龙眼Mn-SOD基因的表达及其启动子功能分析

摘要

Expression of Mn-SOD was analyzed in embryogenic callus (EC) under abiotic stress and exogenous hormone treatment in Dimocarpus longan,and its promoter activities were studied in a tobacco transient expression system.Results showed that the expression of Mn-SOD was inhibited in longan EC treated by red and blue lights;Mn-SOD firstly increased and then decreased in white light treated-EC,and it has contrary expression pattern under green light treatment.The expression of Mn-SOD was inhibited under salt stress,and was not affected by sucrose and days treatment.In a certain range of concentration,exogenous hormones IAA,GA3,MeJA,SA and ETH could induce or inhibit the expression of Mn-SOD in longan EC,suggesting that longan Mn-SOD was involved in the response of exogenous hormones.Four deletions of longan Mn-SOD promoter fused to the GUS reporter gene were constructed and transient transformed into tobacco.The results showed that the activities of the four deletions of longan Mn-SOD promoter were lower than that of CaMV35S promoter,but all of them had the ability to activate the GUS gene.GUS activity and quantitative expression analysis both showed that 3'terminal deletion of MSD-pro4 (-420 bp) promoter is the strongest,followed by the terminal 5'deletions of MSD-pro1(-669～-1 bp),and the weakest were MSD-pro2 (-432 ～-1 bp) and MSD-pro3 (-267～-1 bp),suggesting that longan Mn-SOD promoter within the-669～-432bp region contains important positive regulatory elements,while the -1～432 region contains a negative regulatory element.%以龙眼胚性愈伤组织为材料,研究龙眼Mn-SOD基因在应答非生物因子和外源激素处理下的转录情况,并利用烟草瞬时转化法分析其启动子的表达活性.结果表明:龙眼胚性愈伤组织中Mn-SOD基因的表达受到红光和蓝光的抑制;而在白光处理下,Mn-SOD呈现先上升再下降的趋势,在绿光中,则是先下调再上升.在盐胁迫处理下,Mn-SOD基因的表达受到抑制,在蔗糖和不同天数处理中则不受影响.在一定浓度范围内,外源激素IAA、GA3、MeJA、SA和ETH均能诱导或抑制龙眼胚性愈伤组织中Mn-SOD基因的表达,提示其参与外源激素的应答.构建4个不同启动子缺失片段驱动报告基因GUS的植物表达载体并转化烟草.结果表明,龙眼Mn-SOD基因4个不同启动子缺失片段启动GUS基因的能力均低于CaMV35S启动子,但均可启动GUS基因的表达.瞬时表达和定量表达结果显示,3'缺失的MSD-pro4(-669～-267 bp)启动GUS基因的能力最强,其次为5'缺失的MSD-pro1(-669～-1 bp),最弱的为MSD-pro2(-432～-1bp)和MSD-pro3(-267～-1 bp),提示Mn-SOD启动子-669～-432 bp区段内含有重要的正向顺式调控元件;而-432～-1 bp区段含有负调控元件.