冷藏南果梨香气变淡的分子机制研究

Nanguo Pear (Pyrus ussuriensis Maxim) grows mainly in the Liaoning province. It is normally harvested in the middle of September. After appropriate ripening, the picked fruit present golden color with smooth flesh and rich smell in which the fruit is excellent in quality. The shelf-life of Nanguo pear at room temperature is only 15 to 20 days. Refrigeration can effectively retard the senescence of the fruit. However, in the previous study, it was noted that the aroma of the fruit undegone long-term refrigeration obviously light compared with the fruit without refrigeration, which is mainly caused by the influenced mechanism of esters biosynthesis. Up to now the molecular mechanism of this phenomenon is not clear. In this study RNA-seq and Small RNA-Seq techniques will be used to analyze the transcriptome of ripen fruit with or without refrigeration and identify the different in the expression in mRNA and miRNA between them. Meanwhile, the differentially expressed proteins between the ripen fruit with or without refrigeration will be identified through the analysis by use of iTRAQ techniques. Based on the correlation analysis of the differentially expressed spectrum combined with gene annotation, parsing of expression pattern and related metabolic pathways the metabolic and signaling pathways related to the biosynthesis of esters in refrigerated Nanguo Pear will be predicted. These studies are aimed to explore the molecular mechanism of the weakened aroma in Nanguo Pear stored at low temperature through the analysis of the transcriptome, proteome and metabolism. The findings will be valuable for further improving Nanguo pear storage technology.

南果梨是秋子梨系(Pyrus ussuriensis Maxim)的优质品种，为辽宁的特产水果。9月中旬采收后经适度后熟的果实，金黄艳丽、肉质细腻、香气浓郁、品质极佳。南果梨常温货架期仅有15-20天。冷藏可以延缓其后熟衰老进程，延长供应期。申请者研究发现，冷藏南果梨的香气明显变淡，并证明了其主要影响了酯类香气物质的代谢，但是，其分子机制尚不清楚。本项目拟利用RNA-Seq和小RNA测序技术，对常温自然后熟和冷藏后转入常温后熟的南果梨果实的转录组进行深度测序，发掘差异表达的mRNA和miRNA（microRNA）；同时，利用iTRAQ技术对蛋白质组进行平行分析。在差异表达谱关联分析的基础上，结合基因注释、表达模式和相关代谢通路解析，预测与冷藏南果梨酯类香气合成相关的代谢途径和信号通路，在转录组、蛋白质组和代谢三个层面协同分析冷藏南果梨香气变淡的分子机制，为改进南果梨贮藏保鲜技术奠定基础。

冷藏方式可有效延长南果梨贮藏时间，但经过冷藏的南果梨在常温货架期香气明显变淡，严重影响了南果梨的商品质量。本项目通过转录组测序技术研究发现，与未经冷藏南果梨相比冷藏南果梨中存在3137个差异表达基因可能与冷藏南果梨香气变淡有关。差异表达基因的GO分析表明，这些差异表达基因主要在“代谢过程”，“细胞过程”，“刺激反应”和“催化活性”中发挥作用。差异表达基因KEGG代谢通路分析表明，这些差异表达基因主要通过“脂质代谢”中的脂肪酸合成途径、脂肪酸降解途径、不饱和脂肪酸合成途径、，α-亚麻酸代谢途径和亚油酸代谢途径以及植物激素信号转导途径影响冷藏南果梨香气的形成。通过miRNA测序分析鉴定出南果梨中314个已知的miRNAs。其对应的与香气形成直接相关的靶基因有LOX2S，LOX1_5，HPL，ADH1。本项目还通过iTRAQ差异蛋白质组生物信息学分析发现，由冷藏导致的与可能与香气合成有关的差异蛋白质有290种。KEGG分析表明，与冷藏南果梨香气变淡有关的差异蛋白质被注释到具体的61个代谢通路中，主要分布在碳代谢、能量代谢、脂肪酸代谢和氨基酸等途径。IPP互作分析结果表明，与冷藏南果梨香气变淡有关的差异蛋白质互作数最多的蛋白质主要分布于能量代谢、脂肪酸、氨基酸等代谢。对三大组学高通量分析研究结果筛选出的关键基因及关键代谢途径，利用程序降温、间歇升温、外源ATP处理等手段进行验证，发现脂肪酸代谢途径、能量代谢途径、膜脂代谢途径、乙烯信号转导途径、不饱和脂肪酸合成途径是导致低温后南果梨果实后熟过程中香气变淡的主要原因。此外，在本项目研究结果基础上，还需进一步对其他代谢途径进行深入系统研究，并探讨不同代谢途径的内在关系，建立调控冷藏南果梨香气变淡的代谢通路网络，为探索南果梨冷藏调控技术提供依据。