黄瓜果实中参与韧皮部质外体卸载的关键己糖转运蛋白基因功能鉴定

The fast growth of cucumber fruit, follow by large amounts of photoassimilate consuming, and hexose are the important assimilate for cell division and utilization. So the absorption and transport of hexose play an important role in the growing fruit. Phloem unloading in growing cucumber fruit from blossom to the end of enlargement has been proved following an apoplasmic pathway. However, there was little report about the functional of the hexose transporter (CsHT) in growing fruit. In the prior experiments, we found that there were several CsHT genes shown high expression during the cucumber fruit growth and enlargement. Functional characterization of CsHTs in yeast indicated that some CsHTs have the transport activity of glucose, fructose and galactose. We speculate that CsHTs play an important role in apoplasmic phloem unloading in cucumber fruit, but the specific mechanism is not clear. This subject will use the technology of qRT-PCR, subcellular location, in-situ hybridization, immunohistochemical localization and cucumber GM technology to systematically analyze the expression profile, tissue location and function of CsHTs, for finding out the key CsHT genes that play a role in post phloem unloading and regulate the carbohydrate flow to growing fruit. These results will help to uncover the molecular mechanism that how is the young fruit of cucumber access assimilate efficiently.

黄瓜果实的快速生长与膨大伴随着大量的同化物消耗，而己糖是细胞分裂代谢所利用的重要同化物，因此己糖的吸收和后运输对果实生长至关重要。已有研究表明黄瓜果实从开花到膨大期结束，韧皮部糖卸载采用质外体途径，然而目前关于己糖转运蛋白（CsHT）在果实生长膨大阶段的功能机制鲜有报道。我们前期实验发现，黄瓜果实生长膨大过程中伴随着多个CsHT的表达，酵母的异源吸收功能表明部分CsHT具有葡萄糖、果糖和半乳糖转运活性，推测其在黄瓜果实韧皮部质外体卸载中发挥重要作用，但具体的作用机制尚不明晰。本项目意在综合运用qRT-PCR、亚细胞定位、原位杂交、免疫组织化学染色和黄瓜转基因技术系统研究己糖转运蛋白在黄瓜果实中的表达定位模式和功能，从而鉴定出参与果实韧皮部质外体卸载并调节碳源向果实流动的关键己糖转运蛋白基因及其功能机制，为揭示黄瓜幼果高效获取同化物的分子机制奠定基础。

黄瓜果实等具有短期内快速生长膨大的器官，常常被认为是库强很强的库。库组织器官中韧皮部同化物的卸载、转移和储藏机制往往是限制库器官库强的关键因素。然而，己糖转运蛋白在黄瓜果实韧皮部同化物卸载和维持果实库强过程中的作用机制尚不明晰。本研究我们利用生物信息学分析，荧光定量PCR，亚细胞定位，酵母异源表达系统，免疫组织化学定位和反向遗传学等技术系统的研究了己糖转运蛋白在黄瓜果实及果柄中的功能。结果表明黄瓜基因组中一共有16个可能的己糖转运蛋白基因，其中有6个基因在黄瓜的果实中能够被检测到。RT-PCR和qRT-PCR分析结果表明CsHT3, CsHT12, 和CsHT16 3个己糖转运蛋白基因在黄瓜果实和果柄中的表达量要显著高于其它成员。在酵母中的糖转运功能验证结果表明CsHT12 和 CsHT16 具有葡萄糖、果糖、半乳糖和甘露糖转运活性。因此CsHT12 、CsHT16和之前已经鉴定过的CsHT3一样可能是黄瓜果实中主要发挥作用的己糖转运蛋白。免疫组织化学定位结果表明CsHT3 和 CsHT16都定位在黄瓜果实和果柄韧皮部的特定细胞中，而且定位会随着果实的发育出现转移现象。在黄瓜中过表达CsHT3不仅可以促进果实的膨大，还可以促进果柄中韧皮部纤维的合成从而提高果柄的韧度。而同时下调CsHT3, CsHT12和CsHT16 3个基因的表达则会抑制果实的生长膨大。此外，CsHT3过表达黄瓜中的糖含量显著高于对照，而RNAi果实中的糖含量显著下降。. 综上结果表明，黄瓜果实和果柄中的己糖转运蛋白CsHT3、CsHT12 和CsHT16在黄瓜果实同化物卸载及果实膨大过程中发挥了重要的作用。