蔗糖转运蛋白基因CsSWEET1调控黄瓜亚适温下光合同化物韧皮部装载的机制研究

Cucumber (Cucumis sativus L), a typical thermophilic vegetable, often suffer from suboptimal temperature which has become one of the key limiting factors of cucumber cultivation in protected horticulture. It has been hypothesized that enhancing the capacity of photoassimilates phloem loading may improve the transporting capacity of photoassimilates from source leaves to sink organs, so as to enhance the ability of cucumber adapting to suboptimal temperature. Our preliminary experimental results showed that a novel sucrose transporter CsSWEET1 displayed a sharp difference in expression levels between suboptimal temperature tolerant and sensitive cultivars. In this study, the function of CsSWEET1 on the bidirectional transport of sucrose will be further verified. Furthermore, using a combination of real-time PCR, GUS and immunochemical staining, cucumber protoplast transient expression and immunogold labeling, the spatiotemporal expression, tissue and subcellular localization of CsSWEET1 will be studied. In addition, the differences in sequence and structure of CsSWEET1 between suboptimal temperature tolerant and sensitive cultivars will also be analyzed. These results will determine whether CsSWEET1 is involved in photoassimilates phloem loading in source leaves of cucumber under suboptimal temperature, as well as its mechanism. Furthermore, our data will provide new clues to studies focusing on mechanism of cucumber adapting to suboptimal temperature and give a new theoretical basis for breeding special cucumber cultivars that are suitable for protected horticulture under suboptimal temperature.

黄瓜（Cucumis sativus L.）是典型的喜温蔬菜，在设施栽培中常遭遇亚适温环境胁迫，是限制黄瓜高产高效栽培的关键因子之一。已有研究表明，增强亚适温下叶片同化物向韧皮部的装载能力，可提高同化物从叶片向"库"器官转运的能力，从而提升黄瓜对亚适温的适应能力。预备实验结果表明，最新报道的新型蔗糖转运蛋白基因CsSWEET1在不同亚适温耐性黄瓜品种间存在较大的表达差异。本项目在进一步验证CsSWEET1基因蔗糖双向转运功能的基础上，综合运用荧光定量PCR、GUS和免疫组织化学染色、黄瓜原生质体瞬时表达和胶体金免疫电镜定位等技术开展该基因的时空表达、组织和亚细胞定位分析，并研究该基因在不同亚适温耐性品种间基因序列和结构上的差异，最终明确该基因是否参与了黄瓜亚适温下同化物韧皮部装载及其调控机制，为设施条件下黄瓜对亚适温的适应机制研究提供新线索，为培育耐亚适温设施黄瓜品种提供理论基础。

黄瓜（Cucumis sativus L.）是典型的喜温蔬菜，在设施栽培中常遭遇亚适温环境胁迫，导致同化物积累在叶片不能有效运输至果实，黄瓜果实产量和品质大幅度降低，给生产者带来较大的经济损失。SWEET是近年来才发现的一类新型的糖转运蛋白基因家族，它们通过调控植物体内糖类化合物的运输、分配和贮藏，参与了诸多植物生长发育重要生理过程。目前，葫芦科作物中SWEET基因家族功能相关研究尚属空白。本项目通过同源序列比对在黄瓜基因组中发现了17个SWEET基因家族成员，并对这些成员的序列结构特征进行了分析；进一步通过荧光定量PCR技术研究了这17个CsSWEET基因家族成员对亚适温胁迫的响应特征，筛选出受亚适温胁迫诱导且在典型的耐亚适温和冷敏感黄瓜材料中表达量具有较大差异的Csa4M622870.1；Csa4M622870.1基因全长1324 bp，属于SWEET基因家族的第I亚族，与拟南芥AtSWEET2同源性最高，因此被命名为CsSWEET2 （申报项目时命名为CsSWWET1，此结题报告中均更正为 CsSWEET2）。本项目进一步综合运用荧光定量PCR、GUS组织化学染色、原生质体瞬时表达、酵母异源表达等技术明确了CsSWEET2基因的时空表达、组织定位、亚细胞定位和糖转运特征，结果表明CsSWEET2主要在源叶内质网中表达，具有运输葡萄糖的功能。但是，CsSWEET2基因编码的mRNA序列、DNA序列以及上游启动子序列在不同亚适温耐受性黄瓜品种间没有差异。本项目还成功将35S:CsSWEET2正义表达载体遗传转化拟南芥atsweet2突变体植株。本项目研究结果为明确CsSWEET2基因在黄瓜中的生物学功能奠定了基础；同时本项目研究结果可为进一步阐明其余SWEET基因家族成员在黄瓜和其他葫芦科作物中的功能提供信息参考，为提高葫芦科作物的产量和品质提供理论依据。