ERFs与脂质代谢基因在桃果实采后冷害发生与调控中的作用及其机制研究

As a chilling sensitive fruit, peach is prone to postharvest chilling injury (CI). Pre-storage ethylene treatment was reported to be able to alleviate peach fruit CI, and in our previous study, enhanced ethylene production was found to be related with the alleviation of CI by LTC (pre-storage of fruit at 8℃ for 5 days before 0°C storage). However, the underpinning molecular mechanisms for regulation on peach fruit CI by ethylene are still unknown. Therefore, with ‘Hujingmilu’ and ‘Yulu’ peach as experimental materials, through application of combined lipidomics and transcriptomics analyses, the project plans to mine candidate ERFs and lipid genes regulated by ethylene; then by applying transient and stable transformation in Arabidopsis and tobacco, as well virus induced gene silencing in peach fruit, to evaluate the function of these genes; finally, DNA/protein and protein/protein interaction analyses will be conducted to explore the regulatory roles of ERFs on lipid genes. The study aims to elucidate the molecular basis for alleviation of peach fruit CI by ethylene via ERF. The study can provide in-depth understanding of the biology of CI occurrence and its manipulation, as well as theoretical support for developing postharvest and breeding strategies for reducing postharvest CI.

桃是冷敏性果实，采后冷藏极易发生冷害。前人研究发现贮前乙烯处理可以减轻桃果实冷害，我们先前研究表明LTC（在0℃贮藏前将果实置于8℃锻炼5天）处理减轻桃果实冷害与处理维持了较强的乙烯生成相关，但乙烯调控果实冷害的具体分子机制还不清楚。本项目拟以‘湖景蜜露’和‘玉露’桃为研究材料，运用脂质组和转录组联合分析筛选乙烯调控表达的ERFs和脂质基因，通过拟南芥转基因、烟草瞬时表达和转基因以及桃VIGS分离鉴定参与桃果实冷害发生及其调控的关键ERFs和脂质代谢基因，进而通过DNA/蛋白质和蛋白/蛋白互作技术研究ERFs对脂质代谢基因的调控机制，以阐明乙烯通过ERF调控脂质代谢从而减轻桃果实冷害的分子机制。项目研究对于深入理解冷害发生与调控的生物学机制具有重要意义，也可为开发减轻冷害的贮藏措施与培育抗冷品种提供理论支撑。

随物种不同，乙烯促进或减轻果实采后冷害。乙烯对桃冷害也有影响，但起着促进还是抑制作用则报道不一，且其机制需要探究。脂质代谢关联细胞膜透性，与冷害褐变密切相关。因此，项目围绕乙烯对桃果实冷害和脂质代谢调控这一主题开展了研究工作。研究表明，‘中华寿桃‘果实冷害主要表现为果肉褐变和果实软化受阻，乙烯处理可减轻冷害并伴有乙烯合成能力增强；不同冷藏温度贮藏后果实冷害程度与乙烯合成负相关；贮前果实乙烯释放速率与贮后冷害程度负相关，表明乙烯抑制桃果实冷害具有普适性。转录组分析表明，乙烯处理或非冷害温度下的乙烯加强合成增强了细胞壁修饰基因表达，促进了果实软化；同时，通过调节脂质代谢维持细胞膜透性，减轻了果肉褐变。鉴别了涉及脂质变化和细胞壁修饰的系列关键基因，以及分别参与乙烯调节脂质代谢和细胞壁修饰相关基因表达的ERF成员ABR1和ESE3。不同温度下冷藏桃果实发生不同程度的冷害褐变，以5℃下最重，12℃下也有发生。DNA甲基化组分析表明DNA甲基化很可能参与低温诱导桃果实冷害发生。在乙烯合成和信号转导相关的基因中，分别有5个和4个基因的表达水平与其对应的启动子不同基序甲基化水平表现出明显的负相关性和正相关性。而就差异脂质代谢基因而言，绝大多数基因启动子区DNA甲基化水平在导致严重冷害的温度（5℃和8℃）处理中较高。综上所述，乙烯处理诱导了ABR1和ESE3等ERF成员表达，进而分别调控脂质代谢和细胞壁修饰，最终控制果肉褐变并维持果实软化，减轻果实冷害症状。不同温度下果实冷害程度的差异与乙烯合成、脂质代谢受不同程度影响相关，而DNA甲基化涉及这些代谢途径中关键基因表达的调控。这些研究发现诠释了乙烯在调控桃冷害中的作用及机制，并对于产业上调整控制冷害的措施提供了理论依据。