调控番茄抗坏血酸合成关键基因GME1的转录因子克隆及功能分析

Ascorbic acid is important antioxidant in plants and is also essential nutrient for humans. GDP-mannose-3',5'-phenotype isomerase (GME) catalyzes the first step for ascorbic acid synthesis on glycosides level, and is also involved in gulose pathway. Previous study from our research group have shown that the regulation of GME1 can significantly increase the accumulation of ascorbic acid in tomato fruit, and compared with a enzymatic catalyzation, the transcription factor showed coordinate regulation in multiple sites with more efficiency. In this project, tomato transcription factors regulating tomato GME1 are to be cloned, among which NF-YA has been isolated. The yeast two hybrid is utilized to isolate the transcription factors of GME1, and transcriptional colinearity is also used to screen out those transcription factors with high concurrence with GME1 expression. Then, EMSA and luciferase system is used to verify transcription factors and gfp fusion protein is used to characterize their subcellular localization. By RNAi and overexpression, the function of GME1 transcription factor is to be investigated. Furthermore, Pull-down, yeast two-hybrid and BiFC are used to identify interacting proteins or complex subunits with transcription factors, the function of which is also investigated in transgenic tomato. Cloning and identification of GME1 transcription factor will reveal novel mechanism underlying ascorbic acid regulation in plants, and create new tomato germplasm with high vitamin C level, and thus is of huge scientific and practical significance.

抗坏血酸是植物重要的抗氧化剂，也是人体必需的营养物质。GDP-甘露糖-3’,5’-表型异构酶（GME）催化抗坏血酸生物合成途径中糖核苷水平的第一步，并参与古洛糖途径。项目组前期试验显示调控GME1基因表达可显著增加番茄果实抗坏血酸积累，而转录因子相比催化酶具有多位点协同调控和高效应优势。本项目以番茄GME1为靶基因，在前期GME1转录因子NF-YA的克隆鉴定基础上，通过酵母杂交分离调控GME1转录因子，结合转录谱共线性分析筛选与GME1表达高度相关的转录因子，通过酵母杂交、EMSA和荧光素系统进行验证，分析其gfp核定位特征。通过RNAi和超量表达研究GME1转录因子的功能，利用Pull-down、酵母双杂交、BiFC鉴定转录因子的互作蛋白或复合体亚基，并进行转基因功能分析。GME1转录因子的克隆及鉴定将揭示植物抗坏血酸合成调控新机制，并创制高维生素C的番茄新种质，具有重要科学和实践意义。

抗坏血酸是植物重要的抗氧化剂，也是人体必需的营养物质。GDP-甘露糖-3’,5’-表型异构酶（GME）催化抗坏血酸生物合成途径中糖核苷水平的第一步，并参与古洛糖途径。通过项目执行，本项目研究鉴定了调控GME1 基因的转录因子SlNFYA10，阐明了GME1 基因转录因子SlNFYA10对GME1 负调控机制，创制维生素C 含量显著提高的番茄材料8 份。研究结果对于园艺作物果实品质改良提供了新材料和新理论。项目执行期间，以通讯作者发表标注资助论文12篇，其中SCI收录论文9篇，出版教材1部（副主编），申请专利10项，授权专利6项，获得湖北省科技二等奖和国家科技进步二等奖（序4），培养硕士生5人，博士生2人，博士后1人。