高山红景天UGT72B14-2基因克隆及其原核表达  

Cloning and prokaryotic expression of UGT72B14-2 from Rhodiola sachalinensis

作　　者：胡滢滢 薛飞燕[2] 杨明峰[2] 吕鹤书[2] 柳春梅[2] 马兰青[2] 

机构地区：[1]北京农学院植物科学技术学院,北京102206 [2]农业部都市农业(北方)重点实验室,北京102206

出　　处：《北京农学院学报》2016年第4期7-11,共5页Journal of Beijing University of Agriculture

基　　金：北京市优秀人才基金项目(2013D005021000003);北京市自然科学基金项目(2164059);国家自然科学基金项目(31300620;31370674)

摘　　要：【目的】旨在构建UGT72B14-2的原核表达体系,为进一步研究其功能与应用奠定基础。【方法】利用RT-PCR技术从高山红景天茎叶中分离获取UGT72B14-2基因,在大肠杆菌中表达后,经Ni 2+亲和柱纯化、PD-10柱脱盐处理,利用SDS-PAGE检测目标蛋白。【结果】测序结果表明UGT72B14-2基因长度为1 422bp,预测其编码473个氨基酸,蛋白质相对分子量(Mr)为51.49kDa,等电点(pI)为6.30;SDS-PAGE检测结果表明当初始菌液OD600约0.6时,诱导4h后目标蛋白即可大量表达,纯化、脱盐和浓缩处理后可获得较高纯度的重组蛋白。【结论】UGT72B14-2属于UDP-葡萄糖基转移酶家族的一员,能够在大肠杆菌中表达。【Objective】Salidroside,as a main effective ingredient of Rhodiola sachalinensis,has many pharmacological properties such as hypoxia tolerance,anti-aging,immune enhancement,and so on.Uridine diphosphate glucosyltransferase（UDP-glycosyltransferase,UGT）is the key enzyme of salidroside biosynthesis.In this study,we have constructed prokaryotic expression system which is necessary for further study of the function and application of UGT72B14-2.【Methods】UGT72B14-2 was cloned from the leaf stems of R.sachalinensis by RT-PCR and expressed in a prokaryotic system of Escherichia coli.The recombinant protein was analyzed by SDS-PAGE after treated with a Ni 2＋affinity column and a PD-10 column.【Results】The gene of UGT72B14-2,with 1 422 bp in length,encodes 473 amino acids.The molecule weight（MR）and isoelectric point（PI）of its corresponding protein are predicted as 51.49 kDa and 6.30,respectively.The recombinant protein was expressed abundantly when the induction initiated at OD600-0.6and lasted 4h.And then the recombinant protein with high purity was obtained after purification and concentration.【Conclusion】UGT72B14-2is a new member of UGTs family and can be expressed in E.coli.

关 键 词：高山红景天 尿苷二磷酸葡萄糖基转移酶 原核表达 

分 类 号：Q786[生物学—分子生物学]