跨膜转运SWEET基因对梨果实糖的转运功能及其作用机制研究

The trait of sugar directly affects the sweetness of fruit, it is the core factor to determine the fruit flavor of pear. The trans-membrane transportation of sugar is the key step for sugar accumulation and plays the important role in fruit quality conformation. Based on our previous study, it was interested to find that trans-membrane transporter gene members in PbSWEET family showed high and specific expression pattern during fruit development in pear. However, the sugar transporter function and regulation mechanism of PbSWEET genes in fruit are still not know. In this project, the sequence comparison of PbSWEET gene members from different cultivars were used to clarify the effects of gene sequence and structure on sugar transporter, and the candidate PbSWEET genes were identified by transcriptome analysis between cultivars with high and low sugar content; the gene function were further verified by a series of experiments, including yeast transformation, FRET, transient expression in pear and strawberry fruit, and transformation in strawberry as well; the regulators or interaction genes were identified by Yeast single-hybrid system, Dual luciferase reporter gene system, Yeast two-hybrid system, Pull-down and BiFC analysis. It is expected to firstly clarify the function and the molecular mechanism of PbSWEET genes in sugar transportation. The results will helpful to provide the theory basis and necessary gene resources for genetic improvement of sugar trait in pear.

糖是决定梨果实风味的核心因素，而糖的跨膜运输是影响糖分在果实中积累和风味品质形成的关键步骤。本项目在前期研究中发现糖的跨膜转运PbSWEET基因家族成员在梨果实中高量表达，且具有发育时期表达特异性，但对其参与果实糖转运的功能和作用机制尚未有报道。本项目拟通过对不同梨品种资源PbSWEET基因家族的比较，明确序列结构变异对糖转运的影响；结合高糖与低糖品种转录表达差异分析，筛选出重要功能基因；通过酵母转化、荧光共振能量转移、梨和草莓果实瞬时表达、以及草莓子叶的稳定转化等技术验证PbSWEET基因对糖组份的转运功能和效率；并利用酵母单杂、双荧光素酶报告基因系统、膜蛋白酵母双杂、Pull-down和BiFC等技术鉴定上下游调控因子和互作基因，有望率先阐明PbSWEET基因对梨果实糖转运的功能及其分子作用网络。研究结果将为实现梨果实糖品质性状的遗传改良提供重要的理论依据和必要的基因资源。

糖分的合成与积累是影响果实品质和商品价值的核心因素，也是遗传育种和基础理论研究者普遍关注的科学问题。由于果实糖的合成代谢途径复杂，涉及长距离、跨膜运输，并受多基因协同调控，导致对其作用机制研究难度较大。目前，研究更多的集中在果实糖的合成，对其转运调控的机制研究比较少。本项目通过基因组、转录组以及转基因等研究手段，重点开展了梨SWEET糖转运蛋白基因鉴定和系统的功能研究，研究结果如下：通过对SWEET基因的全基因组分析，在梨中共鉴定到18个PbSWEET糖转运蛋白基因家族成员，其在糖含量不同的野生和地方梨品种资源中存在SNP变异。基因表达分析发现，PbSWEET在叶片（源）和果实（库）中均存在高表达，表明PbSWEET在其糖转运过程中可能起重要作用。通过转录表达和聚类树分析，发现PbSWEET4与已被证明在糖转运中发挥重要作用的AtSWEET15进化关系较近。实验证明，PbSWEET4是在细胞膜上行使功能的糖转运蛋白，过表达PbSWEET4基因促进糖外排，降低了草莓叶片糖含量。进一步研究发现，PbSWEET4与NAC转录因子PbNAP共表达，并且启动子存在多个NAC基因作用位点。实验结果表明，PbNAP可以激活PbSWEET4的启动子，其可能通过上调PbSWEET4基因表达和来促进糖外排。