拟南芥光敏色素A反义RNA载体的构建及转化

光敏色素A是植物远红光信号的关键受体,在植物光信号转导途径中起重要作用。一些具有重要功能的基因(如生长素反应因子8:auxin response factor 8,ARF8)受到PhyA调控。本实验拟通过构建PhyA基因反义株系,研究PhyA调控ARF8等重要功能基因表达的机理。以培养7 d的拟南芥幼苗为材料提取RNA,利用RT-PCR技术扩增出PhyA的全长CDS,将其反向插入表达载体pMD1得到反义表达载体pMD1-PhyA CDSR,并通过农杆菌介导转化拟南芥,经卡那霉素抗性平板筛选和PCR鉴定得到13个PhyA转基因株系。pMD1-PhyA CDSR及其转基因株系的获得为研究PhyA调控ARF8等基因表达的机理奠定了材料基础。

Construction and transformation of antisense RNA expression vector of Arabidopsis phytochrome A gene

Phytochrome A(PhyA)is the specific photoreceptor which receives far-red light in plants.Many critical genes,including auxin response factor 8(ARF8),are regulated by PhyA.Therefore,it is necessary to construct the PhyA-antisence transgenic lines for revealing the mechanism underlying phyA-regulated genes expression.The PhyA CDS was amplified from Arabidopsis total RNA by RT-PCR method.Then,it was inserted reversely into pMD1 to get the antisence expression vector pMD1-PhyA CDSR.The transgenic lines were acquired by agrobacterium -mediated method.13 transgenic lines have been acquired by Kmr screening and PCR analysis.The vector pMD1- PhyA CDSR and the transgenic lines will be useful to study how the genes,such as ARF8,are regulated by PhyA.