水稻SLR1基因的克隆及其植物表达载体的构建

目的]为研究水稻中的SLR1蛋白的功能及寻找与其相互作用的GA信号转导蛋白奠定基础。方法]以水稻品种日本晴基因组DNA为模板,根据水稻SLR1基因cDNA序列设计1对特异引物进行PCR扩增,回收PCR产物连接到pGEMT载体中,经筛选得到水稻SLR1基因的克隆pGEMTSLR。最后采用酚仿抽提和乙醇沉淀的方法构建水稻SLR1基因的正义和反义表达载体。结果]通过PCR扩增获得了约为1.9kb的特异片段。回收PCR产物,将克隆片段和pGEMT载体连接后转化到大肠杆菌DH5α感受态细胞,重组克隆经酶切鉴定后提取重组质粒pGEMTSLR1。该研究成功构建了SLR1基因的正义表达载体pCAMSLR(约0.7kb)和反义表达载体pCAMASLR(约1.2kb)。结论]采用冻溶法可将表达载体pCAMSLR和pCAMASLR导入农杆菌并进行水稻的遗传转化。利用SLR1基因的正义和反义表达载体,可观察转基因植株中该基因的表达上调和下调对植物的影响。

Cloning of SLR1 Gene in Rice and Its Plant Expression Vector Construction

Objective] The aim of the research was to lay foundation for studying the function of SLR1 protein in rice and search GA signal transduction protein with interaction with SLR1 protein. Method] With the genomic DNA of japonica rice variety Nipponbare as templates, a pair of specific premiers was designed according to cDNA sequence of SLR1 gene in rice for PCR amplification. The reclaimed PCR products were connected into pGEMT vector and the cloning pGEMTSLR of SLR1 gene in rice was obtained through screening. Finally sense expression vector and antisense expression vector were constructed by using the methods of phenol-chloroform extraction and ethanol precipitation. Result] About 1.9 kb specific fragment was obtained from PCR amplification. The PCR products were reclaimed, and the cloning fragment and pGEMT vector were connected and transformed into competent cell DH5α from Escherichia coli. The recombinant plasmid pGEMTSLR1 was extracted from the recombinant cloning after enzyme digestion identification. The sense expression vector pCAMSLR(about 0.7 kb)and antisense expression vector pCAMASLR(about 1.2 kb)of SLR1 gene were successfully constructed in this research. Conclusion] Expression vectors Pcamslr and pCAMASLR could be introduced into Agrobacterium tumefaciens to make genetic transformation of rice by frozen-dissolving method. The effects of the up-regulated expression and the down-regulated expression of this gene in transgenic plants on plants could be observed by using sense expression vector and antisense expression vector of SLR1 gene.