小麦TaUBC基因泛素结合酶活性分析  

Ubiquitin Conjugating Enzyme Acitivity Analysis of Wheat TaUBC Gene

作　　者：张祥云[1] 赵思语 温潇 王宁[1] 郭彦[1] 赵庆臻[1] ZHANG Xiang-yun;ZHAO Si-yu;WEN Xiao;WANG Ning;GUO Yan;ZHAO Qing-zhen(School of Life Sciences,Liaocheng University,Liaocheng 252059,China)

机构地区：[1]聊城大学生命科学学院,山东聊城252059

出　　处：《聊城大学学报（自然科学版）》2018年第3期79-85,共7页Journal of Liaocheng University:Natural Science Edition

基　　金：国家自然科学基金项目(31200907);植物基因组学国家重点实验室开放课题(2016A0219-14);山东省自然科学基金(ZR2014CL1009);聊城大学大学生科技文化创新基金(SF2014261;26312160907)资助

摘　　要：已报道的RNA水平研究表明TaUBC可能是小麦产量相关基因,生物信息学分析显示该基因属于泛素结合酶家族.荧光定量PCR显示TaUBC在小麦根茎叶中均有表达.为了验证TaUBC蛋白是否具有泛素结合酶活性,本文利用原核系统表达野生型TaUBC及三种点突变TaUBC^(C21S)、TaUBC^(C85S)、TaUBC^(C107S)蛋白,并对这些蛋白进行体外泛素化活性分析.结果证实TaUBC是一个有活性的泛素结合酶.另外,点突变C85S使得TaUBC丧失E2活性,C21S和C107S则对活性没有影响,证明其UBC结构域中第85位Cys是结合泛素的关键作用位点.Reported results of TaUBC gene in RNA level indicated it may be a yield-relevant gene in wheat.Bioinformatic analysis showed that TaUBC was expressed in root,stem and leaf.In order to verify whether TaUBC protein has ubiquitin conjugating enzyme activity,wild type and three different point mutation types of TaUBC protein(TaUBC C21S、TaUBC C85S、TaUBC C107S)were expressed in bacteria respectively.Ubiquitin conjugating enzyme activity were then detected by in vitro ubiquitination assay.The results showed that TaUBC was an active ubiquitin conjugating enzyme.Point mutation C85S resulted in loss of ubiquitin conjugating enzyme activity of TaUBC,while C21S and C107S had no influence on the activity,proving that C85 in the UBC domain is the crucial catalytic site for ubiquitin binding.

关 键 词：TaUBC 泛素结合酶 点突变 体外泛素化反应 

分 类 号：Q783[生物学—分子生物学]