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.

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.

Gene Mutation Screening by Using Coupled In Vitro Transcription and Translation System

A new method for screening gene mutations by using a Coupled In Vitro Transcription and Translation System is reported in this paper. It includes the following steps: (1) The target DNA fragments with T7T1 sequence at its 5 prime end are amplified (T7T1: GGATCCTAATACGACTCTATAGGGAG ACCACCATG); (2) The RNA and peptide are synthesized and labeled from the PCR product in the coupled In Vitro Transcription-Translation System; (3) The produced peptides are analyzed by using SDS-PAGE and pH Gradient gel focusing electrophoresis. Four peptide products from 4 HB patients with nonsense mutation in Exon H of F IX gene show truncated protein bands with speeded migration in the autoradiography of the SDS-PAGE. Ten out of 11 HB patients with different missense mutations show abnormal patterns in the autoradiography of a pH 4-7 gradient gel focusing electrophoresis. Conclusion: The PCR and the Coupled In Vitro Transcription-Translation System/SDS-PAGE is a good method for proteins truncation test. The PCR and the Transcription-Translation System combined with pH gradient gel focusing electrophoresis is an efficient method for screening the abnormal protein products from DNA fragments with missense mutations.

STUDY ON THE RELATIONSHIP BETWEEN PARAOXONASE GENE POLYMORPHISM AND CORONARY ARTERIAL DISEASE IN NIDDM

Objective To ascertain the relationship between paraoxonase gene (PON) and the morbidity of coronary arterial disease (CAD) in Chinese non-insulin-dependent diabetes mellitus (NIDDM) patients. Methods The exons of PON gene were screened by polymerase chain reaction-denaturing gradient gel elec-trophoresis in 49 NIDDM patients complicated with CAD, 49 NIDDM and 101 healthy control cases of Chinese population. Results Gln-Arg191 polymorphism of the PON gene was detected in Chinese with the AIR allele frequency 0.39 and 0. 61 respectively. The genotype distribution (AA, AR and RB) of the PON gene polymor-phism was significantly different between NIDDM patients complicated with CAD and controls (NIDDM and healthy subjects). The former had a significantly higher B allele frequency (0. 79 vs 0. 62 and 0. 61, P < 0. 01). Conclusion Gln-Arg191 polymorphism of the PON gene is associated with CAD morbidity in Chinese NJDDM patients and B allele might be a risk factor.

COL1A1基因转录调控序列变异与单纯性马蹄内翻足的相关性

采用半定量RT-PCR方法检测20例单纯性马蹄内翻足患儿下肢肌肉及肌腱组织中COL1A1基因mRNA的表达,根据COL1A1基因转录调控区-1031bp～+30bp及第1内含子的序列,设计8对引物,PCR扩增后,采用变性梯度凝胶电泳技术筛查突变并测序。半定量RT-PCR结果表明,与正常对照组相比,单纯性马蹄内翻足患儿患侧肌肉及肌腱组织中COL1A1基因表达水平明显上调(t=12.680,P<0.05);经PCR-DGGE筛查并测序发现1名患者存在-161(C→T)的杂合变异,另1名患者存在+274(C→G)的纯合变异。二者均为新发现的变异。提示COL1A1基因转录调控序列变异可能是单纯性马蹄内翻足的致病原因之一。

红树林混合发酵产氢菌群中梭菌属Fe-氢酶基因(hydA)的克隆及序列分析

采用间歇产气试验方法对红树林淤泥中的混合菌群进行产氢菌群富集,并对混合产氢菌群发酵产氢过程中发酵液的pH值和氧化还原电位(ORP)的变化进行分析。此外,在产氢速率最高时段,发酵液中菌群经过常规的基因组提取,分别采用通用的梭菌属Fe-氢酶基因和16S rRNA基因引物进行PCR扩增,扩增产物经变性梯度凝胶电泳(DGGE)分析。结果发现产氢菌群中至少有6种,而Fe-氢酶基因只含有一种。将Fe-氢酶基因的扩增片段切胶纯化之后,经PCR重新扩增、纯化,克隆测序。NCBI序列比对结果表明,该片段序列与产气荚膜梭菌Fe-氢酶基因的序列相似性达99%。根据已知产气荚膜梭菌Fe-氢酶基因序列设计引物,经两轮PCR扩增获得Fe-氢酶基因全序列。混合菌群中Fe-氢酶基因GenBank数据库的登录号为EU590683。此外,采用Bioedit和Mega2软件构建了Fe-氢酶的NJ系统进化树,结果表明该Fe-氢酶基因与产气荚膜梭菌(Clostridium perfringens)聚为一类。推测的氨基酸序列与产气荚膜梭菌的相应序列相似性达97%—99%。PfamHMM结构域查找结果发现,此氢酶含有五个结构域,分别为1个2Fe-2S铁硫簇结合区域、2个4Fe-4S结合区域、Fe-氢酶大亚基和Fe-氢酶小亚基。

Microbial Community Dynamics During Biogas Slurry and Cow Manure Compost

This study evaluated the microbial community dynamics and maturation time of two compost systems： biogas slurry compost and cow manure compost, with the aim of evaluating the potential utility of a biogas slurry compost system. Denaturing gradient gel electrophoresis （DGGE）, gene clone library, temperature, C/N ratio, and the germination index were employed for the investigation, cow manure compost was used as the control. Results showed that the basic strip and dominant strips of the DGGE bands for biogas slurry compost were similar to those of cow manure compost, but the brightness of the respective strips for each system were different. Shannon-Weaver indices of the two compost systems differed, possessing only 22% similarity in the primary and maturity stages of the compost process. Using bacterial 16S rRNA gene clone library analysis, 88 bacterial clones were detected. Further, 18 and 13 operational taxonomic units （OTUs） were present in biogas slurry and cow manure compost, respectively. The 18 OTUs of the biogas slurry compost belonged to nine bacterial genera, of which the dominant strains were Bacillus sp. and Carnobacterium sp.; the 13 OTUs of the cow manure compost belonged to eight bacterial genera, of which the dominant strains were Psychrobacter sp., Pseudomonas sp., and Clostridium sp. Results demonstrated that the duration of the thermophilic phase （more than 50°C） for biogas slurry compost was 8 d less than the according duration for cow manure compost, and the maturation times for biogas slurry and cow manure compost were 45 and 60 d, respectively. It is an effective biogas slurry assimilate technology by application of biogas slurry as nitrogen additives in the manufacture of organic fertilizer.

变性梯度凝胶电泳装置及其在DNA突变检测中的初步应用

设计了一套适用于变性梯度凝胶电泳 (DGGE)的装置。该装置为带缓冲液循环系统、可精确控制温度的夹芯式双垂直电泳槽 ,其整个凝胶板内的控温精度和均匀度均在± 0 .5℃范围内。对其在DNA突变检测中的应用作了初步的探讨。通过PCR DGGE连用成功地分离了野生型p5 3基因和含点突变的p5 3基因 (第 141密码子由TGC突变为TAC)。

Cultivation-Independent Analysis of the Development of the Lactobacillus spp.Community in the Intestinal Tract of Newborn Piglets

Molecular diversity and development of the Lactobacillus community in the intestinal tract, as influenced by age and intestinal compartment, were studied in one litter of 12 conventionally raised piglets. Piglets were euthanized at each week （3 animals per time）. Digesta and tissue samples from stomach, duodenum, jejunum, ileum, caecum, colon, and rectum were collected and analysed by using 16S ribosomal RNA-based methods. DGGE （denaturing gradient gel electrophoresis） profiles revealed that the Lactobacillus communities throughout the GI tract from duodenum to rectum showed good stability at same age. This indicates that fecal Lactobacillus communities can effectively represent the intestinal community. Two dominant bands were found in tissue samples of the small intestine, suggesting that the lactobacilli can adhere to the small intestinal wall. The Lactobacillus communities in different GI tract compartments developed over time. A successional change of Lactobacillus communities was observed from birth, through creep feeding to one week after weaning, showing a trend from simple to complex and back to simple. Furthermore, a clone library of Lactobacillus spp. 16S rRNA gene sequences were generated from jejunal and colonic chymes. Six dominant DGGE bands generated from jejunal chymes were matched with sequences that show 94-98% similarity to the bands derived from L. reuteri, L. delbrueckii, and L. crispatus. Seven dominant DGGE bands generated from colon chymes were matched with sequences that show 88-99% similarity to those derived from L. reuteri, L. delbrueckii, L. amylovorus/L. sobrius, and L. acidophilus. Amplicons related to L. reuteri were found in all DGGE fingerprints from jejunal digesta of age of weeks 1, 3, and 4. Amplicons related to L. amylovorus/L. sobrius were present in all DGGE fingerprints from colonic digesta of age of week 1, 3, and 4. Amplicons related to L. delbrueckii were found before weaning, L. crispatus after creep feeding before weaning and L. acidophilus after weaning. This indicates that L. reuteri and L. amylovorus/L. sobrius probably belong to the permanent composition, while L. delbruckii, L. acidophilus, and L. crispatus probably belong to the temporal groups of Lactobacillus communities in the GI tract of piglets.

冷却肉中假单胞菌16S rRNA基因不同可变区片段DGGE研究

为揭示变性梯度凝胶电泳(DGGE)技术存在的一些技术缺陷,本实验以冷却肉中不同假单胞菌相关克隆为例,分别在V3和V6～V8两个不同可变区片段比较不同假单胞菌的16S rRNA基因差异。结果表明,以不同可变区为扩增目标进行研究,假单胞菌的DGGE图谱结果存在差异;基因片段序列的G+C含量与图谱中迁移位置并非绝对相关。DGGE技术的这一缺陷很大程度上是由细菌16S rRNA基因序列的自身特性所引起的。

变性梯度凝胶电泳分析PAH基因外显子3突变

采用ＰＣＲ、变性梯度凝胶电泳、限制性内切酶分析及ＤＮＡ直接序列分析等方法，对５８例南方地区中国人苯丙酮尿症（ＰＫＵ）的苯丙氨酸羟化酶（ＰＡＨ）基因外显子３进行了分析，发现９例ＰＫＵ患者外显子３存在突变，占ＰＫＵ患者的１５．５２％（９／５８），外显子３突变占ＰＡＨ基因突变的７．７６％（９／１１６），６例患者存在ＡｒｇｌｌｌＴｅｒ错义突变，该突变位点的基因内频率为５．１７％。结果提示中国南方和北方人群ＰＫＵ分子遗传学可能存在一定程度的差异。

中国汉族人群vWF基因G2805A多态性研究

为研究中国汉族人群vWF基因G2805A多态性特点,应用PCR-DGGE技术结合Nla Ⅳ酶切分析,研究了60名中国汉族人120条染色体vWF基因G2805A的等位基因发生频率及杂合情况,并对28例DGGE电泳行为异常中的4例进行了DNA序列分析。结果发现,在中国汉族人群中存在vWF基因G2805A多态性,其等位基因发生频率分别为0.73(G)和0.23(A),理论杂合率为0.39,实际杂合率为0.40。这表明vWF基因G2805A多态性在中国汉族人群中具有较高的理论杂合率,可用于血管性血友病的遗传咨询和产前诊断。

变性梯度凝胶电泳分析检测结直肠癌APC和p53基因突变

目的:探讨APC和p53基因突变在结直肠癌中的意义.方法:采用变性梯度凝胶电泳(DGGE),DNA测序法分析15例正常人和60例散发性结直肠癌标本的APC基因15外显子和p53基因第5,7外显子的基因突变.结果:在结直肠癌组检出14例APC和16例p53基因突变,测序证实其中13/14例APC发生在突变集中区域;9/16例p53基因突变位于第5外显子,7/16例p53基因突变位于第7外显子,2例同时检出了APC基因和p53基因突变.结论:DGGE是一种快速、简便、高效、灵敏的突变检测技术.同时也证明APC基因突变和p53基因突变均参与了结直肠癌的发生、发展过程.

变性梯度凝胶电泳检测轻型血友病甲的基因突变(Arg1689Cys)

血友病甲是一种常见的遗传性出血性疾病,由因子FⅧ缺乏引起。应用聚合酶链反应和变性梯度凝胶电泳技术,在一例轻型血友病甲患者的因子Ⅷ基因第14号外显子3’端发现碱基替换;直接测序表明密码子CGC突变为TGC,导致FⅧ蛋白Arg1689Cys,使凝血活化FⅧ发生障碍。该突变同时产生一个PstⅠ限制性酶切位点,该突变已证实与血友病相关。

螺旋粉虱成虫体内细菌群落多样性的PCR-DGGE和16S rRNA文库序列分析

为了解螺旋粉虱Aleurodicus dispersus体内细菌多样性和主要优势菌群结构,用PCR-DGGE和16S rRNA文库对采自于海南省番石榴上螺旋粉虱雌、雄成虫体内的细菌群落进行了分析。用PCR扩增体内细菌16S rRNA基因,构建雌、雄虫克隆文库;再用限制性片段长度多态性(restriction fragment length polymorphism,RFLP)方法从文库中筛选不同16S rRNA基因图谱,根据图谱对克隆子进行分型。从螺旋粉虱雌、雄两个样品中共获得10种分类操作单元(operational taxonomic unit,OTUs)。以16S rRNA基因为基础构建系统发育树,系统发育分析表明,螺旋粉虱雌、雄成虫体内优势菌群主要为发酵菌属Zymobacter,杀雄菌属Arsenophonus,泛菌属Pantoea和假单胞菌属Pseudomonas。Candidatus Portiera aleyrodidarum和Arsenophonus sp.可能为其体内共生菌群,在所有样品中均可稳定地检测到。这些微生物可能对螺旋粉虱生长发育、繁殖和性比调控起到重要的协同作用。

我国温带森林立地系统中土壤镉抗性基因cadA的多样性及丰度研究

以土壤中重金属镉的细菌抗性基因cadA的多样性与丰度,间接反映目前我国森林立地环境中重金属镉生物有效态的影响水平.利用变性梯度凝胶电泳及实时荧光定量PCR技术,调查寒温带、中温带及暖温带等温带森林立地系统中土壤重金属污染水平.研究结果表明我国寒温带森林立地样品与中温带森林立地土壤的cadA基因的多样性与丰度水平较为接近,多样性指数均接近2.3,而暖温带森林立地土壤的重金属污染水平较低.

变性梯度凝胶电泳方法在内源微生物驱油研究中的应用

应用基于聚合酶链式反应(PCR)的变性梯度凝胶电泳方法(DGGE)分析了在高温、高压和厌氧条件下富集培养的油藏内源微生物16S rDNA。用化学裂解法(SDS)直接提取了油藏微生物基因组DNA,并以此基因组DNA为模板,用特异性引物对16SrDNA的V8和V9可变区进行聚合酶链式反应扩增。对长约400bp(碱基对)的扩增产物进行DGGE分离后,得到了分离较好的电泳图谱。结果表明,DGGE方法可以分离不同油藏微生物16S rDNA的V8和V9可变区的DNA扩增片断。在内源微生物驱油研究中,DGGE方法是在分子水平上分析油藏微生物群落结构和监测微生物群落动态变化的有效工具。

73例中国人血友病甲基因突变的分析

我们用Ｓｏｕｔｈｅｒｎｂｌｏｔｔｉｎｇ、ＰＣＲ、变性梯度凝胶电泳（ＤＧＧＥ）和ＤＮＡ测序等方法对７３例血友病甲患者（经上海瑞金医院测定血浆ＦＶⅢ：Ｃ和ｖＷＦ：Ａｇ诊断，其中无亲缘关系患者６５例，按ＦＶⅢ：Ｃ水平分为轻、中、重三型。ＦＶⅢ：Ｃ＜２％为重型，共４７例；ＦＶⅢ：Ｃ２％～５％为中型，共９例；ＦＶⅢ：Ｃ５％～２５％为轻型，共１７例）进行ＦＶⅢ基因突变检测。共检出内含子２２倒位２３例，均为重型，约占重型的４９％，与国外报道相似。余下５０例（其中无亲缘关系者４５）用ＰＣＲ－ＤＧＧＥ分析所有外显子及其侧翼内含子序列，发现异常条带则进行ＤＮＡ测序。在１７例患者中检出突变１３种，其中无义突变５种，均为重型；错义突变６种，除１例外都是轻中型；小缺失２例，都是重型；其中，ＡＡ４６６Ｌｙｓ（ＡＡＧ）－Ｔｈｒ（ＡＣＧ），７１９Ｔｙｒ（ＴＡＣ）－Ｓｔｏｐ（ＴＡＧ），ＡＡ８２６Ａｓｐ（ＧＡＣ）－Ｇｌｕ（ＧＡＡ），３１２Ｉｌｅ（ＡＴＣ）－ｘｘＣ及ＡＡ１５５１－１５５２ｄｅｌ（ＡＧＡＡ）为新发现的突变。有亲缘关系的患者都有相同的基因突变，而在无亲缘关系患者未发现相同突变。基因突变与临床表现基本相符。

不同年限毛竹林土壤固氮菌群落结构和丰度的演变

应用变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)和荧光定量PCR(real time fluorescent quantitative PCR,q PCR)方法研究了不同年限毛竹林土壤固氮菌群落结构和丰度的变化。结果表明:土壤p H、有机质、有效磷、速效钾和铵态氮含量在马尾松林改造成毛竹林5 a后明显提高,而后逐渐降低,并趋于稳定;土壤固氮菌多样性和nif H基因丰度也呈现相似的趋势。条带测序分析表明,毛竹林土壤固氮菌均为不可培养的固氮菌,与慢生根瘤菌(Bradyrhizobium sp.)具有较高的相似度。冗余分析结果表明,不同栽培年限毛竹林地土壤固氮菌群落组成发生了明显变化,长期栽培毛竹林引起的土壤养分变化对土壤固氮菌多样性具有重要影响。

PCR-DGGE分析玉溪地区水果泡菜中细菌多样性

【目的】结合自然发酵生产泡菜的原理,分析云南传统水果泡菜的细菌群落组成和结构多样性,探索泡菜发酵过程中的微生物机制。【方法】采用PCR-DGGE和构建16S rRNA基因文库的方法,对从玉溪市场购买的萝卜、黄瓜、梨3种泡菜样品中细菌群落结构进行研究。【结果】玉溪地区自然发酵泡菜中的细菌种类比较丰富,由于不同水果泡菜的营养基质和附着在蔬菜表面及内在的细菌群落结构不同,导致细菌种类在不同泡水果中所占的相对丰度有较大差异。乳酸菌属(Lactobacillus sp.)在泡水果中并非优势种群,但丘状乳杆菌(Lactobacillus collinoides)在发酵中发挥着重要的作用。此外,在散称泡菜液中检测出部分致病菌,主要为肠球菌属(Enterococcus sp.)、葡萄球菌属(Staphylococcus sp.)和不动杆菌属(Acinetobacter sp.),推测市场上散称泡菜容易滋生致病菌,可能存在一定安全风险。【结论】PCR-DGGE技术能够高效、精确地检测到水果泡菜的细菌多样性。泡菜微生物的组成是指示泡菜安全性的一个重要指标。