实验海洋生物学重点实验室知识库

深海是全球最大的生态系统，蕴含着丰富的微生物资源。这里的微生物具有丰富的遗传多样性和代谢特征，它们对深海环境中有机物的分解起着极其重要的作用，并驱动着全球的生物地球化学循环过程。目前由于采样困难、分离培养技术瓶颈等原因，人们对深海微生物的了解与研究还十分有限。获得深海微生物菌株资源是深入发掘深海微生物资源、研究其特殊代谢途径的前提。

本研究中我们使用不同的分离策略和多种培养基从深海冷泉和热液样品中获得了归属于6个门，7个纲，14个目，23个科，39个属的53种微生物（包括123株），其中包含了52种细菌和1种真菌。我们对其中来自深海冷泉的浮霉菌菌株TYQ1和放线菌菌株TYQ2进行了理化性质和基因组分析，揭示其介导的相关代谢途径，并以放线菌菌株TYQ2为例对其代谢特定有机物的能力进行了较深入的研究。

浮霉菌常被认为是糖类物质的分解者，能通过代谢糖类参与海洋碳循环过程。目前关于淡水及浅海浮霉菌的报道很多，但关于深海浮霉菌的研究相对较少，这与深海浮霉菌分离培养的困难有很大关系。本研究中我们采取以N-乙酰-D-氨基葡萄糖（GlcNAc）作为主要碳源，用多种抗生素组合抑制常见菌株生长的方法最终获得一株来自深海冷泉沉积物的浮霉菌菌株TYQ1。结合生理学实验和基因组分析，我们初步研究了菌株TYQ1对N-乙酰-D-氨基葡萄糖（GlcNAc）的代谢能力，发现菌株TYQ1能通过ABC型糖转运体将GlcNAc转运至细胞质中，经糖激酶磷酸化、脱乙酰酶脱乙酰、脱氨酶脱氨等过程进入糖酵解途径和氮代谢过程，最终将GlcNAc代谢利用供自身生长。

放线菌是一大类广泛分布于包括海洋在内的多种环境中的重要原核生物，具有巨大的应用潜力。然而，与其陆源放线菌成员相比，人们分离的海洋放线菌相对较少，尤其是深海。本研究中我们通过使用以牛磺酸为唯一碳源的基础培养基培养了数株深海放线菌，选取其中一株属于诺卡氏菌科的Marmoricola sp. TYQ2进行深入研究，发现其与最近源菌株的16S rRNA基因相似度低于97%，基因组学分析其与近缘菌株基因组相似性均低于新菌临界值，表明其为潜在新种。基于生理生化实验和转录组学分析，牛磺酸的补充能显著促进菌株TYQ2的生长，添加牛磺酸能促使编码与牛磺酸代谢利用以及能量产生相关的基因表达量明显上调。宏基因组分析表明牛磺酸代谢相关基因在深海放线菌中广泛存在。此外，转录组分析表明菌株TYQ2能通过胞外醌酚的氧化还原反应及铁离子与亚铁离子相互转化过程降解聚乙烯醇（PVA），并利用其降解产物产生能量来促进菌体生长。通过16S rRNA扩增子测序分析及前人研究，我们发现放线菌广泛分布于深海中。总体而言，我们的研究结果表明，放线菌可能在深海生态系统的元素（碳、硫等）循环中发挥重要作用，主要归因于它们的广泛分布且具有突出的有机物（牛磺酸、聚乙烯醇等）降解能力。

综上，我们通过物质代谢驱动深海微生物分离培养的策略分离获得微生物并进行相关研究，这有助于我们对深海微生物的开发利用及对深海的物质、元素循环过程的理解。

The deep sea is the largest ecosystem in the world and contains abundant microbial resources. The microbes have abundant genetic diversity and metabolic characteristics here, which play extremely important roles in the decomposition of organic matters in deep-sea environments and drive global biogeochemical cycling processes. At present, due to the difficulty of sampling and the bottleneck of isolation and culture technology, people's understanding and research on deep-sea microorganisms are still very limited. Obtaining deep-sea microbial strain resources is the premise to further explore deep-sea microbial resources and study their special metabolic pathways.

In this study, we obtained 53 species of microorganisms (including 123 strains), belonging to 6 phyla, 7 classes, 14 orders, 23 families, and 39 genera from deep-sea cold seep and hydrothermal samples using different isolation strategies and various media. 52 species of bacteria and 1 species of fungi were included. We analyzed the physiological and biochemical traits and carried out genomic analysis of the Planctomycetes strain TYQ1 and Actinobacteria strain TYQ2 from deep-sea cold seep, revealing the relevant metabolic pathways mediated by them. Taking the actinomycete strain TYQ2 as an example, we conducted a more in-depth study on its ability to metabolize specific organic substances.

Planctomycetes is often considered to be a decomposer of sugars and can participate in the marine carbon cycle by metabolizing sugars. At present, there are many reports on freshwater and shallow sea Planctomycetes, but there are relatively few studies on deep-sea Planctomycetes, which is closely related to the difficulty in isolating and culturing the deep-sea Planctomycetes. In this study, we took N-acetyl-D-glucosamine (GlcNAc) as the main carbon source and used a combination of antibiotics to inhibit the growth of common strains, and finally obtained a Planctomycetes strain TYQ1 from deep-sea cold seep’s sediments. Combined with physiological experiments and genomic analysis, we preliminarily studied the metabolic capacity of strain TYQ1 on N-acetyl-D-glucosamine (GlcNAc), and found that strain TYQ1 could transport GlcNAc into the cytoplasm through the ABC-type sugar transporter, and then enter the glycolysis pathway and nitrogen metabolism through the processes of phosphorylation by sugar kinase, deacetylation by deacetylase, deaminase deamination, etc. Finally, GlcNAc was metabolized and utilized for its own growth.

Actinomycetes are a large group of important prokaryotes widely distributed in various environments including the ocean, and have great potential for application. However, compared with their terrestrial actinomycete members, there are relatively less available marine Actinobacteria isolates, especially deep-sea counterparts. In this study, we cultivated several deep-sea actinomycetes by using taurine as the only carbon source in the basal medium. We selected one of them, Marmoricola sp. TYQ2, belonging to the Nocardiaceae family, for further study. It was found that the similarity of 16S rRNA gene between it and the most recent strain was less than 97%. Genomic analysis showed that the genomic similarity between it and the closely related strains were lower than the critical value of novel bacteria, which indicated that it was a potential novel species. Based on physiological and biochemical experiments and transcriptomic analysis, supplementing taurine can significantly promote the growth of strain TYQ2, and the addition of taurine can significantly up-regulate the expression of genes encoding taurine metabolism and utilization and energy production. Metagenomic analysis indicated that taurine metabolism-related genes were widely present in deep-sea actinomycetes. In addition, transcriptome analysis showed that strain TYQ2 could degrade polyvinyl alcohol (PVA) through the redox reaction of extracellular quinol and quinone and the interconversion of iron ion and ferrous ion, and use its degradation products to generate energy to promote bacterial growth. Through 16S rRNA amplicon sequencing analysis and previous studies, we found that actinomycetes are widely distributed in the deep sea. Overall, our findings suggest that Actinobacteria may play a vital role in elemental cycling (carbon, sulfur, etc.) in the deep-sea ecosystem, mainly due to their widespread distribution and prominent organic substances (taurine, PVA, etc.) degradation capabilities.

In summary, we isolated and obtained microorganisms through the strategy of substance metabolism-driven isolation and culture of deep-sea microorganisms and carried out related research. It will help us to develop and utilize deep-sea microorganisms and understand the cycle process of substances and elements in the deep sea.

第一章  绪论... 1

1.1  深海生态系统概述... 1

1.1.1  深海生态系统及其微生物... 1

1.1.2  冷泉生态系统及其微生物... 1

1.1.3  热液生态系统及其微生物... 2

1.2   微生物多样性与分离培养... 2

1.2.1  微生物多样性检测... 2

1.2.2  微生物分离培养概述... 3

1.2.2.1  微生物分离培养的重要性... 3

1.2.2.2  微生物分离培养现状与限制条件... 4

1.2.2.3  微生物分离培养方法的改良与创新... 4

1.3  深海微生物介导的物质代谢... 6

1.4  海洋放线菌研究进展... 8

1.5  研究内容及意义... 8

第二章  深海微生物的分离培养及两株菌株的生物学特性研究... 11

2.1  实验材料与仪器... 11

2.1.1  实验样品... 11

2.1.2  实验试剂及培养基... 11

2.1.3  实验仪器... 14

2.2  实验方法... 15

2.2.1  深海需氧微生物的分离培养... 15

2.2.2  深海厌氧微生物的分离培养... 15

2.2.3  16S rRNA基因测序与菌种鉴定... 16

2.2.4  一株浮霉菌菌株TYQ1的生物学特性研究... 17

2.2.4.1  菌株TYQ1分离培养... 17

2.2.4.2  菌株TYQ1形态学特征观察... 17

2.2.4.3  菌株TYQ1生理生化特征... 18

2.2.4.4  菌株TYQ1系统发育及基因组测序... 20

2.2.5  一株放线菌菌株TYQ2的生物学特性研究... 21

2.2.5.1  菌株TYQ2分离培养... 21

2.2.5.2  菌株TYQ2形态学特征观察... 21

2.2.5.3  菌株TYQ2生理生化特征... 21

2.2.5.4  菌株TYQ2系统发育及基因组分析... 22

2.3  实验结果与讨论... 23

2.3.1  深海样品分离培养结果... 23

2.3.2  一株深海浮霉菌菌株TYQ1生物学特性研究... 26

2.3.2.1  菌株TYQ1分离纯化与保藏... 26

2.3.2.2  菌株TYQ1形态学特征分析... 27

2.3.2.3  菌株TYQ1生理生化特征分析... 28

2.3.2.4  菌株TYQ1系统发育分析... 29

2.3.2.5  菌株TYQ1基因组测序分析... 30

2.3.3  一株深海放线菌菌株TYQ2生物学特性研究... 32

2.3.3.1  菌株TYQ2分离纯化与保藏... 32

2.3.3.2  菌株TYQ2形态学特征分析... 33

2.3.3.3  菌株TYQ2生理生化特征分析... 34

2.3.3.4  菌株TYQ2系统发育分析... 34

2.3.3.5  菌株TYQ2基因组测序分析... 35

2.4  本章小结... 39

第三章  放线菌菌株TYQ2代谢牛磺酸与聚乙烯醇的机制研究... 41

3.1  实验材料与仪器... 41

3.1.1  实验菌株... 41

3.1.2  实验试剂及培养基... 41

3.1.3  实验仪器... 41

3.2  实验方法... 42

3.2.1  生长情况检测... 42

3.2.2  转录组学分析... 42

3.2.3  实时荧光定量PCR（RT-qPCR）检测验证... 43

3.2.3.1  提取菌株TYQ2总RNA.. 43

3.2.3.2  TYQ2细菌细胞的cDNA合成... 44

3.2.3.3  荧光定量PCR引物设计... 45

3.2.3.4  RT-qPCR实验... 46

3.2.4  其他深海样品宏基因组分析... 46

3.2.5  深海样品扩增子测序分析... 47

3.3  实验结果与讨论... 47

3.3.1  菌株TYQ2生长测定及代谢牛磺酸的转录组学分析... 47

3.3.2  菌株TYQ2代谢牛磺酸的实时荧光定量PCR分析... 53

3.3.3  深海样品的宏基因组分析... 54

3.3.4  其他硫源影响菌株TYQ2生长... 55

3.3.5  菌株TYQ2生长测定及代谢PVA的转录组学分析... 56

3.3.6  菌株TYQ2代谢PVA的实时荧光定量PCR分析... 59

3.3.7  扩增子分析深海冷泉沉积物中微生物的丰度和分布... 60

3.3.8  数据存储... 62

3.4  本章小结... 62

第四章  结论与展望... 65

参考文献... 67

致 谢... 77

作者简历及攻读学位期间发表的学术论文与研究成果    81