泽泻原萜烷型三萜生物合成分子调控机制及其关键前体合成生物学研究

Protostane triterpenes, which are specific active components in a few plants like Alisma orientale (Sam.) Juz., display bioactivities including anti-cancer, anti-hyperlipidemia and anti-HIV. However, this type of compounds cannot meet the demands for its narrowly natural distribution and low contents. Based on our previous research that we have already cloned several key enzyme genes such as AoPSS1and AoPSS2, and candidate transcription factors, this project propose that the key enzymes’ catalytic effect affect the biosynthesis of protostane triterpenes in A. orientale (Sam.) Juz. as well as their expression levels were regulated by transcription factors and elicitors; Furthermore, the construction of the carbon skeleton protosterol in protostane triterpenes is considered as the key step in its biosynthesis pathway; while Synthetic Biology concerned about how to solve the resource scarcity of these active components effectively. The objects of this research subject including: (1) Clarify the regulatory mechanism of the transcription factors which involved in biosynthesis pathway of protostane triterpenes in A. orientale (Sam.) Juz. (2) Study the biosynthesis mechanism of protostane triterpenes induced by elicitors in A. orientale (Sam.) Juz. (3) Build the heterologous expression system to synthesize the key precursor protosterol in A. orientale (Sam.) Juz. This research subject will not only illustrate the biosynthesis regulatory mechanism but also obtain the key precursor of protostane triterpenes in A. orientale (Sam.) Juz, thus provide a foundation to improve the quality of A. orientale (Sam.) Juz. and a clue to solve the resource scarcity.

原萜烷型四环三萜类化合物是泽泻属等少数植物类群中所特有的活性成分，具有抗癌、抗高血脂、抗HIV等生理活性。由于该类资源性成分自然分布窄，含量低，不能满足利用需求。课题组已获得该类化合物生物合成关键酶AoPSS1和AoPSS2等基因及相关候选转录因子，本项目在前期工作基础上提出：原萜烷型三萜类化合物的生物合成取决于关键酶的催化效率，其表达水平受转录因子及诱导子等调控，关键前体原萜烷型三萜骨架原甾醇的形成是其生物合成的核心，而合成生物学研究是解决该类资源性成分稀缺的有效途径。拟开展研究工作：(1)泽泻原萜烷型三萜生物合成相关候选转录因子的调控功能研究；(2)诱导子对该类化合物生物合成诱导机制研究；(3)构建高效异源表达体系生物合成该类化合物关键前体原甾醇。以阐明泽泻原萜烷型三萜生物合成分子调控机制，获得该类化合物关键前体，为改善泽泻品质及解决该类资源性成分稀缺问题奠定基础。

原萜烷型三萜类化合物是泽泻属等少数植物类群中所特有的活性成分，具有抗癌、抗高血脂、抗HIV等生理活性，应用前景广阔。本课题开展了泽泻原萜烷型三萜生物合成调控机制及其关键前体合成生物学研究，取得的成果如下：（1）克隆获得与泽泻原萜烷型三萜类生物合成相关的转录因子AoMYC2a、AoMYC2b、AoMYB4a全长序列，成功构建原核表达载体，诱导转录因子外源表达，并研究其和关键酶基因相互作用关系，揭示了泽泻原萜烷型三萜生物合成相关转录因子的调控机制；（2）在诱导子茉莉酸甲酯和水杨酸作用下，生物合成相关转录因子的表达量显著上调，关键酶基因表达明显增加，同时23-乙酰泽泻醇B含量也明显提高，且转录因子、关键酶基因的表达量与活性成分含量呈显著正相关，阐明了诱导子对泽泻原萜烷型三萜生物合成的诱导作用及机制；（3）克隆获得原萜烷型三萜生物合成关键酶氧鲨烯环化酶全长序列，构建真核表达载体，优化表达条件，获得了高效异源表达体系；并建立标准反应体系，获得了泽泻原萜烷型三萜类化合物生物合成关键前体；（4）建立泽泻转录组学、蛋白组学和microRNA组学平台，构建了“转录因子-差异基因-差异蛋白-活性成分”的调控网络，诱导子通过信号转导系统激活相应转录因子，引发泽泻原萜烷型三萜类化合物生物合成关键酶基因高效表达，提高了原萜烷型三萜类化合物的合成效率，从而调控原萜烷型三萜类化合物生物合成。研究结果为实现泽泻原萜烷型三萜的异源生物合成提供理论与实践依据，为解决该类资源性成分稀缺问题奠定坚实基础。