马铃薯块茎启动子驱动的淀粉合成酶基因RNAi载体构建及遗传转化

Construction and Transformation of Starch Synthases Gene RNAi Expression Vector Regulating by Potato Tuber Specificity Promoter

以马铃薯gbss、ssⅢ与ssⅡ基因的部分c DNA片段拼接成的融合基因gbs_3s_2为靶标,构建以马铃薯块茎组织特异性启动子GBSS驱动的RNAi表达载体p ART-GF。采用农杆菌介导法对NHD3、NF和NC三个马铃薯品种进行了遗传转化,用PCR技术检测转基因植株,采用双波长法测定其微型薯中直链淀粉与支链淀粉的比值。经抗性筛选及PCR检测初步获得10株阳性植株,其中NC、NF和NHD3分别为2株、3株和5株,其微型薯中直链淀粉/支链淀粉比值明显下降。作者成功构建了马铃薯块茎启动子GBSS驱动的淀粉合成酶基因RNAi三价表达载体,初步获得了转基因马铃薯育种材料。

Using the fused gene-gbs3s2 composed of the gbss （ 1-261 ） ,ssⅢ （2164-2407） and ss Ⅱ （161-441） gene cDNA fragments as target,we constructed ihRNAi expression vector pART-GF regulated by GBSS of potato tuber specificity promoter. The inverted repeat construct was transformed to elite potato cultivars NHD3,NF and NC by AgrobacteriumLBA4404-mediated transformation, PCR technology was used to detect transgenic plants, and the ratio of amylose eand amylopectin in transgenic mini-potato were determined by dual-wavelength spectrophotometry. Ten regenerated plants with kanamycin resistance were obtained through resistance screening and PCR detection. Among them,two was NC plants,three was NF plants,and five was NHD3 plants,respectively. The ratio of amylose and amylopectin in transgenic mini-potato were detected to decrease significantly. IhRNAi expression vector pART-GF regulated by GBSS of potato tuber specificity promoter was successfully constructed and breeding material of transgenic potato was obtained.