中蜂囊状幼虫病毒RdRp基因dsRNA干扰的研究
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摘要
中蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)可以引起中蜂幼虫死亡,给中蜂的养殖造成严重的威胁。其RNA依赖的RNA聚合酶(RNA-dependent RNA polymerase,RdRp)是病毒复制中必不可少的中心酶,控制着病毒的复制和翻译过程。本研究以CSBV RdRp基因为靶标,选取两个干扰区域RdRp-1和RdRp-2,并构建相应的dsRNA表达载体,获取dsRNA后进行RNAi实验,通过qRT-PCR检测CSBV RdRp基因的表达情况。实验结果显示:干扰片段RdRp-1不能显著下调RdRp基因的转录,而干扰片段RdRp-2可显著性下调RdRp表达并具有剂量依赖性,当添食2μg dsRdRp-2时,在72 h RdRp基因表达下调了85%,CSBV的衣壳蛋白VP1基因下调表达78%,幼虫死亡率降低60%,表明RdRp基因可以作为RNA干扰的靶标用于CSBV防治,本研究为后期在养蜂场进行蜜蜂病毒病的防治奠定了基础。
The Chinese sacbrood virus(CSBV) is the causative agent of cystic larval diseases in bees. RNA-dependent RNA polymerase(RdRp) is an essential enzyme in viral replication. In the present study, two fragments covering motif A and motif D of RdRp were cloned into the L4440 vector to express dsRNA(dsRdRp-1 and dsRdRp-2) in Esherichia coli HT115. dsRNA was extracted for RNAi experiments and quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR) employed to detect expression of the RdRp gene. Results showed that dsRdRp-2 could down-regulate RdRp expression significantly, whereas dsRdRp-1 could not. Furthermore,silencing of dsRdRp-2 was dose-dependent: the more dsRdRp-2, the better silencing of the RdRp gene. If the amount of dsRdRp-2 was 2 μg, the RdRp gene was down-regulated by 85% after feeding dsRNA for 3 days, the VP1 gene was down-regulated by 78%, and larval mortality was reduced by 60%. These results demonstrate that the RdRp gene can serve as an RNAi target for the prevention of Chinese sacbrood virus disease. Our study provides a new strategy for the control of bee virus disease in the apiculture in future.
The Chinese sacbrood virus(CSBV) is the causative agent of cystic larval diseases in bees. RNA-dependent RNA polymerase(RdRp) is an essential enzyme in viral replication. In the present study, two fragments covering motif A and motif D of RdRp were cloned into the L4440 vector to express dsRNA(dsRdRp-1 and dsRdRp-2) in Esherichia coli HT115. dsRNA was extracted for RNAi experiments and quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR) employed to detect expression of the RdRp gene. Results showed that dsRdRp-2 could down-regulate RdRp expression significantly, whereas dsRdRp-1 could not. Furthermore,silencing of dsRdRp-2 was dose-dependent: the more dsRdRp-2, the better silencing of the RdRp gene. If the amount of dsRdRp-2 was 2 μg, the RdRp gene was down-regulated by 85% after feeding dsRNA for 3 days, the VP1 gene was down-regulated by 78%, and larval mortality was reduced by 60%. These results demonstrate that the RdRp gene can serve as an RNAi target for the prevention of Chinese sacbrood virus disease. Our study provides a new strategy for the control of bee virus disease in the apiculture in future.
引文
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[9] Zhang J, Zhang Y, Han R. The high-throughput product ion of dsRNA against sac brood virus for use in the honey bee Apis cerana(Hymenoptera:Apidae)[J]. Virus Genes,2016, 52(5):698-705.
[10]丁清泉.RNA病毒RNA聚合酶的研究进展[J].中国病毒学,1998, 13(2):107-113.
[11] Timmons L, Court DL,Fire A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans[J]. Gene,2001, 263(1-2):103-112.
[12] Yu N, Christiaens O, Liu J, Niu J, Cappelle K, Caccia S,Huvenne H,Smagghe G. Delivery of dsRNA for RNAi in insects:an overview and future directions[J].Insect Sci,2013, 20(1):4-14.
[13]Aronstein,K A,Pankiw T,Saldivar E. SID-1 is implicated in systemic gene silencing in the honey bee[J]. J Apicult Res,2006,45(1):20-24.
[14] Ganbaatar O, Cao B, Zhang Y, Bao D, Bao W, Wuriyanghan H. Knockdown of Mythimna separata chitinase genes via bacterial expression and oral delivery of RNAi effectors[J]. BMC Biotechnol, 2017, 17(1):9-19.
[15] Pan Z H,Gao K,Hou C X,Wu P,Qin G X,Geng T,Guo X J. dsRNA interference on expression of a RNAdependent RNA polymerase gene of Bombyx mori cytoplasmic polyhedrosis virus[J]. Gene, 2015, 565(1):56-61.
[2]张国只,韩日畴.蜜蜂囊状幼虫病研究进展[J].中国生物防治,2008, S1:130-137.
[3]张炫,陈彦平,和绍禹.蜜蜂病毒学研究进展[J].应用昆虫学报,2012, 49(5):1095-1116.
[4]沈克飞,曹兰,张邑帆,张素辉.重庆中华蜜蜂囊状幼虫病病毒多聚蛋白基因分子特性分析[C]//中国畜牧科技论坛,2016.
[5] Maori E, Paldi N, Shafir S, Kalev H, Tsur E, Glick E,Sela I. IAPV, a bee-affecting virus associated with Colony Collapse Disorder can be silenced by dsRNA ingestion[J]. Insect Mol Biol,2010, 18(1):55-60.
[6] Desai S D, Eu Y J, Whyard S, Currie R W. Reduction in deformed wing virus infection in larval and adult honey bees(Apis mellifera L.)by double-stranded RNA ingestion[J]. Insect Mol Biol,2012, 21(4):446-455.
[7] Chejanovsky N, Ophir R,Schwager M S,Slabezki Y,Grossman S, Cox-Foster D. Characterization of viral siRNA populations in honey bee colony collapse disorder[J]. Virology, 2014, 454-455(1):176-183.
[8] Garbian Y, Maori E, Kalev H, Shafir S, Sela I. Bidirectional transfer of RNAi between honey bee and Varroa destructor:Varroa gene silencing reduces Varroa popula-tion[J/OL]. PloS Pathog, 2012, 8(12):e1003035.
[9] Zhang J, Zhang Y, Han R. The high-throughput product ion of dsRNA against sac brood virus for use in the honey bee Apis cerana(Hymenoptera:Apidae)[J]. Virus Genes,2016, 52(5):698-705.
[10]丁清泉.RNA病毒RNA聚合酶的研究进展[J].中国病毒学,1998, 13(2):107-113.
[11] Timmons L, Court DL,Fire A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans[J]. Gene,2001, 263(1-2):103-112.
[12] Yu N, Christiaens O, Liu J, Niu J, Cappelle K, Caccia S,Huvenne H,Smagghe G. Delivery of dsRNA for RNAi in insects:an overview and future directions[J].Insect Sci,2013, 20(1):4-14.
[13]Aronstein,K A,Pankiw T,Saldivar E. SID-1 is implicated in systemic gene silencing in the honey bee[J]. J Apicult Res,2006,45(1):20-24.
[14] Ganbaatar O, Cao B, Zhang Y, Bao D, Bao W, Wuriyanghan H. Knockdown of Mythimna separata chitinase genes via bacterial expression and oral delivery of RNAi effectors[J]. BMC Biotechnol, 2017, 17(1):9-19.
[15] Pan Z H,Gao K,Hou C X,Wu P,Qin G X,Geng T,Guo X J. dsRNA interference on expression of a RNAdependent RNA polymerase gene of Bombyx mori cytoplasmic polyhedrosis virus[J]. Gene, 2015, 565(1):56-61.