摘要
以棉花曲叶病毒 (CLCuV)侵染的番茄叶片组织总DNA为模板 ,通过PCR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 4类CLCuV分离物的启动子序列的同源性最高为 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 (NicotianatabacumL .)和棉花 (GossypiumhirsutumL .)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 ,在叶肉及维管组织中均有较高的活性 ;病毒链基因方向启动子表达活性较低。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是棉花的遗传转化。
A bidirectional promoter of cotton leaf curl virus (CLCuV) was obtained from the total of DNA CLCuV infected tomato leaves by polymerase chain reaction, and the amplified DNA fragment was cloned into the vector. DNA sequences analysis and homology comparison with the promotor of four kinds of isolates recently found indicated that the cloned promoter fragment composed of 436 bp was 99.32% homolog was up to in nucleotides with that of the isolates. 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 the tobacco ( Nicotiana tabacum L.) and cotton (Gossypium hirsutum L.) leaf cells for transient expression by particle bombardment. The results indicated that complementary sense promoter was a strong promoter with high activity in leaf mesophyll and vascular tissues, but virion sense promoter was weaker. The experiments suggested that isolated bidirectional promoter, as a novel strong promoter, could be used for dicots and especially cotton genetic transformation.
基金
中国科学院重大项目!基金资助项目 (KY95 1_A1_3 0 2_12_0 8)
国家"863"高技术计划基金资助项目!(Z_17_0 1_0 1
BH_0 2_0 1_0 1)
