SWEETs糖转运蛋白在甜瓜果实糖分卸载与累积中的调控作用解析

Sugar transportation is closely related to the yield and quality of fruits, however, the mechanism of how sugars are unloaded from the phloem sieve element to the surrounding parenchyma cells in melon fruit is unclear. In the early stage of our study, we initially learned that the carbohydrate in the melon fruit is unloaded from the parenchyma cells to the sieve tube by an apoplasmic pathway; furthermore we discovered that two SWEETs family members were highly regulated during melon fruit development stages. On the basis, in this project we will firstly further study the unloading pathway of the sugar during melon fruit development; Secondly, the function of the candidate CmSWEET3 and CmSWEET7a will be characterized. The yeast and strawberry plants will be used to verify the sugar transporter ability; Thirdly, their localization will be studied by transient expression of tobacco and in-situ hybridization staining; Fourthly, the biological function by constructing over-expression and CRISPR mutant lines of these two genes will be analyzed; finally, the interaction partners on both transcriptional and translation level will be identified by yeast-one/two-hybrid technologies. This study is of great importance to clarify the mechanism of sugar unloading in melon fruit and dissect the role of SWEETs sugar transporters during the melon fruit development. This study will help improve the fruit quality of melon and ultimately provide a theoretical basis for the production of melon.

糖的运输关系到果实的产量与品质，目前对于甜瓜果实内糖分卸载机理不甚清楚。前期我们初步获悉甜瓜果实内糖分经筛管到达薄壁细胞通过质外体途径进行，并发现两个SWEETs家族基因在果实发育期间大量表达。在此基础上本项目拟对甜瓜果实发育期间糖分的卸载途径进一步深入研究，重点对候选CmSWEET3，CmSWEET7a进行克隆与功能分析，通过在酵母与草莓活体植株中表达验证其运载糖的能力；通过烟草瞬时表达与原位染色明确其亚细胞定位和组织定位；通过构建过表达与CRISPR突变株系解析其生物学功能；最后运用酵母单/双杂交技术从转录水平与翻译水平挖掘其互作调控因子。本研究对于明确甜瓜果实内糖分卸载机理以及SWEETs糖转运蛋白在甜瓜果实发育中起的关键作用具有十分重要的意义，并应用于甜瓜果实品质的改良，最终为西甜瓜生产实践提供理论依据。

甜瓜是我国，也是世界上最重要的夏令水果之一。甜瓜果实内所含碳水化合物的种类和数量很大程度上决定其品质和产量。SWEETs（sugars will eventually be exported transporters）糖转运蛋白具有运输葡萄糖和其它寡糖功能，最近研究表明SWEETs糖转运蛋白在果实发育中可能起调控作用。本研究从甜瓜基因组中鉴定获得18个SWEETs糖转运蛋白基因，进一步通过RT-PCR并结合实时荧光定量PCR（Quantitative Real-time PCR，qPCR）方法，筛选到3个SWEETs基因在整个果实发育发育期内或某个发育时期表达量较高。亚细胞定位显示两个SWEETs基因（CmSWEET3，CmSWEET7a）定位在细胞膜上。进一步通过酵母表达发现，甜瓜CmSWEET7a在体外具有转运葡萄糖和果糖的功能。利用番茄转化体系对CmSWEET3, CmSWEET7a进行过表达研究，成功获得了转基因植株。以甜瓜Vedrantais不同成熟期甜瓜果肉组织构建酵母双杂交ｃＤＮＡ 文库，以CmSWEET7a为诱饵，从文库中筛选到 28个可能与 CmSWEET7a 互作的非重复蛋白，包括激素相关蛋白abscisic acid receptor PYR1-like，auxin-repressed 12.5 kDa protein-like，转录因子F-box/kelch-repeat protein，zinc finger CCCH domain-containing protein 20-like，蔗糖合成酶 Sucrose-phosphate synthase。本研究对于明确甜瓜果实内糖分卸载机理及SWEET糖转运蛋白在甜瓜果实发育中起的关键作用具有十分重要的意义，并为应用于甜瓜果实品质的改良提供重要理论依据。