甜菜M14品系BvM14-RIN4蛋白盐胁迫响应蛋白互作网络的研究

Sugar beet monosomic addition line M14, which contains the Beta vulgaris L. genome with the addition of chromosome 9 of Beta corollif lora Zoss, has exhibited interesting phenotypes, such as tolerance to cold and salt. Therefore, it is an ideal material for studying the mechanism of plants to response to salt stress and exploring high-quality genetic resources of sugar beets. In previous research, transcriptomics and proteomics approaches were used to study the salt tolerance of sugar beet M14 lines at the transcriptional level and protein level. The results showed that BvM14-RIN4 was significantly responsive to salt stress at the transcriptional level and protein level. .On this basis, this research intends to construct a BvM14-RIN4 protein interaction network by bioinformatics methods and validates these interaction proteins by biological experiments. And then the biological function of BvM14-RIN4 and its interaction proteins and their effects on the salt tolerance of sugar beet M14 line will be analyzed. Finally, interaction network of BvM14-RIN4 protein under salt stress was elucidated, which will provided experimental evidence for exploring the salt-resistant molecular mechanism of BvM14-RIN4. This research will provide innovative knowledge for in-depth clarification of the role of BvM14-RIN4 and its interaction proteins in response to salt stress in plants, and will providd a new way for the interdisciplinary combination of computational biology and molecular biology to study the mechanism of plant tolerance.

甜菜M14品系是野生白花甜菜第9号染色体的单体附加系，具有野生白花甜菜的优良性状，是研究植物响应盐胁迫应答机制、发掘甜菜优质基因资源的理想材料。课题组前期以甜菜M14品系为试材，利用转录组测序技术与蛋白质组技术分别在转录水平和蛋白水平对甜菜M14 品系耐盐特性进行了深入研究，结果显示，BvM14-RIN4在转录水平与蛋白水平均显著响应盐胁迫。.在此基础上，本项目拟通过生物信息学方法构建BvM14-RIN4蛋白互作网络，通过生物学实验验证BvM14-RIN4的互作蛋白质。分析互作蛋白的生物学功能及其对BvM14-RIN4耐盐性的影响，阐明盐胁迫下BvM14-RIN4蛋白互作网络，为探讨BvM14-RIN4的耐盐分子机制提供实验证据。本研究将为深入阐明BvM14-RIN4及其互作蛋白在植物耐盐胁迫反应中的作用提供创新性的知识积累，为跨学科结合计算生物学与分子生物学研究植物耐逆机理提供新的范例。

甜菜M14品系是二倍体栽培甜菜附加野生白花甜菜第9号染色体的单体附加系，具有较强的耐盐特性，是研究甜菜耐盐机制的理想材料。RIN4（RPM1-interacting protein）是一个已知的植物先天免疫模型关键蛋白，其抗病机制已经研究的非常透彻，然而其耐盐的分子机制却未见报道。课题组前期研究发现甜菜M14品系中BvM14-RIN4在转录水平与磷酸化蛋白水平均显著响应盐胁迫。.本项目以BvM14-RIN4为研究对象，利用转录组数据库从甜菜M14品系中克隆该基因并进行了生物信息学分析，获得了该基因的cDNA全长及基本序列信息；构建了该基因的植物表达载体，获得了甜菜M14品系BvM14-RIN4基因的转基因阳性植株，通过检测在盐胁迫下转基因植株的表型和生理、生化指标，证实了BvM14-RIN4基因能够增加转基因植物的耐盐性；利用生物信息学方法预测BvM14-RIN4的互作蛋白，结果显示BvM14-RIN4和BvM14-STPK、H+-ATPase 1和H+-ATPase 2等盐胁迫应答基因存在互作关系；蛋白质亚细胞定位结果显示BvM14-RIN4和BvM14-STPK都定位在细胞膜上，证实其可能存在互作关系；盐胁迫下，转BvM14-RIN4基因植株中H+-ATPase含量显著高于野生型和突变体植株，证实BvM14-RIN4可能与质膜H+-ATPase相互作用；转录水平分析的结果显示，盐胁迫下转BvM14-RIN4基因植株中H+ATPase编码基因AHA1、AHA2以及盐胁迫应答相关基因SOS1、AKT1表达水平显著升高，进一步证实BvM14-RIN4可能通过与质膜H+-ATPase互作，调节转基因植株的耐盐性。.本项目构建了甜菜M14品系BvM14-RIN4参与的盐胁迫应答调控网络，初步阐明了甜菜M14品系BvM14-RIN4基因响应盐胁迫的分子机制。本项目的成果为深入研究甜菜M14品系响应盐胁迫应答机制及挖掘甜菜M14品系优质基因资源提供了理论基础，同时为其他作物的抗逆研究提供借鉴。