雷公藤MCT基因RNAi对雷公藤萜类活性成分生物合成的影响  

Effect on biosynthesis of terpenoid active components through RNA interference with MCT gene in Tripterygium wilfordii

作　　者：宋雅迪[1] 赵瑜君 陈上 胡添源 张睿[1] 王家典[1] 卢鋆[1] 王秀娟[1,3] 高伟[1,3] 黄璐琦[2] SONG Ya-di;ZHAO Yu-jun;CHEN Shang;HU Tian-yuan;ZHANG Rui;WANG Jia-dian;LU Yun;WANG Xiu-juan;GAO Weix;HUANG Lu-qi(School of Traditional Chinese Medicine,Capital Medical University,Beijing 100069,China;State Key Laboratory of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Beijing Key Lab of TCM Collateral Disease Theory Research,School of Traditional Chinese Medicine,Capital Medical University,Beijing 100069,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,道地药材国家重点实验室培育基地,北京100700 [3]首都医科大学中医药学院,中医络病研究北京市重点实验室,北京100069

出　　处：《药学学报》2018年第8期1209-1214,共6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 to W.G.)

摘　　要：MCT是萜类生物合成MEP途径上重要的关键酶,本研究利用Gateway克隆重组技术构建TwMCT基因的RNAi表达载体,得到了大小为484 bp的载体片段。通过基因枪技术将TwMCT RNAi载体转入到雷公藤悬浮细胞。采用UPLC测定雷公藤萜类活性成分的含量,结果显示细胞中雷公藤甲素和雷公藤红素的含量相对于对照组p K7GWIWG2D分别下降了23.4%和42.8%。同时利用q RT-PCR检测TwMCT基因及萜类合成途径上主要基因的表达量,结果表明,相对于对照组p K7GWIWG2D,TwMCT表达量下降了29.2%,TwDXR、TwGGPS、TwHMGR和TwHMGS的相对表达量分别下降了36.3%、31.3%、62.2%和29.1%,但TwDXS的表达量上调了114.2%,而TwFPS没有显著性变化。本研究在体内验证了干扰TwMCT的表达对雷公藤萜类活性成分雷公藤甲素和雷公藤红素的积累有显著抑制作用,为进一步探究MCT基因对雷公藤萜类成分生物合成的调控机制奠定基础。MCT is an important key enzyme in the terpenoid biosynthesis in MEP pathway.In this study,Gateway technology was used to construct RNAi vector of Tw MCT,and a vector fragment with a size of 484 bp was obtained.The Tw MCT RNAi vector was transferred into the suspension cells of Tripterygium wilfordiiby gene gun.Accumulation of terpenoids was assayed by UPLC,and the result showed that the content of triptolide and celastrol in cells decreased by 23.4% and 42.8%,respectively,compared with the control group p K7 GWIWG2 D.Moreover,the gene expression of Tw MCT and major genes in terpenoid biosynthesis pathway was detected by q RT-PCR,which demonstrated that the expression of Tw MCT reduced by 29.2% relative to that of the control group p K7 GWIWG2 D,and the relative expression of Tw DXR,Tw GGPS,Tw HMGR and Tw HMGS diminished by 36.3%,31.3%,62.2%,and 29.1%,respectively,but the expression of Tw DXS was up-regulated by 114.2%,and there was no significant change in Tw FPS.Thus,it was verified in vivo that interference with Tw MCT expression significantly inhibited the accumulation of triptolide and celastrol in Tripterygium wilfordii,laying a foundation for further exploring the regulation mechanism of MCT gene on the terpenoid biosynthesis in Tripterygium wilfordii.

关 键 词：雷公藤 TwMCT RNAi 雷公藤甲素 雷公藤红素 

分 类 号：R931[医药卫生—生药学]