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利用PCR方法扩增杨树Poptr;CYCD1;1基因的一段内含子序列,并克隆到pROKⅡ载体中,以构建可用于植物特定基因表达干扰的RNAi载体。为了验证载体的效果,构建pCAMBIA-1303载体转化烟草,经检测,转基因烟草能过量表达外源GUS与GFP融合蛋白。把GUS及GFP片段序列连接于RNAi载体,并导入pCAMBIA-1303载体转化烟草。试验结果表明,GUS及GFP干扰序列的导入均能大大降低外源GUS与GFP融合基因的表达。结果说明构建的载体能有效干扰特定基因的表达。
A partial intron sequence of Poptr; CYCD1; 1 gene was amplified by PCR and cloned into pROKⅡ vector to construct RNAi vector which can be used for plant-specific gene expression interference. In order to verify the effect of vector, we constructed pCAMBIA-1303 vector to transform tobacco and tested that transgenic tobacco can overexpress exogenous GUS and GFP fusion protein. The GUS and GFP fragment sequences were ligated to the RNAi vector and transformed into tobacco using the pCAMBIA-1303 vector. The results showed that the introduction of GUS and GFP interference sequences can greatly reduce the expression of foreign GUS and GFP fusion gene. The results show that the constructed vector can effectively interfere with the expression of a specific gene.