樟树化学型形成关键萜类合酶的系统鉴定  

Systematic identification of chemical forms of key terpene synthase in Cinnamomum camphora

作　　者：马青 马蕊 苏平[1] 申业[1] 陈美兰[1] 靳保龙[1] 欧阳少林 郭娟[1] 崔光红 黄璐琦[1] MA Qing;MA Rui;SU Ping;SHEN Ye;CHEN Mei-lan;JIN Bao-long;OUYANG Shao-lin;GUO Juan;CUI Guang-hong;HUANG Lu-qi(State Key Laboratory Breeding Base of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Henan University of Chinese Medicine,Zhengzhou 450000,China;Ji′an Forestry Science Research Institute,Ji′an 343300,China)

机构地区：[1]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 [2]河南中医药大学,河南郑州450000 [3]江西省吉安市林业科学研究所,江西吉安343300

出　　处：《中国中药杂志》2023年第9期2307-2315,共9页China Journal of Chinese Materia Medica

基　　金：国家重点研发计划项目(2020YFA0908000);中央本级重大增减支项目(2060302)。

摘　　要：樟树Cinnamomum camphora为我国重要的经济树种。根据叶片挥发油中主要成分的种类和含量,樟树通常被分为龙脑型、樟脑型、芳樟醇型、桉叶油型和橙花叔醇型5种化学型,萜类合酶(terpene synthase,TPS)是形成这些化合物的关键酶。虽然多个关键酶基因已得到鉴定,但最具经济价值的(+)-龙脑的生物合成途径未见报道。该研究通过对4种化学型叶片的转录组分析,克隆了9个萜类合酶基因CcTPS1~CcTPS9。经过大肠杆菌诱导表达重组蛋白后分别以香叶基焦磷酸(GPP)和法尼基焦磷酸(FPP)为底物进行酶促反应,结果CcTPS1和CcTPS9均可催化GPP生成冰片基焦磷酸,并在磷酸水解酶的作用下得到(+)-龙脑,产物占比分别为0.4%、89.3%。CcTPS3和CcTPS6均可催化GPP生成单一产物芳樟醇,CcTPS6还可与FPP反应生成橙花叔醇。CcTPS8与GPP反应生成1,8-桉叶油(30.71%)。9个萜类合酶共计产生9个单萜和6个倍半萜类化合物。该研究首次确定樟树中负责(+)-龙脑生物合成的关键酶基因,为进一步阐明化学型形成的分子机制及利用生物工程技术培育高产龙脑新品种奠定了基础。Cinnamomum camphora is an important economic tree species in China.According to the type and content of main components in the volatile oil of leaf,C.camphora were divided into five chemotypes,including borneol-type,camphor-type,linalooltype,cineole-type,and nerolidol-type.Terpene synthase(TPS)is the key enzyme for the formation of these compounds.Although several key enzyme genes have been identified,the biosynthetic pathway of(+)-borneol,which has the most economic value,has not been reported.In this study,nine terpenoid synthase genes CcTPS1-CcTPS9 were cloned through transcriptome analysis of four chemical-type leaves.After the recombinant protein was induced by Escherichia coli,geranyl pyrophosphate(GPP)and farnesyl pyrophosphate(FPP)were used as substrates for enzymatic reaction,respectively.Both CcTPS1 and CcTPS9 could catalyze GPP to produce bornyl pyrophosphate,which could be hydrolyzed by phosphohydrolase to obtain(+)-borneol,and the product of(+)-borneol accounted for 0.4%and 89.3%,respectively.Both CcTPS3 and CcTPS6 could catalyze GPP to generate a single product linalool,and CcTPS6 could also react with FPP to generate nerolidol.CcTPS8 reacted with GPP to produce 1,8-cineol(30.71%).Nine terpene synthases produced 9 monoterpene and 6 sesquiterpenes.The study has identified the key enzyme genes responsible for borneol biosynthesis in C.camphora for the first time,laying a foundation for further elucidating the molecular mechanism of chemical type formation and cultivating new varieties of borneol with high yield by using bioengineering technology.

关 键 词：樟树 化学型 萜类合酶 冰片基焦磷酸合酶 龙脑 

分 类 号：R284.1[医药卫生—中药学]