皱皮木瓜果实有机酸代谢组学研究与调控关键点确定

Present study of fruit physiology showed that effective adjustment and controlling of fruit organic acids content is the key step of improving fruit flavour and quality. But compositive diversity and metabolic complexity of fruit organic acids is the bottleneck problem of fruit organic acids metabolic studying via the traditional routes ,which has been difficult to achieve. Recent development of metabonomics studying techniques provide a new way to explore fruit organic acids metabolic mechanism in depth. Thereby, regarding the typical high-acid fruit tree species wild ‘Chaenomeles speciosa’ which belongs to Maloidae subfamily of Rosaceae in plant taxonomy as the research subject, according to Systems Biological researching thoughts and basing on high throughput assaying techniques, this work plan to carry out screening low-acid,middle-acid and high-acid fruit tree resources from domestic ten natural distributing areas of C. speciosa. Using each two typical strains of them as testing material, we plan to dynamicly monitor the organic acids composition and its content changing law of C. speciosa fruit during its whole developing period. At the same time the 13 key enzymes activity expressing which related to the metabolism law of fruit organic acids would be dynamicly assayed. Upon the basis of that, the fruit organic acid metabolites composing profile and key enzymes activity expressing chart would be built. Chaenomeles speciosa fruit organic acids metabolic mechanism would be perfected and regulating critical points would be analysed. The research results not only would lay the groundwork for futural acidity controlling study of maloideae fruit trees, but also enrich organic acids metabolic theory of fruits. And it also could provide helpful reference for the futural fruit quality improvement utilizing metabonomics studying techniques.

现有果实生理学研究表明，有效调控果实有机酸含量是果实风味改良与品质提高的关键。但果实有机酸的组成多样性和代谢复杂性是传统方法研究果实酸代谢机理的瓶颈问题难以突破。当前代谢组学研究技术的发展为深入探索果实有机酸代谢机理提供了新途径。本项目拟以蔷薇科典型野生高酸果树皱皮木瓜为研究对象，依据系统生物学研究思想，基于高通量检测技术通过对国内十大自然分布区资源筛选，选用高酸、中酸、低酸典型品系在果实全发育期进行有机酸代谢组分变化规律动态监测研究，并通过色谱分离与酶活检测体系相结合，对现有研究发现的13个关键酶活性表达情况进行动态监测，建立全发育期皱皮木瓜果实有机酸代谢组谱和酶活表达谱，通过代谢组学数据分析与生物学解析相结合，完善皱皮木瓜果实有机酸代谢机理并确定调控关键点。研究结果可为苹果亚科果树果实酸度调控研究奠定基础，丰富果实有机酸代谢理论，为利用代谢组学技术探索果实品质改良提供新信息。

有机酸组成与含量是果实品质风味的重要组成因素，因此果实有机酸代谢生理一直是国内外学者的重要研究领域。在前期发现皱皮木瓜为蔷薇科苹果亚科典型高酸果树品种的背景下，本项目：（1）建立了皱皮木瓜多有机酸的溶剂提取HPLC-PDA检测方法，结果表明60%乙醇超声提取，减压浓缩，流动相定容，可实现样品有机酸的高效提取；C18柱10µL进样，0.04 mol•L-1，pH 2.4的KH2PO4-H3PO4缓冲液梯度洗脱，214nm检测32min可同时实现13种有机酸的精确测定。（2）建立了皱皮木瓜多有机酸的甲酯化衍生GC-MS检测方法，结果表明硫酸-甲醇恒温甲酯化衍生，氯仿萃取，可实现有机酸甲酯衍生物的高效提取；Rxt-5MS柱不分流进样，程序升温，33min可实现样品41个有机酸的精确检出。（3）完成了全国十个道地产区12个皱皮木瓜种质资源评价，结果表明样品有机酸组成和含量差异较大，按其总有机酸含量差异，明确了山东沂州为高酸、重庆綦江为中酸、湖北长阳为低酸品系。（4）完成了重庆綦江、山东沂州、湖北长阳皱皮木瓜果实全发育期有机酸检测，綦江样品在果实转色前期总有机酸含量逐渐下降，果实转色后期，又逐渐上升，进入果实成熟期，含量再次呈下降趋势变化，到果实完熟，含量降至最低点；山东沂州和湖北长阳样品数据分析尚未完成。（5）建立了皱皮木瓜果实全发育期有机酸代谢关键酶的提取和活性通量检测方法，结果表明新鲜果肉研磨缓冲液组织破碎、提取缓冲液差速离心，可实现有机酸代谢8个关键酶的有效分离；多孔板反应，多功能酶标仪检测，可实现酶活的通量检测。（6）完成了重庆綦江、山东沂州、湖北长阳皱皮木瓜果实全发育期有机酸代谢8个关键酶的活性检测，但皱皮木瓜样品发育期酶活表达谱数据分析未完成。本项目建立了稳定的有机酸检测方法，全面评价了我国道地产区皱皮木瓜有机酸组成含量差异，系统检测了高酸、中酸、低酸品系果实发育期有机酸代谢谱和酶活表达谱，为我国皱皮木瓜有机酸调控提供了基础数据。但由于数据分析量极大，部分数据分析与生物学意义解析正在进行中。