转录因子HuERF1调控鲜切火龙果酚类物质合成的机制研究

The biosynthesis of phenolic compounds induced by wounding stress in fresh-cut fruits and vegetables is closely associated with the regulation of ethylene signal. As one of the most important transcription factors in the downstream of ethylene signaling pathway, ERF transcription factor plays a crucial role in wounding responses and the biosynthesis of secondary metabolites in plants, but its molecular regulation mechanism involved in the phenolic biosynthesis in fruits and vegetables is still poorly understood. Our previous results indicated that HuERF1 was most likely involved in the biosynthesis of phenolic compounds in fresh-cut pitaya fruit, which remains to be further investigated. In the present study, with pitaya fruit as the test material, the mechanism of transcription factor HuERF1 involved in the biosynthesis of phenolic compounds induced by wounding stress in fresh-cut pitaya fruit will be investigated and served as the core research content. Beginning with key genes cloning of pitaya fruit, the present project is going to analyze the subcellular localization and transcription activity of HuERF1, explore the target genes of HuERF1 and investigate their interaction model by molecular biological techniques including electrophoretic mobility shift assay, dual-luciferase transient expression assay, yeast two-hybrid assay and bimolecular fluorescence complementation analysis, which will finally elucidate the mechanism of HuERF1 involved in the biosynthesis of phenolic compounds in fresh-cut pitaya fruit based on transcription regulation and protein interaction. This project will provide important references for further understanding the mechanism of phenolic regulation under wounding stress and guiding the practice of controlling the product quality of fresh-cut pitaya fruit.

机械损伤胁迫诱导的鲜切果蔬酚类物质合成与乙烯信号分子的调节密切相关。ERF作为乙烯信号通路下游关键的转录因子，在植物伤胁迫应答反应及其次生代谢产物的合成中发挥重要作用，但其调控果蔬酚类物质合成的分子机制尚不清楚。我们前期的研究发现，鲜切火龙果酚类物质合成积累与HuERF1关系密切，其调控机制亟待深入研究。本项目拟以火龙果果实为材料，以HuERF1调控鲜切火龙果酚类物质合成的机制为核心研究内容，克隆火龙果中相关的未知基因，分析HuERF1的亚细胞定位和转录活性，利用EMSA、双荧光素酶报告基因实验、Y2H、BiFC等技术，探索苯丙烷代谢途径关键基因作为HuERF1靶向基因的可能性并解析其中的互作模式，以期从转录调控、蛋白互作等角度深入揭示转录因子HuERF1调控鲜切火龙果酚类物质合成的分子机制。本研究将为进一步认识果蔬伤胁迫下酚类物质调控的机制、指导鲜切火龙果品质控制技术的实践提供科学依据。

鲜切果蔬因新鲜、方便、卫生、营养等优点，已逐渐成为果蔬消费的重要组成部分。但鲜切产品较完整果实更易发生品质劣变，损失严重。当前限制鲜切果蔬保鲜新技术开发的最主要因素是对果蔬伤胁迫响应的关键基础理论仍缺乏深层次的认识。酚类物质是果蔬中重要的抗氧化物质，在鲜切果蔬损伤修复中发挥重要作用。研究表明，酚类物质合成积累与乙烯等信号分子的调节密切相关，但相关研究大多与信号分子对酚类物质积累终端反应的直接调控相关。本项目以火龙果为研究对象，围绕鲜切火龙果酚类物质合成的转录调控这一科学问题，研究了转录因子HuERF1对鲜切火龙果酚类物质合成的调控作用。获得的主要结果如下：1）运用生物信息学分析手段，筛选获得了可能调控鲜切火龙果酚类物质生物合成的HuERF1等关键转录因子。2）结合外源乙烯、1-甲基环丙烯（1-MCP）处理，明确了乙烯信号通路在鲜切火龙果酚类物质合成中的作用，并发现HuERF1在乙烯诱导鲜切火龙果酚类物质积累中发挥重要作用。3）利用凝胶阻滞实验（EMSA）、双荧光素酶报告基因实验（DLR）分析了HuERF1对酚类物质生物合成关键途径苯丙烷代谢相关靶基因的调控作用，发现HuERF1可以与HuPAL、HuC4H的启动子靶向结合并激活其转录活性。该项目研究成果有助于进一步认识鲜切果蔬酚类物质代谢的机理及其调控机制，为今后研发鲜切果蔬品质控制技术提供新的思路。