胸苷酸激酶对泡桐丛枝植原体增殖致病的调控作用研究

To explore phytoplasmas how proliferate rapidly for pathogenicity after infectiong hosts, as a breakthrough poit, the thymidylate kinases (TMK) which are essential for DNA replication were studied. Through early research, we found that the paulownia witches’-broom phytoplasmas could promote Escherichia coli cells proliferation, and the Arabidopsis thaliana in which the PaWB tmk-b gene was overexpressed showed the phenotypes like symptoms induced by the PaWB phytoplasmas. Based on previous researches, this study will reveal the transcriptional regulation mechanisms of the PaWB tmk-b gene in the process of proliferating and showing symptoms of phytoplasmas by studying the spatio-temporal expression pattern of the PaWB tmk-b gene, analyzing the relationships among the expression level of the PaWB tmk-b gene, the concentration of phytoplasmas and degree of the severity of symptoms, seeking transcriptional regulators of the PaWB tmk-b gene, analyzing the patterns of transcriptional regulation of the PaWB tmk-b gene. This study will also define the PaWB tmk-b gene’s pathogenetic roles by immunohistochemical localization in A. thaliana in which PaWB TMK-b proteins were overexpressed specifically in phloem, and subcellular localization in Nicotiana benthamiana in which the PaWB TMK-b proteins were transiently overexpressed. The aim of this study is to explore the regulation functions of TMK-b proteins on proliferation and pathogenicity of PaWB phytoplasmas. It will be able to lay the foundations for revealing the pathogenic mechanism of proliferation and pathogenicity of phytoplasmas, analyzing the structure of the PaWB TMK-b protein, designing and screening inhibitors for the treatment of phytoplasma diseases.

为探索植原体侵染寄主后如何快速增殖以致病，以DNA复制必不可缺的胸苷酸激酶（TMK）为切入点展开研究。前期发现，泡桐丛枝（PaWB）植原体TMK-b能促进大肠杆菌细胞增殖，转化PaWB tmk-b基因的拟南芥能产生类似泡桐丛枝病症状的表型。基于前期基础，项目研究PaWB tmk-b基因时空表达模式，分析PaWB tmk-b表达量、植原体浓度、发病程度的关系，探寻PaWB tmk-b基因转录调控因子并分析其对PaWB tmk-b基因的转录调控模式，以期揭示PaWB 增殖和致病中tmk-b基因的转录调控机制；通过在拟南芥韧皮部特异性表达和在本生烟中瞬时表达PaWB TMK-b并进行免疫组化和亚细胞定位，明确PaWB TMK-b的致病作用。项目旨在探寻TMK-b对PaWB植原体增殖致病的调控作用，为揭示植原体增殖致病机理及解析PaWB TMK-b蛋白结构、设计和筛选抑制剂治疗植原体病害奠定基础。

本项目以泡桐丛枝病植原体为研究对象，以植原体增殖所必须的dTTP合成途径为切入点，对该途径中的tmk-b、tdk和thyA基因序列的多态性和增殖致病性开展了深入研究。采集了我国15个省、直辖市内40多个市县区的90余份泡桐丛枝病样品并风干保存。PCR扩增和测序表明，我国PaWB植原体的tmk-b基因序列单一，tdk和thyA基因均存在两种基因型。诱导表达纯化了SUMO/PaWB TMK-b融合蛋白并制备了兔多克隆抗体。利用本研究中建立的能克服RNA中DNA污染的植原体基因表达定量检测方法，研究了PaWB病状严重程度、植原体浓度、PaWB tmk-b基因mRNA表达量之间的关系，结果显示，植原体浓度与病状严重程度成正相关性，PaWB tmk-b mRNA表达量与植原体浓度成负相关性，这表明植原体定殖后的早期，PaWB tmk-b大量转录以促进病害的发生，随着寄主中植原体含量的增加，病状不断加重， PaWB tmk-b mRNA表达逐渐减弱至不表达。用PaWB TMK-b兔多克隆抗体进行的免疫电子显微镜结果也表明，PaWB TMK-b蛋白在正在出芽的植原体中表达量较高，在不增殖的植原体细胞中表达量低甚至不表达。大肠杆菌生长曲线测定结果显示，在IPTG诱导条件下，相比于含有pET-28a空载体的65#对照菌株，含有pET-28a/PaWB tmk-b的63#菌株生长速率显著加快，这表明PaWB TMK-b蛋白显著促进了大肠杆菌的增殖。在IPTG诱导下，取对数期的63#和65#菌体进行转录组测序，结果表明，相比于65#菌株，63#菌株中绝大多数与DNA合成、RNA转录、蛋白质翻译、蛋白加工等与增殖密切相关的基因表达量显著上调，这进一步证明了PaWB TMK-b蛋白能促进大肠杆菌增殖，也表明PaWB TMK-b可能是通过正向调节增殖相关基因促进细胞增殖。健康、轻度、中度、重度PaWB组培苗转录组测序和抗病性相关的酶活性测定结果显示，寄主染病后一些抗病性相关的基因表达量升高，随着病状严重程度的增加，表达量又呈现下降的趋势；部分抗病性相关的酶活性也表现出相似的变化趋势。另外，原核表达且纯化了PaWB TDK和PaWB thyA蛋白并分别制备了兔多克隆抗体，研究了PaWB tdk和PaWB thyA基因表达量与病状严重程度的关系。该研究对控制植原体增殖以防控泡桐丛枝病具有重要意义。