不同来源酿酒酵母菌株的随机扩增多态DNA分析

研究中试用了20个随机引物对16株不同来源的酿酒酵母菌株全基因组进行了随机扩增多态DNA分析,其中OPG06,OPG11和OPG20三条适宜引物具有鉴别作用,每一引物均可扩增1～10条DNA片段,大多数片段分子量大小在100～2000bp之间,共扩增出34条RAPD谱带,多态性为85.3%,获得了稳定清晰的菌株RAPD指纹图谱。RAPD分析结果表明,不同来源的酿酒酵母菌株之间的遗传相似系数在37.5%～94.1%之间,反映出较高的遗传差异性,并可通过聚类分析将16株不同来源的酿酒酵母菌株按亲缘关系的远近分为6个类群。结果表明,利用RAPD标记技术在基因水平上对酿酒酵母菌株进行分子鉴定和分型是可行的。     

烤烟品种的RAPD引物筛选

本试验筛选出了45个引物,可产生遗传多样性,其中OPA-08、OPA-16、OPC-10、OPC-12、GENMES'S 36号、78号、112号、181号引物其扩增结果稳定性好、多态性强。这些引物的RAPD扩增结果都可作为烟草烤烟品种的遗传多样性分析、基因定位及分子标记等。经凝胶电泳比较,GENMED'S 36号引物可作为烟草品种的最适宜的引物;1.8kb条带被初步认为与抗黑胫病基因相关。这些结果为以后对烟草烤烟品种在分子水平上的研究奠定了基础,加速烟草烤烟品种分子水平上的研究进程      

白平菇不同菌株菌丝体的RAPD分析及系统进化关系的研究

本研究采用RAPD技术分析白平菇四个不同株系的DNA序列多态性,用15个随机引物对菌株基因组DNA进行PCR扩增,其中3个引物可以扩增出较好的多态性条带图谱,共产生66条清晰稳定的带。通过对供试菌株遗传相似系数的计算和系统聚类分析,构建了树状聚类图谱,在分子水平上分析了白平菇的种质遗传学差异,为白平菇菌株鉴定提供快速有效的技术和方法。     

田间不同繁殖代数烟草赤星病菌毒力变异的遗传本质

为弄清烟草赤星病菌毒力与DNA多态性的关系及田间不同繁殖代数烟草赤星病菌毒力变异的遗传本质,分别采用毒力测定和RAPD技术对采集于同一烟田不同生长期的6个烟草赤星病菌株进行测定。结果表明,田间赤星病菌繁殖代数越高毒力越强,毒力差异是病菌基因组DNA致病相关基因差异所致。根据供试菌株在3个烟草品种上的毒力反应,可将其分为2个组,利用RAPD标记可将其分为3个组。12个随机引物扩增供试菌株共产生105个RAPD标记,其中多态性标记占95.24%,表明烟草赤星病菌群体中具有丰富的遗传多样性,毒力多态性与DNA多态性之间存在一致性,但也存在一定的差异。     

PCR技术结合RAPD图谱鉴定食品中双歧杆菌的研究

通过筛选16S rDNA序列库,设计了一对双歧杆菌属特异性引物P175和P874在试验确定的反应条件下,该引物对能正确区分双歧杆菌和非双歧杆菌。RAPD技术用于种及菌株的鉴定。选用10种引物对9株标准菌株基因组DNA进行扩增,对其指纹图谱分析表明S 256引物扩增的图谱可于双歧杆菌菌种及菌株鉴定的依据。通过对图谱相似性进行聚类分析,揭示了双歧杆菌属基因组的多态性及其遗传发育关系。     

烟草品种的DNA指纹图谱和品种鉴定

随机扩增多态性DNA(RAPD)是目前常用的DNA指纹图谱技术.采用改进的"ROSE法"提取DNA,并建立了重复性较好的RAPD标准分析条件.在此标准条件下,检测了烟草10个品种材料基因组的多态性.经10个引物扩增,在得到的53条扩增带中,有10个片段为10个材料所共有,多态性达81.1%.其中引物P7的扩增图谱,各品种间可以互相区别,差异明显,可用作烟草10个品种鉴定的DNA指纹图谱.     

中国猴头菇栽培菌株的系统进化研究

利用随机扩增多态性DNA(RAPD)标记对7 个猴头菇之间的亲缘关系进行研究,获得了猴头菇不同菌株的DNA 指纹图谱。结果显示：14 个随机引物中有3 个随机引物的扩增产物的DNA 条带表现出明显的多态性。这3个引物共扩增出44 条带,其中36 条为多态性条带,多态性位点比率为81.81%。同时利用NTSYSpc 2.1 生物软件分析7 个供试菌株之间的遗传相似性,并绘制系统进化树。     

利用SRAP标记分析花生属花生区组种质亲缘关系

利用54对SRAP引物分析花生属花生区组11个物种28份材料间的遗传变异。结果表明,28份材料共扩增出327条多态性DNA带,平均每对引物扩增出5．95条多态性DNA带,扩增变幅为2—25条。聚类分析表明,28份花生材料间的遗传距离变幅为0．12～0．75,平均为0．42,A基因组物种Arachis duranensis与栽培种花生的亲缘关系最近,可能是栽培种花生的A基因组的供体。28份种质分为两大类,第一大类包含四倍体种质及A基因组的A．villosa和A．duranensis,第二大类包含B基因组、D基因组及其他A基因组。主成分分析结果与聚类分析结果相似,仅将第二大类中的B基因组种质A．batizocoi独立作为第三大类;第一和第二个主成分可以解释81％（57．5％和23．5％）的总变异。     

一株酿酒酵母的特征区域序列扩增标记

以23株不同来源的酿酒酵母为研究对象,分别提取基因组DNA,试用50条随机引物对其进行随机扩增多态性DNA分析,筛选到两条具有菌株鉴别能力的随机引物。P09可以从2.412,ST—01,SK—26,ZD—01四株酿酒酵母基因组DNA中扩增出长度为433bp的Sc—433片断Sc—433;P46可以从2.1882,ST—01,NJ—02三株酿酒酵母基因组DNA中扩增出长度为665bp的Sc—665片断,其中仅有目的菌株ST—01能稳定的扩增出这2个标记。把这2个片断分别克隆到pUCmT质粒载体中,经过酶切鉴定后测序,根据序列设计特异性引物,把RAPD标记转化为特征区域序列扩增标记,为酿酒酵母菌株的分子鉴别提供了新的借鉴。     

烟田杂草小蓟锈病菌遗传多态性的RAPD分析

从50条Random Amplified Polymorphic DNAs(RAPD)随机引物中选取多态扩增性强的15条引物,对蓟柄锈菌(Puccinia obtegens)的22个样品和3个大蓟上采集的锈病菌样品基因组DNA进行扩增,并构建指纹图谱。25个样品包括来自不同地区的9个夏孢子样品,不同孢子类型的6个样品,同一地点不同采集时间的7个夏孢子样品和3个大蓟上采集的样品。结果显示,扩增共产生DNA标记谱带204条,多态性谱带数为160条,多态检测率为78.4%。利用SAS统计分析软件对样品的PCR结果进行了UPGMA(非加权类平均法)聚类分析。结果显示,供试3组样品可分别划分为Ⅰ、Ⅱ、Ⅲ 3个聚类组。研究结果证明,供试样品具有丰富的遗传多态性,且样品间的亲缘关系与来源地、孢子类型和采集时间有一定的相关性。     

Characterization of Penicillium roqueforti strains used as cheese starter cultures by RAPD typing

Seventy-six strains of Penicillium roqueforti used as starter cultures for mould ripened blue cheeses have been analysed for their RAPD genotype by using three different primers. A comparison of the RAPD patterns within each primer group revealed that the genetic constitution of the strains was similar, as most of the strains showed very similar overall patterns. Despite these similarities with each primer, distinct RAPD genotype groups could be identified. With one of the primers, it was possible to detect two heteropolymorphic DNA regions resulting in 13 different groups. With the other two primers, three or four groups could be identified. Between the groups of the different primers marked correspondence with respect to strain distribution could be observed, indicating that the polymorphisms detected by the primers were not independent. The RAPD patterns were compared to the production of secondary metabolites. A correlation was observed between the RAPD patterns of all primers and the production of mycophenolic acid. In addition, one of the primer (ari1) was able to distinguish between P. roqueforti strains producing larger or smaller numbers of metabolites.     

肠出血性大肠杆菌变种的RAPD分型研究

对4株肠出血性大肠杆菌(EHEC)进行随机扩增多态性(RAPD)分析,并结合主要毒力基因如eae和hly,以及志贺毒素滴度,探讨RAPD实验对大肠杆菌致病菌进行基因分型结果的可靠性。实验菌株中有两株变种携带stx基因但不能正常表达志贺毒素,其中一株变种EC169与另两株正常表达志贺毒素的O157菌株具有相似的扩增图谱,而另一株非O157变种EC130与其他菌株聚类明显不同。从20条随机引物中筛选出了重复性强且具有多态性的随机引物G2、G7、G8、G11、G12,可用于大肠杆菌致病菌快速鉴别和食物中毒溯源。     

Discrimination of Tsingtaodahua from Other Hop Cultivars and Its Quality Control by Molecular Analysis

Random amplified polymorphic DNA (RAPD) was used for hop varietal identification, primarily to distinguish Tsingtaodahua, a fine Chinese variety. Eleven typical varieties, including four aroma hops, five bitter hops, Tsingtaodahua and Cluster, were successfully identified on the basis of 28 polymorphic RAPD bands amplified by five random primers. UPGMA analysis of RAPD data showed genetic relationship among analyzed varieties consistent with traditional hop classification. Subsequently, one specific RAPD fragment was converted to a sequence tagged site (STS) marker which can detect as little as a 5% admixture of the variety Kirin 1 in Tsingtaodahua. The RAPD and STS markers can be successfully used for Tsingtaodahua identification and quality control.     

Monitoring the bacterial population dynamics during fermentation of artisanal Argentinean sausages

The dynamics of the microbial community responsible for the artisanal fermentation of dry sausage produced in Argentina was investigated by using classical and molecular approaches. The combined use of RAPD analysis with primers M13, XD9, RAPD1 and RAPD2 and 16S rDNA sequencing were applied to the identification and intraspecific differentiation of 100 strains of lactobacilli and Micrococcaceae. DGGE analysis was used to monitor the dynamic changes in population after total microbial DNA was directly extracted from sausages and subjected to PCR using V3f (GC), Bact-0124f-GC and Univ-0515r primers. The sequence analysis of 16S rDNA of the dominant species was also carried out. Lactobacillus sakei and Lactobacillus plantarum were the dominant lactic acid organisms during the fermentation while Staphylococcus saprophyticus represented the dominant species of Micrococcaceae. It was demonstrated that the ripening process of Argentinean artisanal fermented sausage is driven by a limited number of Lactobacillus and Staphylococcus strains selected from environmental microbiota by the ability to best compete under the prevailing conditions of the ecological niche. The identification of dominant communities present in this artisanal fermented sausage can help in the selection of starter cultures consisting in well adapted strains to the particular production technology.     

Effects of Primers and Taq Polymerase on Randomly Amplified Polymorphic DNA Analysis for Typing Listeria monocytogenes From the Environment of a Shrimp Processing Plant

Ninety-nine randomly selected isolates of Listeria monocytogenes from several processing environment locations, in a shrimp processing plant, obtained during a 5-month sampling period were subjected to randomly amplified polymorphic DNA (RAPD) analysis with the use of four primers. Preliminary studies indicated that the number of DNA bands and their intensity differed greatly with respect to the commercial source of the Taq polymerase used with individual isolates. Eighteen composite RAPD types were discerned with the use of the four primers. Among these 18 composite RAPD types, type 1 comprised 14 indistinguishable isolates, and type 9 comprised 49 indistinguishable isolates. These results indicate that the shrimp processing plant was dominated by these 2 RAPD types that comprised 63.6% of the 99 randomly selected isolates.     

随机扩增多态性法分析西藏牦牛奶酪中乳酸菌的遗传多样性

目的：利用随机扩增多态性（randomly amplified polymorphic DNA,RAPD）法对西藏地区牦牛奶酪中27 株乳酸菌基因型进行同源性分析。方法：利用5 个随机引物对Mg2+浓度、dNTP用量、退火温度、模版用量/引物用量（ng/pmol）4 个条件做单因素梯度试验,建立最佳反应条件,筛选最佳引物,然后对27 株乳酸菌和4 株乳酸菌标准菌株进行随机扩增,用NTsys 2.10e软件对扩增条带进行聚类和遗传相似性系数分析,分析结果与16S rRNA测序得到的菌种鉴定结果进行对比。结果：31 株菌遗传相似系数在0.72～1.00之间,当相似性系数在0.82时,菌株被分成了8 组,菌株按照不同种属聚类,聚类结果同16S rRNA测序结果基本一致,同时成功将Lactobacillus casei和Lactobacillus paracasei两个亚种区分开。结论：RAPD技术可以较好地应用于西藏地区牦牛奶酪中乳酸菌亲缘性关系分析。     

Effects of Primers and Taq Polymerase on Randomly Amplified Polymorphic DNA Analysis for Typing Listeria monocytogenes From the Environment of a Shrimp Processing Plant

Ninety-nine randomly selected isolates of Listeria monocytogenes from several processing environment locations, in a shrimp processing plant, obtained during a 5-month sampling period were subjected to randomly amplified polymorphic DNA (RAPD) analysis with the use of four primers. Preliminary studies indicated that the number of DNA bands and their intensity differed greatly with respect to the commercial source of the Taq polymerase used with individual isolates. Eighteen composite RAPD types were discerned with the use of the four primers. Among these 18 composite RAPD types, type 1 comprised 14 indistinguishable isolates, and type 9 comprised 49 indistinguishable isolates. These results indicate that the shrimp processing plant was dominated by these 2 RAPD types that comprised 63.6% of the 99 randomly selected isolates.     

Molecular typing of the yeast species Dekkera bruxellensis and Pichia guilliermondii recovered from wine related sources

A total of 63 strains of Dekkera bruxellensis and 32 strains of Pichia guilliermondii isolated from wine related environments were identified by restriction analysis of the 5.8S-ITS region of the rDNA. These strains were subjected to intraspecific discrimination using mtDNA restriction and RAPD-PCR analysis. The isolates identified as D. bruxellensis yielded 3 different molecular patterns of mtDNA restriction using the endonuclease HinfI. The pattern A was the most frequent (58 strains) among strains from different sources, regions and countries. Pattern B (4 strains) and C (one strain) were determined in isolates from Portuguese wines. The discrimination among the pattern A strains was achieved by a RAPD-PCR assay with 3 primers (OPA-2, OPA-3 and OPA-9). A total of 12 haplotypes were obtained with the combination of the patterns provided by the 3 OPAs. The pattern 2 was the most frequent and extensively distributed being found in strains from different countries and from different sources like wine, barrique wood and insects. The strains of P. guilliermondii were characterized with restriction of mtDNA using the endonuclease HinfI yielding 7 different restriction patterns. These patterns were associated with different efficiencies of 4-ethylphenol production. Patterns A to D corresponded to 19 strains producing low levels of 4-ethylphenol (<1 mg/l) while patterns F and G grouped 13 strains producing high levels of 4-ethylphenol (>50 mg/l), when grown in synthetic media supplemented with 100 mg/l of p-coumaric acid. The high degree of polymorphism observed shows that intraspecific typing is essential for accurate yeast dissemination studies in wine related environments.     

RAPD with microsatellite as a tool for differentiation of Candida genus yeasts isolated in brewing

Fifteen wild yeast strains were isolated in two factories of a lager brewing company in Poland. Their identification with API 32C system showed mainly the presence of Candida sake species (7/15). To differentiate the isolates, randomly amplified polymorphic DNA (RAPD) with (GTG)(5), (GAC)(5), (GACA)(4) microsatellite primers and M13 core sequence (5'-GAG GGT GGC GGT TCT-3') were chosen. The results of patterns similarity are presented as dendrograms for each RAPD analysis and for overall patterns. On the overall patterns, all isolates identified as C. sake, except Strain No. 1, were regrouped in one cluster. Collection strain C. sake CBS 617 was similar in 46% to the cluster with six isolates (Strain Nos. 3, 6, 8, 11, 13, 14). The second reference strain C. sake CBS 159 and the Strain No. 1 were regrouped with other Candida species (collection strains) showing, respectively, only 20% and 42% of similarity to other C. sake strains. The similarity based on the overall dendrogram between isolate Nos. 3, 6, 8, 11, 13, 14 and C. sake CBS 617 was 49%. Between those strains and other Candida, the similarity was only 37%.