摘要
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 4 36bp ,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。
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.
出处
《高技术通讯》
EI
CAS
CSCD
2000年第4期13-17,共5页
Chinese High Technology Letters
基金
中国科学院重大项目基金!(KY951A13021208)
国家863高技术计划基金!(Z170101
BH-02-01-01)资助项目
关键词
双生病毒
启动子
棉花
烟草
基因枪
Geminivirus
Promoter
Cotton
Tobacco
Particle bombardment
