甜菜M14品系丝氨酸苏氨酸蛋白激酶（BvM14-STPK）响应盐胁迫磷酸化功能位点的研究

Protein phosphorylation is the most common post-translational modification. Many protein kinases are activated by autophosphorylation or phosphorylation by other kinases. Reversible phosphorylation of protein kinases plays a key role in singal transduction, especially in eukaryotic cells. Sugar beet additional M14 line, the material we have studied in this project, is a special germplasm that includes an alien chromosome of No.9 from Beta corolliflora Zoss., and shows more stress tolerance than Beta vulgaris L.. A protein serine / threonine protein kinase, was observed to be increased in expression under 200 and 400 mM NaCl stress conditions in quantitative proteomics experiments. In this project, we aim to isolate and characterize the gene encoding the serine/threonine protein kinase（BvM14-STPK) from the M14 line. The expression pattern and function of the gene in response to the salt stress were researched. Transgenic sugar beet M14 plants in which each of the main phosphorylation sites of BvM14-STPK protein was mutated were obtained. The phenotype and physiological indexes were determined. Finally the key phosphorylation sites of BvM14-STPK in response to salt stress were determined. This study will reveal the mechanisms of phosphorylation sites of BvM14-STPK involved in signal transduction pathway responsive to salt stress in Sugar beet M14.

磷酸化作用是最普遍的转录后修饰，许多蛋白激酶能通过自磷酸化或被其他激酶所磷酸化而被激活。可逆的蛋白质磷酸化作用尤其在调节真核细胞信号转导过程起核心作用。本项目的研究材料M14品系是含有野生白花甜菜第9号染色体的单体附加系，具有比栽培甜菜更高的耐盐性，是一个特殊的甜菜种质资源。前期进行的比较蛋白质组学研究表明，在200、400mmol/L NaCl胁迫下，甜菜M14品系一个丝氨酸/苏氨酸蛋白激酶上调。本项目将从M14品系中分离得到编码该酶的基因；分析该基因的序列特征，表达模式及参与盐应答的功能；获得BvM14-STPK蛋白每个主要磷酸化功能位点都定点突变的转基因甜菜M14品系阳性植株并测定其表型和生理、生化指标，最终确定甜菜M14品系BvM14-STPK蛋白在盐胁迫响应下起主要作用的磷酸化功能位点，初步揭示BvM14-STPK蛋白磷酸化位点参与盐胁迫信号转导途径的机制。

甜菜M14品系具有耐盐特性的优质种质资源。本研究从已经构建的甜菜M14品系盐胁迫RNAseq数据库中获得丝氨酸/苏氨酸蛋白激酶基因（BvM14-STPK）cDNA全长，共计1 688bp，包含1 527bp的最大ORF，编码508个氨基酸。BvM14-STPK具有丝氨酸/苏氨酸蛋白激酶保守结构域，属于RLCK（类受体胞质激酶）类家族。.利用Real-Time PCR技术对BvM14-STPK基因在盐胁迫下甜菜M14品系根、叶组织的表达情况进行了分析，结果表明甜菜BvM14-STPK基因均可以迅速应答盐胁迫。.利用转基因技术获得BvM14-STPK基因拟南芥过表达植株（OX）和BvM14-STPK基因拟南芥恢复植株（CO）。通过测定转基因阳性植株在盐胁迫下的表型、生理生化指标及酶活，结果表明BvM14-STPK基因可以提高拟南芥在盐胁迫下的耐盐性。.为了研究BvM14-STPK蛋白激酶的磷酸化位点，构建了BvM14-STPK的原核表达载体，利用质谱对诱导表达后的BvM14-STPK目的蛋白进行蛋白鉴定和磷酸化位点鉴定，共鉴定了8个关键的丝氨酸磷酸化位点。其中230位丝氨酸位点位于该激酶的激活环附近，可能会对该激酶活性有重要影响。.为了进一步研究230位丝氨酸位点（Ser230）对该激酶活性的影响，将230位丝氨酸位点分别突变为具有持续磷酸化活性的天门冬氨酸（BvM14-STPK S230D）和非磷酸活性的丙氨酸（BvM14-STPK S230A），研究原核表达体系和盐胁迫前后植物表达体系中纯化的BvM14-STPK、BvM14-STPK S230D和BvM14-STPK S230A蛋白激酶的自磷酸化与通用底物磷酸化活性，阐明盐胁迫下BvM14-STPK蛋白激酶230位丝氨酸磷酸化位点的改变对蛋白激酶的自磷酸化与通用底物磷酸化活性的影响。结果表明，230位丝氨酸位点对于BvM14-STPK蛋白激酶的活性具有重要影响，是该激酶活性所必须的位点。盐处理后BvM14-STPK和BvM14-STPK S230A蛋白激酶对通用底物MBP的磷酸化活性增强，发现盐刺激能增强该激酶的活性。.本研究初步揭示BvM14-STPK蛋白激酶磷酸化位点参与盐胁迫信号转导途径的机制，对于阐明植物丝氨酸/苏氨酸蛋白激酶参与盐胁迫信号转导途径的分子机制具有重要科学意义。.