棉花GhCCR4基因表达载体构建及GUS基因的瞬时表达

研究以GUS基因为报告基因,构建CCR4基因的瞬时表达载体,首先酶切带有CCR4基因的 pGEM-T-CCR4载体,获得CCR4基因目的片段,然后将其克隆到瞬时表达载体pGUS/NIa中,获得由CaMV35S启动子调控目的基因的pGUS-CCR4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-CCR4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,表明构建的瞬时表达载体可在植物细胞中高效表达.为进一步在棉花中研究GhCCR4基因的功能奠定了实验基础.

Construction of Cotton GhCCR4 Gene Expression Vector and Transient Expression in GUS Gene

In this study β-glucuronidase gene(GUS) was used as reporter gene in biolistic transformation experiment.Transient expression vector of cotton GhCCR4 gene was constructed.Firstly,the CCR4 gene fragment was obtained from digesting pGEM-T-CCR4,then it was cloned to transient expression vector pGUS/NIa.The transient expression vector pGUS-CCR4 is driven by CaMV35S promoter with GUS reporter gene and the target gene,GUS reporter gene and target gene simultaneously were expressed in single cell.Secondly,verified by digestion with restriction enzymes,the vector pGUS-CCR4 was transformed into onion using PDS-1 000/He biolistic particle delivery system.The GUS gene expression was detected in 24 h after bombardment.Strong GUS gene expression was observed in the epidermal cells of onion under microscope,the results indicated that the transient expression vector pGUSCCR4 could be high efficiency expressed with high efficiency in plant cell.The results of this paper provide a strong foundation for analysis function of CCR4 gene in cotton in the future.