黑木耳菌株酯酶同工酶酶谱多样性研究

目的：为了探索黑木耳菌株之间的遗传距离,构建出供试菌株酯酶同工酶鉴别图。方法：采用垂直板聚丙烯酰胺凝胶电泳对21个黑木耳菌株酯酶同工酶的酶谱多样性进行了研究。结果：酯酶同工酶电泳各个菌株分别具有2~7条酶带,其中在Rf值为0.344处,21个菌株都有谱带出现。21个菌株之间的遗传相似系数在0.167-1.000之间,应用NTSYS软件进行聚类分析,当相似水平为0.73时,可将供试的21个黑木耳菌株分为6个不同的类群。结论：酯酶同工酶可以有效快捷的对黑木耳菌株进行菌种鉴定,是黑木耳菌株遗传多样性研究的理想手段。     

用RAPD分子标记鉴别黑木耳菌种的研究

运用RAPD技术,选用20个寡核苷酸引物对我所供应的8个黑木耳生产菌株进行了分子鉴别。结果表明:在选用的20个随机引物中,有10个引物能对8个供试黑木耳菌株扩增出清晰、稳定的DNA谱带。用NTSYS软件进行聚类分析,在相似水平为0．70阈值时,可将供试的8个黑木耳菌株归为3个组群。     

毛木耳种质资源的RAPD分析

利用22个随机引物对来源不同的56个木耳菌株进行了RAPD分析。结果表明,所有引物的扩增产物DNA片段均表现出明显的多态性,供试菌株总共扩增出164条多态性片段,占总扩增片段的99%;供试菌株两两间的遗传相似系数变化较大(平均GS值0.2143￣0.8764)。采用系统聚类法中的类平均法,对供试的所有菌株两两间相似系数进行聚类,可将它们分为四大类,各大类的类间和类内菌株的遗传变异程度较大,以IV类内各菌株间的最高(平均GS值0.3891),II和III类间的最低(平均GS值0.5887),表明遗传变异也较丰富(总平均GS值0.4918)。将RAPD技术应用于不同菌株间遗传差异的研究,具有反应迅速、不受外界环境条件影响、能从DNA分子水平上揭示菌株间遗传差异等优点,是一种快速准确评估木耳种质资源的有效方法。     

用ISSR分子标记鉴别东北地区黑木耳生产菌株的研究

利用ISSR分子标记对东北地区黑木耳生产菌株进行了分子鉴别,结果表明在选用的20个UBC-ISSR引物中,有10个引物能对供试的27个黑木耳菌株基因组DNA进行扩增,获得的指纹图谱清晰稳定、多态性强。用NTSYS软件进行聚类分析,相似水平在0.75时,可将27个供试黑木耳菌株分为3个组群。研究结果说明ISSR分子标记,可以有效地用于黑木耳生产菌株快速准确鉴别,是黑木耳指纹图谱分析的理想手段。     

用ISSR分子标记鉴别东北地区黑木耳生产菌株的研究

利用ISSR分子标记对东北地区黑木耳生产菌株进行了分子鉴别,结果表明在选用的20个UBC-ISSR引物中,有10个引物能对供试的27个黑木耳菌株基因组DNA进行扩增,获得的指纹图谱清晰稳定、多态性强。用NTSYS软件进行聚类分析,相似水平在0.75时,可将27个供试黑木耳菌株分为3个组群。研究结果说明ISSR分子标记,可以有效地用于黑木耳生产菌株快速准确鉴别,是黑木耳指纹图谱分析的理想手段。     

用ISSR分子标记鉴别东北地区黑木耳生产菌株的研究

利用ISSR分子标记对东北地区黑木耳生产菌株进行了分子鉴别,结果表明在选用的20个UBC-ISSR引物中,有10个引物能对供试的27个黑木耳菌株基因组DNA进行扩增,获得的指纹图谱清晰稳定、多态性强。用NTSYS软件进行聚类分析,相似水平在0.75时,可将27个供试黑木耳菌株分为3个组群。研究结果说明ISSR分子标记,可以有效地用于黑木耳生产菌株快速准确鉴别,是黑木耳指纹图谱分析的理想手段。     

ERIC技术在紫木耳亲缘关系鉴定上的应用研究

为了研究紫木耳与黑木耳和毛木耳之间的亲缘关系,利用ERIC分子标记、Southern杂交技术和形态学分类法,对紫木耳、黑木耳和毛木耳三者进行了详细的研究。在相似性系数为75%的水平上,由ERIC所得的聚类图将7个菌株分为2组,即黑木耳自成一组,毛木耳和紫木耳为一组,两者相似性系数为77%。Southern杂交实验证明毛木耳和紫木耳之间存在较高的同源性。子实体的形态学特征表明紫木耳与毛木耳的亲缘关系较之黑木耳更为密切,同时进一步说明ERIC分子标记是准确可信的。本研究探讨了紫木耳的分类地位,认为在基因组进化上,紫木耳与毛木耳的关系较之黑木耳更为密切。     

木耳属真菌rDNA特异性扩增片段的RFLP研究*

对木耳属8个种25个菌株的ITS和28SrDNA5’端两个区域分别进行了PCR扩增和限制酶切片段长度多态性（RFLP）研究。ITS－RFMP研究结果表明,HaeⅢ可将黑木耳与其它种区分开,MspⅠ可将盾形木耳、角质木耳、琥珀木耳和黑木耳4个种区分开,而供试的HaeⅢ、TaqⅠ、HinfⅠ和MaPⅠ这四种限制酶均不能将皱木耳、大木耳、网脉木耳及毛木耳4个种区分开,表明它们之间的亲缘关系较近;结果还表明,ITS—rDNA拷贝在毛木耳和琥珀木耳种内是异质性的,而在黑木耳种内是同质性的。285rDNA-RFLP研究结果表明,供试的4种限制酶中,仅MspⅠ可将盾形木耳和角质木耳区分开,而不能将其它种区分开,这显示了28SrDNA序列在木耳属不同种间的保守性。     

利用RAPD标记构建木耳属种间关系的研究

摘 要: 利用RAPD标记以银耳属的2个种为外类群研究了木耳属8个种的分子系统发育关系。根据UPGMA 构建的树状图结果表明木耳属(Auricularia)和银耳属(Tremella)分别为单一的分类单元。木耳属的8个种被分成3个明显不同的组,第一组包括盾形木耳(A.peltata Lloyd)和角质木耳(A.cornea Spreng.),第二组包括所有供试的黑木耳(A.auricula Underw.),第三组包括皱木耳(A.delicata Henn.)、琥珀木耳(A.fuscosuccinea Farl.)、毛木耳(A.polytricha Sacc.)、大木耳(A.maxima Y. R. Guo)和网膜木耳(A.reticulata L. J. Li)五个形态种。种间发育关系表明角质木耳和毛木耳应是两个不同的种而非同种异名,子实体横切面中髓层的有无并不表明不同种间亲缘关系的远近。     

木耳属真菌rDNA特异性扩增片段的RFLP研究

对木耳属8个种25个菌株的ITS和28SrDNA5’端两个区域分别进行了PCR扩增和限制酶切片段长度多态性（RFLP）研究。ITS－RFMP研究结果表明,HaeⅢ可将黑木耳与其它种区分开,MspⅠ可将盾形木耳、角质木耳、琥珀木耳和黑木耳4个种区分开,而供试的HaeⅢ、TaqⅠ、HinfⅠ和MaPⅠ这四种限制酶均不能将皱木耳、大木耳、网脉木耳及毛木耳4个种区分开,表明它们之间的亲缘关系较近;结果还表明,ITS—rDNA拷贝在毛木耳和琥珀木耳种内是异质性的,而在黑木耳种内是同质性的。285rDNA-RFLP研究结果表明,供试的4种限制酶中,仅MspⅠ可将盾形木耳和角质木耳区分开,而不能将其它种区分开,这显示了28SrDNA序列在木耳属不同种间的保守性。     

Molecular typing of Vibrio parahaemolyticus strains isolated from the Philippines by PCR-based methods

AIM: The main aim of the present study was to use three PCR-based techniques for the analysis of genetic variability among Vibrio parahaemolyticus strains isolated from the Philippines. METHODS AND RESULTS: Seventeen strains of V. parahaemolyticus isolated from shrimps (Penaeus monodon) and from the environments where these shrimps are being cultivated were analysed by random amplified polymorphic DNA PCR (RAPD-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and repetitive extragenic palindromic PCR (REP-PCR). The results of this work have demonstrated genetic variability within the V. parahaemolyticus strains that were isolated from the Philippines. In addition, RAPD, ERIC and REP-PCR are suitable rapid typing methods for V. parahaemolyticus. All three methods have good discriminative ability and can be used as a rapid means of comparing V. parahaemolyticus strains for epidemiological investigation. Based on the results of this study, we could say that REP-PCR is inferior to RAPD and ERIC-PCR owing to the fact that it is less reproducible. Moreover, the REP-PCR analysis yielded a relatively small number of products. This may suggests that the REP sequences may not be widely distributed in the V. parahaemolyticus genome. CONCLUSIONS: Genetic variability within V. parahaemolyticus strains isolated in the Philippines has been demonstrated. The presence of ERIC and REP sequences in the genome of this bacterial species was confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD, ERIC and REP-PCR techniques are useful methods for molecular typing of V. parahaemolyticus strains. To our knowledge this is the first study of this kind carried out on V. parahaemolyticus strains isolated from the Philippines.     

Genetic characterization of antibiotic-resistant Staphylococcus aureus from milk in the North-West Province,South Africa

Food borne diseases are a major public health concern worldwide. Staphylococcus aureus is one of the potential food borne pathogens which causes nosocomial and community acquired infections. In the present study, 74 representative strains of S. aureus isolated and characterized in previous study from different milk samples were subjected to random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus (ERIC)-PCR to generate fingerprints to determine the genetic relationships of the isolated strains. A total of 20 RAPD patterns were generated and the number of amplified fragments obtained ranged from 0 to 8 with molecular weight ranging from 250 to 2000 bp. A dendrogram based on fingerprinting pattern grouped isolates into twelve major clusters (I–XII). In the case of ERIC-PCR 9 banding patterns were obtained with amplicons ranging from 1 to 8 and band sizes ranging from 250 to 2000 bp. A total of four major clusters (I–IV) were observed in the dendrogram based on ERIC fingerprints. The discrete banding patterns obtained both from ERIC-PCR and RAPD-PCR showed remarkably the genetic diversity of S. aureus. The findings of this study indicate that raw, bulk and pasteurized milk in the North-West Province was contaminated with toxigenic and multi-drug resistant S. aureus strains. This emphasizes the need to implement appropriate control measures to reduce contamination as well as the spread of virulent S. aureus strains to reduce the burden of disease in humans.     

Random and repetitive primer amplified polymorphic DNA analysis of Bacillus sphaericus

A comparative study of 15 strains representing the five homology groups of Bacillus sphaericus was performed by two PCR methods: RAPD and rep-PCR fingerprinting. The PCR analysis performed with primers corresponding to the naturally occurring repetitive sequences REP, ERIC and BOX, as well as with three random primers, showed highly variable patterns and allowed differentiation of the strains studied. This demonstrated the high discriminative power of the methods. The cluster analysis revealed a low level of similarity between the different homology groups, and within groups I and III, which is evidence of the high genetic heterogeneity of the species B. sphaericus. Close genetic relatedness was observed for the representatives of group IIA, pathogenic to mosquitoes, which supports the idea for differentiation of this group as a separate species.     

Evaluation of different DNA-based fingerprinting methods for typing Photobacterium damselae ssp. piscicida

This study evaluates the effectiveness of three different molecular techniques, repetitive extragenic palindromic PCR (REP-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and the random amplified polymorphic DNA (RAPD-PCR) for rapid typing of Photobacterium damselae ssp. piscicida strains isolated from different species of marine fish and geographic areas. The results obtained by the three methods showed that RAPD and ERIC-PCR were more discriminative for suitable rapid typing of Ph. damselae ssp. piscicida than REP-PCR. The analysis of DNA banding patterns generated by both molecular methods (RAPD and ERIC-PCR) clearly separated the strains into two main groups that strongly correlated with their geographic origin. Moreover, the REP-PCR analysis was less reproducible than the RAPD and ERIC-PCR methods and does not allow the establishment of genetic groups. RAPD and ERIC-PCR constitute valuable tools for molecular typing of Ph. damselae ssp. piscicida strains, which can be used in epidemiological studies of photobacteriosis infections.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

Aeromonas hydrophila clinical and environmental ecotypes as revealed by genetic diversity and virulence genes

Aeromonas hydrophila strains recovered from clinical samples and ambient sources were phenotypically and genetically identified. In addition, the distribution of putative virulence factors was assayed. To determine the genetic diversity of these strains, random amplification of polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC)-PCR markers were used. The discriminatory ability of the techniques, using Simpson's index, was 0.96 for both methods. The most consistent dendrogram was obtained when RAPD and ERIC data were combined. The genetic diversity revealed a high intra-specific genetic diversity (h=0.364+/-0.024 and I=0.538+/-0.030). The strains showed a tendency to cluster according to their origin of isolation (best-cut test 0.80 and bootstrap values >50%). The present study demonstrates and quantifies the high intra-specific diversity within this species and reveals a clear differentiation of strains according to their ecological origin. The distribution of virulence-related genes confirm that A. hydrophila is a genetically heterogeneous species that harbour ecotypes which have different pathogenic potential to human and other animals.     

Repetitive elements sequence (REP/ERIC)-PCR based genotyping of clinical and environmental strains of Yersinia enterocolitica biotype 1A reveal existence of limited number of clonal groups

REP- and ERIC-PCR genotyping were used to assess genetic heterogeneity among 81 strains of Yersinia enterocolitica biotype 1A isolated from India, Germany, France and the USA. Although both gave comparable results, ERIC fingerprints discriminated the strains better. The rep- (REP and ERIC) PCR genotyping showed that strains having different serotypes produced identical rep-profiles indicating their limited genetic diversity. The concatenated dendrogram of REP- and ERIC-PCR fingerprints clustered the biotype 1A strains into two major groups. In each group, majority of the Indian, European and American strains exhibited similarities ranging from 85% to >95%. Similarity of rep-PCR fingerprints amongst strains isolated from widely separated geographical regions revealed existence of a limited number of clonal groups of Y. enterocolitica biotype 1A. The present study failed to reveal unequivocal relationships between rep-PCR genotypes and the source of isolation. However, the clinical serotype O:6,30-6,31 strains formed a tight cluster and the aquatic O:6,30-6,31 strains formed a yet another tight cluster.     

胞外漆酶同工酶谱及其在食用菌育种中的应用——Ⅰ.黑木耳及光帽鳞伞不同菌株的鉴别

利用聚丙烯酰胺凝胶等电聚焦圆盘电泳,分析比较了黑木耳(Auricularia auricula)16个菌株和光帽鳞伞(Pholiota nameko)9个菌株的胞外漆酶同工酶酶谱。经多批次重复,得到了所有菌株的恒定酶谱。依照这些酶谱及各酶带等电点(pI)值的不同,确立了它们的漆酶标志位点相似系谱。16株黑木耳被区分成4种类型9个组。9株光帽鳞伞被分成两种类型3个组,但各组内之菌株间仍有异同。试验结果表明,把漆酶同工酶谱应用于菌株鉴别将是一种有意义的方法。