实验海洋生物学重点实验室知识库

        深海是地球最大的独立生态系统，拥有着特殊的地质结构，比如冷泉环境的上涌烷烃渗漏现象等，这些独特的环境条件孕育了丰富的微生物和遗传的多样性。但是受限于采样和培养难度，不可培养的深海微生物高达99%以上，绝大多数的古菌和细菌仍未得到纯培养。通过创新培养方法和技术突破微生物培养障碍，获得某些关键微生物的纯培养，对揭示深海微生物特殊的代谢方式、驱动元素循环的生态作用和微生物资源的深度发掘至关重要。

        本研究基于“科学号”科考船取自深海冷泉环境的沉积物和深层海水样品，通过光照处理、抗生素筛选和厌氧培养等技术，成功获得了11株潜在细菌新种。以16S rRNA序列的相似性和系统发育树的构建为依据，分纯并鉴定了1株潜在新目水平的绿弯菌（Chloroflexi），1株潜在新科水平浮霉菌（Planctomycetes），1株潜在新目的软壁菌（Tenericutes），1株潜在新属水平拟杆菌（Bacteroidetes），1株潜在新种水平的变形菌（Proteobacteria），6株潜在新属种的厚壁菌（Firmicutes）。

        我们聚焦于蓝光处理获得的海绵杆菌新种菌株CSC3.9，对其基本生理生化性质等分类特征、蓝光感受机制和胞外多糖抗肝癌细胞Huh7.5生长增殖的作用进行了系统研究。通过对菌株CSC3.9的多相分类学和进化地位的鉴定分析，将其归为海绵杆菌属的新菌种。菌株CSC3.9表现为革兰氏染色阴性，异养、兼性厌氧生长，透射电镜下的形态表现为杆状，有单鞭毛。其最适生长温度为37℃，能够利用乙酸盐，果糖，葡萄糖，丙酮酸和蔗糖等底物。菌株CSC3.9的主要脂肪酸是C17:1 ω8c，主要的醌类化合物为泛素酮8（Q-8），最终我们将该菌株命名为Spongiibacter nanhainus CSC3.9。

         进而，我们对菌株CSC3.9的蓝光感受机制进行了深入探究。蓝光照射对菌株CSC3.9的生长有明显的促进作用，通过蛋白质组学和体内基因敲除相结合的方法，我们确定了蓝光受体BLUF是菌株CSC3.9关键的蓝光感受蛋白。我们发现蓝光刺激下，蓝光感受蛋白BLUF激活了同一基因簇中趋化和鞭毛运动相关基因的高表达，并进一步改变了菌体细胞的运动行为。通过BLUF蛋白的原核表达，发现其在体外能够特异性结合辅因子FAD，展现出独特的吸收峰，并且在光照射后发生了吸收峰的位移，证实了BLUF蛋白拥有体外的蓝光响应功能。同时BLUF的同源蛋白广泛存在于来自不同环境包括冷泉区的海洋微生物中，且在海绵杆菌属中有较大占比，表明生活在海洋中的非光合细菌感受光的能力普遍存在。

        在对Spongiibacter nanhainus CSC3.9基因组分析时发现，菌株CSC3.9中存在一个完整的多糖合成基因簇。对CSC3.9菌株胞外多糖EPS3.9进行了提取和活性评价，发现胞外多糖EPS3.9可以抑制肝癌细胞Huh7.5的生长。对所产多糖的结构进行了初步鉴定，发现EPS3.9主要是以葡萄糖和甘露糖组成的多糖，分子量是17.14 kDa。随后，对EPS3.9抗肝癌细胞增殖的机制进行了研究，通过蛋白质组学、扫描电镜和超薄切片透射电镜的形态学观察和Western blot的检测初步确定，EPS3.9是通过以GSDME介导的细胞焦亡途径对肝癌细胞Huh7.5进行杀伤。其后用免疫缺陷型肝移植瘤小鼠进行了EPS3.9的体内实验，结果表明EPS3.9在体内也有效的抑制了肝癌细胞瘤重量和体积的增长。

        综上所述，我们通过变换不同的分离策略获得了多个深海冷泉未培养微生物的纯培养代表菌株，对突破难培养微生物培养技术瓶颈做出贡献；非光合微生物新种Spongiibacter nanhainus CSC3.9的感光机制的阐明扩展了对深海微生物特殊代谢和环境适应方式的认知；菌株CSC3.9胞外多糖的抗肿瘤活性研究为深海微生物资源的发掘和利用提供了新的视角。

  The deep sea is the largest independent ecosystem on Earth, with unique geological structures such as upwelling alkanes seeping from cold seeps. These unique environmental conditions have fostered rich microbial and genetic diversity. However, due to the difficulty of sampling and culture, the unculturable deep-sea microorganisms are as high as 99%. The vast majority of archaea and bacteria have not obtained pure culture strains. Innovative culture methods and technologies to break through the barriers of microbial culture and obtain pure cultures of key microorganisms are of great importance to reveal the special metabolic patterns, the ecological role of driving element cycling of deep-sea microorganisms and the deep exploration of microbial resources.

  In this study, 11 potential novel bacteria strains were successfully obtained from sediments and water samples in deep-sea cold seep environments through light incubation, antibiotic screening, and anaerobic culture. Based on the similarity of 16S rRNA sequences, and the construction of the phylogenetic tree, including one strain of Chloroflexi, one strain of Tenericutes, which are potential new orders. Besides, one potential novel family strain of Planctomycetes and one potential new genus strain of Bacteroidetes, one potential new species strain belonging to Proteobacteria, and six potential new species strains of Firmicutes were isolated.

  Subsequently, we focused on a new species strain CSC3.9 obtained by blue light incubation. The basic physiological and biochemical characteristics of strain CSC3.9, the blue light sensing mechanism, and the effect of exopolysaccharide against the growth and proliferation of hepatoma cell Huh7.5 were studied in detail. Strain CSC3.9 was identified as a new species of Spongiibacter by multiphase taxonomy and evolutionary analysis. Cells of strain CSC3.9 were facultative anaerobic, Gram-reaction-negative, rod-shaped with single polar flagellum. The optimum temperature of strain CSC3.9 was 37℃. Growth was stimulated by the supplement of acetate, fructose, D-Glucose, pyruvate or sucrose. Major fatty acids and the main quinone of strain CSC3.9 were C17:1 ω8c and ubiquinone 8 (Q-8). Strain CSC3.9 was named Spongiibacter nanhainus CSC3.9.

  Consistently, the blue light sensing mechanism of strain CSC3.9 was further explored. Growth of strain CSC3.9 was promoted by the illumination of blue light. We next determined that BLUF (a kind of typical blue light photoreceptor) was the most essential factor directing light sensing of strain CSC3.9 through a combined proteomic and genetic method. We demonstrated that BLUF protein activated high expression of chemotaxis and flagellar motility genes in the same gene cluster under blue light stimulation, and further changed the motility behavior of bacterial cells. Through the prokaryotic expression of BLUF protein, it was found that BLUF protein could specifically bind the cofactor FAD in vitro and showed a unique absorption peak, and the shift of absorption peak occurred after light irradiation. It was confirmed that BLUF protein had blue light response function in vitro. Notably, homologs of BLUF widely existed across the marine microorganisms (containing Spongiibacter species) derived from different environments including cold seeps. This strongly indicates that the distribution of light utilization by the non-phototrophic bacteria living in the ocean is broad and has been substantially underestimated.

  The genome analysis of Spongiibacter nanhainus CSC3.9 showed that there was a complete exopolysaccharide synthesis gene cluster in strain CSC3.9. The exopolysaccharide EPS3.9 of strain CSC3.9 was extracted and tested. It was found that EPS3.9 could inhibit the growth of hepatoma cells Huh7.5. The structure of the exopolysaccharide was identified and it was found that EPS3.9 was mainly composed of glucose and mannose. The relative molecular weight of EPS3.9 was 17.14 kDa. Subsequently, the mechanism of EPS3.9 against proliferation of hepatoma cell was studied. Through proteomics, morphological observation of scanning electron microscopy, ultrathin transmission electron microscopy, and Western blot analysis, it was preliminarily confirmed that EPS3.9 killed hepatoma cell Huh7.5 through the DSDME-mediated pyroptosis pathway. The results showed that EPS3.9 also effectively inhibited the growth of hepatoma cell volume in vivo.

  In summary, we obtained a number of pure cultured representative strains from deep-sea cold seep by changing different separation strategies, which contributed to breaking the bottleneck of culture technology for difficult-to-culture microorganisms. Elucidating the photosensitive mechanism of a novel species of non-photosynthetic microorganisms, Spongiibacter nanhainus CSC3.9, has opened up the understanding of the special metabolic and adaptive modes of deep-sea microorganisms. The antitumor activity of exopolysaccharide from strain CSC3.9 opens a new horizon for deep exploration and utilization of deep-sea microbial resources.

目  录
第一章 绪论    1
1.1 深海冷泉生态系统    1
1.1.1 冷泉特殊环境    1
1.1.2 冷泉微生物多样性    1
1.2 未培养微生物    2
1.2.1 未培养微生物    2
1.2.2 微生物纯培养的意义    2
1.2.3 微生物培养的限制和策略    2
1.3 海洋光环境    3
1.3.1 海洋自然光源    3
1.3.2 海洋生物发光    4
1.3.3 深海热液的热辐射光    5
1.4 微生物的光感知    6
1.4.1 微生物的感光行为    6
1.4.2 微生物的感光蛋白    7
1.4.2 微生物的蓝光感受蛋白    7
1.4.3 微生物感光的生理意义    8
1.5 海洋微生物活性物质    9
1.5.1 深海微生物活性物质    9
1.5.2 海洋微生物胞外多糖    9
1.5.3 海洋微生物胞外多糖结构    10
1.5.4 海洋微生物胞外多糖的抗肝癌活性    10
1.6 研究内容及意义    12
第二章 深海冷泉未培养微生物的分离培养    13
2.1 研究背景    13
2.2 实验材料    13
2.2.1 样品来源信息    13
2.2.2 实验试剂    14
2.2.3 主要培养基    15
2.2.4 实验仪器    16
2.3 实验方法    16
2.3.1 好氧微生物的分离培养    16
2.3.2 厌氧微生物的分离培养    17
2.3.3 微生物的多相分类学鉴定    18
2.4 实验结果    20
2.4.1 深海新物种的分离培养    20
2.4.2 蓝光下菌株CSC3.9的富集和分纯    21
2.4.3 海绵杆菌Spongiibacter nanhainus CSC3.9T的多相分类学鉴定    22
2.4.4 冷泉环境绿弯菌门细菌的纯培养    27
2.4.5 冷泉环境浮霉菌代表株的纯培养    29
2.5 结果讨论    36
2.6 本章小结    38
第三章 Spongiibacter nanhainus CSC3.9的蓝光感受机制    39
3.1 研究背景    39
3.2 实验材料    40
3.2.1 实验菌株及质粒    40
3.2.2 实验试剂及主要培养基    40
3.2.3 实验仪器    41
3.3 实验方法    41
3.3.1 不同波长光下菌株CSC3.9的生长检测    41
3.3.2 蓝光刺激下菌株CSC3.9的蛋白组学分析    41
3.3.3 菌株CSC3.9的敲除株构建    41
3.3.4 菌株CSC3.9的趋化运动能力测定    45
3.3.5 荧光定量PCR（qRT-PCR）    45
3.3.6 BLUF蛋白的表达纯化    47
3.3.7 体外BLUF蛋白的蓝光吸收光谱特性检测    49
3.3.8 海洋细菌中BLUF蛋白的同源序列    49
3.4 实验结果    49
3.5 本章小结    65
第四章 Spongiibacter nanhainus CSC3.9胞外多糖EPS3.9的结构解析、抗肿瘤机制揭示及生物合成初探    67
4.1 研究背景    67
4.2 实验材料    68
4.2.1 实验菌株    68
4.2.2 实验试剂    69
4.2.3 实验仪器    70
4.2.4 培养基和所用溶液    70
4.3 实验方法    71
4.3.1 菌株CSC3.9胞外多糖EPS3.9的分离纯化及含量测定    71
4.3.2 肝癌细胞Huh7.5细胞的复苏和培养    71
4.3.3 细胞增殖检测    72
4.3.4 蛋白组学分析    72
4.3.5 Western blot    72
4.3.6 乳酸脱氢酶含量检测    73
4.3.7 扫描电镜    73
4.3.8 透射电镜    73
4.3.9 裸鼠模型    73
4.3.10 高碘酸钠和蛋白酶K处理    74
4.3.11 单糖组成测定    74
4.3.12 分子量测定    75
4.3.13 红外光谱分析    75
4.3.14 扫描电镜和能谱分析    75
4.4 实验结果    75
4.4.1 EPS3.9多糖的制备    75
4.4.2 EPS3.9抑制肝癌细胞Huh7.5生长增殖    76
4.4.3 EPS3.9对肝癌细胞Huh7.5形态学影响    77
4.4.4 蛋白组学分析    79
4.4.5 细胞焦亡分子机制的初步确定    81
4.4.6 EPS3.9体内抑制肝癌细胞Huh7.5的生长    82
4.4.7 菌株CSC3.9基因组中的多糖合成基因簇    84
4.4.8 多糖组分在发挥活性中起关键作用    85
4.4.9 EPS3.9扫描电镜和能谱分析    85
4.4.10 EPS3.9的分子量和多糖组成    87
4.4.11 EPS3.9的红外光谱分析    87
4.5 结果讨论    88
4.6 本章小结    90
第五章 结论及创新点    93
参考文献    95
致谢    107
作者简历及攻读学位期间发表的学术论文与研究成果    109