DsRED红色荧光蛋白植物表达载体的构建和表达

Construction and expression of DsRED red fluorescent protein expression vector in plant

为获得红色荧光蛋白(Red fluorescent protein,RFP)真核表达载体35S-pC1301-DsRED,利用PCR将瞬时表达载体PGDR上的全长DsRED扩增下来并用XbaⅠ和BamHⅠ酶切该片段,同时用相同的内切酶消化中间载体pBluescript SK(+),连接两片段并测序,将鉴定正确的DsRED用相同的内切酶切下并导入双元载体35S-pC1301,利用基因枪将正确质粒转染洋葱内表皮细胞瞬时表达DsRED。结果表明:35S-pC1301-DsRED真核表达载体已成功构建并在荧光共聚焦显微镜下呈现红光,即获得了可产生红色荧光的35S-pC1301-DsRED载体,为今后植物目的基因定位提供了阳性对照,同时中间载体pBluescript SK(+)-DsRED的构建为今后植物目的基因定位提供了实验工具。

In order to obtain the eukaryotic expression vector （35S-pC1301-DsRED） of red fluorescent protein （RFP）, full length DsRED was amplified from transient expression vector PGDR by PCR and was digested by Xba I and BamH I . At the same time, intermediate vector pBluescript SK（＋） was digested by the same enzymes, DsRED and pBlueseript SK（＋） were connected and sequenced. The correct DsRED was digested and connected with binary vector 35S-pC1301. The correct plasmid was bombed and transiently expressed DsRED in the onion emperdiem cells through gene gun transfection method. The result demonstrated that eukaryotic expression vector 35S-DC1301-DsRED was successfully constructed and the red fluorescence could be observed under fluorescent microscopy. Red fluorescent 35S-pC1301-DsRED vector was obtained, which provides positive control for the localization of target genes in the future, while the intermediate vector pBluescript SK（＋）-DsRED serves as an useful experimental tool for the detection of the position of target genes.