拟南芥psy基因cDNA的克隆及其植物表达载体的构建

Cloning cDNA of Phytoene Synthase from Arabidopsis and Construction of Its Plant Expression Vector

为了获得胚乳组织特异性表达八氢番茄红素的转基因小麦,以拟南芥幼叶RNA为模板,由特异型引物通过RT-PCR一步法得到大小约为1.3kb的基因片段,将此片段连接在克隆载体pMD18-T进行测序,结果表明,该基因片段为八氢番茄红素合成酶基因(psy)cDNA片段。将psy基因片段正向插入植物表达载体pLRPT中高分子量麦谷蛋白亚基基因1Dx5启动子与nos终止子之间,pLRPT载体无1Dx5基因开放阅读框,运用菌落PCR对重组子进行筛选与鉴定,说明拟南芥psy基因已正确插入pL-RPT,成功构建了植物表达载体pLRPTPSY。

To obtain transgenie wheat with psy gent in endosperm - specific manner, eDNA fragment about of 1.4 kb was cloned by one - tube RT - PCR reaction with specific primers. Total RNA extracted from young leaf of Arabidopsis thaliana was used as template. The product was inserted to the cloning vector pDM18 - T. After psy DNA fragment was comfirmed by the sequence test, it was subsequently cloned into plant expression vector pLRPT. It was located between wheat high molecular weight glutenin subunit IDx5 promoter and nos terminater, and 1Dx5 promoter was endosperm specific promoter. The resulted construct ion was named pLRPTPSY. The phytocne synthase gene in pLRFF was under the control of wheat endosperm- specific promoter.