广藿香PatTPS启动子功能及DREB转录因子介导的广藿香醇合成调控机制

Patchouli alcohol is an important tricyclic sesquiterpene, which is the main medicinal bioactive component of the famous chinese herbal medicine of Pogostemon cablin. The patchoulol synthase is the key enzyme for the biosynthesis and regulation of patchouli alcohol. However, so far, its studies were restricted to gene clonging, enzyme function, or etabolic engineering application, etc., and the molecular regulation mechansim was limited. On the basis of previous experiment, we found that content of patchouli alcohol, expression level of PatTPS, and promoter of PatTPS were all induced by MeJA and ETH. Many abiotic-induction response elements were predicted through the promoter using database, and a novel transcription factor of PatDREB was screened and identified to bind to it. On the basis of this study, it was suggested that PatDREB probably bound to the DRE element, regulated the transcription of the target gene of PatTPS, and mediated the expression pattern of PatTPS by participating in certain hormonal signaling pathways, and then regulated the biosynthesis of the PatTPS. Our project intends to systematacially study the promoter function of the PatTPS, and dissects the transcriptional activation and expression regulation of PatTPS mediated by DREB, revealing the regulatory mechanism and molecular interactions regulatory network of biosynthesis of patchouli alcohol.

广藿香醇是一种重要三环倍半萜类化合物，是著名中药材广藿香的挥发油主要药用活性成分。广藿香醇合成酶是广藿香醇合成调控的关键酶，但是目前研究局限于基因分离克隆、酶学功能和代谢工程应用等，其分子调控机制鲜有报道。前期实验申请人发现广藿香醇含量、PatTPS表达量以及PatTPS启动子活性均明显响应茉莉酸甲酯和乙烯诱导。数据库预测获得启动子上多个与非生物刺激响应相关序列元件，并通过筛选获得与之结合的新的转录因子PatDREB。研究基础暗示PatDREB很可能结合启动子DRE元件，调节靶基因PatTPS的转录，参与某些激素信号通路影响PatTPS表达模式，进而调控广藿香醇的生物合成。本课题拟系统地展开广藿香醇合酶基因PatTPS启动子功能研究，并且解析DREB转录因子介导的PatTPS的转录激活和表达调控，揭示广藿香醇的生物合成机理，为倍半萜的生物合成通路分子互作和调控网络等提供理论基础。

广藿香醇是一种三环倍半萜，是从广藿香中提取的广藿香油的主要活性成分，在医学和商业上具有重要应用意义。迄今为止，广藿香醇的生物合成途径已被阐明；然而，转录因子对广藿香醇生物合成的转录调控仍然未知。本研究中，我们分离了广藿香醇合酶（PatPTS）基因的启动子，发现茉莉酸甲酯（MeJA）处理可以显著诱导其转录活性。通过酵母单杂交（Y1H）和双荧光素酶（dual-LUC）检测发现，全新的一个核位置的AP2/ERF转录因子PatDREB可直接结合并激活PatPTS启动子。广藿香叶中过表达PatDREB导致广藿香醇产量显著增加，MVA途径基因表达显著上调。酵母双杂交（Y2H）分析也揭示了PatDREB和JAZ4之间的相互作用，表明PatDREB可能在JA信号通路中发挥作用。此外，Y2H和pull down分析进一步发现，PatDREB可以与PatSWC4相互作用，它们协同促进了广藿香醇的生物合成。此外，PatJAZ4可以抑制PatDREB-PatSWC4复合物的转录激活活性，这种抑制作用可以被茉莉酸（JA）逆转。综上，我们鉴定分析了PatPTS启动子的主要功能，解析了PatDREB和PatDREB-PatSWC4复合物在调节广藿香醇生物合成中的积极作用，提高了我们对植物萜类代谢调节网络的理解。