非盐性土壤中嗜盐和极端耐盐原核生物多样性及分类学研究

To investigate the biodiversity, origin and taxonomy of halophilic and extremely halotolerant prokaryotes inhabiting ordinary non-saline soils by using conventional culture-dependent method. After developing an appropriate isolation approach based on the results of a research about the influence of various nutritional factors and growth conditions (salt concentration, temperature and pH value) on the numbers of colony forming unit and the bacterial diversity of the isolates, halophilic and extremely halotolerant prokaryotic strains, both Bacteria and Archaea, would be isolated from non-saline soil samples. Then, the biodiversity of the isolates, including physiological, genetic and species diversity and diversity of communities, would be investigated in detail on the basis of the results of physiological studies and phylogenetic analysis by using the 16S rRNA gene sequence comparing and the multilocus sequence analysis (MLSA) methods, respectively. The distribution and origin of the halophilic and extremely halotolerant prokaryotes inhabiting non-saline soil also would be analyzed based on the data both from this study and from references. Furthermore, some strains representing potential new taxa would be characterized in detail by using a polyphasic taxonomic approach, and then be formally described and validly published, which would be helpful in the conservation and utilization of these microbial resources. Consequently, i) the biodiversity of halophilic and extremely halotolerant prokaryotes inhabiting non-saline soil would be generally recognized, and the potential distribution rules and possible origination patterns of these organisms might be understood to a certain extent; ii) an appropriate isolation approach being used to isolate these microorganisms would be designed; iii) some typical strains would be isolated and then be deposited effectively so as to be used further as model organisms in the study on the mechanism of evolution and adaption of halophilic and extremely halotolerant prokaryotes in non-saline environments.

以我国部分代表性地区非盐性土壤中嗜盐和极端耐盐原核生物为研究对象，深入探讨非盐性土壤中该类微生物的多样性及其来源这个科学问题。在建立有效分离方法的基础上，应用纯培养法和基于DNA序列的系统发育分析法[16S rRNA基因序列分析和多位点序列分析（MLSA）],深入研究非盐性土壤中嗜盐和极端耐盐细菌（含放线菌）和古菌的类群多样性、物种多样性、遗传多样性和生理多样性，并分析其分布特点和可能来源；利用多相分类方法对代表潜在新类群的菌株进行分类学研究。可以较全面地认识该类微生物资源多样性的基本状况，了解其分布特点，初步探讨其多途径来源的可能性，对完善微生物生态学理论体系具有重要意义；建立一套非盐性土壤中嗜盐菌的有效分离方法，分离和保藏一批代表性菌株，确定部分潜在新类群的分类地位，为进一步研究其耐盐机理和进化模式提供模式菌株、并为利用或控制该类微生物资源提供理论指导。

本项目以我国内陆的湖南省湘西州、北部受沙城暴影响较严重的河北省张家口市和南部受海洋风暴影响的海南省文昌市为采样区域，深入探讨了这些代表性非盐环境土壤中嗜盐和极端耐盐原核生物的多样性及其来源这个科学问题。. 建立了一套有效的分离方法，从非盐土样中分离纯化和保藏了720个菌株（嗜盐菌451株、极端耐盐菌309株）。作为对照，从红树林盐性土壤中分离到217株（嗜盐菌203株、极端耐盐菌14株），从吉首市人类粪便分离39株嗜盐菌。. 采用16S rRNA基因序列分析法进行的系统发育多样性分析，结果表明非盐土样分离的720个代表性菌株可以归为104种，归属于3门12科35属。Firmicutes门菌株占绝大多数（678株；94.2%），优势科为Bacillaceae（586株，14属68种），优势属为Halobacillus、Oceanobacillus和Bacillus。建立了基于16S rRNA、gryB和rpoD基因序列的对盐单胞菌科（Halomonadaceae）进行多基因位点序列分析（multilocus sequence analysis；MLSA）的方法；了解了分离菌株的耐盐特性、抗菌活性和产胞外酶特征（淀粉酶、酯酶、明胶酶和脲酶）等生理类型多样性，得到了一批强活性菌株。. 初步探讨了湘西州德夯、张家口天漠和文昌铜鼓岭为三大样区的代表性样点分离菌株的分布特点和来源，发现德夯和天漠样区存在更多的共同分布物种，而铜鼓岭的嗜盐和耐盐细菌分布更为独特；从北方的天漠、到中部的德夯再到南方的铜鼓岭，在属的分布上有一定地理隔离现象；但各样区间在科级分类单元的分布上没有明显差异。. 对部分潜在新类群代表性典型菌株进行了系统分类学研究，有18个典型菌株（type strain）在CGMCC、DSMZ和KCTC进行了妥善保藏，获得了保藏证明。采用多相分类技术（polyphasic approach）完成了6个菌株（JSM 101020、JSM 102062、JSM 104105、JSM 1685098、JSM 1781072、JSM 1782161）的分类学研究，建议代表6个新种。. 综上所述，非盐性土壤中嗜盐和极端耐盐原核生物存在较高的生物多样性和较复杂的分布特点，且蕴藏着较多的新类群（物种）和活性菌株，是一类重要的微生物资源。