固态发酵过程中微生物总DNA提取方法比较

为了分析固态发酵过程中微生物群落的多样性及演替情况,对比研究了从固态发酵中提取细菌和真菌DNA的3种方法--溶壁酶法、超声波法、液氮研磨 CTAB法.使用紫外分光光度计测定了由不同提取方法得到的DNA的产量与纯度;使用细菌16S rDNA基因通用引物(341F和907R)和真菌18S rDNA基因通用引物(NU-SSU-0817和NU-SSU-119)对DNA进行了PCR扩增;采用DGGE(变性梯度凝胶电泳)法对固态发酵中细菌和真菌的多样性进行了分析.结果显示,3种方法得到的粗提和纯化DNA长度均约为23 kb;细菌和真菌PCR产物长度分别约为586 bp和422 bp.细菌和真菌PCR产物的DGGE分析表明,3种方法提取的DNA所反映的微生物多样性比较一致;但紫外分光光度计测定结果表明溶壁酶法提取固态发酵中微生物总DNA产量最高,超声波法次之,液氮研磨 CTAB法最低.     

葡枝根霉(Rhizopus stolonifer)YF6脂肪酶基因的克隆及其序列分析

筛选并鉴定了1株高产脂肪酶菌株葡枝根霉YF6,并采用简并PCR等分子技术从中克隆到1个新的脂肪酶基因lipRs及其对应的全长cDNA,序列分析表明,该基因编码区全长1173bp,不含内含子序列,编码1段由26个氨基酸残基组成的信号肽和1个由365个氨基酸残基组成的成熟蛋白,该成熟蛋白计算分子量(Mr)为39268.27,等电点为pH7.66,有5个可能的N-糖基化位点,含有脂肪酶特征序列GHSLGGA,与来自米根霉(Rhizopus oryzae)的脂肪酶(AAF32408)同源性最高,一致性为83%,相似性为89%.该基因序列已提交GenBank,登录号为DQ139862.     

Detection of dioxygenase genes present in various activated sludge

GOAL, SCOPE AND BACKGROUND: Activated sludge from refineries contains various microorganisms that could utilize aromatics under aerobic conditions due to the oxygenase enzymes. Dioxygenase enzymes are oxygenases, which are involved in the ring cleavage step of aromatic hydrocarbons. In this study, the selected catabolic loci involved in ring cleavage have been monitored in the activated sludge samples at different time intervals. The investigation of the dioxygenase genes in the Effluent Treatment Plants (ETPs) and evaluation of their presence at different time points provides a clue for the aromatic utilizing potential of the inherent microbial flora. METHODS: The catabolic gene loci pheB, xylE, tod-isp, bed and nahG responsible for the enzymes catechol 1,2-dioxygenase, catechol 2,3-dioxygenase, toluene dioxygenase-iron-sulphur protein component, benzene dioxygenase and naphthalene dioxygenase were used respectively. The time dependent change in eubacterial population was demonstrated by the amplification of 16S rDNA product, followed by restriction digestion. The template DNA was obtained from the activated sludge collected from ETPs. The supporting physiological data for the overall performance of sludge was developed using respirometric analysis. The on-site COD and MLSS analysis for ETP was used in final evaluation. The study was carried out with samples collected from three different ETPs and also from a selected ETP at different time intervals. RESULTS AND DISCUSSION: The respirometric studies were carried out with phenol, catechol, toluene, and naphthalene to arrive at the target genotypes for further study by PCR protocol. The respirometric analysis coupled with the COD and MLSS analysis represented the physiological capacity of the various sludges. Initially, the tracking protocol was optimized by using different sludge samples, which were collected from refineries. The selected genotypes were amplified and their presence has been confirmed using Southern analysis. The gene loci tod-isp, bed and xylE were commonly observed at various time intervals of the sludge from the same source. The gene loci pheB and nahG were found to be relatively rare. CONCLUSION: The 16S rDNA PCR products after restriction digestion produced different DNA fingerprint patterns, suggesting that the microbial community composition was diverse in the three sources. Similarly, the presence of the catechol 2,3-dioxygenase, benzene dioxygenase and toluene dioxygenase genes confirmed the aromatic degrading potential in the various sludges. The probes could not pick the nahG and pheB genes. However, the respirometeric assay suggested that the oxidative capacity to use naphthalene as a substrate exists. RECOMMENDATION AND PERSPECTIVE: Our study of the diversity at various time points from the ETP provided an overview of the shifts of the catabolic composition of the sludge. This also depends on the influential parameters like the incoming pollutant level and the environmental conditions that are prevailing and often changing from time to time. The results of direct DNA extraction and PCR amplification do reflect the relative abundance of a particular catabolic genotype, which could be used to monitor the efficiency of treatment.     

环境因素对不同工艺人工湿地反硝化功能基因丰度影响

反硝化细菌在人工湿地对N元素去除的过程中发挥着关键作用.为了解湿地类型、基质、植被和水力负荷及物理参数对人工湿地反硝化功能细菌丰度的影响,构建了9个不同工艺组合的人工湿地小试装置.利用实时荧光定量PCR技术检测湿地基质中反硝化功能基因nirS,nirK和nosZ的丰度.结果显示,湿地中nirS,nirK和nosZ丰度(以每ng总DNA中的拷贝数计)分别为4.88×103~2.13×105 copies/ng,1.2×101~2.14×103 copies/ng和1.98×103~2.09×105 copies/ng, 其中nirS显著高于nirK丰度, nirS丰度与湿地出水的pH显著正相关.分析湿地4种设计参数对反硝化功能基因丰度的影响程度,结果显示,对nirS丰度影响程度大小依次为湿地类型>植被类型>水力负荷>基质类型,水平潜流和上行垂直流人工湿地中nirS丰度显著高于下行垂直流人工湿地,无植物人工湿地nirS丰度显著高于有植物人工湿地;对nirK丰度影响程度大小依次为湿地类型 >植被类型 >基质类型 >水力负荷,水平潜流人工湿地nirK丰度显著高于下行垂直流湿地;对nosZ丰度影响程度大小依次为湿地类型 >水力负荷 >植被类型 >基质类型,但影响都不显著.     

Potential dissemination mechanism of the tetC gene in Aeromonas media from the aerobic biofilm reactor under oxytetracycline stresses

The tetC gene has been found to be one of the most widely distributed tetracycline resistance (tet) genes in various environmental niches, but the detailed dissemination mechanisms are still largely unknown. In the present study, 11 tetC-containing Aeromonas media strains were isolated from an aerobic biofilm reactor under oxytetracycline stresses, and the genome of one strain was sequenced using the PacBio RSII sequencing approach to reveal the genetic environment of tetC. The tetC gene was carried by an IS26 composite transposon, named Tn6434. The tetC-carrying Tn6434 structure was detected in all of the A. media strains either in a novel plasmid pAeme2 (n=9) or other DNA molecules (n=2) by PCR screening. The NCBI database searching result shows that this structure was also present in the plasmids or chromosomes of other 13 genera, indicating the transferability of Tn6434. Inverse PCR and sequencing confirmed that Tn6434 can form a circular intermediate and is able to incorporate into a preexisting IS26 element, suggesting that Tn6434 might be responsible for the dissemination of tetC between different DNA molecules. This study will be helpful in uncovering the spread mechanism of tet genes in water environments.     

荧光定量PCR技术在环境领域的应用

实时荧光定量PCR技术作为一项新兴技术,越来越受到人们的重视,它以快速、准确定量、便捷等优点广泛应用于科学研究各个领域。近年来,实时荧光定量PCR技术不断发展,并应用于环境领域中,大大提高了环境监测水平。综述了实时荧光定量PCR技术的基本原理及其在国内外环境领域中的应用。     

Location and PCR analysis of catabolic genes in a novel Streptomyces sp. DUT AHX capable of degrading nitrobenzene

A novel strain of Streptomyces sp.DUT_AHX was isolated from sludge contaminated with nitrobenzene and identified on the basis of physiological and biochemical tests and 16S ribosomal DNA (rDNA) sequence analysis.The optimal degradation conditions were as follows:temperature 30℃,pH 7.0-8.0,shaking speed 150-180 r/min,and inocula 10%(V/V).The strain,which possessed a partial reductive pathway with the release of ammonia,was also able to grow on mineral salts basal (MSB) medium plates with 2-aminophenol, ph...     

PCR技术检测水中轮状病毒的研究进展

聚合酶链式反应,即PCR(polymerase chain reaction)技术,是一种快速扩增DNA或cDNA序列的方法.水环境中的轮状病毒是导致世界范围内婴幼儿腹泻的主要原因,严重危害着水质安全和人类健康.建立和完善水中轮状病毒灵敏、快速的检测方法对预防和控制水体疾病的暴发具有重要的意义.随着分子生物学技术的发展,PCR及其衍生技术因其具有高度灵敏性、特异性和准确性等特点,成为检测水中轮状病毒最常用的方法.本文综述了利用RCR及其衍生技术,包括逆转录聚合酶链式反应、逆转录半巢式和巢式聚合酶链式反应、多重逆转录聚合酶链式反应、免疫聚合酶链式反应、与免疫磁珠分离相结合的聚合酶链式反应、与细胞培养相结合的聚合酶链式反应及实时定量聚合酶链式反应等,检测水环境中轮状病毒的研究进展,并分析了这些PCR技术在检测水环境中轮状病毒时存在的问题及其应用前景.     

甘蔗蔗糖磷酸合成酶SPSⅡ cDNA片段克隆与表达分析

蔗糖磷酸合成酶(Sucrose phosphate synthase,SPS)是蔗糖合成途径的关键限速酶,在蔗糖积累和碳分配中具有重要作用.在茎组织中SPSⅡ表达量占SPS转录总量的40%,表明SPSⅡ cDNA的克隆与表达对蔗茎中蔗糖的积累有着重要的影响.通过RT-PCR技术克隆分离SPSⅡ基因cDNA片段.序列分析表明该cDNA片段包含1个3 183 bp的开放读码框,可编码1 060个氨基酸.GenBank登录号为EU269038.通过实时荧光定量PCR检测SPSⅡ基因在甘蔗糖分积累的不同时期、不同组织部位的相对表达量.结果表明,在糖分积累初期蔗茎中的SPSⅡ相对表达量最大,在糖分积累中期蔗叶中的SPSⅡ相对表达量达到最高峰;同组织部位中,糖分积累初期SPSⅡ相对表达量高于糖分积累中期、后期.图6参11     

亚硝酸细菌研究进展

从硝化细菌的生长特性 ,分类 ,检测 ,亚硝酸细菌氨单加氧酶等方面概要的叙述了国外在硝化细菌方面研究的进展 ,并展望了以后的研究和应用