紫花苜蓿MsZIP基因RNAi表达载体的构建及苜蓿转化

Construction of RNAi Expression Vector of MsZIP Gene from Medicago sativa L. and Genetic Transformation in Alfalfa

根据已经得到的MsZIP基因序列(GenBank序列号:HQ911778),设计两对含有酶切位点的引物ZIPF1/ZIPF2和ZIPR1/ZIPR2,合成用于构建干扰载体的正反义片段。将正反义片段插入到载体pART27中,构建成为含有发夹结构的RNAi干扰载体pART-F-R。采用农杆菌介导的方法,将该干扰载体成功转化到苜蓿中,得到9株抗性苗,选取其中的3株进行PCR鉴定,结果表明成功得到转基因苜蓿。

According to the sequence of alfalfa transcription factor gene MsZIP（GenBank accession number： HQ911778）,two pairs of specific primers containing different enzyme sites were designed to obtain the positive-sense strand and anti-sense strand,the primers were named ZIPF1/ZIPF2 and ZIPR1/ZIPR2.The positive-sense strand and anti-sense strand were separately inserted into the expression vector pART27 to construct the RNAi vector pART-F-R,which contained a hairpin structure.The RNAi expression vector pART-F-R was transformed into alfalfa by Agrobacterium-mediated transformation system.Nine transgenic alfalfas were got totally,three of them were selected to analyze,the results showed that three transgenic alfalfas were successfully got.