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1.
微囊藻群体总RNA提取方法的比较   总被引:1,自引:0,他引:1  
微囊藻群体富含多糖类物质是影响微囊藻RNA提取的关键因素.为了获得高质量的微囊藻群体总RNA,对比分析4种针对多糖含量较高的方法——方法 1 PGTX-bead法、方法 2 CTAB-bead法、方法 3 Fast RNAPro Blue Kit和方法 4RNeasy Mini Kit对微囊藻群体总RNA的提取效果.采用琼脂糖凝胶电泳检测微囊藻群体RNA的完整性,Nanodrop ND1000分光光度计检测RNA纯度及浓度,并采用q PCR检测DNA污染情况.结果表明,4种方法都能从微囊藻群体中提取获得RNA,并在去除DNA后都可以进行RT-PCR等后续实验.方法 1 PGTX-bead提取的RNA产量最高,纯度好,DNA污染小,成本低,适合从微囊藻群体中大量提取RNA;方法 2 CTAB-bead提取的RNA样品产量也较高,但DNA污染严重,适合需要同时提取DNA和RNA的样本;方法 3 Fast RNAPro Blue Kit和方法 4 RNeasy Mini Kit提取的RNA产量都较低,但方法 4操作简单,耗时短,所检测目的基因的相对表达量较高,更适合从少量的微囊藻群体中提取总RNA.  相似文献   
2.
In order to assess the diversity and spatial distribution of bacterial and archaeal communities in a CO2-rich and meromictic lake, samples were collected along the water column of Lake Monoun and analyzed using PCR-DGGE and quantitative analyses of the 16S rRNA gene. The retrieved sequences were affiliated to 6 bacterial phyla and two archaeal phyla which plausible environmental functions map with the physico-chemical parameters of the lake. Unclassified sequences were also detected. This suggests heterogeneity in community composition and existence of potential candidate divisions. For instance, amongst the bacterial sequences, 18.2% matched with methanotrophic bacteria of the order methylococcales and amongst the archaeal sequences, 16.6% matched with methanogenic species of the order methanomicrobiales. Hence, evidencing the existence of methane-related prokaryotes in the lake, a finding that would play a key role in our understanding of the methane puzzle of Lake Monoun. Other groups capable of a wide range of metal and nutrients transformations were also detected, as well as those of unknown functions. The layering of microbial communities also appeared to directly or indirectly depend on oxygen availability. DGGE and qPCR analyses both suggested a scarcity of archaea in the surface samples. Furthermore, qPCR revealed that bacteria were numerically more important than archaea in all the samples. The general distribution along the water column indicated that archaeal abundance increases with depth.  相似文献   
3.
Aureococcus anophagefferens, a small pelagophyte algae, has caused brown tide blooms in coastal waters of Qinhuangdao in recent years, presenting significant negative impacts on the shellfish mariculture industry. Under standard light microscopy, it is visually indistinguishable from other small algae in field samples due to its extremely small size. In this study, quantitative polymerase chain reaction(q PCR) based on 18 S r DNA sequences was developed and used to detect and enumerate A. anophagefferens. A linear regression(R2 = 0.91) was generated based on cycle thresholds value(Ct) versus known concentrations of A. anophagefferens. Twenty-two field samples collected in coastal waters of Qinhuangdao were subjected to DNA extraction and then analyzed using q PCR. Results showed that A. anophagefferens had a wide distribution in coastal waters along Qinhuangdao. Elevated A. anophagefferens abundance, category 3 brown tide blooms(200 000 cells/m L) occurred at Dongshan Beach and Tiger-stone Beach in August in 2013. In shellfish mariculture areas along coastal waters of Qinhuangdao, 4 stations had category 3 blooms, and 6 stations had category 2 blooms(35 000–200 000 cells/m L) in August and all stations had category 1 blooms(0 to ≤35 000 cells/m L) in October. Quantitative PCR allows for detection of A. anophagefferens cells at low levels in filed samples, which is essential to effective management and prediction of brown tide blooms.  相似文献   
4.
为了探索doublesex1(dsx1)基因是否在蚤状溞的生殖转换过程中发挥作用,利用蚤状溞滤食摄食的特点将体外合成的dsx1双链RNA(ds RNA)通过浸泡得方法分别摄入不同生殖状态的溞体中,实现dsx1基因的表达沉默。随后采用荧光定量PCR(q PCR)以及整体原位杂交分别检测不同生殖状态下蚤状溞在RNAi前后体内dsx1的表达水平变化,进而研究dsx1在蚤状溞生殖转换过程中的作用。结果表明:雄性溞、两性溞、孤雌溞在RNAi后均出现了dsx1 m RNA表达水平的显著下调,且下调量具有显著性差异(P0.05)其中在孤雌溞中下调68%,在三种生殖状态中最为显著,其次是雄性溞(56%)和两性溞(20%)。同时发现,干扰前能够在溞的第一触角、第一胸肢和复眼上检测到明显的信号位点,而在RNAi后,仅在溞体的第一触角上发现少量dsx1基因表达位点,且第一胸肢及复眼上未检测到相关信号。结合未干扰前dsx1在不同生殖状态溞的表达定量以及定位结果,我们推测dsx1可能在蚤状溞的生殖转化基因调控过程中扮演关键角色,并且在维持雄性蚤状溞性别特征的机制中发挥重要作用。  相似文献   
5.
Assessing the impacts of methane released from hydrate-bearing environments on global carbon cycling would require detailed insights into the distributions and capacities of microbial communities at different horizons of sediment column. In this study, we conducted geochemical, gene abundance and diversity analyses for a sediment core retrieved from a potential hydrate-bearing region off southwestern Taiwan. Geochemical profiles were characterized by a sulfate-to-methane transition with decreasing total organic carbon and nitrogen in sediments, and increasing dissolved inorganic carbon, ammonium and total sulfur in sediments. Bacterial and archaeal 16S rRNA and amoA gene abundances decreased with depth. In contrast, ANME-1 and -2 16S rRNA gene abundances increased significantly across the sulfate-to-methane transition and peaked at different horizons below this interface. A total of 124,379 bacterial and 130,351 archaeal reads were recovered through tag-pyrosequencing of 16S rRNA genes and categorized into 9014 bacterial and 6394 archaeal operational taxonomic units on the basis of 97% sequence similarity, respectively. Major bacterial phyla/divisions and archaeal groups (>5% of the total reads) detected included Chloroflexi, Planctomycetes, OP9, Deltaproteobacteria, BHI80-139, MBG-B, Halobacteria, MCG, Thermoplasmata, ANME-1 and MG-I. The abundance variations of most major OTUs (>0.5% of the total reads) were statistically correlated with those of geochemical parameters. These lines of evidence suggest that the populations represented by the major OTUs or detected by group-specific primers were compartmentalized into different horizons and involved directly or indirectly in the cycling of methane, sulfate, organic carbon and nitrogen. Overall, this study demonstrates that the deep sequencing coverage combined with the quantification of gene abundance and geochemical characterization would enable to uncover the detailed distributions and potential metabolic capabilities of specific groups from complexly structured microbial communities in methane-rich marine sediments.  相似文献   
6.
Cyanobacterial blooms in drinking water reservoirs constitute a health risk for humans and animals, especially in tropical zones where climate conditions may favour their prevalence throughout the year. The presence, viability, and toxicity of cyanobacteria were determined in the euphotic zone, in the water-sediment interface, and in the upper sediment fraction of the Riogrande II reservoir (Antioquia, Colombia), which provides drinking water for 1.4 million people. Cyanobacterial identification and quantification were performed in environmental samples of the euphotic zone and of the sediment-water interface. In addition, the effects of environmental parameters on occurrences of cyanobacteria were assessed and two microcystin synthetase (mcy) genes were extracted from water, interface, and sediment samples and quantified using qPCR. The viability of the cells in cultures from these different matrices was determined using chlorophyll-a measurements. Chlorophyll-a concentrations representing the biomass of cyanobacteria were higher at the interface than in the euphotic zone at all sampling sites. The potentially toxic genera Dolichospermum and Microcystis predominated in the euphotic zone and the interface, respectively. Moreover, the copy numbers of mcy genes was higher in cyanobacteria sampled at the interface and the upper sediment layers than in cyanobacteria sampled in the euphotic zone, which were correlated with the order Chroococcales. No impact of variation in environmental conditions on cyanobacterial abundance was observed. Viability of cyanobacterial cultures sampled from the interface and the upper sediment fraction showed the ability of cyanobacteria from several orders to proliferate under lab conditions. These results show that the surface sediment layer from Riogrande II serves as a sink for potentially toxic cyanobacteria which have the potential to recolonize and bloom in the water column.  相似文献   
7.
8.
超氧化物歧化酶(SOD)是植物细胞应对氧化胁迫的第一道,也是最重要的防线。本研究以坛紫菜转录组数据库中的unigene序列为基础,通过RACE扩增技术克隆得到了3条坛紫菜SOD基因的全长序列,分别命名为PhMSD,PhCSD1PhCSD2。序列分析结果表明,PhMSD序列全长973 bp,可编码包含224个氨基酸,等电点为5.75的多肽序列;PhCSD1序列全长1029 bp,可编码包含134个氨基酸,等电点为4.65的多肽序列;PhCSD2序列全长954 bp,可编码包含216个氨基酸,等电点为10.74的多肽序列。根据保守序列和系统进化树分析,可以将三条PhSODs分成两个亚型:其中PhMSD属于锰超氧化物歧化酶,PhCSD1PhCSD2属于铜锌超氧化物歧化酶。采用实时荧光定量PCR技术(qPCR)测定了3条PhSOD基因在坛紫菜不同世代,高温胁迫不同时间水平以及不同失水胁迫梯度下的表达水平变化,结果表明:3条PhSOD基因在叶状体世代中的表达水平均显著高于丝状体世代;在不同水平的高温和失水胁迫下,PhCSD1PhCSD2的表达水平显著上调,而PhMSD则表现出不同的表达模式,高温胁迫下被显著抑制,失水胁迫下没有发生显著变化。  相似文献   
9.
The need for quantitative molecular methods is growing in environmental, food, and medical fields but is hindered by low and variable DNA extraction and by co-extraction of PCR inhibitors. DNA extracts from Enterococcus faecium, seawater, and seawater spiked with E. faecium and Vibrio parahaemolyticus were tested by qPCR for target recovery and inhibition. Conventional and novel methods were tested, including Synchronous Coefficient of Drag Alteration (SCODA) and lysis and purification systems used on an automated genetic sensor (the Environmental Sample Processor, ESP). Variable qPCR target recovery and inhibition were measured, significantly affecting target quantification. An aggressive lysis method that utilized chemical, enzymatic, and mechanical disruption enhanced target recovery compared to commercial kit protocols. SCODA purification did not show marked improvement over commercial spin columns. Overall, data suggested a general need to improve sample preparation and to accurately assess and account for DNA recovery and inhibition in qPCR applications.  相似文献   
10.
近年来,有学者通过古DNA方法成功地重建了海洋沉积记录中古微生物群落变化,并借此反演了当地古环境-气候变化。然而,此种方法对于陆地湖泊沉积记录是否适用仍然有待研究。本文采用聚合酶链式反应(PCR, Polymerase Chain Reaction)、变性梯度凝胶电泳(DGGE,Denaturing Gradient Gel Electrophoresis)与实时荧光定量聚合酶链式反应(Q-PCR,Quantitative PCR)相结合的综合分析技术手段,系统研究青海湖5.8米(时间跨度为~18500年)沉积柱dinoflagellate真核藻类多样性和丰度变化。研究结果显示青海湖Dinoflagellate藻18S rRNA基因序列主要与海洋型藻类Woloszynskia halophila和Scrippsiella hangoei相近(~98%序列相似性)。定量Q-PCR结果显示,每克沉积物含有dinoflagellate藻类18S rRNA基因丰度范围为2.27×103~8.55×106拷贝。另外,Dinoflagellate藻类18S rRNA基因丰度与总有机碳含量成显著正相关(r = 0.408,p = 0.0001)。对比分析揭示较高的藻类丰度对应高总有机碳含量和较低的可溶解性盐电导率;反之,较低的藻类丰度对应较高的可溶解性盐电导率和较低的总有机碳含量。在青海湖区,总有机碳指示着季风降雨变化并间接地指示着外源输入和湖泊营养状况变化,然而可溶性盐电导率则指示着湖泊盐度变化。综上所诉,青海湖沉积柱Dinoflagellate藻类丰度可能响应着历史时期湖泊营养状况和盐度波动情况。  相似文献   
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