桃PpTST1基因可变剪接参与果实糖分积累的调控及机理解析

Soluble sugars are an important component of fruit taste. In peach, sucrose is the predominant component of soluble sugars in mature fruit. We have identified a candidate gene, designated PpTST1, within the chromosomal region harboring a major QTL for fruit sucrose content in peach, and the PpTST1 gene can undergone alternative splicing to produce two mature-mRNAs. In this project, our main objective is to further conduct functional studies for PpTST1 to uncover the mechanism underlying fruit sugar accumulation in peach. Firstly, expression profile of different isoforms of PpTST1 in fruits throughout whole developmental stages will be conducted, and the correlation between each isoform expression level and fruit sugar content will be analyzed. We will identify the core variation sequences of PpTST1 and analyze their potential influence on gene expression or function. Secondly, FRET-sugar sensor will be applied to measure sugar transport activity of different PpTST1 isoforms and their functions will be evaluated using stable transformation of model organisms such as tomato, and/or virus-induced gene transient expression system in peach fruit. These results will be useful for understanding the role of PpTST1 involved in the fruit sugar accumulation. In addition, we will investigate the phosphorylation of PpTST1 protein and its effect on sugar transport activity. Protein Kinase(s) that are responsible for the phosphorylation of the PpTST1 protein will be identified. We will identify transcription factor that controls the transcription of the PpTST1 gene. These results will result in a gene network that controls fruit sugar accumulation in peach. Finally, a series of physiological analysis will be conducted to understand how the alternative splicing of PpTST1 occurs throughout the whole fruit development and to determine whether the alternative splicing of PpTST1 is related to environmental factors and/or hormones. This project not only has a theoretical significance in expanding our knowledge of regulatory mechanism of sugar accumulation in peach fruit, but will also provide molecular tools highly useful for genetic improvement of fruit quality in peach breeding programmes.

糖是决定果实风味品质的一个核心元素。桃果实糖组分以蔗糖为主。本项目拟在前期发现的桃果实蔗糖含量主效QTL区间候选基因PpTST1及其可变剪切基础上，进一步解析它参与果实糖积累调控的分子机理。首先，分析PpTST1不同可变切版本表达与果实糖积累之间的相关性，发掘PpTST1的核心变异并分析其对基因表达或功能的影响。其次，采用FRET-糖感应器测定PpTST1等位基因的糖转运活性，结合PpTST1等位基因在番茄等模式生物中过表达及其在桃果实中瞬时表达等转基因研究，明确PpTST1对果实糖积累的调控作用。再次，发掘调控PpTST1转录及其编码蛋白磷酸化的调节基因，构建PpTST1基因调控果实糖积累的基因网络。最后，分析PpTST1可变剪切在果实发育中的产生规律及其对环境和激素的响应，了解环境对果实糖积累的影响机理。本项目开展对于桃果实含糖量遗传改良具有重要的理论意义和应用价值。

果实甜度与可溶性糖积累有关，在果实风味品质性状形成中起着重要作用。桃果实甜度是受多基因控制的数量性状，其中一个QTL位于第5号染色体的顶端D位点。本项目发现位于D位点的PpTST1基因表达与可溶性糖积累呈现相同的趋势；基于该基因第三个外显子中的一个G/T SNP位点开发了一个dCAPS标记，候选基因关联分析表明该SNP位点与果实糖含量显著关联；明确了PpTST1基因位于液泡膜上，第三外显子的非同义G/T替代对亚细胞定位无影响；在桃果实中瞬时沉默PpTST1基因可显著抑制了蔗糖、果糖、葡萄糖等可溶性糖的积累。这些结果充分表明PpTST1是控制桃果实糖含积累的重要基因。此外，发掘了两个控制桃果实糖分积累的液泡转化酶抑制基因PpINHa和PpINH3，其中前者在桃果实中瞬时过表达促进糖分的积累，而后者在桃果实中瞬时过表达导致可溶性糖含量的下降；明确了PpINHa和PpINH3在糖积累中的作用不能归因于它们与PpVINs的相互作用，揭示了PpINHa和PpINH3通过影响液泡转运体基因PpTST1和PpERD6-Like的表达进而控制果实糖分积累的新机制。本项目研究结果不仅有助于全面了解桃及其它果树果实糖分积累的复杂调控机制，而且为果实甜度性状遗传改良提供了分子工具。