木薯eIF4E7基因RNAi载体构建及沉默效果分析.doc

木薯eIF4E7基因RNAi载体构建及沉默 效果分析 wa bw王健华文蛙昕 中国热带农业科学院热带生物技术研究所农业部热 带作物生物学与遗传资源利用重点实验室海南大学 热带农林学院 由木薯褐色条斑病毒(Cassava brown steak virus, CBSV)和乌干达木薯褐色 条斑病毒(Uganda Cassava brown steak virus, UCBSV)引起的木薯褐色条斑 病毒病(CBSD)严重威胁着非洲木薯种植业的发展，且目前没有十分有效的防 治方法。病毒侵染植物需耍借助植物的翻译和复制系统来完成基因组的翻译及自 身的复制，其中真核翻译起始因子4E (eukaryotic initiation factor 4E, e TF4E)是多种RNA病毒侵染植物的必需因子。e TF4E7是木薯编码的e TF4E基 因家族成员之一，本研究构建该靶标基因的RNAi表达载体pl300-Ca4E7。本生 烟瞬时表达系统结合半定量RT-PCR的方法证明，该载体能高效沉默过量表达载 体p Al-Ca4E7中的e 1F4E7基因在本生烟叶片中的表达。研宄结果为进一步研 究木薯e IF4E7在CBSV及UCBSV侵染中的作用以及利用RNA技术干扰植物基因 的病毒防治新策略奠定基础。 关键词： 木薯;e 1F4E7;干扰载体;本生烟瞬时表达;半定量RT-PCR;沉默效果; 张秀春(1972—)，女，研究生，副研究员；研究方向：事植物分 了?病毒学，E-mail: zhangxiuchun@itbb. org. cno 刘志昕，E-mail: liuzhixin@itbb. org. cn。 2017-03-07 基金：国家自然科学基金项目资助(No. 31461143016, No. Construction of RNAi Vector Targeting Cassava eIF4E7 and Identifcation of Its Interfering Efficiency ZHANG Xiuchun WU Yadan ZHANG Chunwei WANG Jianhua YU Naitong LIU Zhixin Institute of Tropical Bioscience and Biotechnology， Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Biology and Genetic Resources of Tropical Crops， Ministry of Agriculture; Abstract： Cassava (Manihot esculenta Grantz) production in large areas of Africa is severely affected by cassava brown streak disease ( CBSD) which is caused by two virus species, cassava brown streak virus ( CBSV) and Ugandan cassava brown streak virus (UCBSV) . At present, there is no effective strategy for preventing CBSD. To complete the translation and replicaiton of its genome, virus need to employ the translation and replication system of host. During virus infection plant’s eukaryotic initiation factor 4 E ( c IF4 E) has been implicated a necessary factor for a variety of RNA viruses. Cassava e IE4 E7 is one member of Cassava e IE4 E family. At present study we develop a interfering construct pl300-Ca4 E7 which targetting Cassava e IF4 E7 gene and demonstrate the construct can interfere the expression of e IF4 E7 gene efficiently by employing the N.