陆地棉GDP-L-半乳糖磷酸化酶基因启动子的克隆及活性分析

Cloning and expression activity analysis of GDP-L-galactose phosphorylase gene promoter in Gossypium hirsutum L.

抗坏血酸(Ascorbic acid,As A)在植物发育和响应逆境胁迫过程中发挥着重要作用。GDP-L-半乳糖磷酸化酶(GDP-L galactose phosphorylase,GGPase)是As A合成过程中关键酶之一,在As A的生物合成和代谢调控中具有重要作用。为深入了解棉花Gh GGPase基因的表达调控模式,本研究利用PCR技术克隆得到Gh GGPase基因起始密码子上游2 464 bp的启动子序列,序列分析发现该启动子包含多个参与激素信号、光调节和抗逆信号应答的顺式作用元件。构建p Gh GGPase::GFP-GUS过量表达载体,采用农杆菌介导法瞬时转化烟草叶片,进行GUS组织化学染色和酶活性检测,GUS染色实验结果显示,瞬时转化启动子的烟草叶片表现出较深的蓝色,并且具有较高的酶活性表达,这表明Gh GGPase启动子能够显著的驱动GUS的表达。以上结果表明:Gh GGPase启动子是一个完整的功能序列,具有较强的表达活性。

Ascorbic acid（As A） plays important roles in the plant development and response to abiotic stress. GDP-L galactose phosphorylase（GGPase） is one of the key enzymes in As A biosynthesis and has significant function in the As A metabolism and regulation. In this study, toinvestigate the expression and regulation pattern of cotton Gh GGPase gene,the 2464 bp upstream sequences of the start codon of a cotton Gh GGPase was cloned from fiber tissues by PCR method. The plant overexpression vector p Gh GGPase：：GFP-GUS was constructed and transformed into tobacco leaves through Arobacterium mediated transient transformation method, and the transgenic tobacco leaves were used to detect GUS histochemical staining and enzyme activity. The results showed that the promoter region contains multiple cis-acting elements including hormone signal, light regulation and stress signal response, and GUS staining experiment demonstrated that the transgenic tobacco leaves displayed deep-blue appearance and had a high GUS expression activity, suggesting that the Gh GGPase promoter is able to drive GUS expression significantly. All these results suggested that the Gh GGPase promoter is a complete functional sequence with strong expression activity.