摘要
芥子酶是广泛存在于十字花科植物中的一类降解硫代葡萄糖苷的酶。当植物受到病虫侵袭时,芥子酶与其底物硫代葡萄糖苷结合,释放有毒的水解产物形成植物重要的化学防御系统。TGG5是新发现的一类芥子酶基因,对其研究将有利于揭示芥子酶防御系统的功能。试验克隆了拟南芥TGG5基因cDNA,构建酵母表达载体,转化毕氏酵母GS115。经甲醇诱导表达和Ni亲和层析,获得纯化的TGG5重组蛋白。SDS-PAGE分析表明,重组蛋白分子量集中在58kD左右,并有较明显拖尾,呈现典型的糖基化蛋白的特征。进一步探讨了TGG5重组蛋白的酶学特性,结果表明:该芥子酶的Km为1.96mmol/L,Vmax为0.084mmol/(min·mg);酶反应的最适pH为5.5;该芥子酶在37～80℃范围内有较广泛的温度适应性,并具有较好的80℃热稳定性;NaCl对该酶有明显的抑制作用;低浓度抗坏血酸具有激活芥子酶的作用,抗坏血酸终浓度为1.5mmol/L时,酶活力最大。RT-RCR分析,证实了TGG5只在拟南芥根中特异表达,这表明TGG5可能代表了一个芥子酶基因新亚家族。
Myrosinase widely exists in the Cruciferae plant,which can catalyse the hydrolysis of thioglucosides.Myrosinase and glucosinolate are mixed upon infestation by pests,and thioglucosides are hydrolyzed into toxic compounds to serve as defense weapons.A new myrosinase gene was found,denoted as TGG5.The research of myrosinase gene TGG5 could explore the function of myrosinase defense system.In this research,the cDNA sequence of TGG5 was cloned,and the pichia expression vector pPIC3.5K was constructed,then transformation of pichia GS115 was made.The expression of recombinant TGG5 protein was induced by Methanol,and purified by Ni-chelating colum.SDS-PAGE analysis showed that the recombinant protein concentrated in the 58 kD molecular weight around and showed typical features of protein glycosylation with a more visible tail.The myrosinase activity of recombinant TGG5 protein was future researched after separation and purification.The results indicated that the K m and V max of the enzyme were 1.96 mmol/L and 0.084 mmol/（min·mg） respectively.The optimal pH of tested myrosinase was 5.5.The myrosinase showed the wider adaptability in the temperature range of 37～80℃,and took on better stability on 80℃ high temperature.NaCl had an obvious inhibitory effect on activity of tested myrosinase,but low concentration of ascorbic acidcould activate the myrosinase,which reached to a climax with an ascorbic acid final concentration of 1.5 mmol/L.RT-RCR detection confirmed the TGG5 gene only specificlly expressed in roots,which showed that TGG5 represent a novel myrosinase gene subfamily.
出处
《中国农学通报》
CSCD
北大核心
2010年第15期25-31,共7页
Chinese Agricultural Science Bulletin
基金
国家自然科学基金"新型芥子酶基因TGG4和TGG5的表达调控及生物学功能研究"(30571064)
