雷公藤中一个合成香茅醇的单萜合酶基因的克隆与表征  

Cloning and characterization of a monoterpene synthase gene from Tripterygium wilfordii

作　　者：尹艳 郭思远[2] 向安娅[2,4] 李桂林 高伟[2,3] 张夏楠[2] YIN Yan;GUO Si-yuan;XIANG An-ya;LI Gui-lin;GAO Wei;ZHANG Xia-nan(School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 102488,China;School of Traditional Chinese Medicine,Capital Medical University,Beijing 100069,China;School of Pharmaceutical Sciences,Capital Medical University,Beijing 100069,China;School of Pharmaceutical Sciences,Sun Yat-Sen University,Guangzhou 510006,China)

机构地区：[1]北京中医药大学中药学院,北京102488 [2]首都医科大学中医药学院,北京100069 [3]首都医科大学药学院,北京100069 [4]中山大学药学院,广州510006

出　　处：《中国中药杂志》2019年第16期3588-3593,共6页China Journal of Chinese Materia Medica

基　　金：北京市教育委员会科技计划一般项目(KM201710025012);北京市自然科学基金面上项目(7182018)

摘　　要：雷公藤是临床常用于治疗类风湿性关节炎的药用植物,又有抗肿瘤和肥胖症治疗等药理活性。雷公藤中的已知活性成分以萜类为主,但是含量都非常低,因此对雷公藤中萜类化合物生物合成途径的解析成为解决雷公藤资源问题的一大研究热点。而萜类合酶(TPS)是催化形成多种多样的萜类骨架的关键酶。该研究从雷公藤中克隆到一条ORF长度为1 785 bp的基因片段TwMES,利用NCBI的BLASTP,ProtParam,Interpro在线工具以及MEGA 6.0软件对序列进行了生物信息学分析,并利用实时荧光定量PCR检测了该基因对茉莉酸甲酯的响应,同时采用原核表达和体外酶促的方法对其体外催化功能进行了验证。生物信息学分析表明,该基因编码的氨基酸序列同时具有萜烯合酶N-末端结构域、萜环化酶样1 C-末端结构域,以及萜类合酶家族class I的DDxxD保守结构域。在与已知同源萜类合酶序列构建的邻接树中,TwMTS与TPS-b亚家族聚为一支。RT-PCR结果显示50μmol?L^-1 的MeJA 12 h能导致TwMTS的表达量上升到对照组的735倍,24 h达到1 644倍。此外,体外酶促反应结果显示TwMTS能催化GPP生成β-香茅醇,表明TwMTS是一个单萜合酶。以上研究结果为雷公藤萜类成分的生物合成学研究提供了新的元件,为未来生物合成研究奠定了基础。Tripterygium wilfordii is a medicinal plant commonly used in the treatment of rheumatoid arthritis,and with pharmacolog-ical activities in anti-tumor and obesity treatment.The known active ingredients in T.wilfordii are mainly terpenoids,but with very low content.Therefore,the analysis of the biosynthesis pathway of terpenoids in T.wilfordii has become a research hotspot to solve the problem of its resources.Terpenoid synthase(TPS)is a key enzyme that catalyzes the formation of a wide variety of terpenoid skele-tons.In this study,a gene fragment with an ORF of 1 785 bp was cloned from T.wilfordii.Bioinformatics analysis was performed using NCBI's BLASTP,ProtParam and Interpro online tools and MEGA 6.0 software.The response of this gene to methyl jasmonate was also detected by real-time fluorescent quantitative PCR,and its catalytic function was verified by prokaryotic expression and in vitro enzy-matic assay.Bioinformatics analysis indicated that the amino acid sequence encoded by this gene had both N-terminal domain and C-terminal domain of TPS,as well as the DDxxD conserved domain of the class I of TPS family.And TwMTS gathered together with TPS-b subfamily in the Neighbor-Joining Tree constructed with known homologous TPSs.The results of RT-PCR showed that 50μmol·L^-1 MeJA 12 h could increase the expression of TwMTS to 735 times in the control group at 12 h,and 1 644 times at 24 h.In addi-tion,in vitro enzymatic reaction results showed that TwMTS can catalyze the production ofβ-citronellol with GPP as substrate,indica-ting that TwMTS was a monoterpene synthase.The above results provided a new element for the synthetic biology database of T.wilfordii terpenoids,and laid the foundation for future biosynthesis research.

关 键 词：雷公藤 单萜合酶 香茅醇 

分 类 号：R28[医药卫生—中药学]