VvCBF3调控VvGolS1基因表达参与葡萄低温应答的分子基础

Cold stress limits the grape production. In cold signaling pathway, CBF3 is believed a key transcriptional factor. In addition, the raffinose oligosaccharides synthesized by galactinol synthase（GolS） plays a important role in cryoprotection of plants in responding to cold. As found in our former work, hydrogen sulfide (H2S) functions in cold resistance by increasing oligosaccharides levels of GolS and inducing the expression of VvCBF3 and VvGolS1 in grape. Sequencing analysis revealed that there is CBF/DREB binding area in the promoter of VvGolS1, and the yeast one hybrid experiment indicated that VvCBF3 could directly interact with the promoter of VvGolS1. In order to analyze whether VvCBF3 interacts with VvGolS promoter element and its effect on the regulation of gene expression, the interaction between them was tested by yeast one hybrid, EMSA, CHIP and transient transcription activation assays. In further, the tissues from cold resistance cultivar and the VvCBF3 over-expression plants were used to examine the effect of H2S donor and clearing agent on the expression of VvGolS1 and the downstream oligosaccharide production. Our results provide evidence demonstrating the regulated function of VvGolS in H2S-mediated cold stress resistance and cold signaling in grape plants.

低温是限制葡萄生产的主要胁迫因子，CBF是低温诱导信号转导途径的关键转录因子，且肌醇半乳糖合成酶（GolS）途经形成的寡糖是植物重要的低温保护剂。我们前期研究发现H2S介导GolS途径促进寡糖合成，提高了葡萄耐低温胁迫能力；并伴随有VvCBF3和VvGolS1基因表达增加。由于VvGolS1启动子含有与CBF结合的顺式元件，且酵母单杂交显示VvCBF3可以结合VvGolS1的启动子，表明VvCBF3可能转录调控VvGolS1基因表达。本项目拟在以上基础上，利用酵母单杂交、凝胶阻滞、染色质免疫沉淀和瞬时转录激活等方法，剖析VvCBF3与VvGolS1启动子元件的相互作用，以期阐明VvCBF3调控VvGolS基因表达的分子基础；以抗冷葡萄品种、VvCBF3过表达和缺失体植株为材料，检测H2S供体和清除剂对VvGolS1表达和下游寡糖含量的影响，为解析H2S在葡萄低温应答的作用机制奠定基础。

低温是限制葡萄生产的主要胁迫因子，探究其低温响应机制，明晰其调控机理一直备受关注。植物响应环境低温刺激后，CBF级联信号是传递外界刺激的并引起细胞反应的重要途径。植物在低温胁迫应答过程中，肌醇半乳糖合成酶（GolS）途经形成的寡糖发挥了重要作用，但是其中的调控机制并不十分清楚。本项目前期克隆了VvGolS1、VvCBF3、VvWRKY30等基因及其启动子。利用酵母单杂交、EMSA、双荧光素酶系统和烟草瞬时转录激活实验证明转录因子VvCBF3和转录因子VvWRKY30可以与VvGolS1基因启动子结合，表明低温胁迫下，VvWRKY30与VvCBF3作为VvGolS1上游的转录激活因子，激活VvGolS1基因的表达；同时，VvWRKY30还能够作为VvCBF3上游的转录激活因子，激活VvCBF3基因的表达，进而提高抗氧化物酶的活性，棉子糖系列寡糖含量以及抗低温相关基因的表达，降低ROS含量，减少细胞的氧化损伤，从而提高葡萄抵御低温胁迫的能力。进一步研究发现，VvGolS3和VvDREB2A同样受低温诱导表达上调，在低温胁迫下，VvDREB2A结合到VvGolS3启动子的CRT/DRE元件，启动VvGolS3的表达，进而增强棉子糖系列寡糖积累和葡萄低温胁迫耐受能力。本项目研究成果系统深入的解析了VvWRKY30-VvCBF3-VvGolS1-ROFs和VvDREB2A-VvGolS3-ROFs模块调控葡萄抗寒性的分子机制，揭示了多年生果树应答非生物逆境的生理、代谢及其调控机制，为通过栽培技术和遗传手段实现葡萄提质增效，保障我国果树产业健康、稳定发展提供了重要的理论、技术和材料基础。