兼抗玉米矮花叶病毒及粗缩病毒无选择标记siRNA复合表达载体的构建  

Construction of marker-free siRNA complex expression vector against MDMV and MRDV

作　　者：张姣[1] 赵阳[2] 甘德芳[1] 

机构地区：[1]安徽农业大学园艺学院,合肥230036 [2]安徽农业大学生命科学学院,合肥230036

出　　处：《安徽农业大学学报》2011年第2期232-237,共6页Journal of Anhui Agricultural University

基　　金：国家高技术研究与发展计划(863)项目(2008AA10Z408);安徽省高校省级科研项目(KJ2011A110)共同资助

摘　　要：根据GenBank和MaizeSeq等数据库中的玉米矮花叶病毒和粗缩病毒外壳蛋白基因(CP)全序列设计特异性引物,RT-PCR扩增玉米矮花叶病毒及粗缩病毒的CP基因特异性RNA干扰片段,分别构建反向重复片段并串联成pBluscript+SM;Hind III-EcoR I双酶切2T-DNA真核表达载体pDTB,插入Ubiquitin启动子及Nos终止子,构建pDTBU;串联的反向重复片段插入pDTBU,构建兼抗玉米矮花叶病毒和粗缩病毒的siRNA复合表达载体pDTBUSM。酶切结果显示,目的片段均正确插入到相应的载体中。本研究对开展抗病毒RNA干扰调控技术研究,创建高抗病毒玉米新种质具有重要的理论和实践意义。Based on the coat protein gene sequence of MDMV and MRDV in GenBank and MaizeSeq data-base,six pairs of specific primers were designed and six specific fragments were amplified by RT-PCR to prepare siRNA that corresponded to part of the MDMV CP or MRDV CP gene.In the first cloning step,an inverted repeat sequence of pUCCRNAi ＋ 2 F was constructed.Next,two inverted repeat sequences were inserted into pBluscript SK in series to generate pBluscript ＋ SM.Meanwhile,Ubiquitin promoter and Nos termination were cloned into pDTB to generate pDTBU.In the third step,pDTBU and pBluscript ＋ SM plasmids were digested and joined to generate pDTBUSM.The study presented here provides a valuable tool for plant viral control using RNAi and the PTGS approach.

关 键 词：玉米矮花叶病毒 玉米粗缩病毒 外壳蛋白 载体构建 SIRNA 

分 类 号：S513[农业科学—作物学] S503.53