摘要
该研究通过PCR扩增、酶切及连接等分子生物学研究方法分别将β-葡萄糖苷酶基因（gus A）和绿色荧光蛋白基因（gfp）与双向启动子两端融合，构建成双可视报告基因融合双向启动子的植物二元表达载体pBDGG．通过农杆菌介导的叶盘法转化毛白杨叶片以鉴定所构建的双向启动子的功能．对农杆菌侵染3～4d的毛白杨同一叶片进行GUS组织化学染色检测和紫外激发后通过荧光显微镜观测绿色荧光，结果表明gusA基因和疥基因都能够进行瞬时表达，表明该双向启动子可在毛白杨中实现双向表达．该文还讨论了双向启动子在植物基因工程中应用的可能性。为实现植物基因工程的“一箭双星”（即一个启动子同时双向表诀两个或名个基丙）奠定基础．
Simultaneous introduction of multiple genes into plants is a critical step in plant genetic engineering to manipulate multiple functional genes in metabolic engineering and trait stacking. It is important to construct a bidirectional promoter for transforming two or more genes into plants simultaneously. Widely used unidirectional CaMV35S promoter has been modified to a bidirectional promoter in this work by fusing a CaMV 35S minimal promoter （Pmini） at its end in opposite orientation to the original promoter. To test its bidirectional transcriptional activities, two widely used histochemically visible reporter genes, gus A （β- glucuronidase） from Escherichia coli and gfp （ green fluorescent protein） from Aequorea victoria, were fused to the terminus of the bidirectional promoter in different orientations ending with NOS terminator sequences. The transient expression of gus A and gfp genes was detected by histochemical staining for GUS and by fluorescence microscopy for GFP. The diction of transient expression of GUS and GFP in Agrobacterium mediated 3-4 days transformed leaf discs of Populus tomentosa indicated that the promoter did have bidirectional transcriptional activities simultaneously in cells and tissues. It was discussed that this bidirectional promoter could possibly be applied in woody plants engineering.
出处
《北京林业大学学报》
CAS
CSCD
北大核心
2007年第1期119-122,共4页
Journal of Beijing Forestry University
基金
国家自然科学基金项目(30271066
30571512)
关键词
毛白杨
双向启动子
GFP基因
gusA基因
瞬时表达
Populus tomentosa, bidirectional promoter, gfp gene, gus A gene, transient expression
