中蜂囊状幼虫病毒RdRp基因dsRNA干扰的研究  

RNA Interference Against Infection by the Chinese Sacbrood Virus

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作  者:史红霞[1] 党晓群[1] 朱祥龙 马振刚[1] 刘永梅 黄钰彬 周泽扬[1] 许金山[1] SHI Hongxia;DANG Xiaoqun;ZHU Xianglong;MA Zhengang;LIU Yongmei;HUANG Yubin;ZHOU Zeyang;XU Jinshan(Chongqing Key Laboratory of Animal Biology,Chongqing Normal U niversity,Chongqing 401331,China;Sishun Mining and Forestry Farm,Chotigyi County,Ganzhou 341315,China)

机构地区:[1]重庆师范大学重庆市动物生物学重点实验室,重庆401331 [2]江西省崇义县思顺采育林场,赣州341315

出  处:《病毒学报》2019年第1期103-111,共9页Chinese Journal of Virology

基  金:现代农业产业技术体系建设专项资金资助(项目号:CARS-44-KXJ21),题目:蜜蜂健康高效养殖技术集成与示范;重庆师范大学校级项目(项目号:16xzb07),题目:治疗中蜂囊状幼虫病试剂盒的开发与应用;国家自然科学基金项目(项目号:31770160),题目:东方蜜蜂微孢子虫孢壁蛋白及其与侵染功能相关性研究;重庆市教委科学技术研究项目(项目号:KJ703049),题目:东方蜜蜂Ac-NPC1的表达特征分析.

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.

关 键 词:中华蜜蜂 中蜂囊状幼虫病毒(CSBV) RNA依赖的RNA聚合酶(RdRp) dsRNA的体外表达 RNAI 

分 类 号:S895.1[农业科学—特种经济动物饲养;农业科学—畜牧兽医]

 

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