OsKAT3 RNAi转基因水稻植株的构建

发布时间：2018-11-19 14:48

【摘要】：[目的]本研究以水稻‘日本晴’为遗传转化受体材料,利用RNAi技术对水稻气孔调控型钾吸收通道OsKAT3进行功能研究。[方法]通过将OsKAT3与拟南芥和水稻中的KAT类钾通道进行同源性比对,找到编码此蛋白基因C端的一段特异区域,并用于OsKAT3 RNAi表达载体的构建。设计含有相应酶切位点的引物扩增该特异片段,以OsKAT3作为模板进行RNAi-OsKAT3顺式和反式目的片段的PCR扩增。经菌落PCR鉴定挑选阳性克隆后送测序,测序结果表明:RNAi-OsKAT3顺式和反式目的片段均已正确连接到p MD19-T载体上,分别利用SacⅠ/SpeⅠ和Bam HⅠ/KpnⅠ酶切RNAi-OsKAT3顺式和反式目的片段,并将其连接到含有发夹结构的质粒p TCK303上,然后采用农杆菌介导的方法将经酶切验证正确的OsKAT3RNAi表达载体转化到水稻‘日本晴’中。[结果]以表达载体p TCK303多克隆位点两侧的通用引物进行转基因阳性植株鉴定,PCR结果显示OsKAT3顺式和反式特异片段已成功整合到再生水稻植株基因组中,定量PCR结果也证实RNAi转基因植株中OsKAT3基因表达被成功抑制。[结论]通过RNAi技术成功沉默了OsKAT3基因并获得T0代种子,为后续研究该基因功能奠定了一定基础。
[Abstract]:[objective] to study the function of stomatal regulated potassium absorption channel (OsKAT3) in rice by RNAi using rice 'Nippon' as genetic transformation receptor. [methods] by comparing OsKAT3 with KAT potassium channel in Arabidopsis thaliana and rice, a specific region of C terminal encoding this protein gene was found and used in construction of OsKAT3 RNAi expression vector. Primers with corresponding restriction sites were designed to amplify the specific fragment, and OsKAT3 was used as template for PCR amplification of RNAi-OsKAT3 cis and trans target fragments. The positive clones were identified by colony PCR and sequenced. The sequencing results showed that the RNAi-OsKAT3 cis and trans target fragments were correctly ligated to the p MD19-T vector. Sac 鈪，

本文编号：2342665