紫花苜蓿MsCOL1基因RNAi表达载体的构建及转化

Construction and Transformation of RNAi Vector of MsCOL1 Gene from Alfalfa

根据紫花苜蓿CONSTANS类似基因MsCOL1基因(登录号:DQ661682)序列,设计含有酶切位点的两对特异性引物,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,构建含有发夹结构的RNAi载体pART-S-A。利用农杆菌介导方法,将pART-S-A转化到紫花苜蓿中,经PCR检测,获得了7株转基因植株。经RT-PCR检测,证明转基因植株中MsCOL1基因表达量有所下降,其中5株的表达量明显的降低。结果表明,已构建成功具有发夹结构的RNAi载体pART-S-A,它可有效的抑制紫花苜蓿MsCOL1基因。

Based on the sequence of alfalfa Constans-Like 1（MsCOL1） gene（Access No.： DQ661682）,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 restrict sites of expression vector pART27.The RNAi vector of pART-S-A containing a hairpin structure was constructed.Mediated by Agrobacterium tumefaciens,pART-S-A was transformed into alfalfa plants.PCR testing showed that seven transgenic plants were obtained.The result of RT-PCR showed that transgenic alfalfa had lower expression level of MsCOL1 and five of those exhibited obviously low expression levels of MsCOL1.These results indicated that the RNAi vector pART-S-A containing a hairpin structure was constructed successfully and the alfalfa MsCOL1 gene was decreased effectively.