满江红鱼腥藻与其宿主的遗传多样性和协同性的RAPD分析

从16个代表不同种属或地域来源的满江红样本中分离出共生藻并通过处理获得无藻的满江红宿主,对二者同步进行了RAPD扩增,分别得到了大量DNA多态片段。通过建立满江红鱼腥藻及其宿主的UPGMA聚类关系图,看出二者在遗传分支上存在着一定程度的协同对应关系。但在种内的不同品系间,这种协同性有所减少,发现有的品系的共生藻发生了明显的变异。 Abstract:Symbiotic Anabeana azollae and its host plant Anabeana-free Azolla were isolated from 16 Azolla accessions representing different Azolla species or geographic origins.DNA polymorphic fragments were obtained by simultaneous RAPD amplification of both symbiont and host.The UPGMA clusters of Anabeana azollae and its host Azolla were established separately based on Dice coefficient caculation and a coordinated relationship was shown between Anabeana azollae and its Azolla host along both individual genetic divergence,but this genetic homology was reduced among different strains within Azolla species while the obvious mutants of Anabeana azollae were detected in some Azolla tested strains collected from different geographic area in the same host species.     

稗草致病菌——尖角突脐孢菌菌株RAPD指纹图谱的分析

以我国主要稻区的稗草植株上分离的17株尖角突脐孢菌菌株为试验材料,采用改良的SDS法提取其基因组DNA,并运用优化的RAPD分析体系对其进行了分子标记遗传差异研究。从25个随机引物中筛选出20个扩增效果好的引物,对全部试验材料进行了RAPD扩增,共得到239条有效带,其中多态性带229条（占95.8%）。依据扩增结果建立了17株尖角突脐孢菌基因型的DNA指纹图谱并对其进行了有效区分。根据RAPD分析结果计算了菌株间的遗传距离,分析了它们的遗传差异并进行了聚类分析,结果表明,RAPD分子标记技术是能够用于杂草致病菌资源的鉴定的,并可以进一步应用于特定性状的基因标记研究。     

Sensitive and specific detection of Xanthomonas campestris pv. pelargonii with DNA primers and probes identified by random amplified polymorphic DNA analysis.

The random amplified polymorphic DNA method was used to distinguish strains of Xanthomonas campestris pv. pelargonii from 21 other Xanthomonas species and/or pathovars. Among the 42 arbitrarily chosen primers evaluated, 3 were found to reveal diagnostic polymorphisms when purified DNAs from compared strains were amplified by the PCR. The three primers revealed DNA amplification patterns which were conserved among all 53 strains tested of X. campestris pv. pelargonii isolated from various locations worldwide. The distinctive X. compestris pv. pelargonii patterns were clearly different from those obtained with any of 46 other Xanthomonas strains tested. An amplified 1.2-kb DNA fragment, apparently unique to X. campestris pv. pelargonii by these random amplified polymorphic DNA tests, was cloned and evaluated as a diagnostic DNA probe. It hybridized with total DNA from all 53 X. campestris pv. pelargonii strains tested and not with any of the 46 other Xanthomonas strains tested. The DNA sequence of the terminal ends of this 1.2-kb fragment was obtained and used to design a pair of 18-mer oligonucleotide primers specific for X. campestris pv. pelargonii. The custom-synthesized primers amplified the same 1.2-kb DNA fragment from all 53 X. campestris pv. pelargonii strains tested and failed to amplify DNA from any of the 46 other Xanthomonas strains tested. DNA isolated from saprophytes associated with the geranium plant also did not produce amplified DNA with these primers. The sensitivity of the PCR assay using the custom-synthesized primers was between 10 and 50 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

多聚酶链反应技术鉴别肾综合征出血热病毒血清型的初步研究

肾综合征出血热(HFRS)为一组抗原性密切相关的布尼亚科汉坦病毒引起的急性传染病。在我国存在至少两种临床表现、动物宿主及流行特征截然不同的血清型别,即血清Ⅰ型(汉坦型)和血清Ⅱ型(汉城型)。这两型病毒间的血清学定型已有报道。近年来,除啮齿类动物外,从临床病人以及非啮齿类动物体内也分离到了HFRS病毒。同时出现两类型别毒株共存,以及从家鼠体内分离到野鼠型毒株或从野鼠体内分离到家鼠型毒株的复杂情形。为此,准确检定并鉴别不同来源毒株型别,将为深入了解其病原学、流行病学以及制定疫苗生产策略提供重要信息。     

Detection of the patulin-producing potential of some <Emphasis Type="Italic">Paecilomyces variotii</Emphasis> strains isolated from the air samples of Jeddah City,Saudi Arabia,using the RAPD-PCR technique

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis was used to detect the patulin-producing potential among seven different strains of Paecilomyces variotii isolated from air samples collected in Jeddah City, Saudi Arabia. Using five different primers, the strains showed some similarities and distinct RAPD patterns. A correlation between isolation source and clustering was noted in the constructed dendrogram. Patulin-producing strains showed identical RAPD patterns. Primer M13 produced a distinct fragment with toxin-producing strains.     

Differentiation of Erwinia carotovora subsp. atroseptica and carotovora by RAPD-PCR

Six different 10-mer oligonucleotide primers were used to differentiate Erwinia carotovora subsp. atroseptica (Eca) and carotovora (Ecc) using RAPD-PCR. All primers gave different banding patterns for Eca and Ecc indicating their value for identification. UPGMA clustering analysis clearly showed two separate clusters, one for Eca and the other for the Ecc group. Similarity within Eca strains was very high, over 85% among most isolates but within the Ecc group extensive genetic diversity was found and many of the Ecc strains were no more than 50% similar. Similarity between the 10 Eca and 10 Ecc strains was generally only 10–25% based on the results from six primers. Three RAPD fragments from Eca group, which were amplified by three different RAPD primers, were isolated and used as probes for Southern hybridisation to test, if homologous fragments were amplified from Ecc strains. All these probes hybridised only with Eca isolates indicating that these fragments could be useful in order to develop a PCR-based detection system for Eca strains.     

随机扩增多态性DNA技术在鲍氏层孔菌菌株鉴别中的应用

用20个随机引物对7个不同来源的鲍氏层孔菌菌株进行了RAPD分析．结果表明120个随机引物中,有17个引物的扩增产物DNA条带表现出明显的多态性,不同引物对供试菌株扩增出现的DNA条带数目少则10条,多达33条．DNA片段从250bp到2000bp;采用17个引物对7个鲍氏层孔菌菌株共扩增出DNA片段带377条,不同引物扩增出的DNA片段谱带存在较大差异．采用UPGMA系统聚类法,将7个菌株聚类为两大类,能直观准确地揭示菌株间的差异并加以鉴别．     

The uses of AFLP for detecting DNA polymorphism, genotype identification and genetic diversity between yeasts isolated from Mexican agave-distilled beverages and from grape musts

AIMS: The objectives were to determine the variability and to compare the genetic diversity obtained using amplified fragment length polymorphism (AFLP) markers in analyses of wine, tequila, mezcal, sotol and raicilla yeasts. METHODS AND RESULTS: A molecular characterization of yeasts isolated from Mexican agave musts, has been performed by AFLP marker analysis, using reference wine strains from Italian and South African regions. CONCLUSIONS: A direct co-relation between genetic profile, origin and fermentation process of strains was found especially in strains isolated from agave must. In addition, unique molecular markers were obtained for all the strains using six combination primers, confirming the discriminatory power of AFLP markers. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of molecular characterization between yeasts isolated from different Mexican traditional agave-distilled beverages, which shows high genetic differences with respect to wine strains.     

Evaluation of molecular typing techniques to assign genetic diversity among Saccharomyces cerevisiae strains.

Discrimination of strains within the species Saccharomyces cerevisiae was demonstrated by the use of four different techniques to type 15 strains isolated from spoiled wine and beer. Random amplified polymorphic DNA with specific oligonucleotides and PCR fingerprinting with the microsatellite oligonucleotide primers (GAC)5 and (GTG)5 enabled discrimination between the strains tested. Additionally, restriction enzyme analysis, with TaqI and MseI, of PCR-amplified fragments from the complete internal transcribed spacer and nontranscribed spacer, both present in the rRNA-encoding gene cluster, proved to be suitable for generating intraspecies-specific patterns. Random amplified polymorphic DNA with primers 24 and OPA-11 and PCR fingerprinting with primer (GTG)5 appeared to generate the highest degree of diversity. However, the results indicated that there was no single PCR-mediated typing technique enabling discrimination on the strain level. Discrimination of each individual strain was nevertheless possible by combining the results obtained with all typing techniques.     

Identification of a universally primed-PCR-derived sequence-characterized amplified region marker for an antagonistic strain of Clonostachys rosea and development of a strain-specific PCR detection assay

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.     

Specific identification and molecular typing analysis of Lactobacillus johnsonii by using PCR-based methods and pulsed-field gel electrophoresis

A fast and reliable Multiplex-PCR assay was established to identify the species Lactobacillus johnsonii. Two opposing rRNA gene-targeted primers have been designed for this specific PCR detection. Specificity was verified with DNA samples isolated from different lactic acid bacteria. Out of 47 Lactobacillus strains isolated from different environments, 16 were identified as L. johnsonii by PCR. The same set of strains was investigated with five alternative molecular typing methods: enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), repetitive extragenic palindromic PCR (REP-PCR), amplified fragment length polymorphism, single triplicate arbitrarily primed PCR, and pulsed-field gel electrophoresis in order to compare the discriminatory power of these methods. The reported data strongly support the highly significant heterogeneity among all L. johnsonii isolates, potentially linked to their origin of isolation. The use of species-specific primers as well as rapid and highly powerful PCR-based molecular typing tools (namely ERIC- and REP-PCR techniques) should be respectively envisaged for identifying, differentiating and monitoring L. johnsonii strains from various environmental samples, for product monitoring, for species tracing in clinical studies as well as bacterial profiling of various microecological or gastrointestinal environments.     

鱼腥藻类囊体膜及其性质的研究

研究了鱼腥藻(Anabaena azollae Strasb.)的光合膜及其性质,结果如下:(1)以培养液为反应介质,测出鱼腥藻细胞的光合放氧,这种放氧受电子传递抑制剂DCMU的抑制。1mmol/L的NH_4Cl既延迟光合放氧的诱导期又抑制光合放氧速率。(2)在700～300nm波长范围,鱼腥藻出现4个吸收高峰,分别位于680、625、480和440nm处。其中625nm的吸收峰应属蓝绿藻的特有吸收峰。(3)通过高压氮气破碎法,并结合超声波处理,可以破碎鱼腥藻细胞壁,并从中分离出鱼腥藻类囊体膜制剂。测定表明,这种膜制剂具有进行光合磷酸化的能力,同时亦有膜上ATP酶水解ATP的能力。(4)利用此膜制剂,分离并部分纯化出ATP酶,在SDS-PAGE图谱上,此酶的两种小亚基(δ和ε亚基)的分子量分别低于菠菜叶绿体ATP酶的δ和ε亚基,但两种酶的α和β两种大亚基的分子量相近。以上结果提示,鱼腥藻具有进行光合作用的内在机构,这种机构在组分及其性质上与其它种类光合膜的异同是值得深入研究的课题。     

Identification of a Universally Primed-PCR-Derived Sequence-Characterized Amplified Region Marker for an Antagonistic Strain of Clonostachys rosea and Development of a Strain-Specific PCR Detection Assay

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.     

Use of randomly amplified polymorphic DNA analysis to differentiate isolates of Vibrio parahaemolyticus from cockles (Anadara granosa)

A total of 35 Kanagawa-negative strains of Vibrio parahaemolyticus isolated from cockles (Anadara granosa) were investigated by randomly amplified polymorphic DNA fingerprinting with three primers and their plasmid profiles. Eighteen strains carried small plasmid(s) of 2.4 to 7.3kb that enabled the V. parahaemolyticus to be grouped into eight plasmid patterns. The three primers generated polymorphisms in all 35 strains of V. parahaemolyticus tested, producing bands ranging from 0.25 to 3.9kb. The RAPD profiles revealed a high level of DNA sequence diversity within the Vibrio parahaemolyticus strains tested, and that cockles in the study area are populated by genetically polymorphic strains of V. parahaemolyticus.     

两株假单胞菌菌株的RAPD分析及分子生物学鉴定

根据DNA随机扩增多态性(RandomAmplifiedPolymorphicDNA,简RAPD)分子标记技术设计鉴别引物,建立一种快速、准确检测病人体内新发现的假单胞菌菌株的分子生物学方法.采用RAPD分析方法对该菌种的对照菌株AcinetobactercalcoaceticusKHW14(简称A.calcoaceticusKHW14)和新分离的菌株Acinetobactercalcoaceticus(简称A.calcoaceticus)进行指纹分析,依据两菌株的差异序列设计两对引物,并建立最佳的PCR扩增体系,产物经1.2%琼脂糖凝胶电泳得菌株特异性电泳图谱.此图谱可作为鉴定两菌株的标准图谱,RAPD分析方法具有良好的重复性,同时也进一步验证了两菌株的同源性.     

Design of PCR primers and a gene probe for extensive detection of poly(3-hydroxybutyrate) (PHB)-degrading bacteria possessing fibronectin type III linker type-PHB depolymerases

For rapid and sensitive detection of poly(3-hydroxybutyrate) (PHB)-degrading bacteria, a PCR primer set (PHB primers) and a gene probe (PHB probe) were designed, based on the homologous regions of six fibronectin type III linker domain-encoding sequences laid on a variety of PHB depolymerase genes listed in the GenBank. PCR using PHB primers amplified DNA fragments with the expected sizes from all the tested bacterial strains used for primer design; and all of the amplified fragments gave positive signals by Southern hybridization with the PHB probe. No amplified fragments were observed from negative controls. To evaluate the availability of the PHB primers and PHB probe, they were applied to 57 wild-type, PHB-degrading bacteria newly isolated from a variety of environments. The PHB primers amplified DNA fragments with expected sizes from 50 of the 57 wild-type strains, while the PHB probe showed positive signals against the amplified fragments from 47 strains. These results suggest that the primer and probe system established in this study can detect a considerable proportion of the potential PHB-degrading bacteria and can be applied to evaluate PHB-degradation potential in a natural environment, in combination with direct DNA extraction methods.     

无藻萍的RAPD分析及其在三膘组满江红种间关系研究中的应用

从满江红Azolla Lam.萍-藻共生体中提取DNA进行的RAPD系统分析通常忽视了满江红样品的异质性。本研究通过获得无藻的满江红,比较有藻萍、无藻萍和离体藻之间的RAPD指纹图谱。发现从有藻萍中提取DNA的扩增反应来源于萍藻双方DNA的共同影响。依引物和植物样本的不同,共生双方对扩增产物的贡献结果不同,说明了用无藻萍进行RAPD检测的重要性。对满江红三膘组5个种的11个无藻萍样本进行了RAPD分析,由9个引物产生的127个DNA多态片段用于计算样本间的Jaccard相似系数和UPGMA树状聚类图。结果     

应用RAPD标记对斜生褐孔菌菌株亲缘关系的分析

采用RAPD技术对采集不同地区的8个斜生褐孔菌野生菌核分离得到的菌株亲缘关系进行研究,获得了斜生褐孔菌不同菌株的DNA指纹图谱。结果显示:12个引物共扩增出167条带,其中101条为多态性带,多态性比率为60.5%,同一培养时期的各菌株间RAPD图谱表明菌株间存在一定的种内及地理来源差异。若以遗传距离0.508为结合线,可将供试菌株划分为三大类,BCX01、BCX02归为一类,JL01、JL02、JL03、JL04、JL05归为一类,HLJ01单独聚为一类。     

Sequence analysis for the complete proviral genome of reticuloendotheliosis virus Chinese strain HA9901

The genomic DNA extracted from chicken embryo fibroblast (CEF) infected with a Chinese field isolate HA9901 of reticuloendotheliosis virus (REV) was used as the template to amplify the REV proviral genomic cDNA by PCR with 6 pairs of primers according to published sequences. Six overlapping fragments were amplified, cloned into the TA vector and sequenced, including a fragment which was amplified from the circular proviral cDNA and covering both 5′-and 3′-ends. The complete sequence of the whole genome was established and analyzed with a DNAstar software. Comparisons of the sequence with two other strains demonstrated that the genomes of REV were relatively conservative, the homogenecity for all genes or LTR fragments of the 3 strains was over 92%, no matter whether they were isolated from different species and regions in different years. But, the homology of Chinese strain HA9901 to a fowl pox virus-associated strain from Chickens was higher than that to strain SNV isolated from ducks.     

应用RAPD技术鉴定地霉的实验研究

目的探讨RAPD技术在快速鉴定地霉中应用。方法用E.Z.N.A.yeastDNAkit提取地霉菌基因组DNA,采用随机引物AP3(5'-TCGTAGCCAA-3')、ATG(5'-ATGGATCGGC-3')、RP2(5'-AAGGATCAGA-3')、OPA-10(5'-GTGATCGCAG-3')对临床上致病性白地霉、林生地霉皮损株和血液株的基因组DNA进行扩增,对各病原菌的DNA指纹的特征进行分析。结果成功提取了地霉的基因组DNA,其纯度和浓度均能满足PCR反应的要求。利用4种引物对基因组DNA进行扩增,不同种真菌的DNA显示不同的DNA带型,分离自不同感染部位的同种不同株真菌的DNA显示的主要DNA带型基本一致。结论采用E.Z.N.A.yeastDNAkit提取的地霉基因组DNA可以用于PCR反应。RAPD法鉴定地霉菌简单、快速、特异,可用于临床诊断。