三种土壤微生物总DNA提取方法的比较

本文对3种常用的土壤微生物总DNA提取方法Martin法、高盐改进法及试剂盒法进行了比较,并通过DNA得率、纯度及16S rDNA V3可变区的PCR扩增结合DGGE法（denatumg gradient gel electrophoresis）,分别对3种方法进行评价。结果表明,3种方法提取的DNA均能满足土壤微生物多样性分析的要求。其中试剂盒方法操作简单,提取的DNA质量较高,但DNA得率较低且成本昂贵。Martin法和高盐改进法用时较长,DNA得率较高,纯度较低,但对后续PCR扩增和DGGE分析没有明显影响,且成本低廉。     

不同品种水稻土壤氨氧化细菌和氨氧化古菌群落结构组成

本研究通过提取土壤总DNA,利用特异引物进行PCR扩增和变性梯度凝胶电泳(DGGE),研究了不同品种水稻对稻田土壤氨氧化细菌和氨氧化古菌群落结构组成的影响.结果显示:稻田土壤具有丰富的氨氧化细菌和氨氧化古菌,且氨氧化古菌种类更多;不同品种水稻氨氧化细菌群落结构组成差异较大,其中以"天A/Km71"、"闽优1027"和"Km62/1027"3个品种相似性较高,且明显不同于其余3个品种:而氨氧化古菌群落结构组成在不同土层间表现出较大差异,其中以"天A/Km71"和"Km62/1027"的表土与根际土氨氧化古菌群落组成差异最大.研究表明不同水稻品种及土壤层次对氨氧化微生物群落结构组成具有一定影响,证明氨氧化微生物尤其是氨氧化古菌在稻田土壤生态系统中占有重要地位.     

玉米秸秆低温降解复合菌系的筛选

【目的】为加快北方高寒地区玉米秸秆降解速度,筛选低温条件下具有良好降解玉米秸秆的复合菌系。【方法】以富含纤维素的腐烂物为菌源,通过滤纸崩解初筛、酶活和秸秆降解率为指标复筛进行玉米秸秆降解菌系的筛选,并对其成分进行分析。【结果】从腐烂的树叶和高原锯末中筛选到两组玉米秸秆降解复合菌系1号和8号。1号和8号复合菌系在玉米秸秆培养基中,15℃培养15 d,玉米秸秆分解率分别达到30.21%和32.21%;1号复合菌系包含木霉和多种细菌,8号复合菌系含青霉和多种细菌;两组复合菌系优势细菌均为梭菌属和芽孢杆菌属菌种。【结论】筛选到的菌系在低温（15℃）实验室条件下能降解玉米秸秆,菌系主要组成是木霉、青霉、梭菌属（Clostridium sp.）、芽孢杆菌属（Bacillus sp.）和草螺菌属（Herbaspirillum sp.）的细菌。     

应用DGGE分析秸秆日粮对奶牛瘤胃细菌群落的影响

【目的】通过与苜蓿日粮比较,揭示秸秆日粮模式下瘤胃固液相细菌群落的独特结构。【方法】选择12头健康且体重相似的装有永久瘤胃瘘管的中国荷斯坦奶牛,分别饲喂以玉米秸秆（CS）和苜蓿、羊草、青贮玉米三者混合物（MF）为粗饲料组成的日粮,正饲期第13周连续3 d分12个时间点采集瘤胃固、液相内容物样品。提取微生物DNA后,利用PCR结合变性梯度凝胶电泳技术（DGGE）分析细菌群落结构,并进行聚类分析及差异条带的测序分析。【结果】与MF组相比,CS组奶牛瘤胃固、液相细菌DGGE图谱条带的数目和光密度均有一定差异,两处理组都各有一些稍显优势的条带,CS组优势条带要少于MF组。与MF组相比,CS组液相细菌的香浓多样性指数无差异（P＞0.05）,固相细菌香浓多样性指数显著降低（P＜0.01）;通过对差异条带进行测序,发现与MF组相比,CS组中瘤胃细菌Prevotella sp.、Acetivibrio ethanolgignens和Clostridium sp.减少,并且大量来源于Bacteroidetes、Firmicutes、Tenericutes和Proteobacteria等菌门的未培养细菌有所变化。【结论】秸秆日粮条件下奶牛瘤胃存在独特的细菌群落结构,细菌多样性较低。     

淹水培养过程中水稻土地杆菌科微生物群落结构变化特征

采用淹水非种植水稻土微环境模式系统,对水稻土进行1 h和1、5、10、20、30 d淹水培养,利用PCR-DGGE技术检测、多元统计分析淹水培养过程中地杆菌科微生物的群落结构和多样性变化规律及其影响因子。结果表明,淹水培养过程中地杆菌科微生物群落结构发生了明显的演替性变化：由r-对策优势种群演替至k-对策优势种群,且群落结构由不稳定向稳定演变（培养1 h和1 d处理间相似性最低,群落结构变化最大,20 d和30 d处理间相似性最高,群落结构变化最小）;该过程中,地杆菌科微生物物种丰富度指数和Shannon-Weiner指数在1 h处理中均为最小,5 d处理的最大;CCA相关性分析表明,淹水培养过程中Fe（Ⅱ）浓度与群落结构多样性指数呈正相关,证实该类微生物对于异化铁还原能力具有重要作用;测序结果表明,19个优势DGGE条带与来自水稻土中的未培养地杆菌科微生物亲缘关系相近。     

不同施肥处理对农田土壤微生物区系和功能的影响

本研究采用DGGE和Biolog两种方法, 研究了五种不同施肥处理对土壤微生物区系组成和功能的影响。采用UPGMA对DGGE试验结果进行聚类分析得出,小麦季中不施肥对照（T1）和常规氮磷钾肥（T2）聚为一类,相似性为41%,常规氮磷钾肥+秸秆还田（T4）和常规氮磷钾肥+秸秆还田+秸秆腐熟剂（T5）聚为一类,相似性为52%,而70%常规氮磷钾肥+有机肥（T3）单独聚为一类;玉米季也得到类似的结果,只是T1和T2的相似性为68%,T4和T5相似性达78%,而T3依然归为一类,这说明不同施肥处理间土壤微生物区系存在相似性。经过切胶测序及BLAST比对,发现大多为不可培养细菌,可培养细菌中多为芽孢杆菌属和类芽孢杆菌属。Biolog试验采用主成份分析得出,T1和T2土壤微生物功能类似,T4和T5土壤微生物也具有相似的功能,而T3则单独分为一类。其中,T5和T4的土壤细菌群落对底物碳源的代谢活性最强,T3处理次之,T2和T1最低。通过这两种不同试验方法的分析可以看出,不同施肥处理对土壤微生物区系和功能的影响存在关联性。     

石油污染土壤鞘氨醇单胞菌遗传多样性16S rDNA-PCR-DGGE分析

鞘氨醇单胞菌是降解多环芳烃(PAHs)的重要功能微生物。然而,采用属特异引物直接从PAHs污染土壤中检测鞘氨醇单胞菌进而研究鞘氨醇单胞菌在污染土壤中的多样性、种群结构及其与PAHs污染的关系鲜有报道。本研究自行设计鞘氨醇单胞菌属特异性引物,通过16S rDNA-PCR-DGGE技术对沈抚灌区石油污染土壤鞘氨醇单胞菌遗传多样性进行了研究。结果表明,自行设计的鞘氨醇单胞菌属引物732f-982r能够特异、灵敏的从环境样品中检出鞘氨醇单胞菌。当沈抚灌区原位污染土壤的PAHs浓度小于5 mg kg-1时,鞘氨醇单胞菌多样性随着PAHs浓度的增加而增加;而初始PAHs浓度高的实验室模拟实验土壤,其鞘氨醇单胞菌多样性较低。鞘氨醇单胞菌种群结构受污染土壤总石油烃(TPH)含量的影响,TPH含量接近的土壤,鞘氨醇单胞菌的群落结构较为相似。     

Analysis of bacterial communities on alkaline phosphatase genes in soil supplied with organic matter

Abstract

We studied the effects of the application of organic matter (OM) and chemical fertilizer (CF) on soil alkaline phosphatase (ALP) activity and ALP-harboring bacterial communities in the rhizosphere and bulk soil in an experimental lettuce field in Hokkaido, Japan. The ALP activity was higher in soils with OM than in soils with CF, and activity was higher in the rhizosphere for OM than in the bulk soil. Biomass P and available P in the soil were positively related to the ALP activity of the soil. As a result, the P concentration of lettuce was higher in OM soil than in CF soil. We analyzed the ALP-harboring bacterial communities using polymerase chain reaction based denaturing gradient gel electrophoresis (DGGE) on the ALP genes. Numerous ALP genes were detected in the DGGE profile, regardless of sampling time, fertilizer treatment or sampled soil area, which indicated a large diversity in ALP-harboring bacteria in the soil. Several ALP gene fragments were closely related to the ALP genes of Mesorhizobium loti and Pseudomonas fluorescens. The community structures of the ALP-harboring bacteria were assessed using principal component analysis of the DGGE profiles. Fertilizer treatment and sampled soil area significantly affected the community structures of ALP-harboring bacteria. As the DGGE bands contributing to the principal component were different from sampling time, it is suggested that the major bacteria harboring the ALP gene shifted. Furthermore, there was, in part, a significant correlation between ALP activity and the community structure of the ALP-harboring bacteria. These results raise the possibility that different ALP-harboring bacteria release different amounts and/or activity of ALP, and that the structure of ALP-harboring bacterial communities may play a major role in determining overall soil ALP activity.     

不同细菌饲喂细菌线虫对土壤细菌数量、活性以及群落结构的影响

The effects of bacterial-feeding nematodes on bacterial number, activity, and community composition were studied through a microcosm experiment using sterilized soil inoculated with soil bacteria （soil suspension） and with bacteria and three species of bacterial-feeding nematodes （ Cephalobus persegnis, Protorhabditis filiformis, and Caenorhabditis elegans）. Catalyzed reporter deposition-fluorescence in situ hybridization, CO2 evolution, and denaturing gradient gel electrophoresis （DGGE） of PCR ampli- fied 16S rRNA gene fragments were used to investigate bacterial numbers, antivity, and community composition, respectively. Our results showed that bacterial numbers and activity significantly increased in the presence of bacterial-feeding nematodes, which indicated that bacterial-feeding nematodes had a significant positive effect on soil bacteria. The different nematode species had different effects on bacterial numbers and activity. C. persegnis and P. filiformis, isolated from native soil, increased the bacterial number and activity more than C. elegans. The DGGE analysis results showed that dominant bacterial species significantly differed among the treatments, which suggested that bacterial-feeding nematode species modified the bacterial community composition in soil. Further gene sequence analysis results showed that the dominant bacterial species in this study were gram-negative bacteria. Given the completely same conditions except nematode species, the varied selective feeding behavior of different nematode species was the most likely reason for the altered bacterial community composition. Overall, the alteration of bacterial numbers, activity and community composition resulting from the bacterial-feeding nematodes may ult!mately affect soil ecological functioning and processes.     

分子生物学技术在产甲烷古菌研究中的应用

鉴于产甲烷古菌在环境和能源两大方面的特殊作用,对它的研究一直是微生物学研究的焦点之一。现代分子生物学技术的发展与应用,进一步推动了对产甲烷古菌的研究。本文将综述产甲烷古菌基因克隆研究进展以及几项有代表性的分子生物学技术在产甲烷古菌研究中的应用。