利用重组PCR技术构建适合真菌ATMT转化的GFP基因表达载体  

Construction of GFP Gene Expression Vector Suitable for Fungal ATMT Transduction by Recombinant PCR Technology

作　　者：张运峰[1,2] 范永山[2] 冯志娟[2] 林满丽[2] 董金皋[1] 

机构地区：[1]河北农业大学真菌毒素实验室,河北保定071000 [2]唐山师范学院生命科学系,河北唐山063000

出　　处：《安徽农业科学》2009年第27期12972-12974,共3页Journal of Anhui Agricultural Sciences

基　　金：河北省科技厅项目(05547007D-2);唐山师范学院博士资金项目

摘　　要：[目的]构建适合真菌ATMT转化的GFP基因表达载体。[方法]利用含有GFP基因和Nos终止子的A2GFP质粒和含有普遍适于真菌基因表达的构巢曲霉启动子(Ptrpc)的pUCATPH质粒,通过重组PCR技术构建Ptrpc-GFP-Nos重组基因,然后插入到农杆菌质粒pCAMBIA 1300的多克隆位点,构建适合真菌ATMT转化的GFP基因表达载体,并对其进行酶切和测序鉴定。[结果]通过重组PCR技术,仅通过Ptrpc启动子基因扩增、GFP-Nos基因扩增、Ptrpc-GFP-Nos重组基因扩增、1次双酶切、1次连接和转化,就成功地构建了适合真菌ATMT转化的GFP基因的表达载体pCAMBIA 1300-PGN,不需要构建中间载体,并大大简化了连接步骤。该载体经过PCR、酶切和测序鉴定,结构正确,连接准确,序列无误。[结论]该研究为真菌ATMT突变体的构建和快速检测奠定了基础。[ Objective ] The aim was to construct the GFP gene expression vector suitable for fungal ATMT transduction. [ Method ] The A2GFP plasmid contained GFP gene and Nos terminator and pUCATPH plasmid contained Aspergillus nidulans promoter Ptrpc generally suitable for epiphyte gene expression were used for construction of Ptrpc-GFP-Nos recombinant gene by recombinant PCR technology, then the recombinant was inserted into multiple cloning site of agrobacterium plasmid.pCAMBIA 1300, and then the GFP gene expression vector suitable for fungal ATMT transduction was constructed and it was identified by restriction enzyme and sequencing. [ Result] The GFP gene expression vector pCAMBIA 1300-PGN suitable for fungal ATMT transduction was constructed successfully by recombinant PCR technology and only through Ptrpc promoter gene amplification, GFP-Nos gene amplification, Ptrpc-GFP-Nos recombinant gene amplification, one double digestion, one connection and transformation, which did not need construct intermediate vector and simplified the connection steps. The result of identification of PCR, restriction enzyme and sequencing showec that the structure of the vector was right, its connection was accurate and the sequence was right. [ Conclusion] The research laid the foundation for the construction and fast detection of fungal ATMT mutant.

关 键 词：重组PCR技术 绿色荧光蛋白 ATMT转化 表达载体 

分 类 号：S188[农业科学—农业基础科学]