广西红树林海洋放线菌Rpf样蛋白及其生物学活性的研究

The rare and novel species of marine actinomycetes is the source of bioactive metabolites. Currently, only a small portion of marine actinomycetes has been cultivated on the plat. Researchers have tried to mimick the natural habitats of microorganisms to recover new taxa from the marine environment in the laboratory. Some cultivation strategies have been used to promote the number of microorganisms that can be isolated from marine samples but cultivation efficiency keeps a big challenge for microbiologists..The chemical molecules such as Rpf(Resuscitation promoting factor) were reported to be involved in cell-to-cell communication. Rpf from M. luteus was active at picomolar concentrations to boost resuscitation of dormant cell and increase the number of culturable cells. In most actinobacterial species, Rpf-like proteins were highly conserved in the RPF domain comprising 70 amino acids. The function of Rpf-like proteins was speculated to involve facilitating rescue dormant marine actinomycetes. However, it is more likely to use different Rpf-like proteins to rescue the diversity of yet uncultivated populations of marine actinomycetes..The aim of this project is to clone new Rpf-like proteins from marine actinomycetes preserved in our lab and mangrove soil metagenomic library, then the new Rpf-like proteins would be used to resuscitate and isolate novel actinobacteria from mangrove soil. Under the guidance of function-driven screening and sequence-driven screening, marine actinomycetes may contain rpf gene will be screened. The rpf gene of the positive colonies will be amplified by degenerate primers targeting the essential Rpf domains and expressed in Escherichia coli. The Rpf-like proteins were detected by SDS-PAGE and MS/MS-MALDI-TOF. The isolated Rpf-like proteins were purified by a series of filtration columns for crystallization experiments. Crystallization trials were performed at 293 K using thehanging-drop vapor-diffusion method for X-ray diffraction experiments. Structure and stability of Rpf-like proteins were evaluated by Circular Dichroism (CD) spectroscopy..The logarithmic phase and nondividing cells of M. luteus are used to identify the biological activity of Rpf-like proteins by inoculating low concentrations of cells. The correlation research between three dimensional structure and biological actions of Rpf-like domains from marine actinomycetes would be investigated. At last, the viable Rpf-like proteins and marine actinomycetes are used in attempts to resuscitate, promote, isolate novel marine actinomycetes..This study contributes to understand the vital roles played by Rpf-like proteins as intercellular communication among Actinobacteria. In the future, the cultivation strategies of marine actinomycetes depending Rpf-like proteins will be more reflective of the natural habitat. The more new taxa and metabolites will be discovered from the marine environment.

藤黄球菌（Micrococcus lylae）的复苏蛋白Rpf可以复苏“非可培养状态（VBNC）”的藤黄球菌，生物信息学表明Rpf样蛋白同样广泛存在于放线菌中，从红树林放线菌中寻找Rpf样蛋白，并以其复苏海洋放线菌更有针对性。本研究拟以功能驱动筛选和序列驱动筛选方法为指导，从已分离到的海洋放线菌和红树林土壤宏基因组中克隆复苏因子rpf基因，筛选出具有明显活化作用的Rpf样蛋白并进行原核表达，优化Rpf样蛋白最佳结晶条件以获得高质量的Rpf样蛋白晶体，通过蛋白晶体X-ray、圆二色光谱（CD）表征分析等技术解析蛋白质结构，最后评价不同来源Rpf样蛋白的复苏促生长活性，从而阐明其三维结构、来源与功能之间的联系。为通过Rpf样蛋白复苏红树林土壤中处于休眠状态的微生物及获得传统分离培养难以分离纯培养的海洋放线菌奠定基础。

广西北部湾红树林的特殊环境容易造成微生物在VBNC休眠状态和复苏生长状态之间频繁转换。从红树林海洋放线菌中寻找Rpf样蛋白，并以其复苏促生长作用分离获得更多具有新颖生理生化特性或生物活性功能的放线菌存在可能。.本研究分离筛选菌株共计662株菌，通过16S rRNA基因测序分子鉴定后, 得到放线菌81株，在NCBI菌株基因组数据库中，挖掘得到45株菌株全基因组信息，并从中通过生信分析抽取出210个rpf功能基因。对红树林放线菌rpf功能基因进行基因家族分析，得到全rpf基因结构可视化图，其功能结构域有7个，包扩溶菌酶LysM，转糖甘酶Transglycosylas、DUF348、DUF3235、NLPC_P60、SLT和G5结构域，其中LysM区域与细胞壁裂解有关，优先选择处于系统发育树上不同分支，且功能结构组成有明显差异菌株发酵浓缩上清液作为潜在促生因子液，筛查得到9株有明显作用的菌株，其中Glutamicibacter arilaitensis3430菌株对多株潜在新种均有明显活性。以9株放线菌rpf基因为模板分别设计出9对引物，通过基因克隆、T克隆载体、pET30a表达载体构建、诱导表达，目前得到rpf基因克隆载体5株，表达载体菌株3株，成功构建表达载体pET30a-2401rpf、pET30a-14267rpf、pET30a-3430rpf，诱导初步条件为终浓度1mM IPTG，25℃，200rpm/min，诱导4h，目的蛋白成功表达，pET30a-2401rpf表达量最高。经诱导温度、诱导时间、诱导剂浓度实验条件优化，初步确定为1mM IPTG，30℃，200rpm/min，诱导4h。.在诱导条件的基础上进行蛋白分离实验，通过超声波破碎细胞，检测蛋白分布状态，发现pET30a-2401rpf所表达的蛋白多数为包涵体，少部分在上清液，进一步优化诱导条件：终浓度0.2mM IPTG，25℃，160rpm/min，诱导15h，经过镍柱分离得到目的蛋白，蛋白大小为45KDa，与预期大小相符合，开展了酶活检测和复苏活性相关工作。.设计了新型放线菌分离培养基，开展了广西山口红树林土壤可培养细菌的多样性工作，开展了广西北部湾茅尾海红树林根际土壤和红树林根皮放线菌的分离及产酶活性、降解菲活性和抗菌活性的研究；开展了新菌株的发现与鉴定工作。