VvCBF3调控VvGolS1基因表达参与葡萄低温应答的分子基础

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。

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.