%0 Journal Article
%T OsKAT3 RNAi转基因水稻植株的构建
%A 王莉
%A 黄亚楠
%A 郭满园
%A 苏彦华
%J 南京农业大学学报
%D 2016
%R 10.7685/jnau.201603027
%X [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pMD19-T载体上，分别利用SacⅠ/SpeⅠ和BamHⅠ/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pTCK303上，然后采用农杆菌介导的方法将经酶切验证正确的OsKAT3 RNAi表达载体转化到水稻‘日本晴’中。[结果]以表达载体pTCK303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。</br>[Objectives] With rice variety‘Nipponbare’serving as genetic transformation materials,functions of stomatal regulation channel OsKAT3 was studied by RNAi techniques. [Methods] Based on the sequence alignment between the OsKAT3 and KAT-like channels among the Arabidopsis and rice,a specific fragment of the C terminal was chosen for constructing OsKAT3 RNAi expression vector. The primer was designed and the restriction site was inserted,and the OsKAT3 was used as a template for RNAi-OsKAT3 cis- and trans-fragments of PCR amplification.The PCR amplification fragments were connected to the pMD19-T vectors and sequenced after bacterial colony PCR confirmation. The sequencing results showed that RNAi-OsKAT3 cis- and trans-fragments were correctly connected to the pMD19-T vectors. Then the RNAi-OsKAT3 cis- and trans-fragments were digested by restriction enzyme SacⅠ/SpeⅠand BamHⅠ/KpnⅠ,respectively,and connected to the plasmid pTCK303 with a hairpin structure. PCR and double digestion verification showed that constructed pTCK303-OsKAT3(RNAi-OsKAT3) structural integrity was then transformed into rice by Agrobacterium-mediated methods. [Results] Positive recombinant plants were confirmed by PCR with a universal primer pair designed according to the flank sequence of multiple clone site in pTCK303,indicating that the specific cis- and trans-fragments were successfully integrated into the genome of the regeneration rice. Further real-time PCR results also confirmed that the expression of OsKAT3 in the RNAi transgenic plants was successfully suppressed. [Conclusions] The expression of OsKAT3 was suppressed by RNAi technology in the T0 generation RNAi transgenic rice plants,providing a vital foundation for follow-up studies of the function of the OsKAT3
%K 水稻
%K OsKAT3
%K RNAi
%K pTCK303载体
%K 转基因植株&lt
%K /br&gt
%K rice
%K OsKAT3
%K RNAi
%K pTCK303 vector
%K transgenic plants
%U http://nauxb.njau.edu.cn/oa/darticle.aspx?type=view&id=201606013