一种新型双向启动子--棉花曲叶病毒启动子的分离、序列分析与功能初探

以棉花曲叶病毒(CLCuV)侵染的烟草叶片组织总DNA为模板,通过聚合酶链反应扩增CLCuV双向启动子片段并插入克隆载体.序列分析和同源性比较表明,克隆的启动子长436bp,与目前发现的9种CLCuV株系的启动子序列均不相同,同源性最高达99.32%.将启动子片段分别以不同方向与GUS报告基因和nos终止子融合,构建了瞬时表达载体.通过基因枪法将质粒载体导入烟草和棉花叶片细胞中进行瞬时表达,结果表明,互补链基因方向启动子属强启动子,在叶肉及维管组织均有较高的活性;病毒链基因方向启动子表达活性较低.本文初步证实分离的互补链基因启动子可作为新型强启动子应用于双子叶植物尤其棉花的遗传转化.

Isolation, Sequence Analysis, and Preliminary Functional Studies of a Novel Bidirectional Promoter, Cotton Leaf Curl Virus Promoter

The cotton leaf curl virus(CLCuV) promoter were obtained from CLCuV infected tobacco leaves total DNA by Polymerase Chain Reaction, and the amplified DNA fragment were cloned into vectors. DNA sequences analysis and homology comparison indicated that the cloned promoter fragment composed of 436bp is different from that of recently found nine isolates and has 99.32% homologies in nucleotides compared with the highest affinity isolate. Transient expression vectors were constructed by fusing the promoter fragment with GUS reporter gene and nopaline terminator in different orientation. These constructs were delivered into tobacco and cotton leaf cells for transient expression by particle bombardment. Results indicated that complementary sense promoter was a strong promoter with the high activity in leaf mesophyll and vascular tissues. In comparison, virion sense promoter was weaker. Experiments suggested that isolated bidirectional promoter could be applied in dicot transformation, especially cotton genetic manipulation.