基于次生代谢功能基因的广藿香倍半萜类成分形成和积累的分子机制研究

Pogostemon cablin (Blanco) Benth., a well-known Chinese herbal medicine, is well accepted that it can aromatically transform turbid dampness, harmonizes middle jiao, stop vomiting, release exterior dampness and summer-heat attack. Commercial herbs of Pogostemon cablin are divided into Paixiang (cultivated in Guangzhou), Zhaoxiang (cultivated in Zhaoqing), Zhanxiang (cultivated in Zhanjiang) and Nanxiang (cultivated in Hainan). Generally, Paixiang and Zhaoxiang are treated as high quality geoherb, while Zhanxiang and Nanxiang are just used to extract patchouli oil. Patchouli oil is an important ingredient in many fine fragrance products, and the composition of the patchouli oil is distinct because it consists largely of sesquiterpenes. In our previous study, inherent characteristics of Pogostemon cablin in different localities have been elucidated applying GC-MS fingerprint analysis, together with RAPD fingerprint and DNA sequencing analysis. Nowadays, Paixiang is going to be on the edge of extinction because of the rapid urban construction course, and it leads to uncertain clinical effects and application limit. Therefore, it is very important to make it clear that how sesquiterpenes produce and which pathway it comes from. As an attracting result, more appropriate sesquiterpene synthases genes isolated from Pogostemon cablin might catalyze the conversion of farnesyl-diphosphate (FPP) to more sesquiterpene products. In this study, we intend to describe the identification and functional characterization and regulation of the sesquiterpene synthases genes responsible for the production of sesquiterpenes. We also describe the spatial and temporal expression characteristic of key sesquiterpene synthases genes during different development stages of Pogostemon cablin. Besides, the relationship between functional gene expression and active principles accumulation in Pogostemon cablin from different producing regions will be analyzed, which might provide scientific basis for revealing the gene regulation of sesquiterpenes and molecular mechanisms of secondary metabolists accumulating and geoherbs formation.

广藿香具有芳香化浊、开胃止呕、发表解暑之功，为治疗暑湿感冒的良药，传统认为"牌香"是优质的道地药材，发挥主要药效的广藿香油又是工业生产重要的天然香料，其组分中倍半萜类成分多达24种。我们在前期工作中，分析不同产地广藿香的化学和分子特征的异同点，建立了广藿香的GC-MS化学指纹图谱库和DNA指纹图谱库。鉴于"牌香"的濒危已成为严重制约其疗效和开发应用的瓶颈，通过倍半萜类化合物生物合成、代谢途径和调控机理的研究，寻找高效催化FPP向目标产物转化的策略，有望达到缓解和解决此问题的目的。 本项目拟在前期工作基础上，以基因工程技术手段对广藿香挥发油倍半萜类化合物生物合成关键酶基因进行克隆与功能鉴定，对其时空表达特性进行分析，并探讨不同产地广藿香功能基因的表达水平对倍半萜类成分积累的影响，旨在转录水平揭示其生物合成关键酶及其调控机制，为阐明广藿香药材分子标识和道地药材形成的分子机制提供科学依据。

唐宋时期出现了外来药物传入我国的鼎盛阶段，广藿香也在此时期由东南亚引种至我国岭南地区，并因其具有芳香化浊、开胃止呕、发表解暑之功效而得到广泛应用。 传统认为“牌香”是优质的道地药材，“肇香”品质次之，而“湛香”和“南香”因为品质较差，通常只作为提取挥发油的原料，因此发挥主要药效的广藿香精油同时又是工业生产中的重要天然香料，其中作为精油重要组分的倍半萜类成分多达24种。鉴于“牌香”的濒危及其它产区广藿香的病虫害问题已成为严重制约其疗效和开发应用的瓶颈，寻找“牌香”的替代资源、增加广藿香精油的产出途径尤其是倍半萜类成分的产量提升，成为亟待解决的关键问题。本项目从广藿香的本草学考证入手，系统地对记载广藿香的历代典籍进行追溯和述评，阐明了对广藿香引入和应用的演进过程。针对不同产地广藿香的性状特征和品质差异较大的问题，通过田间实地考察对广东及海南不同产区的12个广藿香样品进行性状特征比较，并在直接PCR测序的基础上建立了可以有效区分不同样品的核糖体ITS1区DNA分子标记，可据此开发出用于鉴别上述产区广藿香样品的特异性探针。应用3’和5’快速cDNA末端扩增法对倍半萜合酶cDNA的全长进行扩增，完成了广藿香醇合酶（patchoulol synthase）基因的克隆和原核表达载体的构建，并对其所表达的蛋白进行了性质鉴定和结构分析。研究结果为阐明广藿香药材分子标识和道地药材形成的分子机制，以及在转录水平揭示其生物合成关键酶及其调控机制提供科学依据。