利用GFPRFP双荧光指示载体鉴定特异性启动子功能.docxVIP

生 物 工 程 学 报 journals.im.ac.cn cjb@im.ac.cn Chin J Biotech 2008, December 25; 24(12): 2106-2110 Chinese Journal of Biotechnology ISSN 1000-3061 ? 2008 Institute of Microbiology, CAS CSM, All rights reserved 技术与方法 技术与方法 利用 GFP/RFP 双荧光指示载体鉴定特异性启动子功能 尹涛 1, 秦巧平 2, 张上隆 1, 刘敬梅 3, 陈大明 4 1 浙江大学园艺系, 杭州 310029 2 浙江林学院, 杭州 311300 3 北京市蔬菜研究中心, 北京 100089 4 Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, USA 摘 要 : 在基因表达定位或启动子调控模式的研究中, 多以 gusA 作为报告基因。但由于部分组织中高内源 GUS 背景活 性或转化手段的限制, 使判断基因表达定位或调控时存在很大误差。为了解决上述问题, 本实验将报告基因绿色荧光蛋 白(GFP)和红色荧光蛋白(RFP)融合构建双荧光标记瞬时表达载体pBI221-RFP/GFP。该载体以 CaMV35S 启动子驱动 GFP 确定转化效率, 通过鉴定阳性个体的红色荧光活性分析目的基因或启动子的表达模式。并通过番茄 E8 和西瓜 AGPL1 果实特异启动子验证了该载体在启动子调控模式研究中的应用可行性。结果表明 pBI221-RFP/GFP 是一个可以在基因和 启动子功能验证中应用的高效瞬时表达载体。 关键词 : 绿色荧光蛋白, 红色荧光蛋白, 瞬时表达载体, 特异性启动子 Functional Analysis of Specific Promoter Using Vecotors Harboring GFP/RFP Double Fluorescent Marker Genes Tao Yin1, QiaopingQin2, Shanglong Zhang1, Jingmei Liu3, and Daming Chen4 1 Department of Horticulture, Zhejiang University, Hangzhou 310029, China 2 Zhejiang Foresty University, Hangzhou 311300, China 3 Beijing Vegetable Research Center, Beijing 100089, China 4 Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, USA Abstract: Most studies related to determining the expression profile of genes and specific promoters used histochemical localization of the reporter gene, gusA. While the histochemical method for visualizing gusA expression suffers from several limitations in the determination of gene expression and location, especially in the tissues with high background acitivty. To solve this problem, a transient expession vector pBI221-RFP/GFP, was constructed using GFP and RFP as double fluorescent marker genes. This vector used CaMV 35S promoter to drive GFP and determine the transforming efficiency. It analyzed expression profile of the target gene and promoter through the RFP activities of the tranformed tissues. Through the specific promoter AGPL1 from waterm