紫花苜蓿LEA蛋白基因ihpRNA表达载体构建及烟草转化

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

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NotI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。

During long-term evolution, plant has developed various physiological functions and bio-chemical mechanisms to respond the diverse stresses in different environments. Plant cell accumulates a series of proteins to reduce cell dehydration in the period of water shortage, of all proteins, late embriogenesis abundant LEA protein has been paid attention, which is one of the hot topics in plant stress physiology. In the paper, an RNAi expression vector harboring MsLEA3-1 gene fragment from Medicago sativa L. was constructed. On the basis of the sequence of Medicago sativa LEA protein （MsLEA3-1） gene （GenBank accession number： EU665182）, two pairs of specific primers containing different enzyme sites were designed. 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. The RNAi vector pART-F-R containing a hairpin structure was confirmed by the digestion of restriction enzymes. pART-F-R was transformed into tobacco by Agrobacterium-mediated transformation system. PCR testing showed that 16 transgenic plants were obtained.