To Deltamethrin Resistance In Small Brown Planthopper,Laodelphax Striatellus (FALL(?)N)

Understanding resistance mechanisms is essential to the management of pest and is also key for pest control.Although pests have different genetic background,the resistance mechanism for insecticides is relatively conservative among various species.Nowadays,three major resistance mechanisms have been revealed as:body-wall penetration rate reduction,detoxification metabolism ability enhancement and target enzyme insensitive alternation.For small brown planthopper,little studies on body-wall penetration rate reduction have been studied and reported,perhaps because of its little importance revealed in different insects.So,resistance mechanism mainly involves the other two aspects for small brown planthopper.In previous research,the overexpressions of 6 up-regulated detoxification genes were confirmed by qRT-PCR in deltamethrin-resistant strain.For these six genes,CYP314A1v2,CYP353D1v2,CYP439A1v3,CYP6AY3v2,CYP6FU1 and LSCE12,the expression ratios were found 2.36-,5.33-,4.68-,24-,16-and 11.1-fold higher in deltamethrin-resistant strain than that in sensitive-strain.In this study,we try to find the relationship between the up-regulated detoxification genes and the high level deltamethrin resistance by using RNA interference technology and insect baculovirus expression system.These studies will provide a theoretical basis to clarify the evolution mechanism of insecticide resistance in L.striatellus and draw up effective control measures.The main works are summarized as following:1.Functional analysis of related detoxification genes with RNAi in L,striatellus In this study,the role of related genes in deltamethrin resistance was checked by silencing each of these genes.First,the key conditions for RNA interference in small brown planthopper were optimized,and feeding with the liquid artificial diet with a concentration of 50ng/μl dsRNA for seven days is proper.The interference results indicated that dsRNA feeding depressed the expression levels of CYP314A1v2,CYP353D1v2,CYP439A1v3,CYP4G76,CYP6AY3v2,CYP6FU1 and LSCE12 dramatically(by 51.1%,49.6%,57.3%,51.1%,59.6%,49.1%and 35.3%,respectively)as compared with the control.This indicated that dsRNA feeding silenced these target genes effectively.Furthermore,the healthy female survivals of dsRNA feeding were topically treated with 200 ng of deltamethrin and the mortality was checked after 24 h.The results showed that there were no significant differences in mortality between CK and the treatment feeding with dsGFP or with dsRNA of only one related gene.However,higher mortalities were observed in the groups that fed the diet mixed with several kinds of dsRNAs of different related genes.It indicated that these genes did play a role in deltamethrin resistance,although the impact of a single gene was not obvious.The conclusion came to that insecticide resistance could be resulted only from the overexpression of multiple resistance related genes found previously in this pest.2.Expression of related cytochrome P450s and their founction analysisBased on pFastBacHTA,three recombinated vectors pFastBacHTA-CYP6AY3v2、pFastBacHTA-CYP6FU1 and pFastBacHTA-CPR were constructed,and two cytochrome P450s and a NADPH-cytochrome P450 reductase were expressed in sf9 cells with insect baculovirus expression system.Western blot showed the expected molecular size of CYP6AY3v2,CYP6FU1 and CPR,which were 61kDa,57kDa and 79 kDa,respectively.Then,the enzyme activity of recombinant proteins was checked.The results showed that the enzyme activity of the recombinant proteins for CYP6AY3v2,CYP6FU1 and CPR was 7.9-,11.3-and 5.0-fold higher than the control.Finally,deltamethrin metabolism was assayed by incubation with the recombinant proteins and the results revealed that the tested CYP6AY3v2 and CYP6FU1 both can metabolize deltamethrin to 4-hydroxy deltamethrin.Accordingly,it was thought that the overexpression of CYP6AY3v2 and CYP6FU1 might be involved in deltamethrin resistance in L.striatellus.This work has optimized the conditions for RNAi with dsRNA feeding in S striatellus,and found a proper way to obtain good results.Checking different genes found that a single gene had little effect on insecticide sensitivity,but the combination of multiple of these related genes identified previously could alter insecticide resistance significantly.Functional expression revealed that the tested CYP6AY3v2 and CYP6FU1 both can metabolize deltamethrin to 4-hydroxy deltamethrin,which proved that the over-expression of these two might contribute insecticide resistance in L,striatellus.These results should be significant for understanding the insecticide resistance development and management.