黄瓜己糖转运蛋白CsHT1基因特性及其功能验证

The phloem unloading of photosynthate in sink organs is important for the formation of crop yield and quality. Cucumber is a typical stachyose-transporting species and with an apoplasmic phloem unloading pathway in fruit. However, the primary sugar accumulated in commodity fruit is hexose (glucose and fructose). Whether the hexose transporter is involved in this unloading pathway remains unclear. Therefore we cloned the gene CsHT1 encoding hexose transport protein from cucumber and validated its transport function in yeast. In this project, we will investigate its temporal and spatial expression and its histochemistry localization using Semi-quantitative RT-PCR, Western blot and in-situ hybridization. For the sub-cellular localization, we will use the transient expression of CsHT1-GFP in onion epidermis cells and cucumber protoplast. Furthermore, the over-expression and RNAi vector will be constructed and genetically transformed the Arabidopsis and cucumber, respectively. The sugar accumulation and carbohydrate metabolism during sink organs (fruits, flowers, etc) development of T1 and T2 transgenic plants will be observed and identified. This work will help understand the role and function of CsHT1 in phloem unloading, transport and distribution process at molecular and physiological levels, which will enrich the theory for forming high yield and quality cucumbers.

光合产物在库器官韧皮部的卸载是作物产量和品质形成的重要基础。业已证明，典型的水苏糖运输型植物-黄瓜果实韧皮部卸载为质外体途径，但其商品果实中所积累的糖主要是己糖-葡萄糖和果糖，而已糖转运蛋白是否介导和参与了黄瓜光合产物在库器官的质外体卸载，尚未见文献报道。为此，我们克隆了黄瓜己糖转运蛋白基因CsHT1，并通过酵母异源表达系统初步验证其己糖转运功能。在此基础上，本项目拟运用半定量RT-PCR、Western blot、原位杂交等分子生物学技术研究该基因时空表达特性和组织化学定位；利用洋葱表皮细胞和黄瓜原生质体瞬时表达CsHT1-GFP融合蛋白等方法进行亚细胞定位；构建过量表达和干扰表达载体并分别遗传转化拟南芥和黄瓜，观测T1和T2代转基因植株库器官（果、花等）发育、糖积累和代谢等状况，从分子和生理等方面明确CsHT1在库器官韧皮部卸载、转运与分配过程中的作用和功能，以丰富黄瓜高产优质理论。

黄瓜库器官花粉的形成及花粉管的萌发需要大量而恒定的糖份供应。对于本身属于共质体隔离的库组织，花粉在发育及萌发过程中，糖是如何供应和分配的，目前研究得并不多。本文采用RT-PCR方法从黄瓜雄花中分离出一个的己糖转运蛋白基因CsHT1，并利用在酵母中的异源表达技术、基因枪技术、黄瓜原生质体转染技术、GUS染色技术、拟南芥转化技术、烟草过表达技术等对CsHT1的定位及其在花粉发育过程中的功能进行了系统的研究，结果表明：① CsHT1基因具备己糖载体家族的特有结构并且与其他已知的己糖载体基因具有很高相似性和同源性；② CsHT1在异源酵母表达系统中具有转运葡萄糖的功能；③ CsHT1为定位在质膜上的质膜蛋白；④ CsHT1的表达具有很高的组织特异性，只在发育形成过程的花粉及花粉管中表达；⑤ CsHT1在烟草中的过表达使得烟草花期提前，花朵数量增多；⑥CsHT1反义载体转化拟南芥后使得拟南芥花粉发育异常，最终导致果荚变短，种子数量减少。这些研究结果探明了黄瓜己糖转运蛋白基因CsHT1可以参与拟南芥与烟草器官花中己糖的卸载、转运及分配 ，为我们进一步研究CsHT1在黄瓜库器官花粉中的功能研究提供理论依据。