黄瓜蔗糖转运蛋白基因家族（CsSUTs）的克隆及功能研究

Sucrose is the predominant photosynthetic assimilates and also main carbohydrates of long-distance transport in phloem in plant. Cucumber apoplastic phloem unloading of photosynthate in fruit，which have proved the process in our previous study, require the participation of sucrose transporter(CsSUT) family. It is a pity that it is remain unclear how the CsSUT involved in the opaplastic unloading pathway and affected fruit development. We have cloned the genes CsSUT1,CsSUT2 and CsSUT4 encoding sucrose transporter proteins from cucumber. Here, we will study its spatiotemporal expression, histochemistry localization and sub-cellular localization using real time PCR, Western Blotting, transient expression of CsSUT-GFP in onion epidermis cells and cucumber protoplast, in-situ hybridization and immunogold electron microscopy. We will validate its heterologous expression function using yeast mutant. At the same time, we construct the over-expression and RNAi vector and transform the cucumber and tobacco mediated by agrobacterium respectively and study further the molecular mechanism of apoplastic phloem unloading and fruit development. The response mechanism of CsSUT responding to sucrose and relative hormones will studied also. This will help understand the effects of CsSUT on sucrose apoplastic phloem unloading, carbohydrates distribution, growth and development of cucumber fruit, and enrich the theory for cucumber fruit development.

蔗糖是植物光合产物和长距离运输物质的主要形式，蔗糖从黄瓜（cucumis sativas L.）果实韧皮部质外体卸出（我们前期工作业已证明黄瓜果实光合产物韧皮部卸载为质外体路径）必须依赖蔗糖转运蛋白基因家族（CsSUT），但有关CsSUT对黄瓜果实质外体卸载及其果实发育的影响知之甚少。本项目在克隆了CsSUT1，CsSUT2和CsSUT4的基础上，利用荧光定量PCR、Western Blotting、GFP瞬时表达、原位杂交和免疫胶体金等技术进行CsSUT时空表达、组织化学和亚细胞定位；用酵母突变体进行异源功能验证；构建过表达和干扰载体，利用农杆菌介导转基因技术研究CsSUT在黄瓜和烟草中的表达，研究其对质外体卸载和果实发育的影响；同时，研究CsSUT对蔗糖和激素信号的响应机理，旨在探明CsSUT对黄瓜果实中蔗糖卸载、碳水化合物分配和果实生长发育的影响，丰富黄瓜果实发育和形成理论

蔗糖作为光合作用的主要产物，也是作物运输的主要糖。黄瓜是典型的水苏糖运输型植物，叶片主要以聚合物陷阱模型和质外体装载方式将同化物运输至筛分子，水苏糖运输至黄瓜果柄及果实被降解成蔗糖，在果实主维管束主要利用质外体卸载方式，因此蔗糖代谢在黄瓜中具有特殊性和代表性，研究证明在黄瓜质外体卸载、混合装载过程中，蔗糖转运蛋白发挥了重要作用。.黄瓜蔗糖转运蛋白基因家族（CsSUTs）共有3个亚型，即CsSUT1，2和4。.CsSUT1，虽然在黄瓜各组织中均可表达，但主要在雄花中特异表达，而在其它器官表达水平非常低。对CsSUT1干扰后，雄花中的糖、淀粉含量明显降低，糖代谢相关基因、糖信号下调，多个CsMYBs转录因子上调，并诱导黄瓜的雄性不育。CsSUT1对雄花发育的重要作用在黄瓜育种方面有重要的利用价值。.CsSUT2，主要在黄瓜成熟叶片中表达，通过分子生物学，解剖结构学以及同位素标记实验证明了CsSUT2在黄瓜叶片的质外体装载过程起到了重要的作用。但是，黄瓜叶片利用质外体和共质体混合装载，并且以主动的共质体装载为主，质外体装载为辅。.CsSUT4，它主要在库器官表达，同时在源器官中也有少量表达，且有14个可变剪接体，功能可能具有多样性，就目前的工作来看它主要参与了质外体卸载。