不同年龄段川金丝猴肠道菌群结构差异分析

本试验旨在分析幼年、成年、老年川金丝猴肠道菌群结构以及随年龄变化的趋势。将提取的幼年、成年、老年川金丝猴粪样总DNA进行聚合酶链反应-变性梯度凝胶电泳(PCRDGGE),对结果进行UPGMA聚类分析、主成分分析、生物多样性比较。用荧光定量PCR技术检测肠道菌群中真细菌、乳酸杆菌属(Lactobacillus spp.)、肠杆菌科(Enterobacteriaceae)、双歧杆菌属(Bifidobacterium spp.)的数量。结果显示:无论是UPGMA聚类分析,还是主成分分析以及生物多样性比较均表明,不同年龄段川金丝猴肠道菌群多样性存在明显差异;指纹图谱中成年与幼年川金丝猴的优势条带较多,老年川金丝猴的优势条带则较少。各年龄段川金丝猴肠道菌群中真细菌的数量都为同一数量级(1011);幼年川金丝猴肠道菌群中Enterobacteriaceae的数量显著少于成年和老年川金丝猴(P0.05);成年川金丝猴肠道菌群中Lactobacillus spp.的数量极显著多于幼年和老年川金丝猴(P0.01);老年川金丝猴肠道菌群中Bifidobacterium spp.的数量极显著少于成年和幼年(P0.01)。由此得出,川金丝猴肠道菌群随年龄增长,多样性呈幼年到成年增加、成年到老年降低的趋势,老年时期优势菌群显著减少;此外,川金丝猴肠道各菌群的数量在不同年龄段有较大差异。     

Bacterial communities associated with nodal roots of rice plants along with the growth stages: Estimation by PCR-DGGE and sequence analyses

Bacterial communities in rice roots that developed from different nodes and at different growth stages were compared by using polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA. Rice root samples were collected at three stages, namely tillering (July 2), maximum tillering (July 21), and ripening (September 12). The bacterial diversity in rice roots was found to increase along with the growth stages of the rice plants as well as the root age from the numbers of DGGE bands. The community structure of the bacteria was also found to change with the growth stages and root age from cluster analysis. Sequence analysis of the DGGE bands indicated that the dominant bacteria associated with rice roots were Gram-negative bacteria, especially β-Proteobacteria irrespective of the growth stages and root age. DGGE bands related to Janthinobacterium agaricidamnosum W1r3T and Clostridium sp. FCB90-3 were ubiquitous in many roots irrespective to the sampling date. Principal component analysis enabled to characterize the DGGE bands related to nitrogen-fixing Azoarcus spp., and Azovibrio sp. BS20-3 in the samples collected on July 2 and on July 21, and the myxobacteria collected on September 12, respectively, as representative bacteria in the bacterial communities. The habitat around older rice roots at every sampling date was more reductive than that around younger rice roots, and the DGGE bands related to Spirochaeta spp. were specific in older roots at every sampling date. Some specific bacteria that were most closely related to the DGGE bands were found from principal component analysis to characterize young and old. roots at each growth stage as follows: aerobes Flavobacterium sp. 90 clone 2 and Janthinobacterium agaricidamnosus W1r3T in young roots and facultative anaerobes Dechloromonas sp. MissR and Anaeromyxobacter dehalogenans 2CP-3 in old nodal roots on July 2, strict anaerobe Geobacter pelophilus Dfr2 and aerobes Nitrosospira sp. Nsp17 and uncultured Nitrospira sp. clone 4-1 in old roots on July 21, and different Clostridium spp. in both young and old roots and Desulfovibrio magneticus RS-1 in old roots on September 12, respectively. A larger number of the closest relatives of anaerobic bacteria grew at the late stage than at the early stages, and in old roots than in younger roots. Thus, the environment of paddy roots was remarkably heterogeneous as a bacterial habitat, where not only the whole root system but also a root may create oxic and anoxic environments.     

稻田厌氧氨氧化菌群落结构对氮肥的响应

为探明稻田厌氧氨氧化菌多样性及其对氮肥用量的响应状况,利用厌氧氨氧化菌16S rRNA基因特异引物对定位试验稻田土壤DNA进行PCR-DGGE(聚合酶链反应变性梯度凝胶电泳)并结合DNA克隆测序,研究了氮肥供应量对厌氧氨氧化菌群落结构的影响。DGGE图谱及依据其条带位置和亮度数值计算的多样性指数均显示:高氮处理[N3:225 kg(N).hm 2]的厌氧氨氧化菌群落结构多样性在表层或根层土壤中均显著(P<0.05)高于中、低氮[N2:150 kg(N).hm 2;N1:75 kg(N).hm 2]处理和不施肥对照(CK);同时,高氮处理下表层土壤厌氧氨氧化菌群落多样性指数显著高于根层土壤(P<0.05)。冗余分析(RDA)结果表明,表层土壤中厌氧氨氧化菌群落结构组成与不同氮肥水平处理存在显著相关性(P=0.006)。此外,本试验获得厌氧氨氧化菌DGGE条带DNA序列18条,登录GenBank并获得登录号。研究表明稻田厌氧氨氧化菌群落结构对高氮水平具有较强的响应,尤其是在表层土壤中。     

Suppression of spinach wilt disease by biological soil disinfestation incorporated with Brassica juncea plants in association with changes in soil bacterial communities

Biological soil disinfestation (BSD) is a method of controlling soil-borne pests and diseases through anaerobic decomposition of plant biomass incorporated in field soil with temporary irrigation and covering with sheets. In this study, effects of BSD on suppression of spinach wilt disease were investigated in two different field experiments using mainly Brassica juncea plants as plant biomass. Soil bacterial community compositions were analyzed with clone library analysis based on 16S rRNA gene sequences to determine the relationship between the bacterial composition in the treated soil and suppression of the disease. For the BSD-treated soils, oxidation–reduction potential dropped, and acetate was usually detected at high concentrations. Although the control treatment (irrigation and polythene covering without biomass) decreased the wilt disease incidence in spinach plants cultivated in the treated plot as compared with those for the non-treated plot, BSD-treatments suppressed the disease more effectively. The clone library results showed that both non-treated and control soils contained diversified bacterial communities of various phylogenetic groups, while members of the Firmicutes mainly from the class Clostridia dominated in the BSD-treated soils. The clostridial groups detected were diverse and the major clone groups were closely related to strictly anaerobic fermentative bacteria such as Clostridium saccharobutylicum, Clostridium cylindrosporum, Clostridium sufflavum, and Clostridium xylanovorans. These clostridial groups were almost eliminated from the soil bacterial community when the BSD-treated soil was treated again with irrigation and covering without biomass before the next cropping, in which the wilt disease was hardly suppressed.     

切达干酪成熟过程中细菌群落结构的分析

【目的】研究切达干酪成熟过程（6℃,180 d）中细菌群落的动态变化和组成多样性。【方法】用选择性培养基计数,并对菌落进行总DNA提取和PCR-DGGE分析。【结果】随着切达干酪成熟时间的延长,发酵剂嗜热链球菌的数量显著下降,非发酵剂乳杆菌种类明显增多。对DGGE图谱条带重扩增、测序,并进行BLAST对比分析得出,增多的乳杆菌主要为植物乳杆菌、干酪乳杆菌、副干酪乳杆菌、瑞士乳杆菌和鼠李糖乳杆菌。【结论】切达干酪成熟过程中细菌群落的组成和数量处于动态变化之中,PCR-DGGE用作选择性培养基计数菌落的后续分析,可以明确培养基上长出的菌落属于哪一种或哪一亚种,进而客观和真实地分析出干酪内细菌群落的组成和动态变化。     

磷高效转基因水稻OsPT4种植对土壤细菌群落多样性的影响

为探讨磷高效转基因水稻种植对土壤细菌多样性的影响,在小区试验条件下,以磷高效转基因水稻Os PT4为对象,以非转基因水稻日本晴、磷高效突变体水稻PHO2为对照,设施磷和不施磷两种处理,采用PCR-DGGE测序技术,分析比较了磷高效转基因水稻Os PT4在整个生长期对土壤细菌群落丰富度、均匀度的影响。结果显示,同一处理、同一生长期内磷高效转基因水稻Os PT4、磷高效突变体水稻PHO2与常规水稻日本晴的土壤细菌16S r DNA DGGE指纹图谱基本相似;施磷处理条件下,PHO2在拔节期较Os PT4、日本晴增加一条电泳条带,该条带属于蓝藻菌门(Cyanobactteria)藻青菌属(Cyanobacterium),Os PT4在抽穗扬花期较日本晴缺失一条电泳条带,该条带属于芽单胞菌门(Gemmatimonadetes);不施磷处理时,Os PT4在分蘖期和成熟期较日本晴增加4条电泳条带,测序结果显示分别为变形菌门(Proteobacteria)地杆菌属(Geobacter)、厚壁菌门(Firmicutes)芽孢杆菌属(Bacillus)、绿弯菌门(Chloroflexi)、变形菌门(Proteobacteria)和地杆菌属(Geobacter)。研究表明,在水稻个别生长期,磷高效转基因水稻Os PT4对土壤细菌丰富度(S)、香农-威纳指数(H)和均匀度(EH)产生显著影响。     

玉米-大豆轮作及氮肥施用对土壤细菌群落结构的影响

在大豆开花期分别对3个施氮水平下(0、50和100 kg hm^-2)大豆连作(大豆-大豆-大豆)、玉米-大豆轮作I (大豆-玉米-大豆)及玉米-大豆轮作II (玉米-玉米-大豆),应用PCR-DGGE技术研究了玉米-大豆轮作及施氮对土壤细菌群落结构变化的影响。结果表明,随着施氮水平的提高,3种种植方式土壤中细菌群落多样性、丰富度均呈减少趋势。高氮处理(100 kg hm^-2)明显降低了大豆连作、玉米-大豆轮作II根际土壤细菌群落多样性及丰富度,玉米-大豆轮作I根际土壤细菌群落多样性及丰富度略有降低。玉米-大豆轮作I种植方式可减轻氮肥对其根际细菌群落多样性和丰富度的影响,但施氮明显改变了其细菌群落结构。玉米-大豆轮作II中大豆根际土壤细菌群落结构较为稳定,受氮肥影响较小。在3种种植方式的土壤中,分布着酸杆菌门、变形菌门及厚壁菌门细菌,其中前两门菌群占主导地位。     

栉孔扇贝消化盲囊细菌群落组成的季节变化分析

为分析栉孔扇贝消化盲囊内细菌群落组成及其季节变化,于2009年6月—2010年6月(2月份除外)定期从青岛沙子口流清河湾扇贝养殖海区采集养殖的2龄栉孔扇贝,采用SDS方法提取消化盲囊的总DNA,并通过引物358f和907r采用PCR扩增细菌16S rDNA的V3～V5区序列,利用变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)分离所得扩增序列。DGGE图谱显示,12个月份的样品共有不同位置的谱带11条,其中9条谱带作为共有谱带在所有月份的栉孔扇贝消化盲囊中均有分布,占总谱带数的81.82%,表明整个养殖季节栉孔扇贝消化盲囊内的细菌群落组成相对稳定。对11条DGGE条带进行切胶回收、PCR扩增、纯化、克隆后测序,其中9条谱带克隆测序成功。经BLAST比对搜索,结果显示9条序列与NCBI数据库中的序列相似性达97%～99%,所代表的细菌分别属于4大类群,变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、蓝细菌门(Cyanobacteria)和放线菌门(Actinobacteria);NCBI数据库中与其相近的细菌主要为马赛菌属(Massilia)、芽孢杆菌属(Bacillus)、微小杆菌属(Exiguobacterium)、聚球藻属(Synechococcus)、红球菌属(Rhodococcus)和未培养菌。     

土壤电动修复中匀强电场对土壤细菌群落的影响

对土壤电动修复过程中匀强电场对土壤细菌群落的影响进行研究。土壤细菌计数及PCR-DGGE研究结果显示,在匀强电场中,电场强度与土壤细菌的数量和多样性存在着密切的相关性。在电场强度为3.0V/cm时,电场对土壤细菌的刺激与抑制作用基本平衡。较低的电场强度刺激土壤细菌生长繁殖,高电场强度显著抑制甚至致死土壤细菌。在匀强电场中,中间土壤细菌群落受到的影响较小,电极附近土壤细菌群落受到的影响较大。电场阳极对土壤细菌的数量和多样性产生较大影响,而电场阴极对土壤细菌群落结构的影响较大。     

研究土壤微生物多样性的PCR条件优化

[目的]对PCR-DGGE法的试验条件进行优化,以更好地分析土壤微生物遗传多样性。[方法]改良高盐法提取土壤DNA,改进引物设计,改变PCR反应过程中退火温度、扩增体系,比较PCR扩增结果。[结果]经过改良的高盐法提取的土壤微生物DNA效果更好。PCR扩增选择20μl体系条带单一,易于操作。退火温度选择在55℃时无非特异性扩增,35个循环次数易于后续DGGE分析。[结论]已经优化的PCR基因扩增体系特异性高且稳定可靠。