中国科学院海洋研究所机构知识库

病毒是一种较为原始的只能在生物的活细胞内进行自我复制的非细胞生物，可以感染地球上所有类型的细胞生物。在海洋生境中病毒普遍存在且丰度极高，绝大多数以原核生物为宿主。病毒是海洋微食物网中重要的一环，对地球碳、氮循环有着显著影响。通过裂解宿主，病毒可将原核生物量转化为可溶性有机质重新被原核生物利用，使其无法进入更高的营养级。这一由病毒诱导的有机质流的改变被称为“病毒回路”。此外，病毒对原核生物群落结构、多样性及新陈代谢具有调控作用，并且可作为基因水平转移的媒介。

对海洋病毒的研究迄今已有近30年的时间，但对于海洋沉积物病毒的研究远远少于对浮游病毒的研究，尚有诸多不足之处。首先，目前最为广泛使用的海洋沉积物病毒定量方法——荧光计数法中一些处理方法的定量效能缺乏验证，这会对病毒的准确定量，及病毒-宿主比例(virus-to-prokaryote ratio, VPR)产生直接影响。VPR是描述病毒和原核生物之间关系的基本指标，对于理解病毒的生态作用非常重要。以往的研究显示，沉积物中VPR约为10，且在不同底栖生境内外的波动超过了6个数量级，通常随水深而降低。然而，已有研究发现荧光计数法中常用的离心处理会导致对VPR的显著高估，但这个问题没有引起足够的重视，因为在后续的研究中离心仍然是常用的处理方式。

其次，现有研究多为在某一海域进行单次采样，缺乏更大的时空尺度上的研究。由于海洋沉积物具有较高的异质性，且一些病毒参数的调查方法尚未统一，使得海洋病毒的时空变化特征没有被很好地描述。此外，对于海洋沉积物病毒多样性的研究也较为稀缺。由于病毒难以培养、个体及基因组极小、没有通用引物以及沉积物环境的特殊理化环境等原因，对海洋沉积物病毒多样性的研究困难较多，且缺乏连续性调查数据。

针对这些问题，本文首先对荧光计数法中样品处理流程的主要步骤进行了定量效能评估。在此基础上，调查了从潮间带、陆架海域到深海平原的沉积物病毒丰度、原核生物丰度、VPR及病毒生产力的时空变化。最后，利用病毒phoH基因对潮间带沉积物病毒的多样性及季节变化进行了初步探索。

对荧光计数法定量效能的评估结果表明，常用的离心处理会导致沉积物VPR被高估3—4倍，这显著影响了对沉积物VPR及病毒-宿主关系的认知。结合来自于37份已发表研究的135个VPR数据，以及时空调查数据分析，发现沉积物VPR的均值在2左右，在时空尺度上波动基本在10以内，远低于以往的认知。此外，沉积物中VPR比水体中低一个数量级，且波动远小于后者，表明两种生态系统中的病毒-宿主关系和病毒生态功能可能存在很大差异。

青岛湾潮间带沉积物病毒和原核生物的丰度变化全年高度动态且彼此密切相关，具有与沉积物表层温度显著相关的时间变化模式。相较而言，潮间带VPR仅显示“在高温下更低”的不显著的时间变化，显示了潮间带沉积物病毒对其宿主在数量上的密切依赖以及病毒-宿主关系的稳定。在中尺度范围上，沉积物脱镁叶绿素a含量与病毒、原核生物丰度及VPR的空间分布模式最为相关，表明上层水体的营养条件和有机质向海底的沉积是影响病毒和原核生物群落空间分布的关键因素。

对病毒生产力的研究结果表明，青岛湾潮间带沉积物病毒的活跃程度高，且沙质沉积物中的病毒对其宿主群落的影响高于泥沙质沉积物。从潮间带到深海，沉积物病毒生产力、病毒导致的原核生物死亡比例及有机碳释放量均呈现下降趋势，显示病毒对原核生物群落整体的影响力随水深增加而降低。这可能是原核生物群落中活跃细胞比例随水深增加而减少所致。

对沉积物病毒phoH基因的调查显示，青岛湾潮间带沉积物病毒phoH多样性极高，且具有显著的站位及季节间差异。尽管沙质站位的病毒丰度远低于泥沙质站位，但前者的病毒phoH基因丰富性及均匀度更高，且群落结构在各季节间更稳定。从优势OTU的分布来看，个别优势OTU呈限制性分布，但更多优势OTU在潮间带沉积物中普遍存在。氨基酸水平的系统发育树显示，沉积物中病毒phoH基因多样性远远大于水体，并且发现了4个新的病毒phoH基因群组。

Viruses are primitive non-cellular organism that replicate only in the living cells of organisms and can infect all forms of cellular life in the biosphere. Marine viruses are ubiquitous and highly abundant organisms, most of which infect prokaryotes as hosts. Viruses are an important part of the marine micro-food web, and have a significant impact on the earth's carbon and nitrogen cycles. By lysing their host, viruses convert microbial biomass into dissolved organic matter (DOM), making it inaccessible to higher trophic levels. The viral-induced organic matter flows have been termed the ‘virus shunt’. In addition, viruses have profound effects on the regulation of prokaryotic community structure, diversity and metabolism, and can act as a medium for the genes horizontal transfer.

So far, research on marine sediment viruses is far less than that on planktonic viruses, and there are still many deficiencies. Currently, the most widely used procedure for counting sediment viruses and prokaryotes is epifluorescence microscopy (EFM), where the enumeration efficiency of some steps remained to be evaluated, which would have a direct impact on the accurate quantification of viruses and virus-to-prokaryote ratio (VPR). VPR is a basic index used to describe the virus-host relationship and is important in understanding the role of viruses in the environment. The prevailing studies showed that VPR was approximately 10 in sediments, fluctuating over 6 orders of magnitude within and outside different benthic habitats, usually decreasing with water depth. However, previous studies have shown that centrifugation commonly used in EFM can lead to a significant overestimation of VPR, while it is still commonly used in subsequent studies.

Most of the previous studies examined a small number of samples in a studied area, and there is a lack of study on larger spatial and temporal scale. Due to the high heterogeneity of marine sediments and the un-unified investigation methods of some virus parameters, the spatial and temporal variability of sediment viruses have not been well characterized. In addition, research on the diversity of marine sediment viruses is also inadequate. There are many difficulties in studying the virus diversity of marine sediments especially in larger scales, since viruses are difficult to cultivate and have extremely small genomes lacking universal primers, as well as special physical and chemical conditions in benthic habitats.

According to these, the present study optimized the main steps the most widely used procedure for counting sediment viruses by fluorescence microscopy (EFM). Using optimized EFM procedure, the present study investigated the temporal and spatial variations of sediment viral abundance (VA), prokaryotic abundance (PA), VPR and virus production (VP) from intertidal zone through continental-shelves to deep-sea plains. In addition, the virus phoH gene was used to explore the diversity and seasonal variation of intertidal sediment viruses.

The results of quantitative evaluation showed that the commonly used centrifugation treatment can lead to an overestimation of VPR by 3—4 folds, which affects the cognition of VPR and virus-host relationship in sediments significantly. Based on the collected 135 VPR dataset from 37 publications and the spatio-temporal studied dataset, it was found that the sediment VPR is approximately 2 fluctuating mostly within 10 across both temporal and spatial scales, which was far less than the previous cognition. In addition, sediment VPR is one order of magnitude lower than that in the pelagic habitats and fluctuated to a much less extent, indicating that the virus-host relationship and the ecological function of viruses in the two ecosystems may be very different.

The sediment VA and PA was highly dynamic throughout the year and was closely related to each other in the intertidal zone of Qingdao Bay, with a temporal pattern significantly related to the surface temperature of the sediment. An insignificant seasonal pattern in the VPR was observed in the intertidal zone, with lower VPR values occurring at high temperatures, indicating the close numerical dependence of viruses on their hosts and the stability of the intertidal virus-host relationship. The sediment Phaeophytin a (Pha) content was most relevant to the spatial pattern of VA, PA and VPR on mesoscale, suggesting that the trophic conditions of the upper water column and the sedimentation of organic matter to the bottom are the key factors affecting the spatial distribution of viral and prokaryotic.

The study of viral production showed that the sediment viruses were highly active in the intertidal zone of Qingdao Bay, and the influence of benthic viruses on their host was higher in sandy sediments than that in muddy-sand sediments. Sediment VP, virus-induced prokaryotes mortality (VIPM), and the release of organic carbon by virus lytic infection all showed a downward trend from the intertidal zone to the deep-sea plain, indicating that the impact of the viruses on the prokaryotic community decreases with the decrease of active cells proportion in prokaryotic communities as the sediment nutritional conditions becoming barren.

In the intertidal zone of Qingdao Bay, the diversity of sediment viral phoH gene was extremely high, and there are significant differences between sites and seasons. Although the VA of sandy site S was much lower than the muddy-sand site M-S, the richness and evenness of viral phoH gene of the former was significantly higher than that of the latter, and the community structure of the former was more stable between seasons. Some of the of the dominant OTU of virus phoH gene showed restrictive distribution in different samples, while more were widespread in the intertidal sediments. Amino acid phylogenetic analysis showed that the diversity of viral phoH genes in sediments is much greater than that of water bodies, and four new viral phoH gene groups have been discovered.

摘 要... I

Abstract III

图表目录... XI

第一章............. 绪论... 1

1.1                        海洋病毒简介. 1

1.2 海洋沉积物病毒生态学研究概况. 4

1.2.1 海洋沉积物病毒的研究方法... 4

1.2.2 海洋沉积物病毒的生态学功能... 11

1.3 海洋沉积物病毒多样性研究概况. 14

1.3.1 环境病毒培养... 15

1.3.2 基于TEM的病毒多样性研究... 16

1.3.3 基于基因组大小的病毒群落全基因组图谱分析... 16

1.3.4 基于标记基因的病毒多样性研究... 17

1.3.5 基于宏病毒组的病毒多样性研究... 18

1.4 我国海洋沉积物病毒研究现状. 20

1.4.1 病毒定量研究... 20

1.4.2 病毒多样性研究... 20

1.5 选题意义及研究内容. 21

第二章............. 海洋沉积物病毒荧光计数法的优化... 23

2.1 前言. 23

2.2 实验材料与方法. 25

2.2.1 主要仪器及耗材... 25

2.2.2 试剂准备与配置... 25

2.2.3 研究方法... 27

2.3 结果. 29

2.3.1 定量效能评估... 29

2.3.2 样品处理步骤对VPR的影响... 36

2.4 讨论. 38

2.4.1 焦磷酸钠对病毒和原核生物定量的影响... 38

2.4.2不同条件下超声处理的定量效能... 38

2.4.3 离心与稀释的对比... 39

2.4.4 样品保存方式对计数的影响... 40

2.4.5 沉积物样品处理流程对VPR的影响... 41

2.5 优化的沉积物样品处理流程. 42

本章小结. 44

第三章............. 海洋沉积物病毒动态变化的时空模式及驱动因子    45

3.1 前言. 45

3.2 实验材料与方法. 46

3.2.1 采样站位... 46

3.2.2 样品采集及保存... 47

3.2.3 病毒参数测算方法... 48

3.2.4 环境因子测量... 49

3.2.5实验仪器与试剂... 49

3.2.6 统计分析方法... 49

3.2.7 VPR大数据的收集与筛选... 49

3.3 结果. 50

3.3.1 青岛湾潮间带沉积物病毒的时间变化模式... 50

3.3.2 沉积物病毒的空间变化模式... 56

3.4 讨论. 69

3.4.1 潮间带VA、PA及VPR的季节变化及驱动因子... 69

3.4.2 VA, PA, VPR的空间分布规律及驱动因子... 71

3.4.3浮游及底栖VPR的差异... 72

3.4.4 潮间带沉积物病毒生产力等的季节变化及驱动因子... 73

3.4.5 病毒生产力等的空间变化及驱动因子... 74

本章小结. 76

第四章............. 潮间带病毒phoH基因多样性及季节变化    77

4.1 前言. 77

4.2 实验材料与方法. 77

4.2.1 研究站位及样品采集... 77

4.2.2 实验仪器与材料... 78

4.2.3 DNA提取及扩增... 78

4.3 结果. 78

4.3.1 青岛湾潮间带两站位各季节的OTU构成... 78

4.3.2 各样品间phoH基因的α、β多样性对比... 80

4.3.3 青岛湾潮间带沉积物病毒phoH基因的聚类分析... 83

4.3.4 青岛湾phoH基因优势OTU的群落结构... 85

4.4 讨论. 87

本章小结. 88

第五章............. 结论与创新点... 89

参考文献... 91

附录... 100

致 谢... 101

作者简历及攻读学位期间发表的学术论文与研究成果... 102