海岸带生物学与生物资源保护实验室知识库

海草床是蓝碳的重要组成，然而多种人为及自然因素导致海草床在全球范围内大幅度退化，中国海草亦未能幸免。良好的微生物群落结构会增强植物对环境的适应性，结构失衡则会导致植物的致病衰亡。目前，我们对海草床根际沉积物中细菌多样性及群落结构的了解较为匮乏，尤其是根际微生物随时间和空间变化的演替规律。本研究分别在2017年至2018年间的春（4月）夏（8月）秋（10月）冬（1月）四季，于东营垦利区、大连长海县和威海荣成市三地采集日本鳗草（Zostera japonica）根际及无海草定植的沉积物样品。采用16S rRNA高通量测序分析细菌群落组成和结构。

研究结果显示根际细菌种群丰富，其中变形菌门（42.8%）、拟杆菌门（17.6%）、放线菌门（8.1%）、蓝细菌门（7%）是最主要类群；一方面，优势类群会随季节而演替，放线菌门、绿弯菌门、拟杆菌门、芽单胞菌门的相对丰度分别在春、夏、秋、冬季达到最高值；另一方面，群落组成还会受到区域和样品类型（有无海草定植）的影响，例如Patescibacteria菌门在东营的相对丰度最高，绿弯菌门和变形菌门分别是根际和非根际的主要类群之一。

α多样性分析显示样品内的物种丰富度和多样性受时空影响较强烈，Shannon指数、Simpson指数、Chao1指数、OTU richness指数都在春季最低，而冬季最大；比较区域间的α多样性指数发现：东营>威海>大连。通过α多样性与环境因子相关分析进一步发现，TOC、TOC/TON、SO₄²⁻、钒（V）、锰（Mn）是影响海草根际细菌α多样性的关键因素，说明海草随季节的生长或凋亡或许能影响沉积物中的有机物和无机物含量，进而间接影响细菌群落的演替。

β多样性分析显示，相对于季节的变化，区域和根际效应对海草沉积物细菌群落结构影响更大。首先，空间尺度上，不论有无海草定植，三个区域之间全部差异显著(P = 0.001)。东营海草沉积物细菌群落结构与另外两地差异尤为明显，其原因可能与沉积物质地密切相关，威海和大连为砂质而东营是泥质。其次，样品类型上，根际和非根际之间也存在极显著的差异(P = 0.001)。时间尺度上，发现只有部分季节间的差异不明显，例如夏秋(P = 0.141)、秋冬(P = 0.055)。

样品的理化性质结果显示：有机物、重金属、NO₃⁻及NO₂⁻在夏季普遍较高；地域上东营粒径较小、重金属含量最高；相比于非根际，根际沉积物中的有机物、NH₄⁺、NO₂⁻、重金属(Mn、As金属除外)都呈现出较高含量，且中值粒径更小。细菌群落结合环境因子作RDA分析，发现驱动细菌群落结构的环境因子是粉砂、TOC、铵盐（NH₄⁺）及重金属（As、V）浓度；我们推测沉积物的粒径大小对于群落结构的影响发挥着关键作用，原因在其决定营养物和重金属的吸附，这也解释了为何东营地区的群落结构更为独特。

PICRUSt功能预测了海草根际沉积物细菌群落的功能，结果显示，硝酸盐还原基因（narG、narH、narL）、同化硫酸盐还原基因（cysJ、cysI、cysN）均在春季最低；异化硝酸盐还原基因（nirB、nirD、nrfA、nirK、norZ）、反硝化还原基因（norC、norB、anfG）、固氮基因（nifD、nifH、nifK）在东营相对丰度最高；根际中异化硫酸盐基因（aprA、aprB、dsrA、dsrB）、固氮相关基因（nifD、nifH、nifK、hao）丰度显著高于非根际，这证实了硫酸盐呼吸及生物固氮在海草根际的关键作用。

本研究描述了中国黄渤海日本鳗草生态系统中根际细菌多样性和结构组成特征，发现生长季节、定植区域、根际效应对根际细菌的群落结构的塑造都发挥着重要的作用。

Seagrass bed is an important component of blue carbon. Due to the influence of human and natural factors, seagrass beds have been largely degenerated worldwide, especially in China. Structurally-rational microbial community will enhance the adaptability of plants in the environment, while the imbalance structure will lead to the decline of seagrasses. However, it is not well understood that the diversity and community structure of bacteria in the rhizosphere sediments of seagrass beds, especially that the succession of rhizosphere microorganisms with the time and space are still unclear. In this study, vegetated and unvegetated sediments were collected from Kenli district of Dongying, Changhai county of Dalian and Rongcheng City of Weihai, in spring (April), summer (August), autumn (October) and winter (January) from 2017 to 2018. The composition and structure of bacterial community were determined by 16S rRNA high-throughput sequencing technology.

The results showed that the bacteria in rhizosphere were abundant than the non-rhizosphere, among which Proteobacteria (42.8%), Bacteroides (17.6%), Actinobacteria (8.1%) and Cyanobacteria (7%) were the predominant taxa. On the one hand, the successions of dominant groups changed seasonally. Actinobacteria, Chloroflexi, Bacteroidetes, Gemmatimonadetes were the major phylum of spring, summer, autumn and winter respectively. On the other hand, community composition was also affected by regional and sample types (with or without seagrasses colonization). For example, relative abundances of phylum Patescibacteria occupied high proportion in Dongying, and Chloroflexi and Proteobacteria were one of the abundant groups in vegetated and unvegetated samples, respectively.

Alpha diversity analysis showed that species richness and diversities were strongly influenced by the time and space. Indices Shannon, Simpson, Chao1 and OTU richness were the lowest in spring but the highest in winter. The regional comparisons of alpha diversity index showed that Dongying > Weihai > Dalian. Furthermore, the correlation analysis between alpha diversity with environmental factors showed that TOC, TOC/TON, SO₄²⁻, V, Mn were the key factors shaping the alpha diversity of bacteria in the rhizosphere of seagrasses, indicated that the growth or apoptosis of seagrasses with seasons may affect the content of organic matter in sediments, as well as the succession of bacterial communities.

Compared to seasonal effects, Beta diversity analysis showed that regional and rhizosphere effects posed a greater influence on the bacterial community structure of seagrass sediments. Significant differences were found among the three regions on spatial scale (P = 0.001), no matter seagrasses vegetated or not. The bacterial community structure of the sediments in Dongying were significantly different from those of in Weihai and Dalian. A possibility is that the bacterial community structure may related to the texture of sediments. There is sandy sediment in Weihai and Dalian, but muddy sediment in Dongying. The difference between vegetated and unvegetated of samples was significant (P = 0.001). However, part of seasonal comparisons was not significant, such as summer vs. autumn (P = 0.141) and autumn vs. winter (P = 0.055).

The physical and chemical properties of samples showed that organic matter, heavy metals, NO₃⁻ and NO₂⁻ were generally higher in summer. The smallest particle size and the highest content of heavy metals in Dongying. Compared to unvegetated sediments, contents of organic matter, NH₄⁺, NO₂⁻ and heavy metals (except Mn and As) were higher and particle size was smaller in vegetated sediments. Combining RDA analysis of bacterial communities and environmental factors assay, silt, TOC, NH₄⁺ and heavy metals (As, V) were likely the most important factors shaping community structure. We speculated that sediment size may play a key role in community structure due to that it may be absorb nutrients, heavy metals and so on. This also explained why the community structure of bacteria in Dongying was unique when compared to Weihai and Dalian.

The functional prediction of bacterial community in rhizosphere sediments of seagrasses was conducted by PICRUSt analysis. The results showed that the abundance of nitrate reduction gene and assimilating sulfate reduction gene were the lowest in spring. The relative abundance of dissimilating nitrate reduction gene, denitrifying reduction gene and nitrogen fixation gene was highest in Dongying. The abundance of dissimilating sulfate gene and nitrogen fixation related gene in vegetated was significantly higher than that in unvegetated, this confirmed the key role of sulfate respiration and biological nitrogen fixation in seagrasses rhizosphere.

Summary, this study has described the diversity and structure of bacteria in the ecosystem of Zostera japonica of the Yellow Sea and Bohai Sea in China. It was found that the seasonal, regional and rhizosphere effect played important roles in shaping the community structure of bacteria in the rhizosphere of seagrasses.

第一章 引言... 1

1.1 海草床生态系统... 1

1.1.1 海草及其生态学价值... 1

1.1.2 全球海草退化现状... 1

1.1.3 我国海草床及其退化状况... 2

1.2 国内外海草床研究进展... 3

1.2.1 国外海草研究进展... 3

1.2.2 国内研究进展... 4

1.3 根际微生物及日本鳗草... 5

1.3.1 根际及根际微生物的功能... 5

1.3.2 海草根际微生物多样性研究意义... 5

1.3.3 理化因素对细菌多样性的影响... 6

1.3.4 日本鳗草（Zostera japonica）... 7

1.4 海洋微生物研究方法... 7

1.4.1 分子生物学方法... 7

1.4.2 PICRUSt基因分析... 8

1.5 主要研究内容及技术路线... 9

1.5.1 主要研究内容... 9

1.5.2 技术路线... 10

第二章 研究区域概况和方法... 11

2.1 研究区域概况... 11

2.1.1 东营海草床概况... 11

2.1.2 天鹅湖海草床概况... 12

2.1.3 大连海草床概况... 12

2.2 实验材料和仪器... 13

2.3 实验方法... 14

2.3.1 样品采集与处理... 14

2.3.2 沉积物理化因子测定... 14

2.3.3 DNA提取... 15

2.3.4 PCR扩增... 16

2.3.5 构建文库和高通量测序... 16

2.3.6 数据处理与统计分析... 17

第三章 结果... 19

3.1 海草床沉积物细菌群落组成... 19

3.2 细菌α多样性... 22

3.3 细菌β多样性... 25

3.4 沉积物物理化学性质... 30

3.5 细菌群落与环境变量的关系... 34

3.6 沉积物细菌功能预测... 36

第四章 讨论... 41

4.1 时空及海草定植对细菌群落组成影响... 41

4.2 时空及海草定植对α多样性的影响... 42

4.3 时空及海草定植对β多样性的影响... 43

4.4 不同地点、生长阶段、样品类型（根际非根际）沉积物理化性质不同... 45

4.5 细菌功能预测分析... 47

第五章 结论与展望... 49

5.1 结论... 49

5.2 创新性... 50

5.3 展望... 50

参考文献... 53

致谢... 65

作者简历及攻读硕士学位期间发表的学术论文与研究成果... 67