水稻OsAAA1基因超表达载体构建及遗传转化

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pU1301-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。

OsAAA1 Gene in Rice: Overexpression Vector Construction and Genetic Transformation

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 Real-time PCR results indicated that the expression of OsAAA₁, was up-regulated to different degrees in twenty-three 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.