土壤微生物总DNA提取方法的比较

采用4种土壤DNA提取方法提取了5种类型土壤的微生物总DNA，并对4种提取方法的DNA提取效果进行综合分析，进一步通过PCR—DGGE扩增提取DNA中的细菌16S rDNA片段，并对扩增产物进行DGGE电泳分析。结果表明，SDS-高盐缓冲液法抽提的DNA得率最高，SDS-酚氯仿抽提法的DNA得率最低。DNA的纯度，以BIO-101 DNA Extraction Kit试剂盒法最高，改进试剂盒法纯度最低。通过PCR-DGGE分析土壤微生物多样性结果表明，BIO-101 DNA Extraction Kit试剂盒法提取的DNA代表的微生物多样性最全，而SDS酚氯仿法抽提的DNA代表性最差。     

保护性耕作对丘陵区水稻土团聚体稳定性的影响

长期保护性耕作通过增加土壤有机碳而成为稻田土壤结构改良的一项有效措施,而保护性耕作对土壤团聚体稳定机制的影响尚未完全清楚。本文供试土样采自耕作制定位试验水旱轮作、冬水免耕、垄作免耕和厢作免耕耕层（0～ 20 cm）土壤,土样经过糊化作用、湿润作用和再次糊化作用等预处理,用以阐明稻田土壤团聚体的破碎机制。研究结果表明：糊化作用和湿润作用后紫色水稻土团聚体稳定性差异不明显,而保护性耕作显著影响团聚体的稳定性。糊化作用后团聚体水稳性强弱顺序为：垄作免耕＞厢作免耕＞冬水免耕＞水旱轮作,湿润作用后团聚体水稳性强弱顺序为：厢作免耕＞垄作免耕＞冬水免耕＞水旱轮作。糊化作用下团聚体稳定性与有机碳浓度相关性不显著（r＝0.432,p＞0.05）,湿润作用下团聚体稳定性与有机碳浓度呈极显著正相关（r＝0.626,p＜0.01）。因此,研究结果说明保护性耕作有利于紫色水稻土大团聚体有机碳含量提高,进而增强团聚体的水稳性。     

猪粪和土壤样品中微生物DNA提取方法的比较

猪粪施于土壤可能会对土壤微生物多样性造成影响,为选用同一种DNA提取方法用于土壤和猪粪微生物DNA的提取,该文采用了化学裂解法和试剂盒法同时从土壤和猪粪样品中提取微生物DNA,并对这两种方法的提取DNA的效果进行了比较。结果表明,试剂盒法不能用于提取土壤中的微生物DNA;可以从猪粪中提取到DNA,PCR扩增能得到目的产物,但重复性不高。化学裂解法提取的土壤微生物DNA浓度高但纯度低,纯化后纯度增加,但DNA有所损失,用于PCR扩增时结果不理想;处理猪粪样品,提取的DNA浓度较低但纯度较高,PCR扩增结果比较理想。由此可见,化学裂解法用来提取猪粪样品中的微生物DNA是可行的,但需寻求更好的土壤样品微生物DNA的提取方法。     

猪粪和土壤样品中微生物DNA提取方法的比较

猪粪施于土壤可能会对土壤微生物多样性造成影响，为选用同一种DNA提取方法用于土壤和猪粪微生物DNA的提取，该文采用了化学裂解法和试剂盒法同时从土壤和猪粪样品中提取微生物DNA，并对这两种方法的提取DNA的效果进行了比较。结果表明，试剂盒法不能用于提取土壤中的微生物DNA；可以从猪粪中提取到DNA，PCR扩增能得到目的产物，但重复性不高。化学裂解法提取的土壤微生物DNA浓度高但纯度低，纯化后纯度增加，但DNA有所损失，用于PCR扩增时结果不理想；处理猪粪样品，提取的DNA浓度较低但纯度较高，PCR扩增结果比较理想。由此可见，化学裂解法用来提取猪粪样品中的微生物DNA是可行的，但需寻求更好的土壤样品微生物DNA的提取方法。     

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。     

水稻土水稳性大小团聚体联合测定法及其应用

关于水稻土水稳性团聚体的测定方法,至今尚无统一标准,过去多沿用与旱地相同的方法,而且大、小团聚体的测定是分别进行的。水稻土经过灌水种稻后,特别是受耕作影响频繁的耕层,土壤团聚体的变化很大,且大部分呈微团聚体状态。的试验表明,种稻以后,小于1毫米的水稳性团聚体的数量大大增加^[12].因此,水田与早地团聚体的测定方法应该不同。本文目的在于寻求一种适于水稻土的水稳性团聚体的测定方法,为进一步研究打下基础。     

水分状况对紫色母岩发育的水稻土团聚体及有机碳分布影响

在紫色丘陵区采集了因水分状况影响而形成的4种类型紫色水稻土土样,利用湿筛法获得不同粒径的团聚体,分析了其有机碳和不同土壤发生层中易氧化碳、微生物生物量碳和水溶性碳含量。结果表明,A层土壤中>2 mm的团聚体以潜育型水稻土和渗育型水稻土最高,分别占76.65%和75.92%,其次是潴育型水稻土占43.86%,淹育型水稻土只有13.10%;不同土壤发生层间土壤团聚体的组成也存在较大的差异;除潴育型水稻土的P层外,其余各层土壤的有机碳53.7%~96.2%均分布在>0.25 mm团聚体中;潜育型水稻土的微生物生物量碳和易氧化碳含量在4种水稻土中最高,分别为282.5 mg kg-1和6.59 g kg-1,水溶性有机碳则以渗育型水稻土最高;有机碳含量与>2 mm团聚体含量呈极显著正相关关系,与<0.25 mm微团聚体数量呈显著负相关关系。     

垄作免耕下紫色水稻土有机碳的分布特征

土壤有机碳的空间分布特征是研究土壤有机碳储量及其动态的重要内容之一。本文从土壤团聚体、剖面和田块等尺度研究了四川盆地紫色水稻土在垄作免耕下有机碳的分布特征。结果表明：紫色水稻土团聚体含碳量在大团聚体（2～0．25mm）中最高，但有机碳总量主要分布在0．25～0．02mm。其次是0．02-0．002mm；垄作免耕下团聚体有机碳主要富集在0—10cm土层的大团聚体（〉0．25mm）中（富集系数〉1．5），而常规平作、水旱轮作和垄作翻耕下，剖面中土壤有机碳含量分布自上而下缓慢降低；实验田表层土壤有机碳含量及相关土壤特性的空间分布呈条带状和斑块状格局，具有明显的空间变异性。土壤有机碳含量在垄作免耕处最高，达30．71g kg^-1，在常规平作处最低，为16．50g kg^-1左右。长期垄作免耕会导致有机碳向土壤表层大团聚体的相对富集及在土壤剖面的层次分异。     

西北地区耕地土壤真菌DNA提取方法比较

由于西北土壤理化性质的复杂性和真菌特殊性,所以从土壤中提取真菌基因组DNA就相对细菌更困难。在2种常用的土壤微生物基因组DNA提取方法与在传统提取方法的基础上,结合了一种专门适用于真菌的提取方法进行了比较,并且利用真菌28SrDNA通用引物U1/U2进行扩增。三种提取方法比较结果表明:SDS法提取的DNA纯度最低,传统CTAB-SDS的DNA产量最低,实验室的提取方法既可以提高DNA产量又可以保证DNA的片段完整性,并且本实验室的提取方法扩增效果最好,可广泛应用于西北地区土壤真菌的分子生物学研究。     

稻田垄作免耕对土壤团聚体和有机质的影响

该文以1990年建立的耕作制定位试验田紫色水稻土为研究对象,分析了冬水田（FPF）、水旱轮作（CR）和垄作免耕（RNT）3种耕作方式对土壤团聚体组成和有机质的影响。结果表明,垄作免耕减少了对土壤大团聚体的破坏,在0～10 cm土层,垄作免耕大团聚体含量分别是冬水田和水旱轮作的1.48和1.32倍,微团聚体含量则显著降低;在 ＞10～20 cm土层有相同的趋势。3种耕作条件下,有机碳和氮在团聚体中的分布模式类似,均有向大团聚体富集的趋势,但垄作免耕条件下土壤有机碳和氮质量分数显著高于冬水田和水旱轮作。对土壤颗粒有机质（POM）的分析结果表明,垄作免耕0～10 cm土层轻质组分（LF）的质量分数（1.92 g/kg）与水旱轮作（1.70 g/kg）差异不显著,但显著高于冬水田（1.42 g/kg）。冬水田、水旱轮作和垄作免耕的0～10 cm土层,团聚体内总颗粒有机质（total iPOM）质量分数分别为0.96,1.12,2.14 g/kg;垄作免耕土壤团聚体内细颗粒有机质（fine iPOM）分别为冬水田和水旱轮作土壤的3.02和2.46倍,占总POM差异的57%和66%。垄作免耕土壤团聚体内粗颗粒有机质（coarse iPOM）分别为冬水田和水旱轮作土壤的1.56和1.40倍,占总POM差异的18%和19%。在＞10～20 cm土层有相似的趋势,但在＞10～20 cm层土壤粗iPOM的差异对总POM差异的贡献较0～10 cm层大。垄作免耕减少了对大团聚体的破坏并促进微团聚体向大团聚体团聚;降低了团聚体的周转速率,促进了细iPOM的固定,利于紫色水稻土对碳的固定和积累。     

An improved method for purifying genomic DNA from forest leaf litters and soil suitable for PCR

Background, aim and scope  An improving knowledge of bacterial community within natural environments including forest soils and leaf litters requires extraction of nucleic acids directly from environmental samples since molecular approaches provide less biased access to a larger portion of uncultivable microorganisms. However, when DNA was extracted successfully from these samples, it might still have been difficult to apply it as a template for polymerase chain reaction (PCR) amplifications due to the effect of PCR inhibitors. Various compounds from plant tissues including polysaccharides, phenolic compounds and especially humic acids can inhibit PCR amplification. Some of these inhibitors could inhibit PCR amplification by chelating the Mg²⁺ (cofactor for Taq polymerase), or by binding to target DNA, and PCR amplification would consequently be interfered with. Therefore, eliminating the effects of these PCR inhibitors is one of the most important steps for PCR-based molecular techniques. Four different methods were assessed in this study to purify the genomic DNA extracted from F, L layer leaf litters and forest soil in an exotic pine plantation of southeast Queensland, Australia. Materials and methods  Three samples including two leaf litters and one forest soil were collected with a core (25 × 40 cm) from a 22-year-old slash pine plantation in southeast Queensland, Australia. The DNA fragments were extracted directly using the Ultra Clean™ Mega Prep Soil DNA kit (Mo Bio Labs, Solana Beach, CA). Then, four different purification methods were applied and compared to purify the DNA for PCR amplification, which include PVPP, Sephadex ^TM spin column, low-melting agarose gel and a new modified gel purification method. The purified DNA from these four purification methods was detected by agarose gel electrophoresis, and the purity and usefulness of DNA samples were ultimately determined by successful PCR amplifications. Results and discussion  The DNA was extracted from each sample using the Ultra Clean™ Mega Prep Soil DNA kit, and the DNA eluents were dark in colour and sometimes formed compact aggregates. Subsequently, PCR amplification from such samples failed, although a series of dilutions had been made from neat to 1:10³. The DNA purification step could not, therefore, be avoided. It was observed that both the colour of eluent and the DNA concentration decreased gradually after elution. Considering the difficulties of removing PCR inhibitors and the possibility of high DNA losses, 50–200 μl of sample DNA was used for purification. Four DNA purification methods (the PVPP spin column, Sephadex™ spin column, low-melting agarose gel and the modified gel purification method) were applied and compared on leaf litter and soil samples. The DNA purified by the modified gel purification method provided the best PCR products for 16S rRNA gene amplification, but the other methods, PVPP, Sephadex™ spin column and low-melting agarose gel, produced very weak or no products. Thus, in this study, DNA fragments which were purified by the modified gel purification method were amplified efficiently. This may be attributed to running the low-melting agrose gel for a longer time, which could remove substantial humic substances and also some other compounds from the samples and, thus, prevent them from being involved in PCR amplification. Conclusions  A new modified gel purification method which can improve DNA purification and PCR amplification of environmental DNA is first introduced in this study. Comparing PVPP, Sephadex ™ spin column, low-melting agarose gel and modified gel purification method for the effect of DNA purification, the modified gel purification method is more successful in removing the PCR amplification inhibitors and obtaining the highly purified PCR amplifiable high-molecular-weight DNA. The method described here is cheap, fast and easy to operate. It suggests in this study that the method containing less and easier following steps should be widely used to relieve the heavy working load of molecular-biological researchers. Recommendations and perspectives  This study introduces a new modified DNA purification method, and it is found that this modified gel purification method is effective in removing the PCR inhibitors and obtains highly purified DNA from leaf litters for PCR amplification. The modified gel purification method may have wider applications, although it was only assessed on leaf litter and soil samples. The effect of the modified gel purification method on the DNA purification would need to be further investigated on a variety of samples which suffered from PCR inhibitors, such as clinical samples, plant tissues and environmental samples.     

土壤微生物多样性研究的DGGE/TGGE技术

分子生物学技术比传统的培养方法可得到土壤微生物种群多样性更全面的信息。变性梯度凝胶电泳(Denaturing gradient gel electrophoresis, DGGE)和温度梯度凝胶电泳(Temperature gradient gel electrophoresis, TGGE)可分离PCR扩增的DNA片段,已成为研究土壤微生物群落多样性的重要手段。本文综述了DGGE/TGGE技术在土壤微生物多样性研究中的应用进展,分析了该方法的主要影响因素及其优点和存在的问题。     

Real-time polymerase chain reaction quantification of the transgenes for roundup ready corn and roundup ready soybean in soil samples

A method for quantification of recombinant DNA for Roundup Ready (RR) corn and RR soybean in soil samples is described. Soil DNA from experimental field samples was extracted using a soil DNA extraction kit with a modified protocol. For the detection and quantification of recombinant DNA of RR corn and RR soybean, a molecular beacon and two pairs of specific primers were designed to differentially target recombinant DNA in these two genetically modified crops. Soil DNA extracts were spiked with RR corn or RR soybean DNA, and recombinant DNA was quantified using real-time PCR with a molecular beacon. As few as one copy of RR corn genome or one copy of RR soybean genome was detected in the soil DNA extract.     

Soil aggregation and bacterial community structure as affected by tillage and cover cropping in the Brazilian Cerrados

Microbial-based indicators of soil quality are believed to be more dynamic than those based on physical and chemical properties. Recent developments in molecular biology based techniques have led to rapid and reliable tools to characterize microbial community structures. We determined the effects of conventional and no-tillage in cropping systems with and without cover crops on bacterial community structure, total organic carbon (TOC) and soil aggregation. Tillage and rotation did not affect TOC from bulk soil. However, TOC was greater in the largest aggregate size class (7.98–19 mm), and had greater mean-weight diameter under no-tillage than under conventional tillage in the 0–5 cm soil layer. Soil bacterial community structure, based on denaturing gradient gel electrophoresis of polymerase chain reaction amplified DNA (PCR/DGGE) using two different genes as biomarkers, 16S rRNA and rpoB genes, indicated different populations in response to cultivation, tillage and depth, but not due to cover cropping. Soil bacterial community structure and meanweight diameter of soil aggregates indicated alterations in soil conditions due to tillage system.     

DNA- versus RNA-based denaturing gradient gel electrophoresis profiles of a bacterial community during replenishment after soil fumigation

We compared the responsiveness and sensitivity to soil fumigation of DNA- and RNA-based analyses of a bacterial community. We first established an improved RNA extraction method using DNA as an adsorption competitor, because it is extremely difficult to extract nucleic acids from clay-rich volcanic ash soil (Andisol), which adsorbs nucleic acids. This novel method facilitated RNA extraction from 500 mg of Andisol for molecular analyses. Then we monitored 16S rDNA PCR and 16S rRNA RT-PCR denaturing gradient gel electrophoresis (DGGE) profiles of samples collected from a chloropicrin (CP)-treated field over 2 months. The difference between untreated control and CP-treated plots was detected clearly both in DNA- and RNA-based DGGE profiles after treatment. The temporal changes in DGGE profiles, however, differed between DNA- and RNA-based analyses in CP-treated plots. RNA-based DGGE showed quicker and greater changes in the bacterial community after CP treatment than did DNA-based DGGE, which showed similar trends to RNA-based DGGE but with a time lag. The extent of decrease in the diversity index (H′) and the change in principal response curves was larger in RNA-based analyses. These results indicate that the rDNA PCR-DGGE method also detects DNA of microbes no longer alive after fumigation, and that rRNA provides a more responsive biomarker than rDNA.     

Optimizing the indirect extraction of prokaryotic DNA from soils

The objective of this work was to develop protocols to selectively extract prokaryotic DNA from soils, representative of the whole community, amenable to high-throughput whole genome shotgun sequencing. Prokaryotic cells were extracted from soils by blending, followed by gradient centrifugation. Detergent (sodium deoxycholate) was required for complete dispersion of soil aggregates and detachment of prokaryotic cells from a broad range of soil types. Repeated extractions of a given soil sample were critical to maximize cell yield. Furthermore, cells obtained through repeated extractions captured unique prokaryotic assemblages that would otherwise have been missed in single-pass extractions. DNA was isolated from extracted cells using one of the following treatments: i) lysozyme-SDS-proteinase K (enzymatic) digestion; ii) potassium ethyl xanthogenate digestion; or iii) enzymatic digestion of cells embedded in agarose plugs. In addition, these methods were compared to a commercial bead-beating extraction kit (MoBio UltraClean). Of the indirect DNA extraction methods, plug digestion generated the largest yields (up to 70% of yields obtained by direct DNA extraction) of high-molecular weight DNA (>400 kb). Thus, plug digestion is amenable to large-insert metagenomic library construction and analysis. Comparisons of banding patterns generated by RAPD-PCR and DGGE indicated that sequence composition and inferred community composition of a given extract varied greatly with DNA isolation method. While overall diversity did not change significantly with the cell lysis method, analysis of 16S rRNA gene clone libraries revealed that each extraction procedure produced unique distributions of prokaryotic phyla within the sample population.     

Assessment of nematode biodiversity using DGGE of 18S rDNA following extraction of nematodes from soil

Soil nematodes are both taxonomically and functionally diverse, respond quickly to soil perturbation and have much potential as indicators of soil health. However, because of the perceived difficulty of identifying nematodes to species level morphologically, they are frequently neglected in soil ecological studies. Recently, extraction of soil DNA, amplification of 18S rDNA genes using nematode consensus primers and subsequent separation by denaturing gradient gel electrophoresis (DGGE) has been used to estimate nematode diversity in soil. Here, we investigate an alternative approach whereby nematodes are first extracted from the soil prior the 18S rDNA gene amplification using universal primers. We used this system to estimate nematode species richness in 10 soil samples—five from Scotland and five from the Netherlands. There was no direct correlation between species richness as estimated morphologically and by the PCR-DGGE method. However, inspection of the data suggested that the samples fell into two discrete groups, which was confirmed by canonical and stepwise discriminant function analysis; the values for the Shannon and equitability indices being important discriminators. Further analysis revealed a significant relationship between morphological species richness and DGGE estimates for species that represented greater than 1% of the sample biomass.