紫花苜蓿光敏色素A基因片段的克隆及其RNA干扰载体的构建

cDNA Fragment Cloning of Phytochrome A Gene from Alfalfa and Construction of RNAi Vector

苜蓿(Medicagosativa L.)的秋眠性是一种光周期效应。研究植物体内重要的光受体-光敏色素A(phyto-chromeA,phyA),可能揭示其调控苜蓿秋眠性的机理。为研究其与苜蓿秋眠性的关系,本研究根据RNA干扰(RNAinterference,RNAi)技术原理,以紫花苜蓿幼嫩叶片为材料,提取总RNA,选取基因cDNA序列同源性较低区域设计特异性引物,并引入相应的酶切位点,利用RT-PCR克隆phyA正、反向片段,经过3次亚克隆,构建了phyA的RNAi表达载体,制备了适合转染植物组织的农杆菌菌液,为获得缺失phyA植株创超了条件。

Phytochrome A（phyA） is a significant light receptors in plants and may play an important role in controlling fall dormancy in alfalfa.To investigate the function of phyA regulating photoperiodic reaction in alfalfa,phyA RNAi vectors were constructed in this study.Total RNA was isolated from fresh,young leaves,and a pair of particular primers with double enzyme sites was designed to facilitate the lower homology region of phyA genes.Then,both sense and anti-sense phyA fragments were obtained by RT-PCR.The restriction enzyme sites of vector pHANNIBAL were used to construct the RNAi unit,and inserted into plant vector pART27 to construct the phyA RNAi vector.Results laid a foundation for obtaining transgenic plants with phyA gene silenced.