一种改进的cDNA文库固相构建法

本文介绍一种新的cDNA文库固相构建法。文库构建过程中,cDNA的合成和修饰全部在固相介质――磁珠上完成,简便、迅速、文库质量高,能满足不同目的的cDNA文库构建,是一种值得借鉴和应用的好方法。Abstract：A method for cDNA library construction was introduced.All enzymatic steps of library synthesis were performed on a solid support-magnetic beads.Therefore it combines fast speed and convenient with high quality library,making it ideally suited for most purposes.It is a good method and worth learning and utilization.     

Increasing ecological inference from high throughput sequencing of fungi in the environment through a tagging approach

High throughput sequencing methods are widely used in analyses of microbial diversity, but are generally applied to small numbers of samples, which precludes characterization of patterns of microbial diversity across space and time. We have designed a primer-tagging approach that allows pooling and subsequent sorting of numerous samples, which is directed to amplification of a region spanning the nuclear ribosomal internal transcribed spacers and partial large subunit from fungi in environmental samples. To test the method for phylogenetic biases, we constructed a controlled mixture of four taxa representing the Chytridiomycota, Zygomycota, Ascomycota and Basidiomycota. Following cloning and colony restriction fragment length polymorphism analysis, we found no significant difference in representation in 19 of the 23 tested primers. We also generated a clone library from two soil DNA extracts using two primers for each extract and compared 456 clone sequences. Community diversity statistics and contingency table tests applied to counts of operational taxonomic units revealed that the two DNA extracts differed significantly, while the pairs of tagged primers from each extract were indistinguishable. Similar results were obtained using UniFrac phylogenetic comparisons. Together, these results suggest that the pig-tagged primers can be used to increase ecological inference in high throughput sequencing projects on fungi.     

Organic matter turnover in a sagebrush steppe landscape

Laboratory incubations of¹⁵N-amended soils from a sagebrush steppe in south-central Wyoming indicate that nutrient turnover and availability have complex patterns across the landscape and between microsites. Total and available N and P and microbial C and N were highest in topographic depressions characterized by tall shrub communities. Net and gross N mineralization rates and respiration were also highest in these areas, but microbial efficiencies expressing growth relative to respiration cost were highest in soils of exposed ridgetop sites (prostrate shrub communities). Similar patterns occurred between shrub and intershrub soils, with greater nutrient availability under shrubs, but lower microbial efficiencies under shrubs than between. Surface soils had higher soil nutrient pools and N mineralization rates than subsurface soils, but N and C turnover and microbial efficiencies were lower in those surface soils. All soils decreased in respiration, mineralization, and immobilization rates during the 30-day incubation period, apparently approaching a steady-state substrate use. Soil microbial activity of the high organic matter accumulation areas was apparently more limited by labile substrate.     

Will coral reef sponges be winners in the Anthropocene?

Recent observations have shown that increases in climate change‐related coral mortality cause changes in shallow coral reef community structure through phase shifts to alternative taxa. As a result, sponges have emerged as a potential candidate taxon to become a “winner,” and therefore a numerically and functionally dominant member of many coral reef communities. But, in order for this to occur, there must be sufficient trophic resources to support larger populations of these active filter‐feeding organisms. Globally, climate change is causing an increase in sea surface temperatures (SSTs) and a decrease in salinity, which can lead to an intensification in the stratification of shallow nearshore waters (0–200 m), that affects both the mixed layer depth (MLD) and the strength and duration of internal waves. Specifically, climate change‐driven increases in SSTs for tropical waters are predicted to cause increased stratification, and more stabilized surface waters. This causes a shallowing of the MLD which prevents nutrients from reaching the euphotic zone, and is predicted to decrease net primary production (NPP) up to 20% by the end of the century. Lower NPP would subsequently affect multiple trophic levels, including shallow benthic filter‐feeding communities, as the coupling between water column productivity and the benthos weakens. We argue here that sponge populations may actually be constrained, rather than promoted, by climate change due to decreases in their primary trophic resources, caused by bottom‐up forcing, secondary to physical changes in the water column (i.e., stratification and changes in the MLD resulting in lower nutrients and NPP). As a result, we predict sponge‐dominated tropical reefs will be rare, or short‐lived, if they occur at all into the future in the Anthropocene.     

液相色谱-串联质谱分析化学合成多肽及其主要副产物(英文)

用与反相高效液相色谱偶联的串联质谱 (RPHPLC MS MS)分析一个固相化学合成的九肽H2 N Tyr Val Asn Val Asn Met Gly Leu Lys CONH2 及其 2个主要的副产物 .根据Fmoc化学 ,在Fmoc PAL PEG·PS树脂上从C端至N端手工操作逐步偶联合成九肽 .偶联完成后 ,用试剂R(90 %TFA ,5 %苯甲硫醚 ,3%二巯基乙烷 ,2 %苯甲醚 )室温 (2 0～ 2 5℃ )处理 2 5h ,进行多肽的去保护和切除树脂 .RP HPLC分析结果表明 ,合成粗品中含有 1个主要成分、2个次要成分及多个微量成分 .用RPHPLC MS MS分别对主要成分和 2个次要成分进行了鉴定 .结果证明 ,主要成分即为目标九肽 ;先于目标肽洗脱的副产物分子量比目标肽的分子量多 16 .MS MS证明 ,该副产物包括 2种九肽衍生物 :1种为其Tyr1氧化生成 ,另 1种为Met6氧化生成 ;后于目标肽洗脱的副产物为九肽的Tyr1残基增加 12所致 ,这种现象尚未见报道 .对副产物形成的可能机制进行了讨论     

Gas-Phase Cleavage and Dephosphorylation of Universal Linker-Bound Oligodeoxynucleotides

While base-specific support is commonly used for single-column oligodeoxynucleotide synthesis, the universal linker is critical for high-throughput synthesis of potentially thousands of samples in a single run. Here, we report conditions for cleavage and complete dephosphorylation of two commercial universal linkers, UnySupport and UnyLinker, processed in the gas phase (NH₃) using our custom device. First, we compared the average yield of T10mers over time (15, 30, 60, 120, and 240 minutes, 40 psi, 80°C and 90°C). For samples processed with water added prior to incubation, we discovered a substantial increase in yield compared to those left dry (up to 55%). This was also the case for samples subjected to increases in chamber pressure (10, 20, 30 and 40 psi, 120 minutes, 80°C and 90°C). Next, we compared the effects of increased temperature, pressure and incubation times on the rates of dephosphorylation. We found the optimum conditions to be either 10 psi, 120 minutes at 80°C or 60 minutes at 90°C; in both cases, water added to columns prior to incubation had a substantial effect on rate of reaction as well as overall yield compared with those left dry. Finally, performance between the two linkers was similar enough to conclude each fulfills the desired requirements for mainstream, high-throughput oligodeoxynucleotide cleavage/deprotection and dephosphorylation in the gas phase.     

Some like it toxic

Humans are notorious for disturbing terrestrial ecosystems worldwide, especially those that are in close proximity to urban areas. This disturbance has involved the accumulation of various types of chemical pollutants, of either agricultural or industrial origins, in both soil and water ecosystems. Pollutants have sometimes included essential plant nutrients, such as phosphate and nitrate, which have piled up throughout the years in many ecosystems as a consequence of aggressive agricultural practices, and a number of toxic or trace metals, e.g. iron, nickel or zinc that are important at low levels for the fitness of living organisms, but otherwise toxic at high concentrations ( Ker & Charest 2010 ; Audet & Charest 2008 ). In order to reduce the load of toxic elements, scientists have used the natural capacity of several plant species to sequestrate them from the soil and, ultimately, render them harmless. This process, called phytoremediation, is rather slow, as most plants take years to build up their biomass but has been shown to be ‘boostable’ under experimental conditions in the presence of a particular group of plant symbionts in the soil – the arbuscular mycorrhizal fungi (AMF) ( Gohre & Paszkowski 2006 ). These latter organisms are now widely recognized as being very beneficial for purposes of phytoremediation, but their biodiversity in the most disturbed ecosystems is still virtually unknown. Are these fungi really abundant in heavily polluted soils, or are their communities shrunken down like those of other microorganisms in the presence of heavy pollution? In this issue of Molecular Ecology, the study by Hassan et al. (2011) provides answers to these specific questions by determining the extent of AMF biodiversity across several urbanized areas in the City of Montréal.     

贮藏保鲜中SO2伤害对红提葡萄香气组分的影响

采用固相微萃取技术及气相-质谱联用技术对不同SO2漂白伤害程度的红提果实进行香气成分的提取与鉴定,以探讨SO2漂白伤害对贮藏葡萄果实中香气组分的影响.结果表明,果实经漂白伤害后其具有芳香气味的气体所占比重明显下降,主要成分醇类化合物下降最多,而具有刺激难闻气味的酸类化合物所占比重明显上升;此外,4-萜烯醇、正己醇、月桂醇、松油醇、芳樟醇、橙花醇、乙酸萜烯酯及青叶醛等具有葡萄特征性香味的化合物在香气中所占比例明显下降或消失,而异辛醇、1-辛烯-3-醇、壬酸、辛酸、正癸酸、乙酸、己酸、2,6-二叔丁基对甲酚及2-丁基-5-(2-甲基丙基)-噻吩等具有臭味或刺激性气味的化合物出现或所占比重上升.说明,SO2伤害对红提果实香气的影响主要表现为香味化合物所占比重的减少或消失及异味化合物的产生.