一个新的C2H2型锌指蛋白PhZFP1调控矮牵牛耐寒性的机理研究

Cold tolerance is a vital character affecting the quality of plants. Petunia is an important ornamental plant and model plant, whereas the mechanism which regulates the cold tolerance in petunia still remains obscure. Our previous work showed that PhZFP1 isolated from petunia was a novel C2H2 zinc-finger protein gene. PhZFP1 was strongly and exclusively induced by the cold stress. The freezing tolerance of transgenic petunia plants overexpressing PhZFP1 gene was substantially enhanced compared with that of the control. The analysis of genome data demonstrated that a low temperature responsive pathway of PhZFP1 in petunia is probably different from the well-known CBF pathway in other plants. Based on these data, in this project, we plan to study the mechanism of PhZFP1 which played a role in the regulation of cold tolerance in petunia: using transcriptome sequencing and chromatin immunoprecipitation sequencing techniques, as well as metabolic research methods, the downstream genes regulated directly by PhZFP1 and the metabolic pathways involving the key target genes will be identified. The upstream regulators and the interacting proteins will be screened by yeast one and two-hybrid, respectively, and their biological functions will be characterized, from which a cold-responsive network using PhZFP1 as a node will be set up. The current project is an extension of our earlier work. It will not only enrich the theory of stress adaptation in petunia and other ornamental plants, but also provide gene resource with independent intellectual property rights for the genetic improvement related to cold stress. Therefore, this project holds both theoretical and practical potentials.

耐寒性是影响植物品质的重要性状，矮牵牛是重要的观赏植物和模式植物，其耐寒作用机制还很不明晰。前期我们在矮牵牛中克隆了一个新的C2H2锌指蛋白基因PhZFP1，显著且特异地受低温诱导，PhZFP1超量表达植株的耐寒性明显增强。基因组数据分析表明，矮牵牛中的PhZFP1低温响应途径可能有别于其他植物中广泛存在的CBF途径。本项目拟在此基础上，开展PhZFP1调控矮牵牛耐寒性的机理研究：利用转录组测序和染色质免疫沉淀测序技术，以及代谢组研究方法，筛选PhZFP1直接下游靶基因及其涉及的代谢途径；利用酵母单、双杂交技术，筛选PhZFP1的上游调控因子和互作蛋白，并鉴定互作因子基因的功能，阐明PhZFP1调控矮牵牛耐寒性的分子机理与生理机制。本项目是已有研究的延续和深入，将为矮牵牛等观赏植物的逆境适应提供新的理论依据，并有望为抗寒遗传改良提供具有自主知识产权的基因资源，具有重要理论意义和应用价值。

耐寒性是影响植物品质的重要性状，矮牵牛是重要的观赏植物和模式植物，其耐寒作用机制还很不明晰。国内外近年来对锌指蛋白在提高植物耐寒性方面的重要性已有了较为广泛的共识，但研究大多集中在基因克隆、表达分析及个别基因的转基因功能验证等方面，对其作用机制在生理学和分子生物学上还缺乏系统的研究和深入的解析。课题组前期的研究表明，PhZFP1在矮牵牛在遭受低温胁迫的过程中，扮演了非常重要的角色。本项目在已明确PhZFP1生物学功能的基础上，同时注重对上游调控因子、下游靶基因以及互作蛋白的挖掘。通过生物信息学分析，利用酵母单、双杂交技术，双荧光素酶报告系统，凝胶阻滞迁移（EMSA），双分子荧光互补（BiFC）以及荧光素酶片段互补图像（LCI）等技术，揭示了矮牵牛低温响应途径中多基因的组合调控。通过可溶性糖含量测定，以及GC-MS技术，证实超量表达PhZFP1促进了矮牵牛中肌醇半乳糖苷和棉子糖的积累，解析了由PhDREB1s-PhZFP1- PhGolS1-1组成的调控通路。此外，通过目的基因表达量检测，二氨基联苯胺（DAB）染色，氮蓝四唑（NBT）染色，H2O2含量测定以及超氧化物歧化酶（SOD）活性测定等试验，发现PhZFP1自身也可以感受低温信号，通过提高矮牵牛清除活性氧的能力来增强其耐寒性。值得一提的是，异源表达PhZFP1还能提高其他转基因植物，如番茄和拟南芥的抗寒性。通过该项目的实施，解析了PhZFP1调控矮牵牛抗寒性的分子机理和生理机制，构建了以PhZFP1为节点的转录调控网络，为矮牵牛等观赏植物的逆境适应提供了新的理论依据，同时为植物抗寒基因工程提供了具有自主知识产权的基因资源。