Gene cloning,protein expression,and enzymatic characterization of a double-stranded RNA degrading enzyme in Apolygus lucorum
作者机构：College of Plant ProtectionYangzhou UniversityYangzhouJiangsu ProvinceChina Institute of Plant ProtectionJiangsu Academy of Agricultural SciencesNanjingJiangsu ProvinceChina Department of EntomologyTexas A&M UniversityCollege StationTXUSA Taizhou Customs of the People's Republic of ChinaTaizhouJiangsu ProvinceChina
出 版 物：《Insect Science》 (昆虫科学（英文版）)
年 卷 期：2024年第31卷第1期
页 面：119-133页
核心收录：
学科分类：0710[理学-生物学] 07[理学] 08[工学] 09[农学] 071007[理学-遗传学] 0901[农学-作物学] 0836[工学-生物工程] 090102[农学-作物遗传育种]
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)
主 题：Apolygus lucorum cloning dsRNase enzymatic activity protein expression
摘 要：RNA interference(RNAi)is a powerful tool that post-transcriptionally silences target genes in eukaryotic ***,silencing efficacy varies greatly among different insect ***,we met with little success when attempting to knock down genes in the mirid bug Apolygus lucorum via dsRNA *** disappearance of double-stranded RNA(dsRNA)could be a potential factor that restricts RNAi ***,we found that dsRNA can be degraded in midgut fluids,and a dsRNase of ***(AldsRNase)was identified and *** alignment indicated that its 6 key amino acid residues and the Mg2+-binding site were similar to those of other insects’*** signal peptide and endonuclease non-specific domain shared high sequence identity with the brown-winged green stinkbug Plautia stali *** 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 *** purified AldsRNase protein obtained by heterologously expressed can rapidly degrade *** comparing the substrate specificity of AldsRNase,3 specific substrates(dsRNA,small interfering RNA,and dsDNA)were all degraded,and the most efficient degradation is ***,immunofluorescence revealed that AldsRNase was expressed in the cytoplasm of midgut *** 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 *** and related species.
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