黄瓜CsGol基因在逆境适应中的分工及其上游转录因子鉴定

Alleviating damage through accumulating raffinose family oligosaccharides (RFOs) is a unique mechanism of high plants adaption to abiotic stresses. Galactinol synthase (Gol) is a key enzyme and regulatory point in RFO biosynthesis. Four CsGols were found in the cucumber (Cucumis sativus) genome, all of which can be induced by different abiotic stresses. In addition, cucumber is a RFOs-translocating species, synthesis of RFOs in intermediary cells of leaf minor veins is a key and absolute requirement for phloem loading. Thus, among 4 CsGols, at least one CsGol has both functions of phloem loading and stress adaption. In this study, we intend to identify this “double function” CsGol through in situ hybridization, and then analyze how this CsGol perform the “double function” by treating cucumber plants with certain abiotic stress, and observing the modification of phloem loading and phloem sap components. We also want to differentiate the functions of 4 CsGols in the adaption of 4 common stresses in Chinese greenhouse, i.e., cold, heat, dry and salt stress, by treating cucumber plants with these abiotic stresses and investigating the change of CsGol expressions, enzyme activity and RFOs contents. Finally, we are going to identify upstream transcription factors of these 4 CsGols through electrophoretic mobility shift assay and MS, confirm them by overexpression and suppress expression, and illuminate how these transcription factors determining their functions in phloem loading and different abiotic stress adaption. We hope the results of this study will extend current abiotic stress signal networks by connecting this unique mechanism to other well-known abiotic stress signals and provide new ideas to improve cucumber culture and breeding technology under stress conditions.

通过积累棉子糖家族寡糖(RFOs)减缓伤害是高等植物适应逆境的独特机制。肌醇半乳糖苷合成酶(Gol)是合成RFOs的关键酶。黄瓜基因组中共有4个Gol，可分别被不同逆境诱导。黄瓜同时又是RFOs运输型植物，至少需要1个Gol在小维管束中间细胞中行使韧皮部装载功能，因此该Gol兼具韧皮部装载和逆境适应双重功能。本项目拟通过原位杂交确定该“兼职”Gol；通过能诱导该“兼职”Gol表达的逆境胁迫，分析该逆境下韧皮部装载和汁液成分变化及其与逆境适应的关系；通过低温、高温、干旱、高盐等设施常见胁迫，观测处理后各Gol表达、酶活性和RFOs含量变化，搞清各Gol在不同逆境适应中的分工；通过凝胶阻滞迁移电泳和质谱探明决定不同分工的、调控各Gol表达的转录因子及其启动子结合区域，再通过过量表达和抑制表达加以验证。以期将这一独特的适应机制与现已研明的逆境信号途径联系起来，为设施黄瓜抗逆栽培和育种提供新思路。

黄瓜（Cucumis sativus L.）主要在韧皮部中转运棉子糖家族寡糖（Raffinose family oligosaccharides，RFOs），并在叶片中积累RFOs，通过积累棉子糖家族寡糖减缓伤害是高等植物适应逆境的独特机制。肌醇半乳糖苷合成酶（Galactinol synthase，GolS）是催化RFOs生物合成的关键步骤，测定黄瓜中多个CsGolS异构体的功能多样性具有重要的科学意义。黄瓜基因组中共有4个CsGolSs，可分别被不同逆境诱导，至少需要1个CsGolS在小维管束中间细胞中行使韧皮部装载功能，因此该CsGolS兼具韧皮部装载和逆境适应双重功能。在本项目中，我们发现4个CsGolS亚型在不同的非生物胁迫下都被上调。β-葡萄糖醛酸酶染色和组织分离实验表明，CsGolS1在维管束组织中表达，而其他3个CsGolSs位于叶肉细胞中。进一步研究表明，CsGolS1在小维管束的同化物负载和胁迫响应中具有双重作用，在不利条件下可以增加韧皮部液中RFOs的浓度，进而促进同化物的运输。低温诱导下维管束特异性过表达CsGolS1株系增强了同化物转运效率，加快了冷胁迫下库叶、果实和整株植物的生长速度。我们还通过构建CsGolS1过量表达株系，发现了与野生型植株相比，过量表达CsGolS1株系的抗寒性显著增强，同时进行转录组分析，筛选出肌醇半乳糖苷下游关键基因BIN2（CsaV3_6G015280.1）、CRPK1（CsaV3_4G007620.1）、OST1（CsaV3_7G008840.1）。此外，我们对黄瓜有果无果植株进行全转录组联合分析，筛选出源库调节相关基因。最后，我们的项目结果展示了一种以前未知的对不利环境的反应，并为提高黄瓜的抗逆性提供了一种潜在的生物技术策略。