用无终止子的RFP报告基因捕获水稻终止子研究

PolyA trapping by using terminatorless RFP reporter gene in rice

基因捕获技术已被广泛应用于植物功能基因组学研究。文章构建了水稻PolyA捕获载体pLJ19,该载体含有用于检测转化事件的绿色荧光蛋白报告基因(GFP)及用于进行PolyA捕获的无终止子的红色荧光蛋白报告基因(RFP),在水稻转化愈伤组织阶段筛选同时含有GFP及RFP的转化愈伤组织并进行RFP插入位点T-DNA侧翼序列(FST)分析可获得水稻终止子序列。将pLJ19转化粳稻品种‘日本晴’的胚性愈伤组织,获得了17个具有PolyA捕获事件的愈伤组织。FST序列分析表明6个T-DNA插入在水稻基因组序列中,其中有5个T-DNA插入在水稻基因3’端附近。结果表明,pLJ19载体可用于水稻终止子捕获研究中,可为水稻终止子研究提供研究方法和材料。

Gene trapping method has been widely used in plant functional genomic research. PolyA trapping vector pLJ19 which contains GFP and RFP reporter genes was constructed, GFP reporter gene was used for detecting transformation events and RFP reporter gene was used for PolyA trapping. Calli both with GFP and RFP fluorescence were selected and T-DNA insertion sites were analysed by Genome walking PCR. The plasmid pLJ19 was introduced into calli of Oryza sativa L. ssp. Japonica cv. Nipponbare and then 17 transgenic calli both with GFP and RFP. FST of / T-DNA insertion sites indicated that 6 T-DNA were inserted into rice genome and 5 of 6 them were inserted around rice gene ,3＇ ends. These results indicated that the plasmid pLJ19 was available in PolyA trapping.