现代分子生物学技术在冰川微生物生态研究中的应用

研究冰川中微生物的多样性对于揭示环境气候变迁,研究生物进化,开发微生物资源具有重要意义,现代分子生物学的发展为研究冰川微生物的多样性提供了行之有效的方法.简要综述了16S rDNA文库构建、变性梯度凝胶电泳、限制性片段长度多态性和荧光原位杂交等技术的原理及在冰川微生物生态研究中的应用现状.     

两种粪便保存方法对肠道菌群结构研究的影响

目的比较2种粪便保存方法（室温法和Invitek公司的粪便稳定剂保存法）对菌群结构研究的影响。方法应用PCR-变性梯度凝胶电泳技术（PCR-DGGE）方法,对用2种方法保存的3位志愿者粪便样品进行菌群结构的分析。结果室温法保存粪便样品24h后,S1个体菌群结构与原始样品的菌群结构相似度为83％,S2和S3的菌群结构与其原始样品的相似度仅为77％。而使用粪便稳定剂保存1d,期间各时间点样品菌群结构与原始样品相比变化较小,相似度在80％-90％。结论粪便稳定剂具有一定的稳定样品菌群结构的作用,在新鲜粪佰样品不能寺刻讲行深冻的情况下,使用粪便稳定剂是一种较好的样品保存方法。     

Effects of sampling location and time,and host animal on assessment of bacterial diversity and fermentation parameters in the bovine rumen

Aims: To investigate, using culture‐independent methods, whether the ruminal bacterial structure, population and fermentation parameters differed between sampling locations and time. Methods and Results: The detectable bacteria and fermentation parameters in the digesta from five locations in the rumen of three cows at three time points were analysed. The PCR‐denaturing gradient gel electrophoresis (PCR‐DGGE) profiles were similar among digesta samples from five locations (95·4%) and three time points (93·4%) within cows; however, a lower similarity was observed for samples collected from different host animals (85·5%). Rumen pH and concentration of volatile fatty acids (VFA) were affected by time points of sampling relative to feeding. Conclusions: The detectable bacterial structure in the rumen is highly conserved among different locations and over time, while the quantity of individual bacterial species may change diurnally in response to the feeding. Significance and impact of the study: This study supplies the fundamental understanding of the microbial ecology in the rumen, which is essential for manipulation of ruminal microflora and subsequent improvement in animal production.     

Bacterial and archaeal assemblages in sediments of a large shallow freshwater lake, Lake Taihu, as revealed by denaturing gradient gel electrophoresis

Aims:  To explore the association of microbial community structure with the development of eutrophication in a large shallow freshwater lake, Lake Taihu.
Methods and Results:  The bacterial and archaeal assemblages in sediments of different lake areas were analysed using denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA fragments. The bacterial DGGE profiles showed that eutrophied sites, grass-bottom areas and relatively clean sites with a eutrophic (albeit dredged) site are three respective clusters. Fifty-one dominant bacterial DGGE bands were detected and 92 corresponding clones were sequenced, most of which were affiliated with bacterial phylotypes commonly found in freshwater ecosystems. Actinobacteria were detected in the centre of the lake and not at eutrophied sites whereas the opposite was found with respect to Verrucomicrobiales . Twenty-five dominant archaeal DGGE bands were detected and 31 corresponding clones were sequenced, most of which were affiliated with freshwater archaeal phylotypes.
Conclusions:  The bacterial community structures in the sediments of different areas with similar water quality and situation tend to be similar in Taihu Lake.
Significance and Impact of the Study:  This study may expand our knowledge on the relationship between the overall microbial assemblages and the development of eutrophication in the shallow freshwater lake.     

Molecular typing and identification of Bdellovibrio-and-like organisms isolated from seawater shrimp ponds and adjacent coastal waters

Aims:  To apply and compare two PCR-based methods for typing saltwater Bdellovibrio- and-like organisms (BALOs) and to understand ecological and phylogenetic aspects of the BALOs isolated from shrimp mariculture systems.
Methods and Results:  Using double-layer agar technique, the numbers of culturable BALOs that lyse Vibrio alginolyticus were found to be 10–10³ PFU ml⁻¹ in the surface water samples. A total of 130 BALOs isolates were differentiated into five phylotypes by denaturing gradient gel electrophoresis targeting the 16S rDNA V3 region and four phylotypes by amplified rDNA restriction analysis of the Bacteriovoracaceae -specific 16S rDNA fragment respectively. Phylogenetic analysis of representative isolates showed that all of them were identified as Bacteriovorax spp., but affiliated with four different clusters in the family Bacteriovoracaceae.
Conclusions:  The two PCR-based methods both can be chosen to rapidly type the saltwater BALOs at cluster level. And the relatively large numbers of BALOs with various phylotypes recovered from the same habitats suggested that these predators might play important ecological role in shrimp mariculture environments.
Significance and Impact of the Study:  We proposed two effective methods to distinguish rapidly large numbers of BALOs isolates and our results would be helpful to understand the diversity and function of BALOs in mariculture environments.     

DGGE/TGGE技术在土壤微生物分子生态学研究中的应用

传统的微生物生态学研究方法只限于环境样品中极少部分(0.1%￣1%)可培养的微生物类群,极大程度地限制了对土壤微生物群落结构的研究。综述了以16S rDNA为主要研究对象的DGGE/TGGE(Denaturing gradientgel electrophoresis,DGGE/Temperature gradient gel electrophoresis,TGGE)技术原理,以其为主要手段结合PCR扩增、克隆建库、序列测定以及种系分析对土壤微生物的群落结构和多样性研究的最新动态。DGGE/TGGE技术极大地推动了土壤微生物分子生态学的发展,同时也为实际问题的诊断、作物生长跟踪监测等提供了技术支撑,在土壤微生物分子生态学研究和生产实践中起着越来越重要的作用。     

Denaturing gradient gel electrophoresis analysis with different primers of subgingival bacterial communities under mechanical debridement

DGGE of 16S rDNA is one of the most frequently used methods to study microbial communities. In this study, the DGGE profiles of different 16S rDNA regions of the periodontal pathogens Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella nigrescens were investigated. The results suggested that V3-V5 and V6-V8 fragments may be suitable for community analysis of subgingival bacteria. Further analysis of subgingival samples with V3-V5 and V6-V8 regions as target fragments suggested that, in chronic periodontitis, re-colonization by periodontal bacteria with a population very similar to the baseline may occur by 6 weeks after mechanical debridement.     

基于PCR-DGGE技术的红树林区微生物群落结构

【目的】为了解红树林沉积物中细菌的群落结构特征。【方法】应用PCR-DGGE技术对福建浮宫红树林的16个采样站位样品细菌的群落结构进行了研究。根据DGGE指纹图谱,对它们的遗传多样性进行了分析。【结果】各站位样品细菌多样性指数(H)、丰度(S)和均匀度(EH)均有所不同,这些差异与它们所处站位的不同有关,红树林区细菌多样性高于非红树林区细菌多样性。对不同站位细菌群落相似性分析,它们的相似性系数也存在一定的规律,同一断面的细菌群落结构相近性较高。对DGGE的优势条带序列分析,同源性最高的微生物分别属于变形菌门(Proteobacteria)、酸菌门(Acidobacteria)和绿菌门(Chlorobi),它们均为未培养微生物,分别来自于河口海岸沉积物。【结论】应用PCR-DGGE技术更能客观地反映红树林沉积物中真实的细菌群落结构信息。另外,研究也表明红树林区微生物多样性丰富,在红树林区研究开发未知微生物资源具有巨大的潜力。     

Rumen chemical and bacterial changes during stepwise adaptation to a high-concentrate diet in goats

The correlation between rumen chemical and bacterial changes was investigated during a four periodical stepwise adaptation to a high-concentrate diet (concentrate level at 0%, 30%, 50% and 70% for diet I to IV, respectively) in goats. The results showed that ruminal pH decreased from 6.7 to 5.5 after switching from diet I to II, and was maintained at about 5.5 on diet III. Denaturing gradient gel electrophoresis results showed that the rumen bacterial community was relatively stable during the initial three feeding periods, except for the appearance of three bands when diet changed from I to II, suggesting that an appropriate concentrate level can promote the proliferation of some bacteria. After 12 days of feeding diet III, total volatile fatty acid (VFA) concentration and butyrate proportion decreased. At days 2 and 3 of feeding diet IV, ruminal pH declined sharply to 5.3 and 4.7, respectively, and total VFA concentration decreased further while lactic acid concentration increased markedly, suggesting a relation between lactic acid accumulation and ruminal pH decline. At the same time, many bacteria disappeared, including most fibrolytic-related bacteria while Streptococcus bovis and Prevotella-like species dominated. Interestingly, Succinivibrio dextrinosolvens-like species maintained throughout the experiment, suggesting its tolerance to low pH. In conclusion, rumen bacterial community was relatively stable feeding 0% to 50% concentrate diets, and it was observed that appropriate concentrate levels in the diet could increase the diversity of rumen bacteria. However, concentrate-rich diets caused lactic acid accumulation and low ruminal pH that caused the disappearance of most fibrolytic-related bacteria sensitive to low pH while S. bovis and genus Prevotella persisted.     

非培养方法解析北京地区甘薯叶际细菌的群落结构

利用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术和磷脂脂肪酸(PLFA)分析方法,比较了北京通州、顺义、昌平、延庆地区甘薯叶际细菌的多样性和生物量,并调查了通州地区甘薯叶际细菌群落在不同生长季节的变化情况。PLFA分析结果发现所有检测样品中,革兰氏阳性细菌生物量均高于革兰氏阴性细菌生物量。PCR-DGGE方法与PLFA方法聚类分析结果较一致,甘薯叶际细菌群落结构受到时空因素、甘薯生理特性等的影响,不同地点、不同生长季节甘薯叶际细菌群落结构有较大差异,DGGE条带测序分析表明,Pseudomonas sp.在不同地点的甘薯叶际均为保守菌群,Bacillus sp.,Acinetobacter sp.,