西瓜果实糖积累关键基因ClSTP1和ClSTP2的功能解析

Based on the watermelon whole genome sequencing and fruit development transcriptome sequencing, we found that sugar transport protein was the key factor of sugar transmission in watermelon.Resequencing of the representative watermelon lines further confirmed that sucrose transporter protein ClSTP1 and ClSTP2 were the key candidate genes determing the sugar accumulation in watermelon fruit.We plan to apply the further functional resolution of ClSTP1 and ClSTP2 at transcriptome level, transgenosis level, regulation level and cellular level. Q-PCR strategy will be applied to detect the expression profiles of ClSTP1 and ClSTP2 during watermelon fruit sugar accumulation to clarify the time distribution of them. Then the vector of yeast expression,RNAi and overexpression of ClSTP1 and ClSTP2 will be constructed to verify their biological function. YTH system will be applied to discover the protein interactions between ClSTP1 and ClSTP2 and the related transctiption factors to identify the candidated transcription factors. The subcellular localization of ClSTP1 and ClSTP2 will be perfomred to clarify the spatical distribution in cell level. The clarification of the biological function of the sugar transport protein gene ClSTP1 and ClSTP2 will make the fundation for further research of the gene network mechanism of sugar accumulation in watermelon fruit and for efficient precision quality breeding.

基于已完成的西瓜全基因组测序和果实发育转录组数据，我们发现糖转运蛋白基因对果实糖转运起决定性作用。代表性西瓜重测序进一步证实了蔗糖转运蛋白基因ClSTP1和ClSTP2是影响西瓜糖积累的关键候选基因。本项目从表达、转基因、调控和细胞水平上对ClSTP1和ClSTP2基因进行功能解析。首先利用Q-PCR开展基因表达研究，明确ClSTP1和ClSTP2在西瓜果实糖积累过程中的时间分布；同时构建酵母表达载体、RNAi和过表达载体，开展ClSTP1和ClSTP2的转基因功能验证；利用YTH酵母双杂交系统分析转录因子与ClSTP1和ClSTP2的蛋白互作模式，明确调控ClSTP1和ClSTP2的转录因子；并通过亚细胞定位技术明确ClSTP1和ClSTP2在细胞水平的空间分布。西瓜ClSTP1和ClSTP2基因的功能解析，将为深入探索果实发育糖积累基因网络机制和定向精准品质育种奠定重要的基础。

果实含糖量是西瓜果实品质主要构成因素之一。本项目从转录表达、转基因、调控和细胞水平上对西瓜糖转运蛋白候选基因开展了功能解析。完成了果实发育关键阶段及96份不同含糖量材料qPCR分析，证实了西瓜果实糖转运蛋白基因ClSTP1和ClSTP2的表达水平与西瓜果实含糖量呈正相关趋势；完成了134份代表性西瓜材料全基因组关联分析，证实了目的基因与果实含糖量的之间的关联性；完成了ClSTP1和ClSTP2基因亚细胞定位，明确了ClSTP1在西瓜果实细胞膜和内膜系统表达，ClSTP2在细胞内膜系统表达；完成了目的基因做糖吸收缺陷型酵母菌株上的活性验证，发现ClSTP1和ClSTP2参与果糖和葡萄糖跨膜运输；完成了目的基因启动子区域关键位点筛选及调控因子分析，发现位于QBRIX2-3的转录因子ClPIF3可以结合该ClSTP1 E-box元件, ClSUSIBA2蛋白可以结合ClSTP2糖响应元件SURE；以西瓜半野生材料GS24为材料，转基因验证了西瓜糖转运蛋白基因参与西瓜果实含糖量形成。对阐明西瓜果实品质形成和糖代谢调控网络具有提供了重要信息，同时也为西瓜品质分子辅助育种提供了关键基因资源。