响应茉莉酸诱导GbMYC2转录因子调控银杏萜内酯合成的分子机制

The critical medicinal ingredient terpene trilactones (TTLs) are only identified in Ginkgo biloba, which is one of the most important economic forest species in China. However, the natural content of TTLs in leaf is extremely low, which significantly restricts the development of ginkgo industry. In our previous studies, we identified a transcription factor GbMYC2 that responds to JA signal. The relative expression level of GbMYC2 was positively correlated with the content of TTLs, indicating that GbMYC2 somehow involves in the regulation of TTLs biosynthesis. Based on these work, we use Ginkgo biloba as test materials, and intend to develop the following essays to further elucidate the TTLs biosynthetic pathway. 1) Identify JAZ protein interacting with GbMYC2 via yeast two-hybrid and bimolecular fluorescence complementation technology, thus to reveal the molecule mechanism of GbMYC2 in response to JA signaling. 2) Screen GbMYC2-regulated downstream target genes by yeast one-hybrid technique and dual luciferase reporter assays. 3) Verify the role of GbMYC2 in regulating TTLs biosynthesis by over-expression or repression GbMYC2 (via RNAi) in Ginkgo biloba callus, and further confirm the target genes regulated by GbMYC2. The expected results of this project will provide an important theoretical basis and technical reference for improvement of TTLs content by metabolic engineering in future.

银杏是我国重要的经济林树种，萜内酯是银杏独有的萜类化合物，具有重要的药用价值。然而大多数银杏叶萜内酯含量极低，这严重制约了银杏产业的发展。申请人前期筛选到一个响应茉莉酸诱导转录因子GbMYC2，其在银杏各组织中的表达量与萜内酯含量呈显著正相关，表明GbMYC2可能参与调控银杏萜内酯的生物合成。据此，本项目以银杏为试材，拟开展以下研究：1）通过酵母双杂交和双分子荧光互补技术鉴定与GbMYC2互作的茉莉酸信号途径转录因子JAZ，揭示银杏GbMYC2响应茉莉酸信号的分子机制；2）利用酵母单杂交技术和双荧光素酶报告基因检测实验筛选GbMYC2调控的下游靶基因；3）通过构建GbMYC2超表达和RNAi载体转化银杏愈伤，验证GbMYC2对萜内酯生物合成的调控作用，进一步明确GbMYC2调控的靶基因。项目预期成果可为今后利用遗传改良技术提高银杏萜内酯含量提供重要的理论依据和技术参考。

银杏（Ginkgo biloba L.），又名公孙树，为银杏科银杏属独有的孑遗植物。银杏因含有黄酮类化合物和萜内酯等天然药用活性成分而获得广泛应用。萜内酯化合物是银杏独有的成分，具有抗血栓、抗炎、治疗心脑血管疾病和预防阿尔茨海默病等药理活性，还是血小板活化因子最强的拮抗剂，但因其在银杏中的含量非常低，不足以满足医药行业的需求而应用受限。因此提高银杏中萜内酯含量具有重要意义。前期研究发现MeJA处理后，银杏叶中GbMYC2s表达水平与萜内酯含量具有较高相关性，且萜内酯合成途径中的多个关键酶基因的启动子中存在能够与MYC2结合的基序。本研究以MYC2为切入点，探究了GbMYC2响应茉莉酸信号的表达模式；通过酵母双杂、双分子荧光互补实验验证了GbMYC2与GbJAZs间的互作关系；通过酵母单杂实验筛选并鉴定到GbMYC2的靶基因GbGGPPS；通过银杏叶片瞬时表达GbMYC2试验证明其参与了银杏萜内酯生物合成，且主要促进了二萜类银杏内酯的合成。本研究结果有助于探索转录因子的调控模式，为高萜内酯含量的银杏育种提供有价值的靶点，同时建立了一种有效的银杏转基因体系，为相关研究提供切实可靠的验证手段。