土壤细菌16SrRNA基因变异型及其与植被的相关研究

绕过细菌的分离培养,直接提取土壤DNA,扩谱,克隆土壤细菌群体的16S核糖体RNA基因（16S,rDNA),根据该基因各种变异类型的限制性片段长度多型性,分析土壤细菌分子遗传多样性及其与植被的相关关系,植被的改变影响土壤养分,进而改变土壤细菌群落结构,土壤细菌遗传多样性和分化能反映植被的变化。     

水环境细菌16S rNDA限制性片段长度多型性及群落结构分析

用细菌16S核菌体RNA基因（rDNA）限制性片段长度多型性描述了水环境细菌群落结构。从环境水样中直接分离DNA,以细菌特异的引物扩增16S rDNA。构建质粒文库,随机分离重组质粒,用限制性内切酶消化获得16S rDNA基因型,用基因型的种类及频率描述特定水体生境的细菌群落结构,该方法在分析水体隐含遗传多样性、揭示污染的生物学效应和评价水环境质量等方面具有重要的应用价值。     

油菜内生细菌16S核糖体DNA的RFLP分析

植物内生细菌定殖在植物组织内部但不引起明显的病害症状。从健康油菜植株的不同器官中分离到大量内生细菌,这些细菌菌落形态存在明显的差异,表明油菜组织中存在大量内生细菌,且类群丰富。分离到的122株内生细菌根据菌落形态可以划分为35类;利用细菌通用引物对16S核糖体DNA进行扩增, 获得约15 kb片段,分别用内切酶HaeⅢ和MspⅠ对扩增产物进行限制性酶切,产生不同的酶切图谱,根据酶切图谱聚类分析结果,所有供试菌株被归为39类,这一结果从遗传上显示油菜内生细菌类群的多样性。两种方法归类结果比较发现菌落特征所反映的信息量有限,只能作为初步的参考指标,核糖体DNA的限制性片段长度多态性分析快速、准确,可以作为油菜内生细菌多样性分析的一种有效方法     

日粮中添加鱼油后肉牛瘤胃细菌区系的变化

目的：研究肉牛日粮中添加2%鱼油后瘤胃细菌区系的变化。方法：分别于添加鱼油前后取瘤胃液提取总DNA,根据细菌通用引物27F/1492R扩增16S rDNA基因序列,分别构建2个16S rDNA基因文库;从每个文库中各随机挑取384个克隆,对阳性克隆用HhaⅠ进行限制性片段长度多态性（RFLP）分析、聚类,取各RFLP类群中的代表克隆进行16S rDNA序列测定,构建系统发育树,研究细菌区系多样性的变化。结果：添加鱼油前、后,文库的RFLP图谱分别可以分成74和41个RFLP类群;添加鱼油前后的优势菌群均为噬纤维菌-屈挠杆菌-拟杆菌（CFB）和低（G＋C）含量的革兰阳性菌（LGCGPB）,但添加鱼油后文库中LGCGPB比例降低,而且未检测到纤维菌门细菌;有50%～60%的测定序列与GenBank数据库中的序列相似性高于97%,90%以上为未培养的细菌;添加鱼油后瘤胃细菌的多样性减少。结论：日粮添加鱼油后CFB比例增高但多样性下降,LGCGPB、纤维杆菌比例下降,这一结果为调控瘤胃发酵提供了依据。     

有机污染物对水体真细菌群落结构的影响

为了揭示有机污染物对环境真细菌组成和多样性的影响,应用末端限制性片段长度多态性(tRFLP)和16S rDNA文库技术并结合水质分析方法,比较分析了松花江流域内受不同程度有机污染的4个水体及其沉积物中真细菌的群落结构。tRFLP分析表明各水体及底泥均呈现较为复杂的群落结构模式,不同底泥群落形成的末端限制性片段(TRF)图谱具有很高的相似性,但随着污染程度的加强,部分类群明显富集,而且在水样组和泥样组内,群落结构的相似性同水质相似性是一致的,主成分分析(PCA)显示水样和泥样中的真细菌TRF形成不同的群。16SrDNA文库分析表明松花江哈尔滨段底泥中真细菌分布于10个门,Proteobacteria门占优势,达群落总数的21.92%(β-Proteobacteria亚门占10.96%),而有机染污物严重超标的生活污水排污道底泥中的微生物多样性较低,分布于7个门,Proteobacteria门为优势群,占群落的47.37%(α-Proteobacteria亚门占21.05%,δ/ε-Proteobacteria亚门占15.79%)。该研究表明向水体中长期排放高浓度有机物能使系统中微生物群落多样性降低,与污染物降解相关的功能微生物类群明显富集。     

连作对地黄根际土壤细菌群落多样性的影响

采用末端限制性片段长度多态性(T-RFLP)技术研究地黄连作下根际土壤细菌群落的动态变化.结果表明:地黄根际土壤细菌群落的香农多样性指数、丰富度指数和相似性指数均为对照(CK)1年2年,地黄连作下细菌优势种群的比例显著下降,种植1年土壤中厚壁菌门的芽孢杆菌纲在整个细菌群落中处于主导地位,而种植2年土壤中变形菌门ε-变形菌纲处于主导地位.地黄连作使其根际土壤细菌种类大量减少,群落结构趋于简单.连作后细菌群落多样性水平的变化导致根际土壤微生物群落功能的失调,可能是引发连作障碍的原因之一.     

枯草芽孢杆菌菌剂对烟草根际土壤细菌群落的影响

通过构建16S rRNA克隆文库及采用核糖体DNA扩增片段酶切分析(ARDRA)的方法,研究施用生物防治剂枯草芽孢杆菌菌剂对烟草根际土壤细菌群落结构以及多样性的影响.采用文库库容值(C)、Shannon多样性指数(H)、Pielou 均匀度指数(E)和Margalef丰富度指数(R)对细菌多样性进行评价.系统发育分析表明: 对照及处理样品均检测到12个细菌类群：酸杆菌门(Acidobacteria)、变形菌门(α 、β 、δ 、γ-Proteobacteria)、浮霉菌门(Planctomycetes)、厚壁菌门(Firmicutes)、硝化螺旋菌门(Nitrospirae)、芽单胞菌门(Gemmatimonadetes)、放线菌门(Actinobacteria)、绿弯菌门(Chloroflexi)和拟杆菌门(Bacteroidetes);但各细菌类群的结构组成及所占比例在不同样品间有较大差别.对照土壤中优势菌群为Acidobacteria(27.1%)和Proteobacteria(26.5%);处理土壤中则为Proteobacteria(38.0%)和Acidobacteria(29.6%);菌剂处理后土壤中,γ-Proteobacteria和α Proteobacteria所占比例明显提高,而β Proteobacteria、Planctomycetes和Firmicutes等菌群的数量则相对降低.多样性分析表明,土壤样品均具有丰富的细菌多样性,经枯草芽孢杆菌菌剂处理后,土壤细菌多样性指数和丰富度指数均提高.
     

新疆富蕴地震断裂带植被恢复对土壤古菌群落的影响

选取新疆富蕴地震断裂带8种次生植物根际土壤,以同土层裸地为对照,测量土壤化学性质,利用末端限制性片段长度多态性技术研究塌陷区次生植物对土壤古菌群落的影响.结果表明,多数植物根际土壤养分显著(P＜0.05)高于对照,其主要古菌类群为泉古菌门(Crenarchaeota)和广古菌门(Euryarchaeota),群落间多样性差异大,相似性低.不同植物根际土壤古菌的优势类群数量差异较大,鼠掌老鹳草高达18种,西北绢蒿少为2种.典范对应分析表明,土壤有机质含量对古菌优势类群分布影响最大(Hha Ⅰ酶切:r=0.94;Rsa l酶切:r=0.74),速效磷含量与古菌群落各多样性指数呈显著正相关(P＜0.05),总氮含量与均匀度指数E呈显著正相关(P＜0.05).新疆地震断裂带植被的恢复可影响根际土壤古菌群落的分布、组成和结构,其原因与植物根际土壤化学性质有关,同时在改善土壤肥力方面也有显著效果.     

新疆玛纳斯热气泉免培养土壤细菌多样性分析

【目的】了解新疆玛纳斯热气泉土壤免培养细菌群落组成及多样性。【方法】采用免培养法直接从土壤样品中提取总DNA,利用细菌通用引物对土壤总DNA进行16S rDNA扩增,构建细菌16SrDNA文库。使用HaeⅢ限制性内切酶对阳性克隆进行限制性片段长度多态性分析(restriction fragment length polymorphism,RFLP),挑选具有不同酶切图谱的克隆进行测序、比对并构建16SrDNA系统发育树。【结果】从土壤细菌16S rDNA文库中随机挑选了170个阳性克隆,共得到29个不同的分类单元(operational taxonomic unit,OTU)。系统发育分析归为6个门:酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)和浮霉菌门(Planctomycetes)。其中厚壁菌门(Firmicutes)为绝对优势类群,占整个细菌文库的71%。29个OTUs中有14条序列与GenBank中相关序列的相似性低于97%(序列长度约1.5kb),占序列总数的48%。【结论】热气泉土壤细菌种群多样性较低,但存在大量潜在细菌新种。     

稻瘟病抗性对根际土壤微生物多样性的影响

为了解稻瘟病抗性与根际土壤微生物之间的关系,在云南宜良大田条件下对6个稻瘟病单基因系Piz、Pib、Pikh、Pi19、Pi3、Pita和受体“丽江新团黑谷”(LTH)进行了诱发鉴定,利用ITS1和16SrDNA高通量测序技术对土壤根际真菌的ITS1序列以及细菌16S rDNA区片段进行了分析。结果显示单基因系Piz、Pib、Pikh表现抗病, Pi19、Pi3、Pita和LTH表现感病;子囊菌门(Ascomycota)是真菌的优势门(73.41%),变形菌门(Proteobacteria)是细菌的优势门(32.48%),7个土样在主要微生物种类组成上相似;抗、感单基因系根际土壤真菌在罗兹菌门(Rozellomycota)(分别为7.69%和14.66%, LTH为5.59%)及其下属的罗兹菌种(Rozellomycota＿sp)(分别为6.97%和13.99%, LTH为5.59%)和担子菌门(Basidiomycota)的伞菌纲(Agaricomycetes)(分别为1.2%和0.7%,LTH为1.7%)及伞菌目(Agaricale)(分别为1.1%和0.5%,LTH为1.6%)上...     

砷对土壤脲酶活性影响的研究

采用模拟方法对As污染土壤脲酶特征进行了研究．结果表明,土壤中As浓度在0～200mg·kg^-1浓度范围内,反应初期脲酶活性变化无明显规律;一年后砷激活土壤脲酶活性,二者达到显著或极显著正相关．随As浓度增加,土壤脲酶Km值基本不变或略有增加,Vmax增大,从机制上揭示出As加速脲酶-尿素复合物的解离．厩肥和无肥土样脲酶对As的反应类似,只是变化幅度有所差异．     

Stimulation by nickel of soil microbial urease activity and urease and hydrogenase activities in soybeans grown in a low-nickel soil

Summary The concentration of nickel in some soils may be insufficient to meet the requirements of enzymes such as urease in soybeans and hydrogenase in Rhizobium. In an initial evaluation of nickel availability, several soils were examined for nickel content and microbial urease activity. Total and extractable nickel were determined by atomic emission spectrometry. Purified glucose and urea were added to soils to stimulate microbial growth and urease activity, the latter of which was monitored by the rate of decomposition of¹⁴C urea. Nickel also was added to some samples to determine if the indigenous supply was limiting. In one low-nickel soil (total Ni 13 ppm) urease activity increased 150% in response to additional nickel, while other soils (total Ni 22–3491 ppm) failed to respond to nickel. However, additional nickel did stimulate urease activity (up to 109%) in 3 out of 10 soils to which purified CaCO₃ was added. Presumably the rise in pH associated with this treatment decreased nickel availability. Additions of Co, Mn, Fe, or Cu had no consistent effect on urease activity, thus indicating that the response to Ni was specific. Nickel fertilization increased leaf urease and nodule hydrogenase activity of soybeans grown in low-nickel soil, however, yield was not improved. These results may have practical implications in the nutrition of plants and micro-organisms that metabolize H₂ and urea.     

Inhibition of soil urease activity by aminocresols

Summary The effect on soil urease activity of five aminocresols, at concentrations of 5–100 g/g soil, was examined in the laboratory. Two compounds, 4-amino-o-cresol and 4-amino-m-cresol, significantly inhibited urease activity. The efficacy of 4-amino-o-cresol was compared with that of phenylphosphorodiamidate (PPDA), a known inhibitor, in three U.K. soils. At 50g/g soil 4-amino-o-cresol was as inhibitory as an equivalent concentration of PPDA in a soil with low urease activity, but was less inhibitory in two soils with high urease activity.     

The urease structural gene ureA in Rhizobium meliloti is preceded by an open reading frame necessary for urease activity

Abstract An open reading frame (ORF1) located upstream of the urease structural gene ureA in Rhizobium meliloti strain AK631 was cloned and characterized by DNA sequencing. Comparison of the amino acid sequence revealed partial homology with the urease accessory gene ureD of Klebsiella aerogenes and Proteus mirabilis . Mutational analysis of ORF1 showed that the gene is necessary for urease activity. Its function is still unknown.     

High throughput colorimetric assay for rapid urease activity quantification

A novel high throughput colorimetric urease activity assay was compared to the Nessler method. The new method employs phenol red to determine the urease activity. This method reduces significantly sample processing time and allows real-time investigations. This method is rapid, sensitive, easy, cost-effective, and does not use any toxic chemical reagents.     

Effects of temperature and moisture on urease activity in semi-arid tropical soils

Summary Studies of urease activity in an Indian Vertisol and Alfisol using both buffer (THAM pH 9.0) and non-buffer methods for assay of the urease activity showed that activity increased with increase in temperature from 10°C to a maximum at 60°C (Vertisol) and 70°C (Alfisol). Further increase in temperature decreased urease activity which was nearly totally inhibited at 100°C. Urease activity was not detected in soil samples collected in late summer when the soil moisture content was far below — 15 bar pressure. Urease activity increased with increase in moisture content up to field capacity and remained constant with further increase in moisture content. The relevance of these findings to the ICRISAT improved management practices for Vertisols, which involve seeding of crops into dry soil just before the onset of rains is discussed. Approved as Journal Article No. 288 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT).     

The measurement and distribution of urease activity in a pasture system

Summary Urease activities of soil, litter and plant components of a pasture sward were measured by two methods. The type, height and age of the pasture sward can influence urease activity of the underlying soil. Urease activity of the above-soil plant and litter components is less per unit area of sward (7% of total sward activity) than that of associated soil to 20 cm (93%), although urease activity of the non-soil component is about 9 fold greater per unit mass than that of an equal mass of soil. The implications of the presence of this highly active and accessible urease on transformation and loss of nitrogen on swards topdressed with urea is discussed.     

大气CO2增加对土壤脲酶、磷酸酶活性的影响

1　引　　言自 19世纪 70年代工业化革命以来 ,由于化石燃料燃烧、林草地开垦农用等已引起ＣＯ2 大气排放的不断增加 ,可能使未来的 5 0～ 10 0年内 ,全球大气ＣＯ2 将增加 1倍左右 .许多研究证明 ,大气ＣＯ2 增加可提高植物生长代谢水平[1,8] .其结果是植物代谢分泌物的种类和数量发生变化 ,由植物光合作用强度或速率变化引起的植物枯枝落叶质量也会改变 ,二者均可能在经泌入土壤或凋落进入土壤分解后 ,对植物着生的土壤环境产生直接或间接的影响 .植物对大气ＣＯ2 增加的响应途径还有根圈微生物种群 (植物根圈大量活性Ｃ组分将直接作为微…     

Urea uptake and urease activity in Corynebacterium glutamicum

When Corynebacterium glutamicum is grown with a sufficient nitrogen supply, urea crosses the cytoplasmic membrane by passive diffusion. A permeability coefficient for urea diffusion of 9 × 10^–7 cm s^–1 was determined. Under conditions of nitrogen starvation, an energy-dependent urea uptake system was synthesized. Carrier-mediated urea transport was catalyzed by a secondary transport system linked with proton motive force. With a K _m for urea of 9 μM, the affinity of this uptake system was much higher than the affinity of urease towards its substrate (K _m approximately 55 mM urea). The maximum uptake velocity depended on the expression level and was relatively low [2–3.5 nmol min^–1 (mg dry wt.)^–1]. Received: 11 August 1997 / Accepted: 2 December 1997     

The effect of quinoid and phenolic compounds on urease and dehydrogenase activity and nitrification in soil

Summary 1,4-Naphthoquinone; 2-methyl-1,4-napthoquinone; 2,3-dichlorohydroquinone; 4,6-di-tert.butyl-o-benzoquinone; 4,6-di-tert.butylpyrocatechol and 4-tert.butylpyrocatechol at concentrations of 10 and 20 ppm inhibit mineralization of urea N by reducing urease activity and/or nitrification in soils. Coating of urea with these chemicals was more effective than direct application to the soil. There was no adverse effect of any of the chemicals either on germination or on the growth of wheat plants at concentrations of 20 and 50 ppm added to soil.