水稻OsAAA1基因超表达载体构建及遗传转化  

OsAAA1 Gene in Rice: Overexpression Vector Construction and Genetic Transformation

作　　者：宋斯敏 郑启明 李世娇 邓仕琪 李琨 刘新琼 Song Simin;Zheng Qiming;Li Shijiao;Deng Shiqi;Li Kun;Liu Xinqiong(Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China/Key Lab for Biotechnology of State Ethnic Affairs Commission/College of Life Science,South-Central University for Nationalities,Wuhan 430074)

机构地区：[1]中南民族大学生命科学学院/武陵山区特色资源植物种质保护与利用湖北省重点实验室/生物技术国家民委重点实验室,武汉430074

出　　处：《中国农学通报》2020年第12期91-96,共6页Chinese Agricultural Science Bulletin

基　　金：国家自然科学基金面上项目“水杨酸应答新基因OsAAA-ATPase-1在水稻广谱抗病反应中的分子机理研究”(31370306)。

摘　　要：为了构建带Flag标签的OsAAA1超表达载体,获得阳性转基因植株并分析其OsAAA1基因表达情况。以日本晴的cDNA为模板,将OsAAA1克隆至p U1301-Flag载体;重组质粒通过PCR、酶切及测序鉴定正确后,以农杆菌为介导进行遗传转化至日本晴;转基因植株通过分子鉴定正确后,采用Real-time PCR检测OsAAA1基因的mRNA表达水平变化。成功构建了OsAAA1-pU1301-Flag重组载体;遗传转化后获得33株转基因植株;通过分子鉴定筛选出26株阳性转基因植株;荧光定量PCR结果分析发现23株阳性转基因植株OsAAA1基因的表达出现不同程度上调。与日本晴相比,有8株表达量达到30倍以上,其中有5株表达量超过40倍。成功构建了带Flag标签的OsAAA1超表达载体;遗传转化结果表明OsAAA1-Flag能整合到日本晴的基因组DNA中,从而使OsAAA1基因的表达水平增加。本研究为后续通过Flag标签,在水稻植物体内直接挖掘与OsAAA1互作的功能蛋白奠定了基础。The objectives are to construct the overexpression vector with Flag tagged OsAAA1, obtain positive transgenic plants and detect expression of OsAAA1, in positive transgenic rice. The sequence of OsAAA1, was amplified by polymerase chain reaction(PCR) using the cDNA template of Nip. The PCR product was cloned into Flag-tagged pU1301;after confirmation of PCR, enzyme digestion and sequencing, recombinant plasmid was transformed to Nip by agrobacterium;when positive transgenic plants were verified by molecular identification, the expression of OsAAA1, was detected by Real-time PCR. The recombinant plasmid of OsAAA1,-pU1301-Flag was successfully constructed;after genetic transformation, thirty-three plants were differentiated;twenty-six transgenic plants were verified to be positive by molecular identification;the Realtime PCR results indicated that the expression of OsAAA1, was up-regulated to different degrees in twentythree plants. Compared with the Nip, the expression of OsAAA1, was over 30 times in eight positive transgenic plants, in five of which the expression was even over 40 times. The overexpression vector with Flag tagged OsAAA1, was successfully constructed. Genetic transformation results showed that OsAAA1, with Flag-tag could be integrated into the genomic DNA of Nip, which could increase the expression of OsAAA1,. This study provides a basis for discovering functional proteins interacting with OsAAA1, in rice by using the Flag tag.

关 键 词：水稻 OsAAA1基因 pU1301-Flag载体 遗传转化 分子鉴定 

分 类 号：S511[农业科学—作物学]