桃PpOA3基因参与果实酸度调控的功能及机理解析

Organic acids are an important component of fruit taste. In peach, organic acid accumulation in fruit is mainly controlled by the D locus on chromosome 5. We have identified a candidate gene in the D locus, designated PpOA3, which is responsible for fruit acidity in peach. In this project, our main objective is to further conduct functional studies for PpOA3 and to understand the mechanism underlying fruit acidity in peach. Firstly, expression profile of PpOA3 in fruits will be conducted, and the correlation between the PpOA3 expression level and fruit acidity will be analyzed. We will identify the core variation sequences of PpOA3 and analyze their potential influence on gene expression or function. The identification of core variation sequences of PpOA3 can help us to understand the genetic basis of low-acid trait that is assumed to be an important even during peach domestication, and can be used to develop functional makers for marker assistant selection in peach programmes. Secondly, gene functionalities will be evaluated for the PpOA3 gene using stable transformation of model organisms such as tomato, and/or virus-induced gene transient expression system in peach fruit. Finally, a series of physiological analysis, including subcellular pH fluorescent display, protein fragmentation mutation, and patch clamp electrophysiology, will be conducted to understand how the PpOA3 gene involves in transmembrane transport of organic acid, and to understand the mechanism by which the PpOA3 gene regulates the accumulation of organic acids in fruits. This project not only has a theoretical significance in expanding our knowledge of regulatory mechanism of organic acid accumulation in peach fruit, but will also provide molecular tools highly useful for genetic improvement of fruit quality.

有机酸是影响果实风味品质的重要因素之一。桃果实有机酸积累主要受第5号染色体D位点控制。本项目拟在前期发现D位点候选基因PpOA3的基础上，进一步解析其调控果实酸度的分子机理。首先，构建PpOA3基因的果实表达谱，并分析其表达水平与桃果实酸度性状之间的相关性；发掘PpOA3基因的核心变异序列并用于分子标签的开发，分析核心变异序列对基因表达或功能的影响，明确桃果实低酸驯化性状形成的遗传基础。其次，通过PpOA3基因在番茄等模式生物中过表达及其在桃果实中瞬时过表达或抑制等转基因研究，明确PpOA3基因参与果实有机酸积累的调控作用及分子机制。最后，开展膜片钳电生理、亚细胞pH荧光展示和蛋白分段突变等研究，解析PpOA3基因参与有机酸跨膜运输的生理机理。本项目的开展既能解析控制桃果实有机酸积累的分子机理，又可获得控制果实酸度性状的功能分子标记用于桃品质遗传改良，具有重要的理论意义和应用价值。

果实酸度是决定水果口感品质的一个重要因素。桃果实酸度受第5号染色体上的一个主效D位点控制。本项目通过遗传作图将D位点定位于509 kb的区间，获得了与桃果实的非酸/酸性状共分离的分子标记5dC720和5C1019。鉴定了D位点候选基因PpRPH，该基因编码一个小肽，其表达水平与果实酸度呈负相关，在低酸品种果实中的表达水平显著高于高酸品种。与空白载体转化的对照相比，PpRPH在烟草叶片中的瞬时异位表达导致pH值增加约40%，同时柠檬酸和苹果酸含量分别比空载体对照显著降低了22%和37%。这些结果表明PpRPH是D位点的候选基因。此外，对75个桃品种成熟果实中的有机酸进行了测定，发现桃果实有机酸主要成分以苹果酸和柠檬酸为主，但奎宁酸含量也较高。在果实发育早期有机酸积累增加，但在果实发育后期，低酸品种比高酸品种表现出显著的减少。低酸品种由于GABA途径基因GAD和苹果酸转运蛋白基因ALMT9的上调和下调，导致柠檬酸降解和苹果酸向液泡的转运减少，也就是说苹果酸的积累是在代谢和液泡贮藏水平上控制的，而代谢对柠檬酸的积累是至关重要的。本项目研究结果不仅有助于全面了解桃及其它果树果实酸度的复杂机理，而且为果实酸度性状遗传改良提供了分子工具。