灯盏花黄酮6-羟化酶（F6H）和类黄酮7-O-葡糖醛酸转移酶（F7GAT）基因克隆与功能分析

Erigeron breviscapus (Vant.) Hand.-Mazz. is one of medicinal plants with regional feature of Yunnan Province, which particularly and highly accumulates scutellarin, a kind of 6-OH flavone glucuronide derivatives. Even the biosynthesis of flavonoids is well elucidated in plant, the biosynthesis and regulation mechanism of scutellarin is still unclear especially on the last two key reactions, hydrolation at 6-C and glucuronidation at 7-C, both of which is catalyzed by flavone 6-hydroxylase (F6H) and ?avonoid 7-O- glucuronosyltransferase (F7GAT), respectively. To elucidate the biosynthesis pathway of scutellarin in Erigeron breviscapus, the genes of two key enzymes (F6H & F7GAT) will be cloned by the methods of homologous cloning，DD-RT-PCR, and cDNA library screening. The catalytic characteristics of the enzymes and the functions of their genes will be detected by heterologous expression in yeast expression system and RNA interference using Agrobacterium rhizogenes-mediated genetic transformation, respectively. Furthermore, the temporal and spatial expression of two genes and the effects of environment factors on their expression will be detected by Real-Time PCR. The results of this study will provide a full understanding of biosynthesis of scutellarin and helpful information on molecular breeding of Erigeron breviscapus.

灯盏花[Erigeron breviscapus (Vant.) Hand.-Mazz.]是最具云南地方特色药用植物之一，能够特异性地积累高含量的灯盏乙素(6-OH黄酮的葡糖苷酸) 。尽管植物类黄酮的生物合成途径已清楚，但是灯盏乙素的生物合成机理仍尚未完全明确，特别是最后两步关键反应，即6-位羟化和7-位葡糖苷酸化，其分别由黄酮6-羟化酶（F6H）和类黄酮7-O-葡糖醛酸转移酶（F7GAT）催化。本项目拟利用同源克隆、DD-RT-PCR、cDNA文库筛选、RACE等技术克隆灯盏花F6H基因和F7GAT基因，酵母表达检测酶催化底物特异性，RNA干涉验证基因功能；并利用定量PCR方法检测两个基因的时空表达模式，及其表达对环境因素的响应和与灯盏乙素积累的关系，明确灯盏乙素生物合成最后两步反应的分子机理与调控机制，阐明灯盏乙素完整的生物合成途径，为灯盏花分子育种奠定基础。

灯盏花[Erigeron breviscapus (Vant.) Hand.-Mazz.]是最具云南地方特色药用植物之一，能够特异性地积累高含量的灯盏乙素(6-OH 黄酮的葡糖苷酸) 。尽管植物类黄酮的生物合成途径已清楚，但是灯盏乙素的生物合成机理仍尚未完全明确，特别是最后两步关键反应，即6-位羟化和7-位葡糖苷酸化，其分别由黄酮6-羟化酶（F6H）和类黄酮7-O-葡糖醛酸转移酶（F7GAT）催化。.本项目利用转录组测序、基因组测序、以及酵母合成等方法，克隆到灯盏花的F6H和F7GAT基因，并且完成了灯盏乙素的酵母全合成，为灯盏乙素工业生产提供了可能。同时，建立了灯盏花的转基因体系和基因编辑技术体系。研究还发现菊科植物灯盏花的F7GAT基因的保护序列与已知的唇型目植物不同，说明二者催化特性的形成的分子机理不同，但还需要进一步的研究。