青海地区典型盐湖嗜盐菌群落结构与盐激转录应答机制研究

Qaidam Basin located in the Qinghai-Tibet Plateau has many supernormal brine lakes in which numerous extremely halophilic bacteria inhabit here. Domestic previously reports is less, hence it has important scientific value on carrying out those studies involved in the population structure and biodiversity. Recently halophiles as coming stars for industrial biotechnology, pay more attention in basic and applied research. Based on typical salt lake (Qarhan Salt Lake[QSL], Chaka Salt Lake[CSL] and Koke Salt Lake[KSL]) in the Qinghai Province, the purpose in this study is to interpret the microbial community diversity and abundance using the metagenomic technology and microbial 16S rRNA/ITS analysis, and to understand the adaptive relationship between microorganism and environment, and to dig the specific new genes. According to the transcriptomic analysis and comparative transcriptome, we should discuss the gene expression and difference expression under the salt-shock model, and explores the master regulatory gene of compatible solutes ectoine biosynthesis, and comprehensively analyze the potential relationship between salt-shock response and molecular mechanism of ectoine accumulation switch. Subsequently, new up genes are clone and express in order to strengthen ectoine biosynthetic pathways. The entire study in here will provide the solid theoretical support for prospective over mass production, optimization control of fermentation engineering and application of biological medicine.

青海柴达木盆地的极端盐湖资源丰富，富集多种嗜盐微生物，国内的相关研究报道尚少，因而开展其种群结构与多样性研究具有重要的科学价值。近年来，嗜盐微生物作为极端环境微生物的主要类群，在基础与应用研究方面倍受关注。本课题旨在利用高通量宏基因组技术和微生物16S rRNA/ITS多态性分析，以此解读青海三大典型极端盐湖(察尔汉盐湖、茶卡盐湖和柯柯盐湖)的嗜盐微生物群体多样性及丰度，并探求微生物与环境适应性的关系，发掘和研究新型的特定功能基因。基于转录组学和比较转录组学分析，研究盐激条件下模式菌株的基因表达和差异表达，重点挖掘相容溶质四氢嘧啶(Ectoine)合成调控相关的新型基因，以此关联分析盐激应答启动与Ectoine积累响应的分子机制。通过分子克隆新型上调基因(Up gene)，此强化Ectoine生物合成代谢途径，此为后续过量化生产、发酵工程控制优化和生物医药应用开发等研究提供坚实的理论支撑。

柴达木盆地的极端盐湖资源丰富，富集多种嗜盐微生物。利用16S rRNA高通量测序分析察尔汉、茶卡和柯柯盐湖的嗜盐微生物多样性及丰度。结果显示：极端盐湖细菌的主要类群依次是Firmicutes、Proteobacteria、Bacteroidetes、Actinobacteria和Cyanobacteria等；古菌主要类群是Euryarchaeota、Woesearchaeota和Nanohaloarchaeota。超盐细菌的优势属群进化相似性聚类明显，分为两个热度区域，与环境因子TS、Cl-、Mg2+、K+和CO32-呈正相关作用。古菌优势属群有Halonotius、Halorubrum、Haloarcula、Halapricum等。基于比较转录组学，在HS 2.5M、MS1.0 M和LS0M盐激条件下，进行H. campaniensis XH26的基因表达差异分析。结果显示HS vs LS、HS vs MS和MS vs LS组间的up gene个数分别为519个、436个和155个，而down gene分别为712个、659个和117个。KEGG Pathway分析表明表达差异基因主要参与的物质代谢和信号转导途径，涉及Flagellar assembly、Two-component system、Bacterial chemotaxis、Phenylalanine metabolism、Arginine biosynthesis(P-Value>0.9)等。新转录本预测发现Novel gene有455个。利用分子克隆技术成功构建重组E.coli BL21/pCold I DNA-promoter+ectABC，实现了ectA、ectB与ectC基因的异源表达。结果显示：E.coil BL21/pCold I DNA-[promoter+ect ABC]耐受盐度为1.3 M NaCl，高于E.coil BL21(0-0.8 M)。优取上调SDR家族氧化还原酶基因nug89(292bp)、nug90(285bp)和nug91(759bp)，成功构建重组E.coli BL21 pET-28a+nug89/nug90/nug91，SDS-PAGE分析Nug89，Nug90和Nug91的目的大小分别为10.1kDa、10.3kDa和27.7kDa，功能研究有待深入。