Cloning And Functional Analysis Of Genes Encoding RNases In Codling Moth

The codling moth(Cydia pomonella)is a member of the Lepidopteran family Tortricidae.Their larva has a strong adaptability and reproductive ability and feeds on fruits such as apples and pears.In China,they are mainly distributed in Xinjiang and Gansu province and have a tendency to expand to the east.However,our current conventional control strategies have little killing effect on codling moth,which constitutes a severe threat to the output,quality,and trade of fruits in China.RNA interference(RNAi)is a gene silencing technique that has emerged in recent years.In the field of agricultural pest control,the RNAi technique is used to kill the pests mainly by feeding a double-stranded RNA(dsRNA)containing food that silences the expression of key genes.However,this technique demonstrates different silencing efficiencies in different insects.For example,the efficiency is very poor in some lepidopterous insects such as codling moth,bombyx mori,and ostrinia furnacalis.The main reason for the low efficiency is due to degradation of a large amount of dsRNA by some ribonucleases(RNases).The purpose of this study is to investigate the relationship between the RNases and RNAi efficiency,and establish an RNAi system in codling moth for the purpose of killing the pests.Firstly,we screened out four genes RNase 1,RNase 2,RNase 3,and RNase 4 from the transcriptomic data of codling moth and cloned the genes successfully.Quantitative Real-time PCR results showed that the RNase1 and RNase4 genes were highly expressed in the midgut of the codling moth,the RNase3 gene was mainly expressed in the salivary gland,whereas the RNase2 gene showed differential expression in different tissues.Bioinformatic analysis showed that the sequences of these four RNase genes evolutionarily shared a certain conservation in Lepidopteran insects and encoded a common structural domain responsible for the cleavage and degradation of nucleic acid sequences.Secondly,degradation of double-stranded EGFP RNA(dsEGFP)by the RNases was evaluated in vitro.Data from dissecting experiments demonstrated that the tissue fluids from salivary glands,intestinal tract,and hemolymphs of codling moth possessed significant activity to degrade dsEGFP.Futhermore,we constructed four recombinant plasmids to prokaryotically expressed the four RNases.The supernatant of the bacterial lysate expressing RNase proteins was incubated with dsEGFP and then subjected to agarose gel electrophoresis analysis.The results showed that the lysate containing the RNase 2 protein has the function of degrading dsEGFP.Finally,we took the Inhibitor of apoptosis proteins(IAP)gene as an example to evaluate the RNAi efficiency of target genes when the expression levels of different RNases were downregulated.The results showed that when the expression of RNases was maintained at a normal level,no significant change was observed in the expression level of the IAP by injecting or feeding 1 ?g of IAP double-stranded RNA(dsIAP)into the codling moth.Subsequently,we injected synthesized small interference RNAs(siRNAs)of RNases into codling moth to evaluate the silencing effect of these siRNAs.The results showed that the expression level of RNase 1 was increased to 152%,and those of RNase 2,RNase 3,and RNase 4 were reduced to 31%,39%,11%compared to the control.Furthermore,after the RNase 2,RNase 3,and RNase 4 genes were knocked down,we fed the codling moth with an equal dose of dsIAP again and found that 2 d later,the expression level of IAP was reduced to 38%,28%,and 56%,respectively,2 d later,when compared to the control.These data indicated that silencing of genes RNase 2 and RNase 3 can effectively enhance the interference effect of feeding dsIAP.In summary,we analyzed the structure and function of RNase genes of codling moth,and proved that knockdown of RNase 2 and RNase 3 genes can significantly improve the interference effect of feeding dsRNA,thus establishing an effective gene silence system in codling moth.The system provides an important support for future research on the gene function and innovative strategies for prevention and control of codling moth,and also a reference for the gene silencing of other Lepidoptera insects.