紫花苜蓿锌指蛋白基因RNAi表达载体的构建及在苜蓿的转化

根据紫花苜蓿锌指蛋白MsZFN基因(GenBank登录号为EU624138)序列,设计两对含有酶切位点的特异性引物,以紫花苜蓿cDNA为模板,分别合成用于构建干扰载体的正反义片段,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。利用农杆菌介导方法,将pART-F-R转化到紫花苜蓿中,经过PCR检测,获得了3株转基因植株。经过RT-PCR检测,证明转基因植株中MsZFN基因表达量明显低于未转基因的植株。结果表明,已构建成功具有发夹结构的RNAi载体pART-F-R,它可有效的抑制紫花苜蓿MsZFN基因。

Construction and Transformation of RNAi Vector of MsZFN Gene from Alfalfa (Medicago sativa L.)

Based on the sequence of Medicago sativa Zinc Finger Protein (MsZFN) gene (GenBank accession No. EU624138), two pairs of specific primers containing different enzyme sites were designed. With the template of full-length cDNA , 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 constructed. Mediated by Agrobacterium tumefaciens, pART-F-R was transformed into alfalfas. PCR testing showed that three transgenic plants were obtained. Result of RT-PCR showed that transgenic alfalfas had lower expression level of MsZFN gene than wild alfalfas. Those results indicated that the RNAi vector pART-F-R containing a hairpin structure was constructed successfully and highly efficient for the simultaneous silence of MsZFN gene.