MALBAC技术扩增微生物群落基因组的效率评估

环境样品的低生物量是微生物宏基因组学研究面临的首要挑战,通过基因组扩增技术来满足高通量测序对DNA样品量的需求是最常用的解决策略。MALBAC(Multiple Annealing and Looping Based Amplification Cycles)基因组扩增试剂盒最初为扩增和研究哺乳动物的单细胞基因组而研发。本文中,我们通过人工构建的微生物群落来检测该试剂盒在微生物宏基因组扩增方面的效率和应用可行性。结果表明,每个标准反应中,10 pg的DNA模板量足以满足MALBAC试剂盒对样品扩增的需要。每个标准反应DNA模板用量为10和100 pg时,所扩增DNA样品的基因组覆盖度与原始未扩增样品表现出高度的一致性,证明MALBAC试剂盒扩增效果的高度稳定性和一致性。常用的GenomePlex全基因组扩增试剂盒使我们可以在每个标准反应DNA模板量为100 pg的条件下扩增获得足够的DNA样品,但是结果表明该参照试剂盒无法有效的实现对群落中低丰度细菌菌株基因组的线性扩增。对于MALBAC试剂盒和参照试剂盒而言,在扩增高GC含量的微生物物种基因组DNA方面效率低下。我们的实验结果表明MALBAC试剂盒在高效扩增环境样品宏基因组DNA方面的可行性,但对该试剂盒在扩增环境样品中高GC含量微生物物种方面的适用性存在疑虑。     

A seasonal comparison of prokaryote numbers,biomass and heterotrophic productivity in waters of the KwaZulu-Natal Bight,South Africa

The KwaZulu-Natal Bight is a shallow indentation of the eastern seaboard of South Africa, characterised by a narrow (45 km wide) extension of the continental shelf, with a shelf break at about 100 m. It has a complex hydrography: the waters of the bight are derived from the fast-flowing, southward-trending Agulhas Current, which is fed mostly by the tropical and subtropical surface waters of the South-West Indian Ocean subgyre, which are generally oligotrophic in nature, notably depleted in reduced nitrogen and phosphate except at river mouths and during periodic upwelling of deeper nutrient-rich water. Despite this, the bight is believed to be relatively productive, and it is suggested that efficient nutrient recycling by prokaryotes may sustain primary productivity efficiently, even in the absence of new nutrient inputs. Here we have measured bacterial numbers, biomass and heterotrophic productivity during summer and winter in conjunction with phytoplankton standing stock and factors that influence it. Bacterial distribution closely matched phytoplankton distribution in surface waters, and was highest close to the coast. Bacterial standing stocks were similar to those of oligotrophic systems elsewhere (0.5–5.0 × 10⁵ cells ml^–1; 1 × 10^–8 to 1.25 × 10^–7 g C ml^–1) and increased in association with the development of phytoplankton blooms offshore and with inputs of allochthonous material by rivers at the coast. Heterotrophic productivity in summer was lowest in the far south and north of the bight (0.5 × 10^–10 g C ml^–1 h^–1) but higher close to the shore, over shallow banks, and in association with increased phytoplankton abundance over the midshelf (1.0–3.5 × 10^–9 g C ml^–1 h^–1). There were marked seasonal differences with lower bacterial standing stocks (5 × 10⁴ to 2 × 10⁵ cells ml^–1; 4–5 × 10^–9 to 1–2 × 10^–8 g C ml^–1) and very low bacterial productivity (4 × 10^–11 to 1 × 10^–10 g C ml^–1 h^–1) in winter, probably resulting from lowered rates of primary productivity and dissolved organic matter release as well as reduced riverine allochthonous inputs during the winter drought.     

A new tribe of Galerucinae leaf beetle (Insecta: Coleoptera: Chrysomelidae) from the Upper Cretaceous Taimyr amber

The new tribe Taimyralticini trib. nov., new genus Taimyraltica gen. nov. and new species Taimyraltica calcarata sp. nov. (Coleoptera: Chrysomelidae: Galerucinae) are described from Upper Cretaceous (Santonian) Taimyr amber of northern Siberia (Yantardakh locality). The new genus shares characters with genera of the tribe Alticini (small size, transverse antebasal groove on pronotum, regular punctate-striate elytra) and the Galerucini (narrow, non-swollen metafemora). A possible apomorphy of the new genus and tribe is a large and acute metatibial spur, erect and oriented transversely to the longitudinal axis of tibia. This character state is unknown in recent and fossil genera of Galerucinae. Early evolution and divergence of leaf beetles are discussed, and a Jurassic and Early Cretaceous subfamily radiation is proposed. Fossil galerucines are reviewed. The oldest Phyllotreta is recorded from the lower Miocene of Izarra (Spain). Our findings show an extreme rarity of Phytophaga in Taimyr amber from Yantardakh. Absence of Phytophaga was recorded earlier at Obeschayuschiy (Santonian-Campanian of the Magadan Region) and compared with their much larger share in Khetana (north of Khabarovsk Krai); we find nearly the same differences between Taimyr amber from Yantardakh and Canadian amber. In Obeschayuschiy this appears due to the Mesophytic character of the flora. It is assumed that the Santonian amber gymnosperm forest of the Khatanga Basin had the same character.     

微生物对地下水中萘的降解特性及动力学研究

针对我国东北某石油烃污染场地地下水中的萘污染,筛选出了Acinetobacter和Pseudomonas菌属的高效萘降解菌群。该菌群对萘的耐受性较高,且具有良好的乳化性,能够自动调节细胞表面疏水性和自聚集性。不同pH值、萘初始浓度、温度和菌接种量对萘降解效率的影响研究表明:最适菌群生长的pH范围为7.08.0;萘降解效率和速率在1.005.00 mg/L范围内与其初始浓度成正比;在1030 ℃温度范围内均表现出较高的萘降解效率。在此基础上,利用Guass、GuassAmp、LogNomal、Poisson和Pulse数学模型对萘的降解过程进行拟合,其降解速率更符合GuassAmp模型。通过将降解动力学模型中的常数与影响因素相关联,推导出了拟合度较高的多因素影响下的萘降解动力学方程。     

细菌反硝化法同时分析天然水中硝酸盐氮、氧同位素组成研究

细菌反硝化法是目前同时分析天然水中硝酸盐氮、氧同位素组成的最新方法。该方法包括反硝化菌的选取与培养,利用反硝化菌将硝酸根完全转化成N2O气体以及N2O气体的提取、纯化和同位素测定。该方法采用硝酸盐标准,对测试结果需进行试剂本底、同位素分馏、同位素交换校正。与传统方法相比,细菌反硝化法可同时分析低浓度微量水中硝酸盐的氮、氧同位素组成,且速度更快捷,结果更可靠。     

末次冰期低纬度西太平洋硅藻席沉积与生态特征

硅藻在全球碳循环中发挥着重要的作用。"树荫种"硅藻在次表层水体中的勃发成席,并迅速埋藏成为硅藻席沉积,使人们逐渐意识到次表层生产在整个生产力及输出生产中起着重要作用。介绍了首次在低纬度西太平洋区域发现的硅藻席沉积的分布特征,硅藻席发现站位呈带状分布,大致呈北西-南东向展布,大部分散布在17.5°～20°N之间。采集到硅藻席沉积物的站位其水深在碳酸钙补偿深度(CCD)以下4837～6150m的深水区,多分布在较平坦的海底,且受风力和陆源物质输入影响相对较强的区域。该区域硅藻席的形成可能是由于末次冰期时该海区有大洋锋面的形成所致。该区域的成席硅藻Ethmodiscus rex(Wallich)Hendey为典型的"树荫种"硅藻,可以通过自身调节浮力的作用,在水体相对稳定的贫氧大洋中生存并勃发成席。末次冰期低纬度西太平洋硅藻席沉积的发生,可能使该区成为CO2的汇。     

海水循环水系统中环境因子对大西洋鲑体内土腥味物质含量的影响

为获知不同时期封闭式海水循环水养殖系统中水质因子、浮游植物和细菌群落结构对大西洋鲑(Salmo salar)体内土腥味物质含量变化的影响,于2018—2019年4次采集封闭式海水循环水养殖系统的水样和大西洋鲑。测定水体的氨氮、亚硝氮、硝氮、活性磷酸盐等指标,分析水体浮游植物群落结构并通过16S高通量测序测定水体中细菌群落结构,通过微波蒸馏-固相微萃取-气相色谱-质谱联用(MD-SPME-GC-MS)方法,分析大西洋鲑背肌中土臭素(Geosmin,GSM)和二甲基异冰片(2-methylisoborneol,MIB)的含量。研究发现:测定水质指标中温度、溶解氧、盐度、pH、氨氮、亚硝氮、硝氮、活性磷酸盐、总氮、总磷、总悬浮物和溶解性总有机碳在不同时期差异显著。共鉴定出浮游植物7门、13目、26属,蓝藻门(Cyanophyta)、硅藻门(Bacillariophyta)和绿藻门(Chlorophyta)为优势门;不同时期细菌群落中的优势菌门均为变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes),但细菌群落结构存在差异,这主要是由水质差异引起的。在不同时期,鱼体MIB含量均高于GSM含量,MIB为鱼体中主要的土腥味物质,在7和10月MIB含量较高。鱼体土腥味物质含量受浮游植物和细菌群落影响,而水质因子可能通过影响浮游植物和细菌群落的结构,来影响养殖鱼体内土腥味物质的含量。     

海鞘血细胞培养和细菌污染控制

以海鞘(Ciona savignyi)为研究对象,考察了海鞘血细胞原代培养方法及培养中细菌污染的鉴定和控制.血细胞在培养1 h后贴壁,细胞基本以圆形细胞为主,变形细胞一般呈不规则形.变形细胞存活时间较短,而圆形细胞存活时间较长.对于培养过程中的细菌污染,通过细菌分离、培养和纯培养发现两类菌株检出率较高,均为革兰氏阴性菌.经PCR扩增16S rDNA基因序列片断,结果显示这两类菌株分别属于弧菌属(Vibrio)和施万氏菌属(Shewanella).药敏试验结果显示,弧菌属对氯霉素和环丙沙星等具有较强的敏感性;对施万氏菌属较敏感的药物依次为亚胺培南和氯霉素等.最后比较了几种抗生素组合控制血细胞培养中细菌污染的使用效果,其中氯霉素、亚胺培南加双抗的组合有较好的抑菌效果并对培养细胞的贴壁和生长没有影响.     

Biogeochemical Variation in Dimethylsulfide, Phytoplankton Pigments and Heterotrophic Bacterial Production in the Subarctic North Pacific during Summer

Dimethylsulfide (DMS), chlorophyll a (Chl-a), accessory pigments (fucoxanthin, peridinin and 19-hexanoyloxyfucoxanthin), and bacterial production (BP) were measured in the surface layer (0–100 m) of the subarctic North Pacific, including the Bering Sea, during summer (14 July–5 September, 1997). In surface sewater, the concentrations of DMS and Chl-a varied widely from 1.3 to 13.2 nM (5.1 ± 3.0 nM, mean ± S.D., n = 48) and from 0.1 to 2.4 µg L^–1 (0.6 ± 0.6 µg L^–1, n = 24), respectively. In the subarctic North Pacific, DMS to Chl-a ratios (DMS/Chl-a) were higher on the eastern side than the western side (p < 0.0001). Below the euphotic zone, DMS/Chl-a ratios were law and the correlation between DMS and Chl-a was relatively strong (r ² = 0.700, n = 27, p < 0.0001). In the euphotic zone, DMS/Chl-a ratios were higher and the correlation between DMS and Chl-a was weak (r ² = 0.128, n = 50, p = 0.01). The wide variation in DMS/Chl-a ratios would be at least partially explained by the geographic variation in the taxonomic composition of phytoplankton, because of the negative correlation between DMS/Chl-a and fucoxanthin-to-Chl-a ratios (Fuc/Chl-a) (r ² = 0.476, n = 26, p = 0.0001). Furthermore, there was a positive correlation between DMS and BP (r ² = 0.380, n = 19, p = 0.005). This suggests that BP did not represent DMS and dimethylsulfoniopropionate (DMSP) removal by bacterial consumption but rather DMSP degradation to DMS by bacterial enzyme.     

基于高通量测序的象山港水域细菌多样性的研究

象山港位于浙江省宁波市东南部沿海,属于半封闭式海湾,是以海水养殖区和海洋牧场示范区为主的重要多功能水域,近年来生态环境问题日益突出。为研究象山港海域在早春低水温环境中细菌的多样性,通过高通量测序方法对3月份的象山港水域进行检测。沿象山港南部海湾随机选取6个取样点(G04,G06,G08,G09,G011,G012),共检出836属和2 166种细菌,隶属33个门、91纲、186目和352科。其中优势细菌类群主要包括变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)和蓝细菌门(Cyanobacteria)。变形菌门在G04中占比最高(91.45%),拟杆菌门在G09中占比最高(18.05%);而放线菌门在G12中占比最高(7.95%)。在经度较高且离海岸较远的水域,细菌丰富度也越高;同时随着取样位点纬度的增加,γ-变形菌丰度逐步增高,其中在G04中最高(72.76%),在G12中最低(29.11%)。各取样点的细菌分离培养结果也表明弧菌属丰度较高,且主要集中在G04中。利用高通量测序方法揭示了象山港早春全水域的细菌结构组成框架图,可为象山港的水产养殖和可持续发展提供参考依据。