采后损伤加速杨梅乙烯生成及果实衰老进程的分子调控机制

Postharvest injury happened easily during Chinese Bayberry storage, preservation and transportation process, which would cause a decline in quality. Postharvest injury can stimulate the bayberry ethylene production and accelerate the senescence process of fruit. However, the molecular basis and regulation mechanism are still rarely reported. This project intends to research the transcriptional regulation of ethylene generation and aging process accelerated by postharvest injury in Chinese Bayberry, using high-throughput sequencing RNA-Seq and real-time fluorescence quantitative PCR. The expression level of ethylene synthesis and senescence-related genes and transcription factors, etc. would be accurately analyzed by real-time quantitative PCR technique. Combined the data of RNA-Seq and real-time qPCR study, the impact of postharvest injury to ethylene production, intracellular level of gene transcription and signal transduction networks in Chinese Bayberry would be researched. The molecular basis and regulation mechanism of postharvest injury accelerating Chinese Bayberry ethylene production and senescence process would be explored. These work would provide a theoretical basis of screening new preservatives and treatment methods, and bring benefit to the research and development of new bayberry preservation technology system.

杨梅是我国特有的浆果，口味独特，花色苷含量丰富，经济价值高，但杨梅果实采后极易受到损伤发生腐败。现有杨梅采后研究主要集中在贮藏保鲜技术，研究内容多是活性成分及生理分析，损伤引发腐败的内在机理仍未得到阐释，初步认为损伤引发的腐败与乙烯生成和衰老调控有关，而损伤后乙烯生成规律及其如何参与衰老进程尚不清楚。本项目将围绕采后损伤如何加速杨梅乙烯生成和衰老进程的科学问题，运用高通量测序RNA-Seq、实时荧光定量PCR等转录组学和核酸定量技术，分析乙烯合成和衰老进程相关的基因和转录因子的表达变化，在整体和分子水平对转录活动及调控规律开展研究。研究杨梅采后损伤对乙烯合成基因转录和信号转导网络的影响，探索采后损伤加速杨梅乙烯生成及衰老进程的分子机理及调控机制，为探索更适宜的杨梅保鲜方法和技术体系提供理论依据。

本研究选取了浙江省杨梅主要栽培品种“东魁”为实验材料，以高通量测序技术RNA-Seq和实时荧光定量PCR技术为主要的技术手段，研究杨梅采后损伤引发的生理生化反应、乙烯代谢相关基因转录变化和果实衰老进程的变化。主要在以下方面开展了研究工作：.（1）采后损伤杨梅转录组的RNA-Seq高通量测序及生物信息学分析.提取纯化了杨梅果实采后损伤不同时期各样品的mRNA，构建了cDNA文库，并进行了RNA-Seq高通量测序及生物信息学分析工作。.（2）采后损伤对杨梅乙烯生成、信号传导和衰老进程相关基因的转录调控.克隆得到了杨梅乙烯生物合成途径中的关键酶ACO和ACS的基因，并研究了该基因在损伤后的转录变化，及其在脱落酸的影响下的转录规律。.研究得到以下主要成果：.（1）通过RNA-Seq技术获得杨梅机械损伤后转录组的Unigene数据库。该数据库包含110,732条Unigene。.（2）通过转录组学研究和生物信息学比对，明确了机械损伤后杨梅转录组主要在转录相关、蛋白质合成相关、类固醇和脂类代谢、抗氧化酶类代谢以及碳源氮源代谢和植物激素信号转导等途径发生了基因转录调控。.（4）结合转录组数据及表达量数据，提出了杨梅采后损伤加速乙烯生成和果实衰老进程的可能机理：机械损伤直接导致杨梅果实宏观结构和细胞微观结构的破坏，细胞壁损伤和细胞膜通透性变化引起细胞的胁迫应激反应，细胞内发生大量遗传信息相关的变化，表现为基因损伤修复（Nucleotide excision repair）、转录活性增强（RNA polymerase）以及活跃的蛋白质合成（Ribosome biogenesis），细胞的生理活动发生大量变化。类固醇（Steroid biosynthesis）和磷脂类（Glycerophospholipid metabolism）代谢提高，用以强化和修复细胞膜结构；碳源代谢（Pyruvate metabolism）旺盛，分解糖类以提供能量；氨基酸合成旺盛（Biosynthesis of amino acids），用以提供蛋白质合成；抗氧化酶基因上调，应对氧自由基升高。随后植物激素代谢和调控（Plant hormone signal transduction）被激活，脱落酸ABA可能早于乙烯被激活，随后调控乙烯生成，把细胞损伤信号传递给相邻细胞直至整个果实，引起更加复杂的变化