Characterization of depth-related microbial communities in lake sediment by denaturing gradient gel electrophoresis of amplified 16S rRNA fragments

The characterization of microbial communities of different depth sediment samples was examined by a culture-independent method and compared with physicochemical parameters, those are organic matter （OM）, total nitrogen （TN）, total phosphorus （TP）, pH and redox potential （Eh）. Total genomic DNA was extracted from samples derived from different depths. After they were amplified with the GC-341 f/907r primer sets of partial bacterial 16S rRNA genes, the products were separated by denaturing gradient gel electrophoresis （DGGE）. The profile of DGGE fingerprints of different depth sediment samples revealed that the community structure remained relatively stable along the entire 45 cm sediment core, however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident. The principle coordinates analysis suggested that the bacterial communities along the sediment core could be separated into two groups, which were located 0-20 cm and 21-45 cm, respectively. The sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belonged to Bacillus, Bacterium, Brevibacillus, Exiguobacterium, γ-Proteobacterium, Acinetobacter sp. and some uncultured or unidentified bacteria. The results indicated the existence of highly diverse bacterial community in the lake sediment core.

Analysis of microsatellite instability in stool DNA of patients with colorectal cancer using denaturing high performance liquid chromatography

AIM： To evaluate the usefulness of denaturing high performance liquid chromatography （DHPLC） for analyzing microsatellite instability （MSI） status in stool DNA of patients with colorectal cancer. METHODS： A total of 80 cancer tissues from patients with primary sporadic colorectal tumor （proximal cancer： 27, distal cancer： 53） and matched stool （which were employed for comparison with the tissues） were analyzed for MSI status in BAT 26. DNA samples extracted from stool were evaluated by nested polymerase chain reaction （PCR） and DHPLC for MSI analysis. RESULTS： Six cases （7.5%） of MSI were identified in BAT 26 from 80 cancer tissues. All the stool DNA samples from patients whose cancer tissue showed IVlSI also displayed MSI in BAT 26. CONCLUSION： As MSI is one of the established fecal DNA markers to screen colorectal cancer, we propose to use DHPLC for the IVlSI analysis in fecal DNA.

STUDY ON THE RELATIONSHIP BETWEEN MELTING POINT AND ACID DENATURING ACIDITY OF DNA BY ULTRAVIOLET SPECTROMETER

The relationship between meltillg point and acid denaturing acidity of DNA was studied by ultraviolet spectrometer. According to our investigation there exists a good linear relationship between melting point and acid denaturing acidity of DNA. The acid denaturing acidity. which could be obtained easily. may be used in the field of biochemistry as an alternative to melting point.

Variations in Laboratory-Scale Actinomycete Communities Exposed to Cadmium as Assessed by Denaturing Gradient Gel Electrophoresis Profiles

The actinomycete populations and functions in cadmium （Cd） contaminated soil were investigated by the cultivation- independent molecular methods. The genomic DNA was extracted and purified from soil adulterated with various con- centrations of Cd in the laboratory. The partial 16S rDNA genes were amplified by polymerase chain reaction （PCR） using specific primers bound to evolutionarily conserved regions within these actinomycete genes. The diversity in PCR- amplified products, as measured by denaturing gradient gel electrophoresis （EGGE）, was used as a genetic fingerprint of the population. Principle component analysis and Shannon-Weaver diversity index （H） analyses were used to analyze the DGGE results. Results showed that the two principal components accounted for only a low level of the total variance. The value H in contaminated soil was lower than that in the control at later stages of cultivation, whereas at earlier stages it was higher. Among the six sampling time points, the first, fifth and sixth weeks had the highest values of H. Significantly negative correlations between bioavallable Cd concentration and H values existed in the samples from weeks 2 （R = 0.929, P 〈 0.05） and 4 （R = 0.909, P 〈 0.05）. These results may shed light on the effect of Cd on the soil environment and the chemical behavior and toxicity of Cd to actinomycetes.

Signal Peptide and Denaturing Temperature are Critical Factors for Efficient Mammalian Expression and Immunoblotting of Cannabinoid Receptors

Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e.,≥95°C), forming a large molecular weight band when analyzed by immuno-blotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immuno-blot analysis. These findings provide very useful information for efficient mammalian expression and immuno-blotting of membrane receptors.

Monitoring of microbial community structure and succession in the biohydrogen production reactor by denaturing gradient gel electrophoresis(DGGE)

To study the structure of microbial communities in the biological hydrogen produc-tion reactor and determine the ecological function of hydrogen producing bacteria,anaerobic sludge was obtained from the continuous stirred tank reactor(CSTR)in different periods of time,and the diversity and dynamics of microbial communities were investigated by denaturing gra-dient gel electrophoresis(DGGE).The results of DGGE demonstrated that an obvious shift of microbial population happened from the beginning of star-up to the 28th day,and the ethanol type fermentation was established.After 28 days the structure of microbial community became stable,and the climax community was formed.Comparative analysis of 16S rDNA sequences from reamplifying and sequencing the prominent bands indicated that the dominant population belonged to low G+C Gram-positive bacteria(Clostridium sp.and Ethanologenbacterium sp.),β-proteobacteria(Acidovorax sp.),γ-proteobacteria(Kluyvera sp.),Bacteroides(uncultured bacte-rium SJA-168),and Spirochaetes(uncultured eubacterium E1-K13),respectively.The hydrogen production rate increased obviously with the increase of Ethanologenbacterium sp.,Clostridium sp.and uncultured Spirochaetes after 21 days,meanwhile the succession of ethanol type fer-mentation was formed.Throughout the succession the microbial diversity increased however it decreased after 21 days.Some types of Clostridium sp.Acidovorax sp.,Kluyvera sp.,and Bac-teroides were dominant populations during all periods of time.These special populations were essential for the construction of climax community.Hydrogen production efficiency was de-pendent on both hydrogen producing bacteria and other populations.It implied that the co-metabolism of microbial community played a great role of biohydrogen production in the reactors.

Denaturing gradient gel electrophoresis fingerprinting of soil bacteria in the vicinity of the Chinese Great Wall Station, King George Island, Antarctica

Bacterial diversity was investigated in soil samples collected from 13 sites around the Great Wall Station, Fildes Peninsula, King George Island, Antarctica, using denaturing gradient gel electrophoresis （DGGE） of 16S rRNA genes. The classes α-, β-, and γ-Proteobacteria, as well as the phylum Actinobacteria, were found to be the dominant bacteria in the soils around the Great Wall Station. Although the selected samples were not contaminated by oil, a relationship between soil parameters, microbial biodiversity, and human impact was still seen. Sample sites in human impacted areas showed lower bacterial biodiversity （average H′= 2.65） when compared to nonimpacted sites （average H′= 3.05）. There was no statistically significant correlation between soil bacterial diversity and total organic carbon （TOC）, total nitrogen, or total phosphorus contents of the soil. Canonical correlation analysis showed that TOC content was the most important factor determining bacterial community profiles among the measured soil parameters. In conclusion, microbial biodiversity and community characteristics within relatively small scales （1.5 km） were determined as a function of local environment parameters and anthropogenic impact.

Denatured proteins show new vitality:Green synthesis of germanium oxide hollow microspheres with versatile functions by denaturing proteins around bubbles

The hollow structure has long attracted great attention because of its excellent properties.However,this special structure is usually synthesized through some complex approaches.Herein,we discovered that denatured bovine serum albumin(BSA)can trigger unusual biomineralization for the simple,green and shape-controllable synthesis of germanium oxide(GeOx)hollow microsphere(HMS).At high temperature(60℃),BSA was denatured,and a compact BSA layer was formed around the H2 bubbles.The denatured BSA layer was stable and suitable for anchoring and growing GeOx.By simply changing the BSA concentration and temperature,various morphologies of GeOx could be obtained.Due to the denatured protein skeletons and microenvironment-regulated collapse,GeOx HMS showed great potential for intelligently responsive pesticide delivery in the insect gut,showing superiority over traditional delivery systems,which early release pesticides in the mouth and stomach.Inspired by its large specific surface area,excellent biocompatibility,modifiable functional groups,and high electrocatalytic activity,GeOx HMS was also applied to versatile sensors for H_(2)O_(2) assays at physiological pH and rapid coronavirus COVID-19 detection.This work not only provides some evidence for understanding proteins in depth but also paves a new avenue for the biomineralization-inspired synthesis of hollow structures with versatile functions.

Optimization of the Conditions of SRAP Molecular Marker Used in Analysis of Fagopyrum tataricum

[Objective] The aim was to investigate the optimal conditions of SRAP molecular marker used in the analysis on Fagopyrum tataricum（L.）Gaertn.[Method]SRAP-PCR amplification system on Fagopyrum tataricum was optimized by interactive orthogonal design L27（313）in 5 elements（Mg2＋,dNTP,Taq DNA polymerase,template DNA and primer）at 3 levels.And the non-denaturing and denaturing PAGE detection methods were compared.The comparative test of DYCZ-24F and DYCZ-20C electrophoresis operating systems was carried out.[Result]The effects of four single-factor（Mg2＋,dNTP,Taq DNA polymerase and primer）and two interactions（Mg2＋×dNTP,Mg2＋×Taq DNA polymerase）on tartary buckwheat SRAP-PCR were significant.An optimal reaction system was established containing 1.5 mmol/L Mg2＋,0.2 mmol/L dNTP,1.5 u Taq DNA polymerase,40 ng DNA,0.25 μmol/L primer and 2 μl 10×buffer.Seven samples of tartary buckwheat were amplified using this system,and electrophoresis results showed clear bands,high level of polymorphism and good reproducibility.The PCR products were tested by denaturing and non-denaturing PAGE,and the results showed that the non-denaturing PAGE,DYCZ-24F operating system was more suitable for SRAP analysis.[Conclusion]This study established a foundation for the construction of SRAP genetic map of tartary buckwheat.

不同性状窖泥的细菌群落结构与酸酯含量分析

为了解不同性状窖泥细菌群落结构及酸酯代谢的差异,分别选取新窖泥、趋老熟窖泥和老熟窖泥,对其细菌16S r DNA的V3区进行变性梯度凝胶电泳分析和同源性比较,并结合窖泥主要有机酸及有机酸酯含量进行了典型相关分析。结果表明,老熟窖泥的细菌多样性指数及均匀度指数高于新窖泥和趋老熟窖泥,得到的39个优势条带,进行细菌DNA测序可分为14类;Clostridium XIVa、Aminobacterium均只在老熟窖泥中检测到;新窖泥和趋老熟窖泥与Lactococcus、Lactobacillus、乳酸、乳酸乙酯含量正相关,老熟窖泥与Clostridiales、己酸、己酸乙酯和丁酸含量正相关。

多功能农药降解基因工程菌株m-CDS-1环境释放安全评价

【目的】为了评价多功能农药降解基因工程菌株m-CDS-1的环境释放中间试验水平的安全性。【方法】通过农药检测、平板计数、Most probable number-PCR(MPN-PCR)和Denaturing Gradient Gel Electrophoresis(DGGE)等方法在江苏大丰进行了工程菌株m-CDS-1田间降解农药效果、定殖动态和对土壤微生物群落结构影响的研究。【结果】投加1.01×107CFU/g干土的工程菌株m-CDS-1在30d时均能完全降解10.71mg/kg的甲基对硫磷和1.29mg/kg的呋喃丹。平板计数表明工程菌株m-CDS-1在土壤中快速下降;MPN-PCR检测结果显示,在4d,15d和30d时,0～10cm混合土壤中该工程菌株的数目分别为2.15±0.98×106CFU/g干土,3.70±4.66×104CFU/g干土和检测不出。工程菌株m-CDS-1的投加不会对土壤可培养三大微生物菌群数量产生显著影响;基于细菌16S rDNA V3区的DGGE分析结果表明,施加农药对细菌菌落结构有显著影响,4d,11d,30d时农药施用区与空白对照区的图谱相似性分别为17.16%,49.81%和75.01%,但到60d时的相似性为98.62%。工程菌株m-CDS-1释放在前期对细菌群落结构有一定影响,4d,11d和30d工程菌株释放区相对于空白的相似性分别为49.57%,38.30%和83.30%。在60d时,空白、施药和施菌小区的图谱相似性都在90%以上。【结论】工程菌株m-CDS-1不仅可同时高效降解甲基对硫磷和呋喃丹,仍保持了实验室内的原有特性,而且不会成为优势菌群长期在土壤环境中存在,也不会对土壤微生物群落结构造成长期影响。

Applications of a Single Molecule Theory of Protein Dynamics

A single molecule theory for protein dynamics has been developed since 2012. It consists of the concepts of conformational Gibbs free energy function (CGF) and single molecule thermodynamic hypothesis (STH) that claims that all stable conformations are (local or global) minimizers of CGF. These are enough to give a unified explanations and mechanisms to many aspects of protein dynamics such as protein folding;allostery;denaturation;and intrinsically disordered proteins. Formulas of CGF in water environment had been derived via quantum statistics. Applications of them to soluble proteins are: docking Gibbs free energy difference formula and a practical way to search better docking site;single molecule binding affinity;predicting and explaining why structures of a monomeric globular protein looks like a globule and is tightly packed with a hydrophobic core;a representation of the hydrophobic effect;and a wholistic view to structures of water soluble proteins.

PCR指纹图谱技术在酸奶质量监测中的应用

用PCR指纹图谱技术对市售某品牌酸奶进行了短期动态监测,对于收集的3个生产批次的9个酸奶样品,用ERIC(EnterobacterialRepetitiveIntergenicConsensus,肠杆菌基因间保守重复序列)-PCR和PCR-DGGE(DenaturingGradientGelElectrophoresis,变性梯度凝胶电泳)指纹图谱技术对其菌种组成及其稳定性进行评价。ER-IC-PCR指纹图谱聚类分析结果显示,同一生产批次内3个不同包装的样品ERIC-PCR指纹图谱相似性为90%左右,而不同生产批次样品之间ERIC-PCR指纹图谱相似性有所下降,为82%。该结果进一步用DGGE指纹图谱进行证实。DGGE分析结果表明,G2样品明显缺少一条主带,经割胶回收测序发现,这条主带代表的微生物是Lacto-bacillusdelbrueckiisubsp.Bulgaricus,说明该产品不同生产批次间的稳定性不是很好。DGGE图谱和测序结果还显示,除G2样品外,该酸奶样品的菌种组成与产品标签说明是一致的。

一种快速的酸奶质量跟踪监测检验新方法

分析酸奶中的菌种组成是酸奶质量控制的重要指标。本文用PCR-DGGE(denaturinggra-dientgelelectrophoresis,变性梯度凝胶电泳)指纹图谱技术对市售某品牌酸奶在1个月内进行了动态监测,共收集了6个生产批次的18个酸奶样品,通过建立16SrDNAV3区PCR-DGGE分析、DGGE图谱条带割胶回收测序等一套完整的检测评价方法,结合常规分离培养方法,对样品微生物组成及其稳定性进行跟踪监测。结果显示,所有供试样品在整个动态监测期内DGGE指纹图谱都出现2条带,显示其微生物组成比较稳定,割胶回收测序结果表明其微生物组成与厂家标注完全一致,分别为Lacto-bacillusdelbrueckiisubsp.bulgaricus(保加利亚乳杆菌)和Streptococcusthermophilus(嗜热链球菌)。分离计数结果表明前者比后者低3个数量级,二者的DGGE条带亮度也有显著差别。本研究用菌种组成及其稳定性两个指标对酸奶样品质量进行评价,建立了DGGE评价方法,结果表明,该技术可以作为酸奶质量控制和动态监测的快速简便的新方法。

PCR-DGGE Analysis of Bacterial Communities Structure in Babylonia areolata Culture Systems of The Subtidal Zone and The Pond Mulched Plastic Film and Sand in Bottom

To know the bacterial communities structure in Babylonia areolata culture systems and to research and optimize the management pattem of Babylonia areola-ta culture systems of the pond mulched plastic film and sand in bottom, the bacte- rial communities in Babylonia areolata culture systems of the sub-tidal zone and the pond mulched plastic film and sand in bottom were analyzed at molecular level by adopting the denaturing gradient gel electrophoresis （DGGE）. The results indicated that the dominant bacterial communities in Babylonia areolata culture systems of the sub-tidal zone and the pond mulched plastic film and sand in bottom, which were built on the basis of the seawater in East-island of Zhanjiang, included Proteobac- teda Chloroflexi, Cyanobacteria and Actinobacteria. The dominant bacterial groups in the above pond culture system were Garnmaproteobacteria, Alphaproteobacteria, Deltaprotecbacteda, Epsilonproteobacteda, Anaerolineae, Cyanobacteria and Acti- nobacteda. The dominant bacterial communities in the subtidal zone culture system were Gammaprotecbacteda, Alphaproteobacteria, Deltaproteobacteria, Anaerolineae and Cyanobacteda, and there were less Epsilonproteobacteria and Actinobacteria in the culture system. The higher diversity was detected in the above two culture sys- tems. The results of unweighted pair group method with arithmetic average （UPG- MA） showed that the bacterial communities of the sediment samples S1 and S2 in the above two culture systems were a cluster, the similarity of bacterial communities was 54.5%. The bacterial communities of seawater samples S3 and S4 in the above culture systems were in clusters, and the similarity of the bacterial communi- ties was 84.0%. The results showed that the microorganism ecological level in the Babylonia areolata culture systems of the pond mulched plastic film and sand in bottom could be similar to the sub-tidal zone culture systems through changing the pond seawater and monitoring the microbial population.

变性梯度凝胶电泳技术在微生物多样性研究中的应用

变性梯度凝胶电泳是不依赖于培养的、依据DNA分子的大小和所带电荷分析微生物多样性和动态变化的分子生物学技术,具有检测极限低、分析速度快及重复性好等优点。主要对变性梯度凝胶电泳原理、特点及其在微生物多样性应用方面进行综述。

Plankton community composition in the Three Gorges Reservoir Region revealed by PCR-DGGE and its relationships with environmental factors

To explore the relationships between community composition and the environment in a reservoir ecosystem, plankton communities from the Three Gorges Reservoir Region were studied by PCR-denaturing gradient gel electrophoresis fingerprinting. Bacterial and eukaryotic operational taxonomic units （OTUs）, generated by DGGE analysis of the PCR-amplified 16S and 18S rRNA genes, were used as surrogates for the dominant ＂biodiversity units＂. OTU composition among the sites was heterogeneous; 46.7% of the total bacterial OTUs （45） and 64.1% of the eukaryotic OTUs （39） were identified in less than half of the sampling sites. Unweighted pair group method with arithmetic averages （UPGMA） clustering of the OTUs suggested that the plankton communities in the Xiangxi Rive sites were not always significantly different from those from the Yangtze River sites, despite clear differences in their environmental characterizations. Canonical correspondence analysis （CCA） was applied to further investigate the relationships between OTU composition and the environmental factors. The first two CCA ordination axes suggested that the bacterial community composition was primarily correlated with the variables of NO3^--N, dissolved oxygen （DO）, and SiO3^2--Si, whereas, the eukaryotic community was mainly correlated with the concentrations of DO, PO4^3--P, and SiO3^2--Si.

Phylogenetic diversity and specificity of bacteria associated with Microcystis aeruginosa and other cyanobacteria

Interactions between bacteria and cyanobacteria have been suggested to have a potential to influence harmful algal bloom dynamics; however, little information on these interactions has been reported. In this study, the bacterial communities associated with five strains of Microcystis aeruginosa, three species of other Microcystis spp., and four representative species of non-Microcystis cyanobacteria were compared. Bacterial 16S rDNA fragments were amplified and separated by denaturing gradient gel electrophoresis （DGGE） followed by DNA sequence analysis. The similarities among bacterial communities associated with these cyanobacteria were compared to the digitized DGGE profiles using the cluster analyses. The bacterial community structure of all cyanobacterial cultures differed. Cluster analysis showed that the similarity values among M. aeruginosa cultures were higher than those of other cyanobacterial cultures. Sequence analysis of DGGE fragments indicated the presence of bacteria including, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacteroidetes and Actinobacteria in the cyanobacterial cultures. Members of the Sphingomonadales were the prevalent group among the Microcystis-associated bacteria. The results provided further evidence for species-specific associations between cyanoabcteria and heterotrophic bacteria, which are useful for understanding interactions between Microcystis and their associated bacteria.

Comparison of intestinal bacterial communities in grass carp,Ctenopharyngodon idellus,from two different habitats

The intestinal bacteria of vertebrates form a close relationship with their host.External and internal conditions of the host,including its habitat,affect the intestinal bacterial community.Similarly,the intestinal bacterial community can,in turn,influence the host,particularly with respect to disease resistance.We compared the intestinal bacterial communities of grass carp that were collected from farm-ponds or a lake.We conducted denaturing gradient gel electrophoresis of amplified 16S rRNA genes,from which 66 different operational taxonomic units were identified.Using both the unweighted pair-group method with arithmetic means clustering and principal component analysis ordination,we found that the intestinal bacterial communities from the two groups of pond fish were clustered together and inset into the clusters of wild fish,except for DF-7,and there was no significant correlation between genetic diversity of grass carp and their intestinal bacterial communities(Mantel one-tailed test,R=0.157,P=0.175).Cetobacterium appeared more frequently in the intestine of grass carp collected from pond.A more thorough understanding of the role played by intestinal microbiota on fish health would be of considerable benefit to the aquaculture industry.

Community analysis of ammonia and nitrite oxidizers in start-up of aerobic granular sludge reactor

A lab-scale sequencing batch reactor （SBR） was set-up and the aerobic granular sludge was successfully incubated using anaerobic granular sludge as seed sludge. Nitrogen was partially removed by simultaneous nitrification and denitrification （SND） via nitrite with free ammonia （FA） of about 10 mg/L. The denaturing gradient gel electrophoresis （DGGE） method was used to investigate community structure of α-Proteobacteria, β-Proteobacteria, ammonia oxidizing bacteria （AOB）, and Nitrospira populations during start-up. The population sizes of bacteria, AOB and Nitrospira were examined using real-time PCR method. The analysis of community structure and Shannon index showed that stable structure of AOB population was obtained at day 35, while the communities of α- Proteobacteria, β-Proteobacteria, and Nitrospira became stable after day 45. At stable stage, the average cell densities were 1.1× 10^12, 2.2×10^10 and 1.0×10^10 cells/L for bacteria, AOB and Nitrospira, respectively. The relationship between characteristics of nitrifying bacteria community and nitrogenous substrate utilization constant was discussed by calculating Pearson correlation. Certain correlation seemed to exist between population size, biodiversity, and degradation constant. And the influence of population size might be greater than that of biodiversity.