苹果两个clade-III SWEETs基因在果实糖积累中的功能解析及其调节基因的鉴定

Sugar is one of the core elements that influence the quality flavors of fruits. The process of sugar accumulation in fruits is extremely complex, which involves in the biosynthesis of photosynthates, and their transportation, unloading, metabolism, transformation and storage. Candidate genes association analysis indicates that both MdSWEET9b and MdSWEET15a genes are tightly associated with sugar accumulation in apple fruit, but the underlying molecular regulatory mechanism remains unknown. In this project, we intend to analyze the function of the two genes through approaches including yeast expression system, HEK-293T cells and patch-clamp technique, subcellular localization and transgenic technology. Thereafter, regulatory genes of MdSWEET9b and MdSWEET15a are further identified by yeast one-hybrid assay, electrophoretic mobility shift assays and chromatin immunoprecipitation. Meanwhile, experiments of yeast two-hybrid, firefly luciferase complementation assay and GST-pull down are carried out to validate the interaction proteins of MdSWEET9b and MdSWEET15a. In the end, we expect to build a MdSWEET9b- and MdSWEET15a-mediated regulatory network and clarify the molecular regulatory mechanism of sugar accumulation in apple fruit. The results of the project will have important theoretical significance and practical application value on the quality genetic breeding of apple fruits .

糖是影响果实风味品质的核心元素之一。果实中糖的积累涉及光合产物的合成、运输、卸载、代谢、转化和储存，过程极其复杂。前期通过候选基因关联分析发现MdSWEET9b和MdSWEET15a是调控苹果果实糖积累的关键基因，但其分子机调控理尚不清楚。本项目拟利用酵母表达系统、HEK-293T细胞及膜片钳技术、亚细胞定位和转基因等手段分析其基因功能；并进一步借助酵母单杂交、凝胶阻滞和染色体免疫共沉淀等技术鉴定其上游调节基因；同时开展酵母双杂交、荧光互补和GST-pull down等实验筛选MdSWEET9b和MdSWEET15a的互作蛋白，最终构建MdSWEET9b和MdSWEET15a介导的果实中糖积累的调控网络，解析苹果果实中糖积累的分子调控机理，项目预期成果将会对苹果果实品质遗传育种具有重要的理论意义和实际应用价值。

苹果是世界重要经济果树，我国是世界苹果生产和出口第一大国。近年来，我国苹果产业问题日益凸现，主要是品种结构不合理，优质果率偏低，国际市场竞争力较差。因此，改善与提高苹果果实品质已成为我国苹果产业发展的主要任务。糖是影响果实风味品质的核心元素之一。果实中糖的积累涉及光合产物的合成、运输、卸载、代谢、转化和储存，过程极其复杂，至今关于果实内糖分是如何运输及其分配规律，糖代谢相关酶与果实糖积累的具体关系，果实发育过程中糖代谢关键酶、糖转运蛋白等是怎样协同调控果实糖的积累等相关机理仍知之甚少。本项目在研究团队前期通过候选基因关联分析发现MdSWEET9b和MdSWEET15a是调控苹果果实糖积累的关键候选基因的基础之上，首先通过稳定转化苹果愈伤和番茄，明确了MdSWEET15a基因过表达可以显著促进蔗糖、果糖和总糖的积累，而MdSWEET9b基因的过表达并不能显著影响糖的含量；同时，亚细胞定位的结果表明MdSWEET15a蛋白主要定位在液泡膜上面，而利用酵母表达系统，通过功能互补实验鉴定到MdSWEET15a 转运底物为蔗糖，以上这些结果表明MdSWEET15a在苹果果实糖积累的过程中起着重要的作用；进而结合酵母单杂交筛库和染色体免疫共沉淀等技术鉴定到MdSWEET15a上游的调节基因MdWRKY31，并且LUC/REN实验也证实了MdWRKY31可以激活MdSWEET15a基因的转录；利用酵母双杂交、萤火虫荧光素酶互补等技术筛选到一些MdSWEET15a的互作蛋白。综合上面研究结果最终构建了MdSWEET15a介导的苹果果实中糖积累的分子调控网络。本项目的开展，可鉴定控制苹果果实糖积累的关键基因并构建其调控网络，有利于解析苹果果实中糖积累的分子遗传机理，对于苹果果实品质遗传育种及改良，具有重要的理论意义和实际应用价值，符合我国苹果产业发展的战略需求。