百合AGPase基因RNAi载体构建及遗传转化研究  

Construction of RNAi Vector and Genetic Transformation of Lilium AGPase Gene

作　　者：张进忠 孙嘉曼[3] 李朝生[2] 刘挺燕[2] 范燕萍 ZHANG Jin-zhong;SUN Jia-man;LI Chao-sheng;HU Ting-yan;FAN Yan-ping(College of Forestry and Landscape Architecture,South China Agricultural University,Guangdong Guangzhou 510642,China;Insti-tute of Biotechnology,Guangxi Academy of Agricultural Sciences,Guangxi Nanning 530007,China;Guangxi Crop Genetic Improvenment and Biotechnology Laboratory,Guangxi Nanning 530007,China)

机构地区：[1]华南农业大学林学与风景园林学院,广东广州510642 [2]广西农业科学院生物技术研究所,广西南宁530007 [3]广西作物遗传改良生物技术重点开放实验室,广西南宁530007

出　　处：《西南农业学报》2018年第6期1097-1103,共7页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金(31660560);广西农业科学院基本科研业务专项(桂农科2017YM32);广西南宁市科学研究与技术开发计划项目(20133164)

摘　　要：【目的】AGPase基因可在百合鳞茎膨大发育过程中影响淀粉的合成代谢,从而调控鳞茎发育,构建干扰AGPase基因RNAi载体并遗传转化进行反向下调作用研究,可为该调控机制的研究提供更多信息。【方法】克隆300 bp的AGPase基因保守序列,利用Gateway技术通过BP反应将该序列插入入门载体p DONR221,进行LR反应将该序列正反向插入干扰载体p Jawohl8-RNAi中,经过酶切鉴定所构建RNAi载体的正确性;并通过农杆菌介导法转入百合组培苗中,PCR检测RNAi载体转化农杆菌,荧光定量PCR检测农杆菌转化植株的AGPase相对表达量变化。【结果】成功构建p DONR221-AGPase入门克隆与p Jawohl8-RNAi-AGPase表达载体,遗传转化百合愈伤诱导出AGPase表达量下调的转化植株。【结论】采用Gateway技术可方便构建RNAi表达载体,选择的AGPase保守序列能作为干扰片段对百合AGPase自身转录的mRNA进行下调。[ Objective] AGPase gene can influence the synthesis and metabolism of starch so as to regulate the intumescentia and development of lily bulb. Studying the down-regulation ofAGPase gene through construction of RNAi vector interfering with AGPase gene for genetic trans- formation can provide more information for the research of the regulation mechanism. [ Method] Conserved 300 bp sequence of AGPase gene has been cloned, and it has been connected to the entry vector pDONR221 by Gateway BP reaction. In the LR reaction, the sequence was inserted forward and backward into the interference vector pJawohlS-RNAi. The correctness of the constructed RNAi vector was identified by enzyme digestion and transfen＇ed into the tissue culture seedlings of Lilium by Agrobacterium tumefaciens mediated. Detection of the transfor- mation of the RNAi vector into Agrobacterium tumefaciens by PCR, the relative expression of AGPase in transformed plants through Agrobacte- rium tumefaciens was detected by fluorescence quantitative PCR. [ Result] The entry vector pDONR221-AGPase and expression vector pJa- wohlS-RNAi-AGPase have been constructed successfully, by genetic transformation, the lily seedlings grow from the callus with down regula- tion expression of AGPase. [ Conclusion] The Gateway technology can construct RNAi expression vector conveniently. And the selected AG- Pase conservative sequence can be used as an interference fragment to down regulate the mRNA transcript of Lilium AGPase gene.

关 键 词：百合 AGPase基因 RNA干扰 载体构建 遗传转化 

分 类 号：Q812[生物学—生物工程]