小麦RCA的cDNA片断克隆和反义表达载体的构建

Cloning of Wheat Rubisco Activase cDNA Fragment and Construction of Antisense Expression Vector

目的:试图分离和克隆小麦Rubisco活化酶cDNA片断并构建反义表达载体。方法:采用RT-PCR技术克隆cDNA片断,对序列用Blast等软件进行分析,并将该片断反向连接于植物表达载体pROK2的CaMV35S启动子下游,构建反义表达载体。结果:获得了小麦Rubisco活化酶(RCA)的cDNA片断(GenBank注册号:DQ984669);小麦RCA的cDNA片断推导的氨基酸序列与其它植物的RCA氨基酸序列高度同源。序列比较表明,用本实验所得的cDNA序列推导出的氨基酸序列与GenBank登录的大麦(AAA63163)、南极银须草(AAP83928)、水稻(AAX95414)、玉米(AAC97932)、拟南芥(NP 850321)和烟草(CAA78703)等的RCA序列的同源性分别为97%、95%、88%、83%、82%和82%;分析表明,该序列为新的小麦RCAα的cDNA序列;通过选择和引入合适的酶切位点进行载体构建,构建了小麦RCA的cDNA反义表达载体pR-AntiRCA。结论:构建成了小麦RCA的cDNA反义表达载体。

Objective：The cDNA fragment of wheat Rubisco acfivase （RCA） was cloned and antisense expression vector was constructed. Methods：RT- PCR （Reverse Transcription Polymerase Chain Reaction） method was used to clone the cDNA frament. The Blast software was used to analyse the sequence. The fragment was ligated to the CaMV35S promoter downstream to construct antisense expression vector. Results： The cDNA fragment of wheat RCA was cloned （GenBank accession humor： DQ984669）. The result of domain analyze showed that it is a member of RCA family. Compared puptafive amino acid sequence to other higher plants showed that it belonged to RcaA subunit family and has high homology with other plants such as Hordeum vulgare （97%）, Deschampsia aatarctica（95% ）, Oryza sativa （88%）, maize （83%）, Arabidopsis thaliana （82%）and Nicotiaria tabactan （82%）. Using appropriated sites of restriction endonueleaso, the antisense expression vector of wheat RCA was constructed. Conclusion：The vector for wheat RCA anfisense expression was constructed.