KMS XINJIANG INSTITUTE OF ECOLOGY AND GEOGRAPHY，CAS
| 拟诺卡氏菌 YIM 90087T盐胁迫下的转录组及四氢嘧啶代谢通路的研究 | |
| 韩剑 | |
| Subtype | 博士 |
| Thesis Advisor | 李文均 |
| 2018-06-05 | |
| Degree Grantor | 中国科学院大学 |
| Place of Conferral | 新疆乌鲁木齐 |
| Degree Discipline | 理学博士 |
| Keyword | 耐盐菌 嗜盐菌 拟诺卡氏菌 转录组分析 四氢嘧啶 halotolerant bacteria halophilic bacteria Nocardiopsis transcriptomic analysis ectoine |
| Abstract | 嗜盐放线菌具有独特形态结构和特殊代谢机制，能产生多种具有独特性质和应用前景的生物活性分子，对高盐环境有极强的适应性，是一类极具研究价值的极端微生物类群。采用先进的多组学手段对其生态适应机理和分子机制进行深入研究，有助于阐明嗜盐微生物的嗜极机制和生命进化机制，并为嗜盐放线菌的资源收集与开发利用提供新的理论依据和科学指导。基于前期，对拟诺卡氏菌的比较基因组学分析，发现四氢嘧啶合成途径是古老的、进化过程中长期存在于嗜盐拟诺卡氏菌株中的；长期盐胁迫下膜蛋白的比较蛋白组学研究发现，随着外界盐浓度的增加，四氢嘧啶 ABC 转运蛋白显著上调。因此，推测四氢嘧啶在拟诺卡氏菌适应高盐环境的过程中发挥了极其重要的作用。本研究利用转录组技术对耐盐拟诺卡氏菌 YIM 90087T盐胁迫下转录组的变化进行了分析，并对其四氢嘧啶代谢通路进行了研究，主要的研究成果如下：通过对 YIM 90087T菌株长期盐胁迫下转录组的比较分析发现，在 0-15%盐浓度胁迫下基因的转录主要受细胞生长发育和盐胁迫两方面的影响。0-5%的盐浓度范围内，与细胞生长发育相关的调控是主要因素；10-15%NaCl 浓度范围内，与细胞生长发育相关的基因表达水平开始下调，细胞生长缓慢；与盐胁迫调控相关基因的表达逐渐上调。盐胁迫下，YIM 90087T 菌株的差异表达基因主要集中在 ABC 转运蛋白(ko02010) 和甘氨酸、丝氨酸以及苏氨酸代谢途径(ko00260)等代谢途径中。与本课题组研究的一株嗜盐拟诺卡氏菌 YIM 90004T盐胁迫下的转录组比较发现，1、在高盐胁迫下，两株拟诺卡氏菌的四氢嘧啶羟化酶(EctD)基因和相容性溶质（四氢嘧啶、胆碱）转运蛋白的基因均发生了显著上调；耐盐菌 YIM 90087T中转录调控因子(WhiB)基因呈现明显上调；2、耐盐菌 YIM 90087T的丙酮酸脱氢酶基因显著下调；而嗜盐菌 YIM90004T中磷酸盐转运和脯氨酸分解途径的相关基因显著下调。四氢嘧啶的合成和转运途径是 YIM 90087T高盐胁迫下上调的主要代谢途径，对其研究发现，四氢嘧啶是其胞内最主要的相容性的溶质，胞内积累量随盐浓度的升高而增加；高盐胁迫下，外源添加四氢嘧啶/羟基四氢嘧啶可以缩短停滞期，显著提高菌株的生长速率。YIM 90087T的四氢嘧啶合成基因簇具有独特的遗传结构，将 ectABC基因导入到大肠杆菌 BW25113 中，通过全细胞催化的方式成功合成了四氢嘧啶。通过不同盐浓度下重组菌株合成四氢嘧啶的产量发现，其初始合成速率和持续合成能力受外界盐浓度的影响，同时表明来源于耐盐菌 YIM 90087T的 EctABC 的酶活和稳定性也受外界盐浓度的影响。本研究通过对耐盐拟诺卡氏菌 YIM 90087T长期盐胁迫下的转录组研究，探讨了YIM 90087T 的盐环境适应机制；对其四氢嘧啶代谢通路及其合成途径中相关蛋白功能的研究，揭示了四氢嘧啶在其盐环境适应过程中发挥的作用及其变化机制，为研究盐胁迫下拟诺卡氏菌调控进化、蛋白功能和物种多样性奠定基础。 |
| Other Abstract | Halophilic actinomycetes, an important group among extremophiles, which due to itsspecial physiological structrue, mechanism of metabolic, numerous metabolites and salineenvironmental suitability, become more glamorous both in theory and application. Itsenvironmental adaptive mechanism and molecular mechanism have been studiedthoroughly by using the method of multi-omics technology, which provide theory gist andscientific guidance for adaptation and evolution of extremophiles and resource collectionand development of halophilic actinomycetes. Based on comparative genomics andproteomics, we found that synthesis pathway of ectoine/hydroxyectoine was very old andalways presented in Nocardiopsis, and its ABC transports appeared significantlyupregulated under high salt stress. Therefore, we presumed that synthesis pathway ofectoine/hydroxyectoine play an imporetant role in Adaption to high salt environment. Westudy the morphological, transcriptomic and synthesis pathway of ectoine of halotolerantNocardiopsis gilva YIM 90087Tunder salt stress, and the main results are as follows:The RNA-req results analysis showed that: regulations of cell growth and salttolerance are two important factors to N.gilva YIM90087T with growth and developmentunder 0-15% NaCl circumstances. In 0-5% NaCl concentration, the cell growth anddevelopment regulation paly important roles. Whereas, in 5-15% NaCl ranges, regulationsof salt tolerance will replace cell growth. Under salt treatment, N.gilva YIM 90087Tshowed significant deference in ABC transporters (ko02010), Glycine, serine and threoninemetabolism (ko00260). Comparison with transcriptomic of halophilic N. xinjiangensis90004Tunder salt stress, we found that ectD and ABC thansport gene appearedsignificantly up-regulated with 15% NaCl in the two strains. The differences betweenN.gilva YIM 90087Tand N. xinjiangensis 90004Tare that: 1. Expression of transcriptionalregulation factor WhiB gene, related to process of hypha formation, is obviously increasedin N.gilva YIM 90087T; 2. The noteworthy down-regulated gene were pyruvatedehydrogenase complex genes in N.gilva YIM 90087T, and phosphate ABC transporter gene, proline dehydrogenase gene in N. xinjiangensis 90004T, respectively.Synthesis and transportation of ectoine are key up-regulation pathway under high saltstress in N.gilva YIM90087T. Ectoine is the vital compatible solute in the cells.Accumulation of ectoine increase with the increase of salt concentration. Addition ofexogenous ectoine and hydroxyectoine were helpful to protect N. gilva YIM 90087T undersalt stress. The organizational types of ect gene cluster from N. gilva YIM 90087T wasclearly different from others Nocardiopsis strains. Ectoine was successfully synthesized byusing the method of whole-cell catalysis in recombinant E.coli which were transferdectABC from N. gilva YIM 90087T. The yield of ectoine in different salt stress showed thatInitial production and processivity were affected by NaCl concentration, which impliedthat activity and stablility of EctABC were also affected by NaCl concentration.Our study explored the salt-adaptive mechanisms of halotolerant N. gilva YIM 90087Tat the transcriptional level and clarified function of metabolism pathway of ectoine undersalt stress, so as to establish a foundation for study of evolution of transcriptionalregulation, protein function, and diversity of species. |
| Subject Area | 植物学 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | http://ir.xjlas.org/handle/365004/14926 |
| Collection | 研究系统_荒漠环境研究室 |
| Affiliation | 中国科学院新疆生态与地理研究所 |
| First Author Affilication | 中国科学院新疆生态与地理研究所 |
| Recommended Citation GB/T 7714 | 韩剑. 拟诺卡氏菌 YIM 90087T盐胁迫下的转录组及四氢嘧啶代谢通路的研究[D]. 新疆乌鲁木齐. 中国科学院大学,2018. |
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