土壤微生物分子生态学研究方法

主要介绍了目前在土壤微生物生态学研究中的常用分子生物学方法,包括核酸探针杂交技术、基于PCR技术的研究方法、特异DNA片段的序列分析、DNA扩增片段梯度凝胶电泳检测技术等。     

Physical, chemical and microbiological properties of an Andisol as related to land use and tillage practice

The effects of land use and management practice on soil physical, chemical and microbiological properties may provide essential information for assessing sustainability and environmental impact. This study compared the effects of 41 years of no-tillage (NT) with continuous apple orchard, with those of conventional tillage (CT) with wheat–soybean rotation and another of puddling (PD) with continuous rice on the characteristics of a pumice Andisol in a temperate region of northern Japan. Higher values for bulk density, penetration resistance, pH, C/N ratio, exchangeable Na (X-Na), Fe, and Mn were observed for PD than NT and CT. On the other hand, organic matter, EC, N, exchangeable K (X-K), exchangeable Ca (X-Ca) and Cu were significantly higher for NT than CT and PD. Highest content of Zn was found in CT compared to other practices. The three-phase composition at pF 2.0 was significantly affected by land use and tillage practices. The solid phase and liquid phase were greater under PD than under NT and CT, while air phase was greater under CT than under NT and PD. Significantly higher values for saturated hydraulic conductivity was found in CT than NT and PD. Total phospholipid fatty acid (PLFA) and PLFA for bacteria, aerobes and cyanobacteria were remarkably higher in NT than CT and PD, regardless of depth. On the other hand, PLFA for methane-oxidizing bacteria, sulfate-reducing bacteria and mycorrhizae were significantly higher in CT than NT and PD. PLFA for fungi was significantly higher in surface (0–10 cm) soils than subsurface (10–20 cm) soils regardless of treatments. Highest bacterial and fungal diversity evaluated by DNA band number in DGGE analysis based on PCR amplification of 16S rDNA and 18S rDNA fragments, respectively, were observed in surface soil of PD. The result suggests a linkage between microbial community and tillage practices in temperate Andisol. This study also justifies the need of measuring soil characteristics based on soil microbial communities.     

AHP中判断矩阵一致性的可控标准

将不完全一致性矩阵产生的＂非一致性＂偏差作为随机变量来对待,构造相应统计量,得出判定一致性的一个新标准,使决策者可以根据实际问题及个人偏好确定适当的置信水平进行一致性检验.     

分子动力学模拟解析脉冲电场对类PSE鸡肉肌球蛋白凝胶特性作用规律

【目的】以类PSE鸡肉肌原纤维蛋白为研究对象,通过分子动力学模拟技术研究脉冲电场处理对其凝胶特性和构象修饰的影响,并对凝胶特性改变的潜在机制进行阐述,以此拓宽这种新兴食品加工技术的工业应用,为禽类加工方向提高异常肉制品的经济效益提供一种新的方法。【方法】本试验选取类PSE鸡肉为原料并提取其肌原纤维蛋白,对比8、18和28 kV·cm^-1电场强度下脉冲电场(脉冲电场频率为800 Hz,占空比47%)处理及非脉冲电场处理的类PSE鸡肉肌原纤维蛋白不稳定动力学指数、流变学特性及通过分子动力学技术建立电场强度18 kV·cm^-1的脉冲电场模拟体系,通过收集模拟过程中的均方根偏差(RMSD)、均方根波动(RMSF)和对蛋白质总体构象的观察的变化来阐明在该条件下脉冲电场处理对类PSE鸡肉肌原纤维蛋白分子构象动态变化的作用机制。研究脉冲电场作用对类PSE鸡肉肌原纤维蛋白构象稳定性、保水性、流变特性的影响。【结果】不同电场强度的脉冲电场处理后的类PSE鸡肉肌原纤维蛋白体系不稳定动力学曲线斜率更小,蛋白体系相较于未经处理的样品更加稳定。蛋白凝胶的保水性结果为1...     

微卫星PAGE银染法及其干胶的简易制备

为了使微卫星PCR扩增的DNA片段能得到较好的分离和长期保存实验胶片 ,研究利用 1 5对猪的微卫星引物 ,经PCR扩增 ,非变性聚丙烯酰胺凝胶电泳 ,银染法显色 ,然后制成了干胶。结果表明 ,聚丙烯酰胺凝胶具有很好的分离微小差异DNA片段的能力 ,而且银染后制成的干胶可长期保存。此法尤其适用于不具备凝胶成像系统和干胶机的实验室使用。     

利用PCR-DGGE技术研究长期自然恢复、种植作物和裸地条件下黑土土壤细菌群落结构变化

Soil microbial biomass and community structures are commonly used as indicators for soil quality and fertility. A investigation was performed to study the effects of long-term natural restoration, cropping, and bare fallow managements on the soil microbial biomass and bacterial community structures in depths of 0--10, 20--30, and 40--50 cm in a black soil (Mollisol). Microbial biomass was estimated from chloroform fumigation-extraction, and bacterial community structures were determined by analysis of 16S rDNA using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Experimental results showed that microbial biomass significantly declined with soil depth in the managements of restoration and cropping, but not in the bare fallow. DGGE profiles indicated that the band number in top 0--10 cm soils was less than that in depth of 20--30 or 40--50 cm. These suggested that the microbial population was high but the bacterial community structure was simple in the topsoil. Cluster and principle component analysis based on DGGE banding patterns showed that the bacterial community structure was affected by soil depth more primarily than by managements, and the succession of bacterial community as increase of soil depth has a similar tendency in the three managements. Fourteen predominating DGGE bands were excised and sequenced, in which 6 bands were identified as the taxa of Verrucomicrobia, 2 bands as Actinobacteria, 2 bands as α-Proteobacteria, and the other 4 bands as δ-Proteobacteria, Acidobacteria, Nitrospira, and unclassified bacteria. In addition, the sequences of 11 DGGE bands were closely related to uncultured bacteria. Thus, the bacterial community structure in black soil was stable, and the predominating bacterial groups were uncultured.     

Effects of coated and non-coated ZnO nano particles on cucumber seedlings grown in gel chamber

Zinc oxide-engineered nanoparticles (ZnO ENPs) have received the most attention in recent years. This increasing interest has been directed towards studying the environmental fate and effects of ZnO ENPs on ecological terrestrial species. In this study, ZnO NPs were synthesized by atmospheric pressure solution evaporation method and were coated or uncoated with humic acid (HA). The root uptakes of uncoated and HA-coated ZnO NPs and zinc (Zn) were investigated by gel-grown cucumber. Two ZnO levels (1 and 200 µM) were applied in the form of coated (T₃) and non-coated (T₂) NPs or bulk particles (T₁). The results showed that coating NPs by HA increases zeta potential of NPs and decreases their aggregation size due to the increase in the repulsion forces among the particles. Addition of 1 mgL^?1 ZnO into gel chamber enhanced root and shoot biomass; however, the shoot growth was higher in the presence of NPs compared to its bulk counterpart. Moreover, greater phytotoxicity of ZnO from the source of NPs than bulk particles in shoot was observed. Scanning electron microscopy results showed a clear evidence of the penetration of NPs into root cells.     

Identification of serum protein markers for early diagnosis of pregnancy in buffalo

Improper or delayed pregnancy diagnosis has significant impact over animal production, particularly in buffaloes which inherently suffer from several reproductive inefficiencies. Thus the present study has undertaken to identify serum protein markers pertaining to early pregnancy diagnosis in buffaloes. Serum samples were collected from 10 pregnant Murrah Buffalo heifers at weekly intervals from days 0‐35 post‐artificial insemination and from 12 inseminated non‐pregnant cyclic buffalo heifers on days 0, 7, 14 and 21. Two‐dimensional gel electrophoresis and densitometric analysis revealed the presence of five protein spots showing average density fold change of ≥4 during early pregnancy. Mass spectrometry analysis identified these up‐regulated proteins as anti‐testosterone antibody light chain, apolipoprotein A‐II precursor, serum amyloid A, cytokeratin type II, component IV isoform 1, which are have established roles in embryogenesis, but over‐expression of the fifth identified protein immunoglobulin lambda light chain in pregnancy has been elucidated as a novel finding in the current study. Further, with bioinformatics analysis, potential antigenic B‐cell epitopes were predicted for all these five proteins. An antibody cocktail‐based approach involving antibodies against all these five up‐regulated entire proteins or their epitopes could be developed for early detection of pregnancy in buffaloes. © 2016 Japanese Society of Animal Science     

超声波对肌原纤维蛋白热诱导凝胶化学作用力与保水性的影响

【目的】研究超声波对肌原纤维蛋白凝胶化学作用力与保水性的影响,并探讨化学作用力与保水性之间的内在联系。【方法】取活AA鸡屠宰,提取肌原纤维蛋白(myofibrillar proteins,MP),用不同超声时间处理MP并制成热诱导凝胶,运用凝胶中总巯基含量的变化来反应二硫键的形成;凝胶的表面疏水性S0和Zeta电位值来表征疏水作用力和静电斥力;用拉曼光谱的I850/I830比值大小反映凝胶氢键的变化;凝胶保水性用高速离心机测定。【结果】超声波处理0—6 min时,MP凝胶的总巯基含量随时间的增加而减少,活性巯基含量显著增加;处理6—15 min时,MP凝胶的总巯基以及活性巯基均随时间延长而显著减少,而对MP原料进行同样处理时,处理时间在0—6 min内,MP总巯基和活性巯基变化趋势与MP凝胶相同;超过9 min后,活性巯基和总巯基没有显著变化,且二者含量逐渐接近。表明短时间超声波处理促进了MP分子内部巯基转变成二硫键,长时间超声波处理和加热成胶的共同作用促进了活性巯基转变成二硫键。MP凝胶的表面疏水性先随超声时间延长从1 194.1显著上升到1 489.5(6 min),随后逐渐降至1 230.8,表明超声波产生的空穴效应能够使MP分子内部疏水基团暴露到分子表面,但超过6 min后,增加的蛋白表面疏水性又被包裹在凝胶网络中。MP凝胶的Zeta电位绝对值随超声时间的增加从6.03显著增加到7.68(P0.05),超过6 min后显著减少,表明超声波处理使MP分子逐渐展开,蛋白质分子间静电作用增强,但过度展开后对其形成凝胶的静电作用力不利。适度的超声时间(0—6 min)使MP凝胶的归一化强度I850/I830比值从0.9805增加至1.023(P0.05),表明MP与水分子形成的"蛋白-水分子"氢键增多,6 min过后比值减少,蛋白与水分子之间的氢键减弱;超声波处理0—6 min后的MP凝胶保水性从47.5899%快速升至72.9855%(6 min)(P0.05),之后随时间延长,凝胶的保水性减少至44.356%。相关性分析结果表明,保水性与总巯基的相关性不大(P0.05),与活性巯基显著相关(P0.05),与表面疏水性、电位绝对值和氢键都极显著相关(P0.01)。【结论】超声波显著影响肌原纤维蛋白凝胶化学作用力与保水性;疏水作用力、静电斥力和氢键是决定保水性的主要作用力,而二硫键次之;超声波处理时间为6 min时为最优条件,在此条件下,凝胶的疏水作用力、氢键和静电斥力均达到最大,致使凝胶网络结构均匀致密,能最大限度的保留水分。     

不同相对湿度对间歇性26℃环境下肉鸡盲肠菌群多样性的影响

本试验旨在研究不同相对湿度(RH)对间歇性26℃环境下肉鸡盲肠菌群多样性的影响。选取29日龄爱拔益加(AA)肉鸡180只转入环境控制舱,随机分成3个组(RH分别为30%、60%和85%),每组6个重复,每个重复10只鸡(公母各5只)。从29日龄开始,每天10:00—16:00(6 h)温度维持26℃,RH分别为30%、60%和85%,剩余时间温度为21℃,RH为60%。试验共14 d。采用16S rDNA的变性梯度凝胶电泳(DGGE)技术,结合特异性和共性条带割胶回收DNA进行克隆和测序,分析RH在间歇性26℃偏热处理第7天和第14天时对盲肠内容物菌群结构和多样性的影响。结果表明:1)试验第7天,30%RH组肉鸡盲肠DGGE条带数(菌群丰富程度)高于60%RH组,而85%RH组低于60%RH组;试验第14天,30%、85%RH组肉鸡盲肠DGGE条带数均高于60%RH组。2)聚类分析显示,试验第7天,85%RH对盲肠菌群影响明显;试验第14天,30%RH对盲肠菌群影响明显。但随着处理时间推移,30%RH对肉鸡盲肠菌群影响越大。3)间歇性26℃环境下不同RH组肉鸡盲肠内共性菌群是Faecalibacterium prausnitzii;在试验第7天,30%、85%RH组肉鸡盲肠中特异性菌群是Stomatobaculum longum。结果提示:间歇性26℃环境下,低湿(30%RH)和高湿(85%RH)影响肉鸡盲肠菌群的结构和多样性,且不同处理时间RH的影响不同