芡实种仁类黄酮合成关键基因EfPAL功能解析

Euryale ferox Salisb is an important characteristic vegetable in China. It is widely used in traditional Chinese medicine and health food because it is rich in flavonoids and other functional substances. At the same time, It ishe medicine food homology often known as "water ginseng". The transcriptome sequencing and iTRAQ proteomics were used to study the different developmental stages of Euryale ferox Salisb, accurately obtain the key gene of flavonoid synthes is EfPAL. However, the regulation mechanism of EfPAL in the synthesis of flavonoids in Euryale ferox Salisb has not been reported. In this study, we use genetic transformation, VIGS, subcellular localization and transgene technology to verified functional the system select candidate genes. The regulation network of candidate gene EfPAL involved in flavonoid anabolism was investigated by yeast single-, double-hybrid, Chip, EMAS, pull-down and electrophoretic mobility shift assays. This study will help to promote the molecular breeding process of Euryale ferox Salisb with high flavonoid content and serves the high-quality development of Euryale ferox Salisb industry.

芡实是我国重要的特色蔬菜，因富含类黄酮等功能物质而广泛用于中药和保健食品，常被誉为“水中人参”，是我国重要的药食同源植物。我们通过对芡实种仁发育过程的转录组和iTRAQ蛋白质组学分析，确定EfPAL是芡实类黄酮合成关键基因，但其对芡实种仁中类黄酮的合成调控机制尚未见报道。本项目拟利用酵母单、双杂交、染色质免疫共沉淀、EMAS、pull-down和凝胶阻滞电泳技术等，探讨EfPAL调控芡实类黄酮合成的代谢网络；挖掘该网络中与EfPAL协同起重要作用的关键上下游基因；并利用VIGS、亚细胞定位和转基因等技术对相关候选基因进行系统功能验证，探明EfPAL调控芡实类黄酮合成的分子机制，促进高类黄酮含量的芡实分子育种进程，服务芡实产业的高质量发展。

类黄酮是植物中最重要的次生代谢产物之一，苯丙氨酸解氨酶(PAL)是类黄酮生物合成的第一限速酶。然而，关于PAL功能的研究还很少。芡实种仁芡米中含有丰富的类黄酮，是我国的一种重要的特色保健蔬菜，也是我国许多中药的重要原料。通过全基因组鉴定，我们获得了12个芡实的PAL基因，除了基因结构上的差异外，它们在蛋白质结构上都是严格保守的。系统发育分析表明，EfPAL1-6在进化过程中是完全保守的。进一步与转录组中检测到的PAL进行比较，发现EfPAL1和EfPAL2是类黄酮生物合成的关键候选基因。随后，酶活性测定和高效液相色谱表明，EfPAL1和EfPAL2都催化苯丙氨酸生产肉桂酸。拟南芥过表达EfPAL1和EfPAL2可促进类黄酮的生物合成。此外，Y1H实验确定转录因子EfZAT11和EfHY5与EfPAL2的启动子结合，进一步荧光素酶(LUC)活性分析表明，EfZAT11促进EfPAL2的表达，EfHY5抑制EfPAL2的表达。这些发现明确了黄酮生物合成过程中涉及的关键PALs，并建立了上游调控网络。为黄酮类化合物生物合成机理的研究提供了新的信息。