含有Rubisco小亚基启动子和转运肽序列的通路克隆入门载体的构建和应用(英文)

Construction and Application of a Gateway Entry Vector With Rubisco Small Subunit Promoter and Its Transit Peptide Sequence

Gateway(通路克隆)技术是最近开发出来的一种分子克隆技术,其特点是操作简单、省时高效,已经成功应用于很多基因表达载体的构建.然而,现有的通路克隆植物表达载体不包含任何将表达蛋白定位到叶绿体中的序列.将通路克隆入门质粒载体pENTR-2B的XmnⅠ位点改造成HindⅢ位点,产生入门载体pENTR*-2B,然后将番茄1,5二磷酸核酮糖羧化酶(Rubisco)小亚基3C的启动子(PrbcS)及其转运肽序列(*T)和绿色荧光蛋白(GFP)报告基因亚克隆到pENTR*-2B中,构建通路克隆入门载体pENTR*-PrbcS-*T-GFP.实验结果证实,用pENTR*-PrbcS-*T-GFP和通路克隆的植物表达载体进行LR反应,构建GFP的光诱导型植物表达载体,可以成功地将表达的GFP定位到转基因植物的叶绿体中.利用β-葡糖苷酸酶(GUS)报告基因替代该入门载体中的GFP基因做试验也得到相似的结果.这说明用目的基因替换该入门载体中的GFP可以构建目的基因的入门载体,然后用通路克隆技术可以快速构建其光诱导型植物表达载体,将表达的目的蛋白定位到转基因植物或组织细胞的叶绿体中.

Gateway technology has been demonstrated to be easy and successful in construction of expression vectors for genes of interest. However, the present Gateway plant destination vectors do not contain any sequence for targeting expressed proteins of interest to chloroplasts. The Xmn Ⅰ restriction site was converted into a HindⅢ site in the Gateway entry vector pENTR-2B to generate pENTR＊-2B and a Gateway entry vetcor designated as pENTR＊-PrbcS-＊T-GFP was constructed by subcloning the tomato Rubisco small subunit 3C promoter （PrbcS） and its transit peptide sequence （＊T） as well as a GFP （green fluorescent protein） reporter gene into pENTR＊-2B. These results demonstrated that the pENTR＊-PrbcS-＊T-GFP could be used to generate the plant expression vector via Gateway technology for targeting the expressed GFP into the chloroplasts of transgenic plant leaves. Similar results were also obtained for GUS （[3-glucuronidase） reporter gene when it was used to replace the GFP gene in pENTR＊-PrbcS-＊T-GFP. These results indicated that pENTR＊-PrbcS-＊T-GFP can be generally applied to generate an entry vector for a target gene by replacement of the GFP with the target gene and the plant expression vector that serves to localize the expressed target protein in chloroplasts can be achieved rapidly via Gateway technology.