脉冲场凝胶电泳技术及其在真菌学研究中的应用*

脉冲电泳是用于分离大分子量DNA的一种电泳技术,已广泛用于真菌的核型分析,种群特异性鉴定,基因定位及遗传分析的研究。介绍了脉冲电泳的原理,发展和基本操作程序,并阐述了脉冲电泳技术在真菌分子生物学研究领域的应用。     

毛细管电泳在基因研究中的应用

讨论用于基因研究的各种方法,并指出毛细管电泳技术的优点。评述毛细管电泳(CE)用于基因分析的原理,详细介绍毛细管电泳在基因研究中的应用新进展。展望CE及其在基因研究的应用前景。     

利用脉冲电泳技术转化玉米获得转基因植株的研究

本研究采用脉冲电泳技术将外源Bar基因和B-t基因转化玉米种胚并直接成苗,通过除草剂抗性初步筛选,再进行PCR检测、Southem杂交验证,探讨了脉冲电泳介导外源基因直接转化玉米种胚的有效性,结果表明：脉冲电泳技术介导玉米转基因是有效可行的,其T0代植株PCR阳性率与T1代植株转化率分别为11．36％和1．04％。在外源基因的转化中,处于不同启动子下的B-t基因和Bar基因共转化频率为90．9％。T1代PCR表现为阳性的植株Southem杂交结果显示脉冲电泳法介导的外源基因在受体中多为单拷贝,且已遗传给后代,并获得稳定表达。     

酿酒酵母染色体DNA样品制备和CHEF分析

等高锁状均匀电场（contolllsclampedhomogeneouselectncfield,CHED电泳技术与分子杂交、酵母人工染色体（yeastartificialchromosome,YAC）等分子生物学技术的有机结合,推动了真菌电泳核型、染色体作图和染色体DNA长度多型性（chromosomalDNAlellgthpolymorphism,CLP...     

基于智能控制的微管电泳检测技术

国内外毛细管电泳(CE)产品深受电泳焦耳热存在的影响,使得毛细管电泳试样的流通量非常少,严重影响到了CE发展与应用。本文对毛细管电泳技术存在的问题进行进一步的量化分析,利用智能控制技术来构造微管电泳检测系统,采用内制冷的方式,及时原地的带走电泳过程中产生的焦耳热,采用多通道高压电源作为整个电泳系统的驱动力,通过对运行缓冲液的合理调控,实现对待分离组分的高效分离。研究出高效微管电泳技术的全分析系统模型,以期能给生命科学、材料科学、环境科学提供又一强有力的研究手段。     

自动化激光散射外差电泳术

在经典电泳法中,电泳技术分成大分子电泳及显微(细胞)电泳两大类,实验装置及观察方法都有区别。而自动化激光外差电泳术却可用同一装置完成上述两类测量,除能同时测量扩散系数外,还可能完成反应动力学方面的测量。自动化激光散射外差电泳术是由激光散射外差探测术及电泳术组合而成的。70年代初期Ware及Uzgiris以略为不同的方法独立发表了实验及理论研究。按经典分类法,Ware是从大分子电泳开始,而Uzgiris则是从显微电泳开始,并已用于蛋白质、胶体、细胞学及免疫学研究。最近,Malher等报告了结构略为不同的类似装置。     

薄膜电泳

电泳方法是对生物组成成份的分离、分析不可缺少的技术。近年来相继出现了薄膜电泳,聚丙烯酰胺凝胶电泳,等电聚焦等新的电泳技术。应用有支持物的电泳技术,不仅能鉴别和分析构成生物体的成份和生物大分子,同时对细胞的分离和纯化方面的应用也不断有发展。由于薄膜电泳所用的装置和用具可以自己制做,加上操作简便迅速,因而这样的电泳方法就成为生化研究中的常规重要技术之一。这种用支持物的电泳也叫区带电泳。薄膜电泳主要是用纤维素薄膜     

PFGE在真菌基因研究中的应用

IPFGE与连锁群从酿酒酵母脉冲电场电泳研究到医学上重要酵母类真菌的分类,人们已逐渐建立了参数随菌种各异的PFGE电泳条件,以适于不同大小染色体的分离,获得了一系列酵母类及丝状真菌,包括四大纲以及非典型真菌的染色体核型及基因组大小（表1）,并利用已知基因探针进行染色体连锁群的定位。1987年,Sndth等l’吩离得到了粟酒裂殖酵母的3条巨大染色体,使PFGE的分辨能力达到Tgmb,极大促进了该菌株的基因图谱工作的进展。紧接着,他们又得到了假丝酵母类具有种特异性的电泳核型,并成为分子流行病学中酵母类致病真菌的分型依据。…     

双水相电泳分离蛋白质的研究

近几年来,随着生物技术的迅速发展,制备型电泳技术的研究得到了重视。然而由于技术上的原因,大规模的制备型电泳技术的研究还未能取得突破。阻碍电泳放大的一个主要问题是由于电加热作用而导致的热对流对电泳分离的破坏。为解决这一问题,人们提出了许多方法。例如,在太空的微重力环境下进行电泳,应力稳定自由流动电泳,循环等电聚焦和区带电泳,色谱电泳和等电膜等电聚焦等。这些方法在电泳放大上都取得了一定的进展,但各有其局限性。最近,Clark提出利用双水相的液液界面阻止热对流的设想,为开发大规模的制备型电泳技术开辟了一条新途径、Raghava Rao等在两种双水相体系上施加电场后成倍地缩短了分相时间。Levine和Bier采用U型管电泳装置研究了双水相体系中血红蛋白的电泳迁移率,观测到界面有阻滞作用。Clark在柱型电泳装置中进行了一组双水相萃取肌红蛋白的简单实验。在10mA的恒电流下电泳40min之后,肌红蛋白的分配系数为7.5,而当电场反向后,分配系数变为0.04,界面阻力并不显著,两者结论并不一致。     

SDS-PAGE、同工酶等电聚焦和RAPD标记鉴定两系杂交水稻F1种子纯度对比研究

目的：探讨利用种子贮藏蛋白SDS—PAGE电泳、酯酶同工酶超薄等电聚焦电泳和RAPD分子标记鉴定五个两系杂交水稻组合F_1种子纯度的可行性。方法和结果：利用SDS—PAGE电泳技术未能找到所试五个组合各自的父本特征蛋白质带;利用酯酶同工酶超薄等电聚焦电泳技术和RAPD分子标记可找到所试五个组合的父母本特征酶带和RAPD标记,但酯酶同工酶的多态性同时也受种子萌发时间的影响。酯酶同工酶超薄等电聚焦电泳和RAPD分子标记可用于所试五个两系杂交水稻组合F_1种子纯度的鉴定。     

Genetic diversity in Swiss cheese starter cultures assessed by pulsed field gel electrophoresis and arbitrarily primed PCR

AIMS: To assess intraspecific genetic heterogeneity among commercial Swiss cheese starter culture strains of Lactobacillus helveticus, Streptococcus thermophilus and Propionibacterium freudenreichii and to compare the efficacy of two genetic typing methods. METHODS AND RESULTS: Two genetic typing methods, pulsed field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR), were used. Nine Strep. thermophilus strains revealed eight PFGE and five AP-PCR genotypes. Seventeen Lactobacillus strains yielded 16 and five genotypes by PFGE and AP-PCR, respectively. Eleven Propionibacterium strains yielded 10 PFGE genotypes. Cluster analysis of PFGE profiles generated similarity coefficients for Strep. thermophilus, Lact. helveticus and Prop. freudenreichii strains of 29.5%, 60.3%, and 30.5%, respectively. Milk acidification rates for Strep. thermophilus and Lact. helveticus were determined. CONCLUSIONS: Pulsed field gel electrophoresis is more discriminatory than AP-PCR. The Lact. helveticus group is more homogeneous than the other species examined. Strains with > 87% similarity by PFGE consistently had the same acidification rate and AP-PCR profile. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial strains sold for Swiss cheese manufacture in the United States are genetically diverse. Clustering of genetically related bacteria may be useful in identifying new strains with industrially relevant traits.     

Pulsed-field gel electrophoresis in the study of mesophilic and psychrophilic Aeromonas spp.

Pulsed-field gel electrophoresis (PFGE) was used to study the genetic diversity of mesophilic Aeromonas hybridization group (HG) 1, HG 2, HG 3, HG 4, HG 5, HG 6, HG 7, HG 8/10and HG 11, psychrophilic Aeromonas salmonicida subsp. salmonicida and atypical Aerom. salmonicida strains. Xba I was chosen for restriction because it producedfragments whose numbers and size were appropriate for PFGE analysis of all studied HGs. Allmesophilic Aeromonas strains within an HG had different banding patterns. No sharedbands which could be used for identification of an HG were found. Pulsed-field gelelectrophoresis analysis further confirmed the known genetic homogeneity of Aerom.salmonicida subsp. salmonicida . Pulsed-field gel electrophoresis pattern analysissuggested that the genomic size of Aerom. salmonicida subsp. salmonicida issmaller than that of mesophilic Aeromonas spp. or atypical Aerom. salmonicida . Aeromonas salmonicida subsp. salmonicida had only one large restriction fragment (310kb) and lacked other large fragments (>160 kb). Although the PFGE patterns of atypical Aerom. salmonicida resembled the banding patterns of mesophilic Aeromonas spp.they had several small fragments (15–50 kb) shared with Aerom. salmonicida subsp. salmonicida suggesting genetic relatedness.     

Rapid discrimination of environmental Vibrio by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

The aim of this study was to discriminate 30 Vibrio strains isolated from two wastewater treatment plants from Agadir, Morocco by two molecular typing methods, pulsed-field gel electrophoresis (PFGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Out of the 30 strains of Vibrio examined in this study, 5 isolates could not be typed by PFGE and consistently appeared as a smear on the gel. In general, high genetic biodiversity among the Vibrio strains was found regardless to the isolation source. The results of MALDI TOF analysis show a high congruence of strain grouping demonstrating the accuracy and reliability of MALDI-TOF MS.     

Molecular characterization of chromosome termini of the arbuscular mycorrhizal fungus Glomus intraradices (Glomeromycota)

The minimum chromosome number of Glomus intraradices was assessed through cloning and sequencing of the highly divergent telomere-associated sequences (TAS) and by pulsed field gel electrophoresis (PFGE). The telomere of G. intraradices, as in other filamentous fungi, consists of TTAGGG repeats, this was confirmed using Bal31 nuclease time course reactions. Telomere length was estimated to be roughly 0.9 kb by Southern blots on genomic DNA and a telomere probe. We have identified six classes of cloned chromosomal termini based on the TAS. An unusually high genetic variation was observed within two of the six TAS classes. To further assess the total number of chromosome termini, we used telomere fingerprinting. Surprisingly, all hybridization patterns showed smears, which demonstrate that TAS are remarkably variable in the G. intraradices genome. These analyses predict the presence of at least three chromosomes in G. intraradices while PFGE showed a pattern of four bands ranging from 1.2 to 1.5 Mb. Taken together, our results indicate that there are at least four chromosomes in G. intraradices but there are probably more. The information on TAS and telomeres in the G. intradicies will be essential for making a physical map of the G. intraradices genome and could provide molecular markers for future studies of genetic variation among nuclei in these multigenomic fungi.     

Characterization of Erwinia amylovora strains from Bulgaria by pulsed-field gel electrophoresis

The aim of this study was to characterize genetically Bulgarian Erwinia amylovora strains using pulsed-field gel electrophoresis (PFGE) analysis. Fifty E. amylovora strains isolated from different hosts, locations, as well as in different years were analysed by PFGE after XbaI, SpeI, and XhoI digestion of the genomic DNA. The strains were distributed into four groups according to their XbaI-generated profile. About 82% of the strains displayed a PFGE profile identical to that of type Pt2. Three strains belonged to the Central Europe Pt1 type. Two new PFGE profiles, not reported so far, were established--one for a strain isolated from Malus domestica and another for all Fragaria spp. strains. The same grouping of the strains was obtained after analysis of the SpeI digestion patterns. On the basis of PFGE profiles, after XbaI and SpeI digestion, a genetic differentiation between the strains associated with subfamily Maloideae and subfamily Rosoideae was revealed. The presence of more than one PFGE profile in the population of E. amylovora in Bulgaria suggests a multiple source of inoculum.     

Genotypic and phenotypic differentiation of an antifungal biocontrol strain belonging to Bacillus subtilis

Physiological and molecular fingerprints of biotechnologically relevant rhizobacteria are necessary for registration, patenting, recognition and quality checking of the strains. To characterize the biological control agent, Bacillus subtilis B2g, the strain was compared with other plant-associated B. subtilis isolates. Phenotypic characterization included biochemical and nutritional properties, in vitro activity and analysis of potential antagonistic mechanisms towards several plant pathogenic fungi. According to the phenotypic characteristics, it was not possible to differentiate the biocontrol agent from the other strains, although the enzymatic fingerprint was unique. Genotypic diversity among the isolates was characterized by molecular fingerprinting methods using REP-PCR (repetitive extragenomic palindromic PCR), and macrorestriction of genomic DNA and electrophoretic separation of DNA fragments by pulsed-field gel electrophoresis (PFGE). A protocol for PFGE analysis using restriction enzyme SfiI for B. subtilis was developed. PFGE typing of B. subtilis B2g resulted in a unique fingerprint. Therefore, it was possible to differentiate B. subtilis B2g, the biocontrol agent of Phytovit, from other antifungal B. subtilis isolates.     

Genetic diversity of Salmonella enterica serovar Paratyphi A from different geographical regions in Asia

AIMS: Subtyping of Salmonella Paratyphi A isolates from India, Pakistan, Indonesia and Malaysia was carried out by pulsed-field gel electrophoresis (PFGE) to assess the extent of genetic diversity of these isolates from different endemic countries. METHODS AND RESULTS: A total of 39 human isolates of Salmonella Paratyphi A from Pakistan, India, Indonesia and Malaysia were studied using PFGE analysis following digestion of chromosomal DNA with XbaI. Seven isolates from Pakistan were resistant to ampicillin, tetracycline and cotrimoxazole. It was noted that Salmonella Paratyphi A isolates obtained from outbreaks in India had limited genetic diversity and probably belonged to closely related clones. Significant genetic homogeneity was observed among antimicrobial-resistant isolates from Pakistan and antimicrobial-sensitive isolates from Pakistan and Indonesia, respectively. CONCLUSIONS: PFGE was a useful subtyping technique to differentiate Salmonella Paratyphi A from different endemic countries. However, it fails to differentiate the antimicrobial-resistant and -sensitive strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the present study verify the usefulness of PFGE in characterizing and comparing strains of Salmonella Paratyphi A. Our study suggests that a limited number of clones are responsible for paratyphoid fever in these countries.     

Differentiation of Citrobacter strains using chromosomal DNA restriction patterns

The aim of this study was checking of the usefulness of chromosomal DNA restriction patterns in differentiation of Citrobacter strains. Molecular characterization of total 56 isolates of Citrobacter from Poland and Czech Republic, was performed by pulsed-field gel electrophoresis after digestion of chromosomal DNA with restriction endonuclease Xba I (5'-TCTAGA-3'). Chromosomal DNA of all tested Citrobacter strains gave after electrophoresis 12 to 21 bands and patterns consisting of 12 to 21 fragments ranging in size from 790 kb to 48.5 kb and smaller, which where not distinguishable. Pulsed-field gel electrophoresis patterns were useful for comparing Citrobacter strains. Identical restriction patterns generated by PFGE were observed in the case of selected strains e.g. strains C. sedlakii studied in this study, coming from an outbreak, having the some phenotype. In addition, PFGE patterns can be used to evaluate the clonal relatedness among bacterial isolates. PFGE can be helpful for assessing genetic relatedness among strains epidemiologicaly unrelated e.g. C. werkmanii strains tested in this study. The sum of DNA fragments after Xba I digestion indicates the genome size of Citrobacter strains. This suggests that PFGE should be useful for epidemiological investigations of Citrobacter strains.     

Pulsed-field gel electrophoresis and PCR characterization of environmental Vibrio parahaemolyticus strains of different origins

The present study used pulsed-field gel electrophoresis (PFGE) characterization to examine the intraspecies variability and genetic relationships among environmental isolates of Vibrio parahaemolyticus from different European countries. This is first study performed on environmental V. parahaemolyticus that included more than one European country.