根癌农杆菌vbp2基因启动子转录调控的探析  

Exploration of the Transcriptional Regulation of Agrobacterium tumefaciens vbp2 Promoter

作　　者：徐楠 徐宇娟 孙盼[1] 宗仁杰 郭敏亮[1] XU Nan;XU Yu-juan;SUN Pan;ZONG Ren-jie;GUO Min-liang(College of Bioscience and Biotechnology,Yangzhou University,Yangzhou 225009)

机构地区：[1]扬州大学生物科学与技术学院,扬州225009

出　　处：《生物技术通报》2021年第12期41-49,共9页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31870118,21808196,31170073);中国博士后基金(2018M632389);江苏省高校面上基金(18KJB180030)。

摘　　要：根癌农杆菌(Agrobacterium tumefaciens)能够将自身质粒上的部分DNA片段(简称T-DNA)以T-复合物的形式转运、整合至宿主基因组,而用作高效的植物转基因工具。vbp2是编码VBP蛋白的3个同源基因之一,VBP蛋白通过与T-复合物上的VirD2结合参与T-DNA的转运过程。根据生物信息学预测,vbp2基因的启动子区可能含有多个转录调控元件。通过同源重组的方法,将A.tumefaciens染色体上的vbp2基因替换成编码红色荧光蛋白的rfp基因,使vbp2的启动子控制rfp的表达,因此,可通过测定所表达的RFP的荧光强度来表征vbp2启动子的活性。结果表明:乙酰丁香酮(AS)和鼠李糖(Rha)均能诱导vbp2启动子表达RFP,AS和Rha的最佳诱导浓度分别为150μmol/L和100μmol/L。通过截短vbp2启动子DNA片段的方法,证明响应AS和Rha诱导的调控区域分别位于上游的165-260 bp和126-165 bp。Agrobacterium tumefaciens is able to transfer DNA fragments(T-DNA)from its plasmid to plant cells in the form of T-complex and to integrate the T-DNA into the genome of host plants,thereby it has been used as an effective transgenic vector for plant.vbp2 is one of the three homologous genes encoding VBPs,and VBPs are involved in the transport of T-DNA by binding with the VirD2 in the T-complex.Bioinformatics analysis showed that there might be several transcriptional regulation elements in the promoter region of vpb2 gene.The vbp2 in the chromosome of A.tumefaciens was in situ replaced by the rfp encoding red fluoresce protein(RFP)using homologous recombination method,so that promoter of vbp2 controlled the expression of rfp.Therefore,the activity of vbp2 promoter can be characterized by the fluorescence intensity of the expressed RFP.The results showed that acetosyringone(AS)and rhamnose(Rha)both were able to induce the vbp2 promoter to expresse RFP.The optimal concentrations of AS and Rha for the induction were 150μmol/L and 100μmol/L,respectively.The test of the transcriptional activity of the truncated vbp2 promoter regions revealed that transcriptional regulation elements induced by AS and Rha were located on the regions of 165-260 bp and 126-165 bp upstream of the vbp2 gene,respectively.

关 键 词：根癌农杆菌 vbp2基因 转录调控 转录活性 荧光蛋白 

分 类 号：Q78[生物学—分子生物学]