深海热液区管状蠕虫Ridgeia piscesae中Ricin B-lectin型结构域基因的克隆与表达

作为一种重要的模式识别因子,凝集素在多个领域中均有着广泛的应用价值,因此,开发和利用新型凝集素将具有重要意义.本研究以生活在深海热液区极端环境条件下的管状蠕虫Ridgeia piscesae为材料,首次从管状蠕虫中克隆获得Ricin B-lectin基因rgal.序列分析表明,RGAL与已知序列的相似性较低,其含有2个Ricin B-lectin型结构域,并且该结构域具有该家族所特有的β-三叶草形三维结构.多方面的分析结果显示RGAL可能是一个新颖的凝集素蛋白.对RGAL进行了原核重组表达,并对其复性条件进行了摸索优化,成功获得了其可溶性表达产物,为后续RGAL的生物活性分析奠定了坚实的基础.     

热液区管状蠕虫几丁质结合蛋白的克隆表达及功能鉴定

本文应用PCR技术对四种管状蠕虫几丁质结合蛋白进行了扩增,进而连接到pIZ-FLAG构建了重组表达载体,并在昆虫细胞中实现了几丁质结合蛋白的异源重组表达.我们对几丁质结合蛋白的功能进行了初步研究,亲和活性实验结果表明这四种几丁质结合蛋白对α-chitin具有亲和活性,并且可以辅助几丁质酶,对降解α-chitin和β-chitin均有不同程度的促进作用,且作用效果明显.结果表明,这四种几丁质结合蛋白均为α型,且可以促进几丁质酶的活性,对降解几丁质多糖具有巨大的应用潜力和应用价值.     

Microbial community structure and diversity in deep-sea hydrothermal vent sediments along the Eastern Lau Spreading Centre

The aim of this study is to investigate microbial structures and diversities in five active hydrothermal fields’ sediments along the Eastern Lau Spreading Centre (ELSC) in the Lau Basin (southwest Pacific). Microbial communities were surveyed by denatured gradient gel electrophoresis (DGGE) and clone library analysis of 16S rRNA genes. The differences in microbial community structures among sediment samples from the five deep-sea hydrothermal sites were revealed by DGGE profiles. Cluster analysis of DGGE profiles separated the five hydrothermal samples into two groups. Four different 16S rRNA gene clone libraries, representing two selected hydrothermal samples (19-4TVG8 and 19-4TVG11), were constructed. Twenty-three and 32 phylotypes were identified from 166 and 160 bacterial clones respectively, including Proteobacteria, Bacteroidetes, Firmicutes, Nitrospirae and Planctomycetes. The phylum Proteobacteria is dominant in both bacterial libraries with a predominance of Gamma-Proteobacteria. A total of 31 and 25 phylotypes were obtained from 160 and 130 archaeal clones respectively, including Miscellaneous Crenarchaeotic Group, Marine Group Ⅰ and Ⅲ, Marine Benthic Group E, Terrestrial Hot Spring Crenarchaeota and Deep-sea Hydrothermal Vent Euryarchaeota. These results show a variety of clones related to those involved in sulfur cycling, suggesting that the cycling and utilization of sulfur compounds may extensively occur in the Lau Basin deep-sea hydrothermal ecosystem.     

Microbial diversity in polluted harbor sediments II: Sulfate-reducing bacterial community assessment using terminal restriction fragment length polymorphism and clone library of dsrAB gene

Sulfate-reducing bacteria (SRB) are important regulators of a variety of processes in coastal marine sediments regarding organic matter turnover, biodegradation of pollutants, and sulfur and carbon cycles. Yet their community compositions have not been investigated in polluted harbor sediments. This study described the diversity and spatial variation of SRB communities in surface sediments in Victoria Harbor, Hong Kong. The spatial variation of SRB communities was described by terminal restriction fragment length polymorphism (T-RFLP). The results showed that the most diversified terminal restriction fragments were found at polluted sites. In addition, cluster analysis indicated that although the SRB communities were different at the two polluted sites, they were still more similar to each other than to the two more distant reference sites. Based on a dsrAB clone library constructed at a polluted site, diversified SRB were found, represented by 30 Operational Taxonomic Units (OTUs). Upon comparisons among the SRB sequences detected from this study and those in the GenBank, five clades of SRB were found. Three clades belonged to the known families Desulfobacteraceae, Desulfobulbaceae, and Syntrophobacteriaceae. The majority of sequenced clones, which distantly related to sequences in the GenBank, constituted the remaining two unclassified groups, suggesting unique SRB members related to the polluted harbor environment. Statistical analyses indicated that estimated SRB richness correlated with environment factors such as sulfur content, acid volatile sulfate, and redox potential.     

南极纳尔逊冰川土壤中微生物的群落结构及其代谢特征研究

冰川土壤中的微生物是冰冻圈生态系统中的重要组成部分。南极纳尔逊冰川四周环海,临近海洋的物质输送和其他因素扰动改变了近岸土壤中部分理化因子,从而对土壤中的微生物群落产生影响。本研究采集了南极纳尔逊冰川不同近海距离处的土壤样品,并对其进行了细菌和古菌V4区扩增子测序以及宏基因组测序,探讨了不同近海距离的冰川土壤中的微生物群落结构和代谢潜能。物种多样性结果显示,不同位点的土壤微生物群落组成有所差异,但变形菌门、放线菌门、拟杆菌门等在冰川土壤样品中普遍存在且相对丰度较高。宏基因组分析结果显示,不同近海距离的冰川土壤微生物群落的功能基因分布不同,且能量代谢和跨膜运输等代谢途径的基因的丰度随着采样位点远离海洋而降低。冰川土壤中碳、氮、硫代谢分别以还原性柠檬酸循环、反硝化、同化硫酸盐还原途径为主,其中反硝化途径基因在所有样品中丰度较高。通过分箱组装获得了含有反硝化功能基因的基因组bin＿71,并重构了其核心的代谢通路。本研究初步揭示了南极纳尔逊冰川土壤中微生物的群落结构及代谢潜能,为后续南极冰川土壤新物种的发现、功能基因的挖掘、以及探究全球气候变暖下海洋对沿海生态系统的影响提供了基础数据。     

渤海沉积物中细菌群落结构和基因丰度的空间分布特征

This study investigated differences in the community structure and environmental responses of the bacterial community in sediments of the Bohai Sea.Illumina high-throughput sequencing technology and real-time PCR were used to assay the bacterial 16S rRNA genes in the surface sediments of 13 sampling stations in the Bohai Sea.The results showed that sediments at the majority of the 13 sampling stations were contaminated by heavy metal mercury.The main phyla of bacteria recorded included Proteobacteria(52.92%),Bacteroidetes(11.76%),Planctomycetes(7.39%),Acidobacteria(6.53%)and Chloroflexi(4.97%).The genus with the highest relative abundance was Desulfobulbus(4.99%),which was the dominant genus at most sampling stations,followed by Lutimonas and Halioglobus.The main factors influencing bacterial community structure were total organic carbon,followed by depth and total phosphorus.The content of lead,cadmium,chromium,copper and zinc had a consistent effect on community structure.Arsenic showed a negative correlation with bacterial community structure in most samples,while the impact of mercury on community structure was not significant.The bacterial community in sediment samples from the Bohai Sea was rich in diversity and displayed an increase in diversity from high to low latitudes.The data indicated that the Bohai Sea had abundant microbial resources and was rich in bacteria with the potential to metabolize many types of pollutants.     

Determination of nitrate in the blood of the hydrothermal vent tubeworm Riftia pachyptila using a bacterial nitrate reduction assay

The vestimentiferan tubeworm Riftia pachyptila derives most or all of its nutrition from intracellular chemosynthetic bacterial symbionts. Because purified preparations of symbionts respire nitrate, possibly nitrite, and oxygen, host transport of nitrate is a topic of interest. In the present study, we have developed a nitrate detection assay that utilizes a nitrite reductase-deficient Escherichia coli strain for the reduction of nitrate to nitrite, which is then determined spectrophotometrically. Nitrate and nitrite concentrations were measured in the blood and coelomic fluids of R. pachyptila collected from hydrothermal vent sites at 9°N and 13°N. The blood was shown to have nitrate concentrations up to one hundred times that of ambient sea water (40 μM). Blood nitrate levels reached concentrations of>1 mM, while nitrite was measured in the range of 400-700 μM. The concentrations of nitrate and nitrite in the coelomic fluids were 150-240 μM and <20 μM, respectively. The nitrate determination technique we present here is simple, applicable for laboratory and shipboard use on sea water or biological fluids, and works reliably within the 0.5 to 2000 μM range.     

胶州湾海域表层沉积物细菌多样性

采用16S rDNA文库结合PCR-RFLP分析的手段,对胶州湾4个站位沉积物中的细菌多样性和群落特征研究分析。结果表明,沉积物中细菌种类丰富,最多包含分布于14个已知门类的细菌,和一些未被认知的序列;各站位沉积物中优势菌群均为变形菌门和酸杆菌门,其中γ-和δ-变形菌纲为变形菌门中的绝对优势类群,在4个文库序列中平均占42%和16.75%;此外,拟杆菌门、浮霉菌门和放线菌门的种类也较为大量存在。各细菌种群有较明显空间分布差异,可能与不同区域胶州湾环境条件相关。     

Sponge heterotrophic capacity and bacterial community structure in high‐ and low‐microbial abundance sponges

The ecological and evolutionary forces that shape interactions between marine sponges and their symbiotic microbiota remain poorly understood. Considerable work has been done to characterize the sponge microbiome, and this research has shown that there are two distinct types of sponges: those with high microbial abundances (HMA) and those with low microbial abundances (LMA). There is no clear evolutionary driver for this distinction, and we have only a limited understanding of how HMA or LMA status affects host phenotypes (e.g. feeding behavior, capacity for nutrient cycling). We had two primary goals with the present study. First, we used a variety of microscopic techniques to compare aspects of host biology (e.g. choanocyte chamber morphology and density) in the context of symbiont status. Secondly, we used molecular approaches to uncover components of ecological structure of bacterial communities in HMA versus LMA sponges (e.g. species richness, evenness). We found that choanocyte chamber density is greater in LMA sponges than in HMA sponges. We also found distinct patterns of organization for bacterial communities in HMA and LMA sponges, although environmental factors, to a lesser degree, also influence community structure in the Floridian sponges we examined. These results suggest that the large bacterial communities found in HMA sponges may allow the host to decrease their heterotrophy versus that of LMA sponges with minimal bacterial communities.     

北黄海微表层细菌丰度与可培养细菌群落结构分析

为分析海洋微表层这一特殊生境中的细菌类群,于2010—2011年4个航次对北黄海微表层和次表层海水中的总菌丰度、可培养细菌丰度和群落结构进行了分析。采用流式细胞仪测定总菌丰度,平板计数法测定可培养细菌丰度,PCR-16SrDNA分析可培养细菌的群落结构。结果表明,调查海域微表层海水总菌均值为1.76×106 cell/mL,次表层海水总菌均值为1.07×106 cell/mL。可培养细菌丰度范围是1.00×102~1.70×106 CFU/mL,微表层和次表层可培养细菌所占总菌数量的百分比分别为13.05%和0.45%。微表层对总菌的富集因子(EF)均值为2.02,可培养细菌的EF均值为74.16。PCR-16SrDNA序列分析结果表明,该海域可培养细菌分属变形菌门(Proteobacteria)(94.34%)、厚壁菌门(Firmicutes)(1.89%)、拟杆菌门(Bacteroidetes)(1.89%)和放线菌门(Actinobacteria)(1.89%)4个类群。本研究初步发现,微表层对细菌具有较强的聚集作用,尤其对可培养细菌聚集作用更为明显。微表层中可培养细菌的群落结构与次表层有所不同,其种类丰富,来源和功能多样。由此可见,微表层独特的生境成就了其独特的微生物类群,其生态功能有待于进一步研究。     

急性盐度胁迫对缢蛏肠道菌群结构及功能的影响

盐度是水产动物肠道微生物稳态的重要影响因子,其对水产动物的生长和健康有着重要影响。缢蛏（Sinonovacula constricta）是一种埋栖类的滩涂养殖贝类,常受到盐度波动的影响。然而,盐度是否对缢蛏肠道组织及其肠道菌群造成不利影响尚不清楚。为此,本研究设置了低盐（5）、正常盐度（20）和高盐（35）3个不同的盐度胁迫组,分别在胁迫前（正常盐度20,0 d）和胁迫15 d后收集缢蛏样品,利用组织病理学、16S rRNA高通量测序技术以及PICRUSt2工具对各处理组样品的肠道组织结构、菌群构成及其潜在功能进行比较分析。结果显示,15 d的急性盐度胁迫对缢蛏肠道组织造成了不同程度的损害,表现为细胞空泡、组织坏死和绒毛杂乱等。在所有组中共鉴定到712个细菌操作分类单元（OTUs）,其中共有OTUs占细菌总数的6.2%。低盐和高盐胁迫下缢蛏肠道主要优势菌分别隶属于γ-变形菌纲（γ-Proteobacteria）和α-变形菌纲（α-Proteobacteria）。急性盐度胁迫改变了肠道细菌α-多样性,且在低盐组中物种丰富度最低。低盐和高盐胁迫下缢蛏肠道细菌群落结构均发生了显著改变（p < 0.002）,同时伴随肠道菌群种间互作的降低。3种盐度胁迫下缢蛏肠道菌群组装的生态过程主要是确定性过程,而在低盐胁迫下其确定性过程有所降低。功能预测结果表明,急性盐度胁迫显著改变了缢蛏肠道菌群介导的营养物质和能量代谢等相关的功能通路丰度,而与免疫相关的功能通路丰度在高盐组中显著升高。本研究结果表明,急性盐度胁迫可引起缢蛏肠道发生病理损伤并伴随肠道菌群及其潜在功能的明显改变,这些改变可能影响缢蛏的健康。     

Relationship between metal levels in the vent mussel Bathymodiolus azoricus and local microhabitat chemical characteristics of Eiffel Tower (Lucky Strike)

The turbulent mixing of hydrothermal hot fluid with cold seawater creates large chemical gradients at a small spatial scale that may induce variable physiological and biochemical adaptations within the vent fauna. The adaptation to such a variable environment by the vent mussel Bathymodiolus azoricus relies on a dual symbiosis hosted in the gills, and digestion of particulate organic matter. The surrounding environment not only provides the necessary energy sources and suspended organic particles for the vent mussel nutrition, but also potentially toxic compounds such as metals. Our main goal was to see if there is a relation between metal accumulation in mussel organs and the chemical characteristics of their close environment. Mussels were collected at six locations in a cold part of the Eiffel Tower fluid-seawater mixing zone, characterized by distinct chemical compositions. Metals (Cd, Cu, Fe and Zn) and metallothioneins were quantified in the gills and digestive gland. The physiological condition of the sampled mussels was also evaluated using tissues and gill indices. Our study indicates that the accumulation of metals in B. azoricus is related to their spatial distribution and linked to fine scale environmental conditions that influence the physiological status of the organism.     

Spatial distribution, diversity and composition of bacterial communities in sub-seafloor fluids at a deep-sea hydrothermal field of the Suiyo Seamount

Spatial distribution, diversity, and composition of bacterial communities within the shallow sub-seafloor at the deep-sea hydrothermal field of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific Ocean, were investigated. Fluids were sampled from four boreholes in this area. Each borehole was located near or away from active vents, the distance ranging 2–40 m from active vents. In addition, fluids discharging from a natural vent and ambient seawater were sampled in this area. We extracted DNA from each sample, amplified bacterial 16S rRNA genes by PCR, cloned the PCR products and sequenced. The total number of clones analyzed was 348. Most of the detected phylotypes were affiliated with the phylum Proteobacteria, of which the detection frequency in each clone library ranged from 84.6% to 100%. The bacterial community diversity and composition were different between hydrothermal fluids and seawater, between fluids from the boreholes and the vent, and even among fluids from each borehole. The relative abundances of the phylotypes related to Thiomicrospira, Methylobacterium and Sphingomonas were significantly different among fluids from each borehole. The phylotypes related to Thiomicrospira and Alcanivorax were detected in all of the boreholes and vent samples. Our findings provide insights into bacterial communities in the shallow sub-seafloor environments at active deep-sea hydrothermal vent fields.     

海南西北部近岸海域浮游动物群落结构

为探讨海南西北部近岸海域浮游动物群落结构,根据2016年11月(秋季)、2017年2月(冬季)、2017年5月(春季)和2017年8月(夏季) 4个季节的浮游动物调查数据,对该海域浮游动物的种类组成、优势种及其生态类群、丰度和生物量进行了分析。结果表明:4个季节共鉴定浮游动物7门113属215种(含未定种),秋季最多(134种),冬季(113种)和春季(111种)较为接近,夏季(94种)最少,各季节均以桡足类和水螅水母类占优势。浮游动物种类的空间分布上,秋季和冬季整体呈现近岸和远岸较中间高,而春季和夏季由近岸到远岸呈逐渐增加的变化趋势。浮游动物种类随季节变动不大,但优势种更替较为明显,仅亚强次真哲水蚤(Subeucalanus subcrassus)和肥胖箭虫(Sagitta enflata)为4个季节共有优势种。优势种生态类群主要可分为暖温种、广温广盐种、近岸暖水种、热带暖水种及暖水广布种。浮游动物丰度均值秋季(124. 75 ind./m3)与冬季(152. 43ind./m3)相近,春季(64. 76 ind./m3)和夏季(74. 44 ind./m3)相近,春季、夏季的丰度均值要明显低于秋季、冬季,平面分布上秋季和冬季呈现近岸高远岸低,但不同的是水深在大于20 m以上的海域,冬季平均丰度要稍高于秋季,而春季和夏季呈现近岸低远岸高的变化特点。浮游动物生物量冬季(263. 68 mg/m3)最高,秋季(147. 38 mg/m3)次之,春季(59. 13 mg/m3)和夏季(61. 45 mg/m3)相近,平面分布上与丰度分布趋势相似。     

海水养殖蟹体内与外部环境中菌群结构的PCR-DGGE比较——以三疣梭子蟹和锯缘青蟹为例

利用PCR-DGGE指纹图谱技术和UPMGA聚类分析,对三疣梭子蟹Portunus trituberculatus、锯缘青蟹Scylla serrata的鳃组织及养殖环境中的菌群结构进行了对比分析。结果表明:三疣梭子蟹、锯缘青蟹鳃组织及养殖环境水样在指纹图谱上分别显示出21,21,23和21条信号强度不同的条带,条带的数量反映出三疣梭子蟹养殖环境中的菌群最为丰富,条带信号的强度,反映出不同样品中存在各自的优势菌种;可将4个样品中的菌群划分为2个群落,其中三疣梭子蟹和锯缘青蟹鳃组织中的菌群聚为一类,其相似度为75%,2种蟹养殖环境中的菌群聚为一类,相似度为69%。首次尝试利用16S rDNA的PCR-DGGE指纹图谱技术分析了2种健康蟹体内和各自养殖环境中微生物结构的差异性,从而寻找蟹体内的益生菌,将有助于建立一种不依赖于分离培养的原位研究蟹体内菌群组成的分子诊断技术,为今后研究锯缘青蟹、三疣梭子蟹与养殖水体微生物及相关蟹病害的关系提供基础科学依据。     

南麂岛邻近海域底栖生物群落结构分析

本文首次报道南麂岛邻近海域底栖生物的种类组成及数量分布规律，调查海域共获底栖生物１９９种，其中，多毛类占３９％，软体动物占２３％，种类组成上存在明显的季节演替现象。年平均生物量为４５．０４ｇ／ｍ２，秋季高于春季；平均密度为３８４个／ｍ２，春季高于秋季，南麂岛西北面有一个高生物量区。本文还应用群落特征指数及多维调节（ＭＤＳ）方法分析了底栖生物群落结构，探讨环境因子对群落结构的影响。结果表明，除了可分出代表该海域底栖生物群落的基本特征的沿岸区外，尚可再分出４个特殊的区域，各区有自己特有的优势种；各区的群落结构与水深有一定关系。     

Spatial-temporal variation of phytoplankton community structure in Jiaozhou Bay,China

To better understand the spatial-temporal variation in phytoplankton community structure and its controlling factors in Jiaozhou Bay, Qingdao, North China, four seasonal sampling were carried out in 2017. The phytoplankton community structure and various environmental parameters were examined. The phytoplankton community in the bay was composed of mainly diatoms and dinofl agellates, and several other species of Chrysophyta were also observed. Diatoms were the most dominant phytoplankton group throughout the year, except in spring and winter, when Noctiluca scintillans was co-dominant. High Si/N ratios in summer and fall refl ect the high dominance of diatoms in the two seasons. Temporally, the phytoplankton cell abundance peaked in summer, due mainly to the high temperatures and nutrient concentrations in summer. Spatially, the phytoplankton cell abundance was higher in the northern part of the bay than in the other parts of the bay in four seasons. The diatom cell abundances show signifi cant positive correlations with the nutrient concentrations, while the dinofl agellate cell abundances show no correlation or a negative correlation with the nutrient concentrations but a signifi cant positive correlation with the stratifi cation index. This discrepancy was mainly due to the diff erent survival strategies between diatoms and dinofl agellates. The Shannon-Wiener diversity index ( H′) values in the bay ranged from 0.08 to 4.18, which fell in the range reported in historical studies. The distribution pattern of H′ values was quite diff erent from that of chlorophyll a , indicating that the phytoplankton community structure might have high biomass with a low diversity index. Compared with historical studies, we believe that the dominant phytoplankton species have been changed in recent years due mainly to the changing environment in the Jiaozhou Bay in recent 30 years.     

象山港人工鱼礁区浮游动物种类组成及群落结构特征

2011年和2012年期间两个夏季(7月)在象山港人工鱼礁区及其邻近海域进行了浮游动物及主要环境因子调查,根据采集的浮游动物样品的分析鉴定及其他环境因子测试结果,对调查海域的浮游动物群落进行研究。结果表明,象山港人工鱼礁区及其邻近海域有浮游动物6大类36种,优势种为短尾类幼体(Brachyura larvae)、汤氏长足水蚤(Calanopia thompsoni)、真刺唇角水蚤(Labidocera euchaeta)、太平洋纺锤水蚤(Acartia pacifica)等。两个航次鱼礁区浮游动物生物量无显著差异性,与对照区相比,2012年鱼礁区明显较高;浮游动物丰度则出现明显的差异(2011年较大),与对照区相比,鱼礁区无明显差异性;多样性和均匀性变化一致,且出现显著差异(投礁后,多样性明显升高,且高于对照区)。通过对调查水域浮游动物群落聚类分析发现,鱼礁区浮游动物的类群与对照区存在明显的差异,主要分为两个类群,以鱼礁区和对照区的典型两个区域。     

秦皇岛海域浮游植物的群落结构特征

2014年4~9月对秦皇岛海域进行了调查,共鉴定出浮游植物3门33属70种,以硅藻门为主,7、8月份浮游植物种类数最多,约40种。在整个调查期间,浮游植物丰度在不同站位和月份之间差异较大,平均值为2.42×108个/m3,浮游植物优势种有北方娄氏藻、尖刺拟菱形藻、丹麦细柱藻、微小原甲藻等,优势种种类数9月最多,6月最少。浮游植物多样性指数为1.27~3.6,均匀度为0.31~0.73,6月份群落结构较单一,其他月份群落结构较稳定。     

中国近海区域古菌群落结构研究概述

古菌作为海洋微生物的重要组成部分,是海洋生态系统中物质与能量传递的重要贡献者,在全球生物地球化学循环中扮演着不可或缺的角色。虽然目前绝大多数的古菌尚未获得纯培养,但近年来分子生物学技术的发展使人们认识到古菌参与甚至主导了海洋生源要素循环的关键过程。本文介绍了目前研究海洋古菌多样性的主要手段,包括:荧光定量PCR、16S rRNA基因扩增子高通量测序、宏组学以及单细胞测序等技术,系统地阐述了中国近海不同区域(包括辽河口、黄河口、渤海、南北黄海、长江口、东海北部、珠江口以及南海区域)海水及沉积物中的古菌群落结构和多样性特征。研究表明我国近海地区的主要古菌类群为广古菌门(Euryarchaeota)、奇古菌门(Thaumarchaeota)、乌斯古菌门(Woesearchaeota)和深古菌门(Bathyarchaeota)等,其生物地理学分布主要受到环境因子和距离因素的影响,且在河口和近海海域呈现出完全不同的分布特点。在此基础上,对未来的研究方向进行展望,为全面深入了解我国边缘海生态系统中古菌群落及其参与的生物地球化学循环奠定了基础。