Construction of ihpRNA Expression Vector of MsLEA3-1 from Medicago sativa L. and Genetic Transformation in Tobacco

Late embriogenesis abundant (LEA) protein is one of the hot topics in plant stress physiology. In this study, an RNAi expression vector harboring MsLEA3-1 gene fragment from alfalfa (Medicago sativa L.) was constructed. Two pairs of specific primers with different enzyme sites were designed based on the sequence of MsLEA3-1 (GenBank accession number EU665182). With the template of PMD-LEA plasmid constructed, positive-sense strand and antisense strand were obtained, which were separately inserted into the expression vector pART27. A hairpin structure in the RNAi vector pART-F-R was confirmed by the digestion of restriction enzymes. pART-F-R was transformed into tobacco via Agrobacterium-mediated transformation system, and 16 transgenic plants were obtained after PCR validation.

摘 要根据紫花苜蓿LEA蛋白MsLEA3-1基因(GenBank登录号为EU665182)序列, 设计两对含有酶切位点的特异性引物LEAf1/ LEAf2和LEAr1/LEAr2, 以构建好的PMD-LEA质粒为模板, 分别合成用于构建干扰载体的正反义片段pMD-F和pMD-R, 将正反义片段分别插入表达载体pART27的相应位置, 构建成含有发夹结构的RNAi载体pART-F-R, 经过Not I酶切鉴定, 证明载体构建成功。通过农杆菌介导的方法, 以干扰表达载体pART-F-R转化烟草, 经过PCR检测, 得到16株阳性转基因植株, 为MsLEA3-1基因的功能研究奠定基础。