MYB和bHLH转录因子对天山雪莲类黄酮生物合成途径协同调控机理研究

Saussurea involucrata is a Chinese precious medicinal plant (from genus Saussurea, Asteraceae)，ranked as level 3 of threatened species, with the function of rheumatism and chills relieving, blood activating, anti-inflammatory and analgesia. Flavonoids are significantly active compounds in Saussurea involucrata. It is essential for Saussurea involucrata serving as traditional Chinese medicinal materials to regulate the flavonoid biosynthetic pathway and attain the higher medicinal composition, and so as to meet with modern market need and industrialization. Single or several transcription factors can often activate the cooperative expression of many genes in the secondary metabolism pathway, thus regulating the pathway effectively. The biological synthesis of flavonoid in Saussurea involucrata was regulated molecularly in our lab. The transgenic Saussurea involucrata with red phenotype, higher anthocyanin and flavonoid accumulation, was acquired through simultaneously transforming the Arabidopsis PAP1 (MYB) and the Maize Lc (bHLH) transcription factor, while the green material through transforming separately PAP1 and Lc. Therefore, it is inferred that PAP1 and Lc regulate cooperatively the biosynthetic pathway of anthocyanin and flavonoid in Saussurea involucrata. The main goal of the project is to find out how PAP1 and Lc regulate coperatively the biosynthetic pathway of anthocyanin and flavonoid in Saussurea involucrata; the interaction between PAP1 and Lc; and the regulation mechanism to structual genes of the transcription factors.Thus this will be of important value for finally clarifying the regulation mechanism of the flavonoid biosynthesis in Saussurea involucrata and developing the resources.

天山雪莲是我国名贵药用植物，属国家三级保护濒危物种,具有散寒除湿、活血通经、抗炎、镇痛等功能。雪莲主要有效成分为类黄酮化合物，对其进行有效调控，获得更高含量目标产物是雪莲作为中药材适应现代市场和产业化的必由之路。而单个或几个转录因子常可激活次生代谢物合成途径中多个基因协同表达，从而有效调控次生代谢途径。本实验室开展了雪莲类黄酮生物合成分子调控研究。将拟南芥PAP1（MYB）和玉米Lc（bHLH）基因共转化天山雪莲得到了花青素以及类黄酮含量等药用成分提高的红色转基因雪莲，而单独转二者任何一个基因所得到的雪莲都是绿色表型，因此推测PAP1和Lc协同调控雪莲花青素及类黄酮生物合成途径。本项目主要研究PAP1和Lc基因是如何协同调控雪莲花青素及类黄酮生物合成途径，特别是转录因子之间相互作用以及转录因子对结构基因的调节机理。对最终解析雪莲类黄酮生物合成调控机理及有效开发天山雪莲资源具有重要意义。

天山雪莲品质优良，是用药最广泛的雪莲，但野生资源远远不能满足市场需求，因此人工种植被寄予厚望。为人工种植提供活性成分高产的雪莲种质，开展天山雪莲的代谢基因工程研究势在必行。.本研究将拟南芥PAP1、玉米Lc以及它们的双价载体导入天山雪莲，获得了相应的转基因愈伤组织和植株，其中共转化PAP1和Lc启动了天山雪莲幼苗的花青素合成和积累；同时也不同程度提高了其他苯丙素成分含量，如绿原酸(酚酸)、紫丁香苷(木脂素)、1,5-二咖啡酰奎宁酸(酚酸)、芦丁(类黄酮)等；基因表达分析显示，绝大多数的苯丙素代谢相关的基因表达被上调，尤其是与类黄酮以及花青素代谢相关的基因，如PAL、CHS、F3H、DFR3、ANS、F3′H等。由此可见，共转化PAP1(MYB)和Lc(bHLH)能够调控天山雪莲苯丙素的生物合成，提高了苯丙素活性成分含量。此外，抗氧化活性评价显示，随着苯丙素含量的提高，共转化PAP1和Lc天山雪莲的抗氧化能力也明显增强。. 针对雪莲苯丙素代谢的基因工程调控的目标，首先开展了天山雪莲苯丙素代谢途径解析的工作，对天山雪莲进行了转录组测序，获得了与苯丙素代谢相关酶和转录因子基因序列相似性极高的45条Unigenes，并根据基因功能注释分别命名。通过高效液相色谱-质谱联用(HPLC-MS)和荧光实时定量方法对天山雪莲不同其器官和生长阶段的苯丙素代谢产物和相关基因进行了定量分析。综合分析基因表达与代谢产物的关系，推测：1) 天山雪莲MYB2与拟南芥AtMYB 90功能同源，能够调控其花器官中花青素合成相关基因CHS1、DFR1、ANS表达，积累花青素；2) 天山雪莲FLS、DFR3、GT2和CHS5等基因在花器官中大量表达，可能在其类黄酮合成中发挥了重要作用。而天山雪莲MYB3和bHLH表达模式与这些酶基因类似，有可能MYB3和bHLH通过对上述类黄酮合成相关酶基因的调控，控制天山雪莲花中类黄酮合成和积累。. 我们克隆了天山雪莲的MYB2、MYB3、bHLH和WDR基因，对最终解析雪莲类黄酮生物合成的调控机理，对有效开发天山雪莲资源具有重要意义。