RNA interference(RNAi)is a powerful tool that post-transcriptionally silences target genes in eukaryotic cells.However,silencing efficacy varies greatly among different insect species.Recently,we met with little succe...RNA interference(RNAi)is a powerful tool that post-transcriptionally silences target genes in eukaryotic cells.However,silencing efficacy varies greatly among different insect species.Recently,we met with little success when attempting to knock down genes in the mirid bug Apolygus lucorum via dsRNA injection.The disappearance of double-stranded RNA(dsRNA)could be a potential factor that restricts RNAi efficiency.Here,we found that dsRNA can be degraded in midgut fluids,and a dsRNase of A.lucorum(AldsRNase)was identified and characterized.Sequence alignment indicated that its 6 key amino acid residues and the Mg2+-binding site were similar to those of other insects’dsRNases.The signal peptide and endonuclease non-specific domain shared high sequence identity with the brown-winged green stinkbug Plautia stali dsRNase.AldsRNase showed high salivary gland and midgut expression and was continuously expressed through the whole life cycle,with peaks at the 4th instar ecdysis in the whole body.The purified AldsRNase protein obtained by heterologously expressed can rapidly degrade dsRNA.When comparing the substrate specificity of AldsRNase,3 specific substrates(dsRNA,small interfering RNA,and dsDNA)were all degraded,and the most efficient degradation is dsRNA.Subsequently,immunofluorescence revealed that AldsRNase was expressed in the cytoplasm of midgut cells.Through cloning and functional study of AldsRNase,the enzyme activity and substrate specificity of the recombinant protein,as well as the subcellular localization of nuclease,the reason for the disappearance of dsRNA was explained,which was useful in improving RNAi efficiency in A.lucorum and related species.展开更多
The efficiency of RNA interference(RNAi)varies substantially among different insect species.Rapid degradation of double-stranded RNA(dsRNA)by dsRNA-degrading nucleases(dsRNases)has been implicated to cause low RNAi ef...The efficiency of RNA interference(RNAi)varies substantially among different insect species.Rapid degradation of double-stranded RNA(dsRNA)by dsRNA-degrading nucleases(dsRNases)has been implicated to cause low RNAi efficiency in several insect species.In this study,we identified four dsRNase genes(OfdsRNaseL Ofd-sRNase2,OfdsRNase3 and OfdsRNase4)from the Asian corn borer(Ostrinia furnacalis)transcriptome database.Bioinformatic analyses showed that each deduced protein sequence contained endonuclease NS domains and signal peptides.Gene expression analysis revealed that OfdsRNase2 was exclusively expressed in the midgut of larvae.RNAi efficiency was investigated in 2-d-old fifth-instar larvae(high expression of dsRNase2)and 2-d-old pupae(low expression of dsRNase2)by feeding or injecting dsRNA targeting a marker gene that encodes the lethal giant larvae protein(OfLgl).Our results showed that OfLgl only partially silenced the expression of OfLgl in pupae,but not in larvae,suggesting that OfdsRNase2 could contribute to lower RNAi efficiency in larval stages.This hypothesis was supported by our RNAi-of-RNAi experiment using a tissue culture technique where the silencing efficiency against the reporter gene,OfHexl,was significantly improved after knockdown of OfdsRNase2.When double luciferase assays were performed to evaluate the role of the four dsRNases in vitro,only OfdsRNase2 expressed in S2 cells significantly affected RNAi efficiency by degrading dsRNA.Taken together,our results suggested that the degradation of dsRNA by OfdsRNase2 in the midgut contributed to low RNAi efficiency in O.furnacalis larvae.展开更多
基金This study was supported by Jiangsu Agricultural Science and Technology Innovation Fund[CX(21)3088,CX(22)2038]Jiangsu Province Key R&D Program(Modern Agriculture)Project:Surface Project(BE2021303).
文摘RNA interference(RNAi)is a powerful tool that post-transcriptionally silences target genes in eukaryotic cells.However,silencing efficacy varies greatly among different insect species.Recently,we met with little success when attempting to knock down genes in the mirid bug Apolygus lucorum via dsRNA injection.The disappearance of double-stranded RNA(dsRNA)could be a potential factor that restricts RNAi efficiency.Here,we found that dsRNA can be degraded in midgut fluids,and a dsRNase of A.lucorum(AldsRNase)was identified and characterized.Sequence alignment indicated that its 6 key amino acid residues and the Mg2+-binding site were similar to those of other insects’dsRNases.The signal peptide and endonuclease non-specific domain shared high sequence identity with the brown-winged green stinkbug Plautia stali dsRNase.AldsRNase showed high salivary gland and midgut expression and was continuously expressed through the whole life cycle,with peaks at the 4th instar ecdysis in the whole body.The purified AldsRNase protein obtained by heterologously expressed can rapidly degrade dsRNA.When comparing the substrate specificity of AldsRNase,3 specific substrates(dsRNA,small interfering RNA,and dsDNA)were all degraded,and the most efficient degradation is dsRNA.Subsequently,immunofluorescence revealed that AldsRNase was expressed in the cytoplasm of midgut cells.Through cloning and functional study of AldsRNase,the enzyme activity and substrate specificity of the recombinant protein,as well as the subcellular localization of nuclease,the reason for the disappearance of dsRNA was explained,which was useful in improving RNAi efficiency in A.lucorum and related species.
基金supported by National Natural Science Foundation of China(Grant No.31730074,31901953)2017 Special Talents Projects in Shanxi Province,China(201705D211027)Key Research and Development Program of Shanxi Province(201803D221004-5).
文摘The efficiency of RNA interference(RNAi)varies substantially among different insect species.Rapid degradation of double-stranded RNA(dsRNA)by dsRNA-degrading nucleases(dsRNases)has been implicated to cause low RNAi efficiency in several insect species.In this study,we identified four dsRNase genes(OfdsRNaseL Ofd-sRNase2,OfdsRNase3 and OfdsRNase4)from the Asian corn borer(Ostrinia furnacalis)transcriptome database.Bioinformatic analyses showed that each deduced protein sequence contained endonuclease NS domains and signal peptides.Gene expression analysis revealed that OfdsRNase2 was exclusively expressed in the midgut of larvae.RNAi efficiency was investigated in 2-d-old fifth-instar larvae(high expression of dsRNase2)and 2-d-old pupae(low expression of dsRNase2)by feeding or injecting dsRNA targeting a marker gene that encodes the lethal giant larvae protein(OfLgl).Our results showed that OfLgl only partially silenced the expression of OfLgl in pupae,but not in larvae,suggesting that OfdsRNase2 could contribute to lower RNAi efficiency in larval stages.This hypothesis was supported by our RNAi-of-RNAi experiment using a tissue culture technique where the silencing efficiency against the reporter gene,OfHexl,was significantly improved after knockdown of OfdsRNase2.When double luciferase assays were performed to evaluate the role of the four dsRNases in vitro,only OfdsRNase2 expressed in S2 cells significantly affected RNAi efficiency by degrading dsRNA.Taken together,our results suggested that the degradation of dsRNA by OfdsRNase2 in the midgut contributed to low RNAi efficiency in O.furnacalis larvae.
