桃果实内酯类香气物质的生物合成及其调控机制研究

Peach is a worldwide fruit species, and deeply loved by consumers because of its strong aromas. Lactone was considered as 'peach-like' compound and it is the most important characteristic aroma component for peach fruit.It play decisive role in aroma of ripen peach. My previous work showed that the first enzyme in the process of β-oxidation,acetyl coenzyme A(ACX), involved in lactone biosynthesis. However, it was not well known synthesis step, especially downstream ACX. On the base of our previous study，'zaolu' flat peach was used to study lactones biosynthesis and regulation mechanism of postharvest temperature.13C stable-isotope technique was used to mark fatty acid of the precursor of lactones. The mark system from fatty acid to lactone was established. Metabolic intermediate were indentified and molecular structure were characterized by using Ultra-Performance Liquid Chromatography tandem Mass Spectrometry (UPLC-MS/MS) and Nuclear Magnetic Resonance(NMR). Differentially expressed gene were identified by using deep sequencing and qPCR. And which was combined with metabolic intermediate to infer possible key gene involved in lactones biosynthesis. Based on verifying results by using yeast expression, the biosynthetic linear model was constructed, which make the mechanism of lactone biosynthesis clear. The effect of postharvest temperature on all key steps of lactone biosynthesis was analized systematically to know the reason that chilling injury leading to lactone loss and to understand mechanism of regulation of intermittent warm to lactone biosynthesis. These results will help us to understand the concrete pathway of lactone biosynthesis, and provide theoretical and practical ground for fruit quality breeding and regulation of fruit aroma quality for peach fruit.

桃是一种世界性水果，香味浓郁，深受消费者喜爱。内酯属"桃味"化合物，是桃果实最重要的特征香气物质，对成熟桃的香味具有决定性作用。申请人研究发现，脂肪酸β-氧化的起始酶ACX参与了桃内酯的合成，但其下游具体合成途径尚不清楚。故本项目拟在前期研究基础上，以'早露蟠桃'为材料，通过稳定同位素13C饲喂技术标记内酯的前体物质脂肪酸，建立从脂肪酸到内酯的线性标记体系，使用UPLC-MS/MS及核磁共振技术鉴定内酯合成的中间产物，利用Deep sequencing/qPCR技术，发掘参与内酯合成的关键基因，并使用酵母表达蛋白进行验证，在上述基础上，建立桃果实内酯合成的线性模型，明确其生物合成机制。通过分析采后温度对内酯合成关键步骤的影响，揭示冷害抑制内酯合成的原因，阐明间歇升温对桃果实内酯合成的调控机制。本项目研究结果将有助于明确桃果实内酯合成的具体途径，为桃品质育种和香气品质调控提供理论和实践依据。

内酯被称为“桃味”化合物，是桃、杏、草莓等多种水果特征香气成分，也是重要的香料。本项目主要针对植物中内酯合成途径不清楚这一学术空白，以富含内酯的“早露蟠桃”果实为研究材料，开展了桃果实内酯类香气物质的生物合成机制研究，通过四年的研究完成了全部计划任务。研究计划包括：获得内酯合成关键基因及其家族成员，建立内酯合成的生化反应模型；解析这些关键基因在内酯合成中的具体生物学功能；探索冷害导致桃果实内酯丧失的原因和间歇升温对桃果实内酯的调控机制。本研究按计划开展了研究内容的相关实验，建立了利用13C标记脂肪酸的技术体系，可以直接用于果实的相关研究。棕榈酸（PA）、亚油酸（LA）和亚麻酸（LeA）处理能显著增加γ-癸内酯等8种内酯的含量。使用高效液相色谱/电喷雾串联质谱（UPLC-MS/MS）技术分离鉴定到脂肪酸代谢的中间产物。基于转录组数据的基因共表达网络分析（WGCNA）共挖掘到了5个结构基因和1个乙烯信号转导元件参与桃果实内酯的合成调控。酵母转基因结果发现，PpFAD3具有脂肪酸脱饱和活性，能够特异结合LA，PpEH1具有环化酶特性，PpACX1能特异氧化脂肪酸，PaAAT1具有催化合成内酯的活性。本研究建立了从“脂肪酸—中间产物—内酯”的内酯合成生化反应模型，明确了冷害导致桃果实香味丧失和间歇升温处理（IW）处理对桃香气品质的调控机制。. 在计划的研究内容之外，在本基金资助下，我们还使用cytoscape构建了桃果实内酯合成相关候选基因的调控网络，为后续开展相关研究奠定了基础。. 项目执行期间，共发表本研究相关（标注本项目资助）的论文3篇，1篇SCI论文在Food chemistry发表，申请专利1项。本项目共培养研究生3名，本科生2名。