TwHMGR过表达对雷公藤甲素和雷公藤红素生物合成的影响  

The effects of TwHMGR overexpression on the biosynthesis of triptolide and celastrol in Tripterygium wilfordii

作　　者：王家典[1,2] 赵瑜君 张逸风 胡添源[1] 卢鋆 周家伟[1] 马宝伟[1] 张睿 高伟[1] 黄璐琦[2] WANG Jia-dian;ZHAO Yu-jun;ZHANG Yi-feng;HU Tian-yuan;LU Yun;ZHOU Jia-wei;MA Bao-wei;ZHANG Rui;GAO Wei;HUANG Lu-qi(School of Traditional Chinese Medicine,Capital Medical University,Beijing 100069,China;National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]首都医科大学中医药学院,北京100069 [2]中国中医科学院中药资源中心,北京100700

出　　处：《药学学报》2018年第8期1225-1232,共8页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81773830);北京市自然科学基金项目;北京市教育委员会科技发展计划重点项目(KZ201710025022);北京市属高校长城学者培养计划(CIT&TCD20170324)

摘　　要：雷公藤3-羟基-3-甲基戊二酸单酰辅酶A还原酶(Tripterygium wilfordii 3-hydroxy-3-methylglutaryl coenzyme-A reductase,Tw HMGR,Gen Bank:ALU11310.1 3)是MVA途径中的第一个限速酶,是细胞质中萜类代谢途径中的重要调控位点。为探讨Tw HMGR对雷公藤甲素和雷公藤红素生物合成的影响,对其进行了过表达研究。利用Gateway技术将Tw HMGR的开放阅读框(Open Reading Frame,ORF)构建到过表达载体中,通过基因枪介导过表达载体转入雷公藤悬浮细胞。q RT-PCR结果显示Tw HMGR的基因相对表达量在过表达组显著提高,是空载体组基因相对表达量的1.75倍;UPLC结果显示雷公藤甲素及雷公藤红素含量在过表达组中分别提高为空载组的163.93%和190.04%。本实验中Tw HMGR在雷公藤悬浮细胞中成功过表达,表现为基因相对表达量的上升和雷公藤甲素及雷公藤红素含量的上调,证明了Tw HMGR在雷公藤萜类成分生物合成途径上的正向调节作用,为雷公藤重要活性萜类成分的合成生物学研究奠定了基础。Tripterygium wilfordii 3-hydroxy-3-methylglutaryl coenzyme-A reductase（Tw HMGR） is an important regulation site in terpenoids metabolic pathway in cytoplasm which is the first speed limit enzyme of MVA pathway.In order to investigate the effects of Tw HMGR on the biosynthesis of triptolide and celastrol in Tripterygium wilfordii,the overexpression of Tw HMGR（OE-HMGR） was studied in this paper.We cloned the full-length of Tw HMGR to construct overexpression vector by Gateway technology then delivered the expression vector into Tripterygium wilfordii suspension cells by gene gun.q RT-PCR was used to detect the expression of Tw HMGR： the expression of Tw HMGR was increased to 1.75 folds over the control group（empty vector： p H7 WG2 D） in the overexpression group.The accumulation of triptolide and celastrol in the suspension cells of Tripterygium wilfordii was detected by UPLC,revealing that： the contents of triptolide and celastrol were increased to 163.93% and 190.04% of over the control group in the overexpression group.Based on these findings,the positive effect on the accumulation of active terpenoids,triptolide and celastrol in Tripterygium wilfordii was found and the results laid a foundation of the synthetic biology research on important active terpenoids in Tripterygium wilfordii.

关 键 词：TwHMGR 过表达 雷公藤甲素 雷公藤红素 基因枪 

分 类 号：R931[医药卫生—生药学]