拟南芥AtZFN3基因RNA干扰载体的构建与表达

Construction and Expression of RNA Interference Vector of AtZFN3 Gene in Arabidopsis thaliana

为了构建具有发夹结构的RNAi表达载体来研究拟南芥AtZFN3基因表达的效果。根据拟南芥(Arabidopsis thaliana)锌指蛋白AtZFN3基因序列,设计含有酶切位点的两对特异性引物。以拟南芥cDNA为模板,分别合成用于构建干扰载体的正、反义DNA片段,将正、反义片段分别插入表达载体pART27的相应位置上,构建含有发夹结构的RNAi载体pART-ZFN3。利用农杆菌介导法,将pART-ZFN3转化到拟南芥中,PCR检测证实获得了5株转基因植株,经荧光定量PCR检测,证明了转基因植株中AtZFN3基因表达量显著低于未转基因的植株。结果表明:具有发夹结构的RNAi载体pART-ZFN3已构建成功,它对拟南芥AtZFN3基因的表达具有抑制作用,为深入分析拟南芥AtZFN3基因功能提供了基础。

In order to construct an RNAi expression vector with hairpin structure to achieve the effect of AtZFN3 gene expression in Arabidopsis thaliana.According to the Arabidopsis thaliana,zinc finger protein AtZFN3 gene sequence,two pairs of specific primers containing the restriction sites were designed.The Arabidopsis thaliana cDNA was used as a template to synthesize the positive and antisense DNA fragments for constructing the interference vector.The positive and antisense fragments were inserted into the corresponding positions of the expression vector pART27 to construct the RNAi vector pART-ZFN3 containing the hairpin structure.Transformation of pART-ZFN3 to Arabidopsis using Agrobacterium-mediated methods and PCR detection confirmed that 5 transgenic plants were obtained.The expression of AtZFN3 gene in transgenic plants was significantly lower than that of non-transgenic plants by real-time PCR.The results showed that the RNAi vector pART-ZFN3 with hairpin structure was successfully constructed,which inhibited the expression of AtZFN3 gene in Arabidopsis thaliana,which provided a basis for further analysis of Arabidopsis AtZFN3 gene function.