摘要
【目的】以GUS基因为报告基因,构建GhCAD6基因的瞬时表达载体。【方法】采用PCR方法和基因枪法。【结果】用PCR法,以pGEM-T-CAD6质粒为模板,获得GhCAD6基因目的片段。然后将其克隆到瞬时表达载体pRTL2-GUS/NIa中,获得由CaMV35S启动子调控目的基因的pGUS-CAD6融合表达载体,使目的基因能够和GUS基因同时表达。采用基因枪法将pGUS-CAD6转化到洋葱表皮细胞中,暗培养24 h,经GUS组织化学染色,检测到多个洋葱细胞呈现蓝色。【结论】构建的瞬时表达载体可在植物细胞中高效表达,为进一步研究棉花GhCAD6基因的功能奠定了实验基础。
[ Objective and Method ] In this study β - giueuronidase gene （GUS） was used as reporter gene in biolistic transformation experiment. Transient expression vector of cotton ChCAD6 gene was constructed. [ Result] Firstly, the GhCAD6 gene cDNA was obtained from pGEM - T- CAD6 by PCR, then it was cloned into transient expression vector pRTL2 - GUS/NIa. The transient expression vector pGUS- CAD6 is driven by CaMV35S promoter with GUS reporter gene and the target gene, GUS reporter gene and target gene simultaneously express in single cell. Secondly, verified by digestion with restriction enzymes, the vector pGUS- CAD6 was transformed into onion using PDS- 1000/He biolistie particle deliver）, system. The GUS gene expression was detected in 24 h after bombardment, strong GUS gene expression was observed in the epidermal ceils of onion under microscope, the results indicated that the transient expression vector pGUS- CAD6 could be highly effectively expressed in plant cell. [Conclusion]The results of this paper provide a strong foundation for analyzing fimcfion of GhCAD6 gene in cotton in the future.
出处
《新疆农业科学》
CAS
CSCD
北大核心
2010年第1期20-24,共5页
Xinjiang Agricultural Sciences
基金
国家自然科学基金项目(30660088)
国家"863"项目(2006AA10Z184)
新疆维吾尔自治区高技术研究发展计划项目(200611101)
农业部转基因重大专项课题(2009ZX08005-011B)
