蜡梅CpAGL6基因启动子的克隆及功能初步分析

Cloning and Preliminary Functional Analysis of Cp AGL6 Promoter from Chimonanthus praecox

利用hi TAIL-PCR法,从蜡梅(Chimonanthus praecox)基因组中克隆到花发育相关基因Cp AGL6翻译起始位点上游1 266 bp的启动子序列。生物信息学分析表明,该启动子序列中存在启动子的基本元件TATA-box和CAAT-box及多个与植物非生物胁迫相关的响应元件。为进一步分析Cp AGL6启动子的功能,构建该基因启动子与GUS基因融合的植物表达载体,并用农杆菌介导法转化烟草。对转基因烟草进行GUS组织化学染色及GUS酶活性定量检测,结果显示,Cp AGL6基因启动子能够驱动GUS基因在转基因烟草的叶、茎、花中表达,并且在不同花期表达强度存在差异,在根中几乎不表达。转基因植株经黑暗、赤霉素和4℃低温处理后,GUS酶活性均有所增加。结果表明,Cp AGL6启动子主要驱动GUS报告基因在花器官和绿色器官组织中表达,推测其在抵抗非生物胁迫中具有重要作用。

The regulative sequence(1 266 bp)of the flower development-related gene Cp AGL6 promoter was cloned from genomic DNA of Chimonanthus praecox by hi TAIL-PCR. Bioinformatics analysis revealed that the promoter sequence contained basic cis-elements,such as TATA-box and CAAT-box and many elements involved in the plant abiotic stress. In order to study the function of Cp AGL6 promoter,a promoter-reporter vector was constructed,and introduced into tobacco by Agrobacterium-mediated method. Then the transgenic tobacco with GUS histochemical staining and quantitative detection of GUS enzyme activity were analyzed,the results showed that Cp AGL6 promoter could drive the GUS gene exclusively express in leaves,stems and flowers of transgenic tobacco,and there were significant differences of GUS enzyme activity among the flowers in different stages,almost no expression in roots. GUS enzyme activity of transgenic plants have increased under different treatments,including darkness,gibberellin(GA3)and 4 ℃ low temperature. The results indicated that Cp AGL6 promoter mainly driven GUS reporter gene expression in floral organs and green organs or tissues of tobacco,playing a very important role in abiotic stress resistance.