【摘要】：[目的]本研究以水稻‘日本晴’為遺傳轉(zhuǎn)化受體材料,利用RNAi技術(shù)對水稻氣孔調(diào)控型鉀吸收通道OsKAT3進行功能研究。[方法]通過將OsKAT3與擬南芥和水稻中的KAT類鉀通道進行同源性比對,找到編碼此蛋白基因C端的一段特異區(qū)域,并用于OsKAT3 RNAi表達載體的構(gòu)建。設計含有相應酶切位點的引物擴增該特異片段,以OsKAT3作為模板進行RNAi-OsKAT3順式和反式目的片段的PCR擴增。經(jīng)菌落PCR鑒定挑選陽性克隆后送測序,測序結(jié)果表明:RNAi-OsKAT3順式和反式目的片段均已正確連接到p MD19-T載體上,分別利用SacⅠ/SpeⅠ和Bam HⅠ/KpnⅠ酶切RNAi-OsKAT3順式和反式目的片段,并將其連接到含有發(fā)夾結(jié)構(gòu)的質(zhì)粒p TCK303上,然后采用農(nóng)桿菌介導的方法將經(jīng)酶切驗證正確的OsKAT3RNAi表達載體轉(zhuǎn)化到水稻‘日本晴’中。[結(jié)果]以表達載體p TCK303多克隆位點兩側(cè)的通用引物進行轉(zhuǎn)基因陽性植株鑒定,PCR結(jié)果顯示OsKAT3順式和反式特異片段已成功整合到再生水稻植株基因組中,定量PCR結(jié)果也證實RNAi轉(zhuǎn)基因植株中OsKAT3基因表達被成功抑制。[結(jié)論]通過RNAi技術(shù)成功沉默了OsKAT3基因并獲得T0代種子,為后續(xù)研究該基因功能奠定了一定基礎。
[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，

本文編號：2342665