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.
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.
Zhongguo Zhong Yao Za Zhi. 2023 May;48(9):2307-2315. doi: 10.19540/j.cnki.cjcmm.20230213.103.
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, linalool-type, 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.
樟树是中国重要的经济树种。根据叶片挥发油中主要成分的类型和含量，樟树被分为五个化学型，包括冰片型、樟脑型、芳樟醇型、桉叶素型和橙花叔醇型。萜类合酶（TPS）是这些化合物形成的关键酶。虽然已经鉴定出几个关键酶基因，但具有最高经济价值的（+）-冰片的生物合成途径尚未见报道。在本研究中，通过对四种化学型叶片的转录组分析，克隆了九个萜类合酶基因CcTPS1-CcTPS9。重组蛋白经大肠杆菌诱导后，分别以香叶基焦磷酸（GPP）和法呢基焦磷酸（FPP）为底物进行酶促反应。CcTPS1和CcTPS9都能催化GPP生成冰片基焦磷酸，冰片基焦磷酸可被磷酸水解酶水解得到（+）-冰片，（+）-冰片产物分别占0.4%和89.3%。CcTPS3和CcTPS6都能催化GPP生成单一产物芳樟醇，CcTPS6还能与FPP反应生成橙花叔醇。CcTPS8与GPP反应生成1,8-桉叶素（30.71%）。九个萜类合酶产生了9种单萜和6种倍半萜。该研究首次鉴定出樟树中负责冰片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以及利用生物工程技术培育高产冰片新品种奠定了基础。
