Study On RNAi Efficiency Of Double-Stranded Ribonuclease(dsRNase) In Apolygus Lucorum(Meyer-D(?)r)

Green Mirid Bug(Apolygus lucorum)is an important worldwide agricultural pest with wide distribution,serious damage and complicated feeding habits.It damages the juice of young parts by pricking and sucking,resulting in a significant decrease in the yield and quality of economic crops.At present,chemical pesticides are the main control measures.This technology will cause problems of pesticide residues,environmental pollution and safety.In recent years,RNA interference(RNAi)technology of double-stranded RNA(dsRNA)has been successfully applied in pest control,which provides a new control strategy and theoretical basis for pest control.However,dsRNA is highly susceptible to degradation.How to improve the stability of dsRNA is the key to breakthrough the inefficiency of RNAi technology.Double-stranded ribonuclease(dsRNase)is an endonuclease in insects that can degrade dsRNA.The activity of this enzyme is the key to improve the efficiency of RNAi in insects.This paper takes A.lucorum as research object,explores the enzymatic properties of dsRNase,clarifies the degradation of dsRNase on dsRNA,clarifies the optimal degradation of dsRNA conditions by dsRNase,and reveals its effect on the RNAi efficiency of housekeeping genes.The results can enrich the molecular mechanism of insect RNAi and help to broaden the practical application of RNAi technology in pest control.The research results presented in this paper are mainly as follows:1.Cloning,Spatio-temporal expression analysis and tissue localization of double-stranded ribonuclease gene in A.lucorumBased on the laboratory transcriptome data of A.lucorum,a double-stranded ribonuclease gene AldsRNase(GenBank accession number:ON973858)was cloned by RACE technology.The full-length open reading frame was 1227 bp,encoding 408 amino acids(aa).The predicted molecular weight of ExPASy was 44.7 kD and theoretical isoelectric point was 8.71.The gene contains a 19 aa signal peptide,a Mg2+binding site(N269),three substrate binding sites(A236,R237,R281)and six key amino acid residues(A236,R237,H239,N269,E277,R281).It contains a non-specific endonuclease(Endonuclease NS,pfam01223,135-382 aa)domain with the typical characteristics of DNA/RNA non-specific endonuclease,which can be used to cleave and degrade nucleic acid sequences.Amino acid sequence alignment showed that AldsRNase had the closest relationship with PsdsRNase from Plautia stali(Hemiptera:Miridae),with the highest sequence identity of 52.62%,and identity with homologous genes of other insects ranging from 30%to 40%,such as Bemisia tabaci(BtdsRNase,35.22%)in Hemiptera,and Diabrotica virgifera virgifera(DvdsRNase,33.16%)in Coleoptera belongs to lower genetic level.Phylogenetic tree analysis showed that the dsRNase of insects in Hemiptera,Orthoptera and Hymenoptera belonged to different branches.AldsRNase had the closest evolutionary relationship with PsdsRNase,which may belong to the same ancestor.Real-time quantitative PCR(qRT-PCR)was used to analyze the spatio-temporal expression of AldsRNase in A.lucorum.AldsRNase was continuously expressed in the life cycle from 1 to 16 days of age,and the mRNA expression level showed a fluctuating overall upward trend.The expression level in the last instar was generally higher than that in the initial instar,and reached the peak at the 13th day(i.e.,the 4th instar molting).In addition,the expression of AldsRNase was tissue-specific in different tissues,with high expression in salivary glands and midgut,and low expression in the cuticle,foregut and hindgut.Subcellular localization of the double-stranded ribonuclease protein by immunofluorescence found that AldsRNase was distributed and expressed in the midgut cytoplasm.2.Study on the enzymatic characteristics of double-stranded ribonuclease and its influence on RNAi effect in A.lucorumTo clarify the enzymatic characteristics of double-stranded ribonuclease in A.lucorum,the purified fusion protein AldsRNase by heterologous expression of insect baculovirus cell protein expression system.By incubating dsRNA gel electrophoresis in vitro,AldsRNase could degrade dsRNA effectively,and the optimum pH and temperature of its enzyme activity were 5 and 30℃,respectively.To clarify the substrate specificity of double-stranded ribonuclease in A.lucorum,dsGFP was used as the tested object.It was found that the fusion protein AldsRNase had the function of degrading dsRNA,siRNA and dsDNA,and the degradation efficiency of dsRNA was the highest.At the same optimum pH=5 and temperature 30℃,the recombinant protein AldsRNase only requires 10 ng that can completely degrade GFP of dsRNA,While GFP of siRNA(1000 ng)and dsDNA(100 ng)for completely eliminating need 200 ng and 100 ng,respectively.In this study,ultraspiracle protein(AlUSP)was used as a target gene to detect the transcription level of AlUSP when the expression level of AldsRNase decreased,and analyze the RNAi efficiency of AlUSP.Compared with the control group treated with dsGFP,dsAldsRNase treatment significantly reduced the content of dsRNase in A.lucorum and enhanced the stability of dsGFP in midgut fluid.The artificial synthetic dsRNA mixture(dsAldsRNase+dsAlUSP)was injected into A.lucorum,and the knockout efficiency of USP was detected after 1 d,2 d,3 d and 4 d.Compared with dsUSP treatment group,the interference efficiency of USP was increased by 63.79%,52.70%and 60.00%after 2 d,3 d and 4 d,respectively;Meanwhile,the lethal rate of A.lucorum was significantly increased by 26.67%and the fresh larvae weight was decreased by 15.49%after 4 d of dsRNAs treatment.In summary,the full-length ORF sequence of AldsRNase from A.lucorum,with typical characteristics of DNA/RNA non-specific endonuclease,was cloned and identified for the first time in this study;AldsRNase showed developmental specificity and tissue specificity in spatio-temporal expression analysis;At the same time,a polyclonal antibody against AldsRNase was successfully prepared,and AldsRNase was highly expressed in the midgut cytoplasm by immunofluorescence analysis;The recombinant protein systematically expressed in eukaryotic cells is obtained,and the physiological pH,temperature,time and the like regulate the stability of dsRNA by affecting the activity of AldsRNase,and have wide degradation effects on substrates dsRNA,siRNA and dsDNA;After exogenous RNAi treatment,the expression of AldsRNase is inhibited,thereby improving RNAi efficiency.