几株酵母菌的分子系统学鉴定

通过对酵母菌5.8S核糖体RNA的ITS区域PCR扩增及限制性酶切片段多态性(RFLP)的图谱分析以和26S rDNA D1/D2区及5.8S-ITS区的序列分析,共鉴定13株分离自新疆鄯善地区的葡萄酒相关酵母菌。5.8S-ITS区的4种内切酶(HaeⅢ、Hin6Ⅰ、HinfⅠ、AluⅠ)的酶切分析产出4种特异性图谱。根据26S rDNA D1/D2区的序列分析和BLAST比对,将供试菌株鉴定为4个属4个种,分别为Saccharomyces cerevisiae、Candida zemplinina、Hanseniaspora uvarum、Pichia kluyveri var. kluyveri;而根据5.8S-ITS-RFLP及5.8S-ITS序列分析,将供试菌种鉴定为Saccharomyces cerevisiae、Candida zemplinina、Hanseniaspora uvarum、Issatchenkia terricola 4个属4个种。3种方法对供试的10个菌株的鉴定结果一致,而对另外3株的鉴定结果不同。     

应用PCR-RFLP技术鉴别区分中国沿海四种主要养殖扇贝

扇贝的物种鉴定,通常主要是依据形态学标准来进行。然而,当用于形态辨别的部分被去除时,对扇贝的物种鉴定工作则显得十分困难。本研究利用对扇贝核糖体ITS的PCR-RFLP分析,首次从分子水平上鉴定区分了中国沿海四种主要的养殖扇贝(栉孔扇贝、华贵栉孔扇贝、虾夷扇贝及海湾扇贝)。根据四种扇贝ITS的测序结果,选取三种内切酶(SmaI、MseI及TaqI)应用于PCR-RFLP分析。根据三种内切酶产生的酶切图谱,四种扇贝可被有效鉴别区分。酶切结果在四种扇贝各群体间的稳定性,证明了利用该技术对四种扇贝进行物种鉴定的可行性。本研究同时成功地对扇贝加工品-干贝进行了物种鉴定,证明了该技术在实践中应用的可行性。     

河南省烟草赤星病病原鉴定

为明确河南省烟草赤星病病原菌的种类,从河南省不同烟区采集烟草赤星病典型病斑,分离病原菌并且做致病性测定,对确定为病原菌的菌种按国际标准方法进一步做形态和分子鉴定。结果显示分离到的85株菌株中60株被证明是烟草赤星病病原菌,从形态学上鉴定为三个种,分别是链格孢Alternaria alternata、长柄链格孢Alternaria longipes和鸭梨链格孢Alternaria yaliinifciens。其中鸭梨链格孢Alternaria yaliinifciens引起烟草赤星病属首次报道。三个种的代表菌株的5.8S rDNA ITS基因序列间及已报道的链格孢Alternaria alternata,长柄链格孢Alternaria longipes和鸭梨链格孢Alternaria yaliinifciens的5.8S rDNA ITS区均没有较大差异,证明该基因在链格孢小孢子种间较保守,不适合区分这些链格孢种。     

气调包装技术在鲜切肉苁蓉保鲜中的应用

针对鲜切肉苁蓉易出现褐变和品质劣变的问题,该研究以新疆荒漠肉苁蓉为研究试材,在(4±0.5)℃条件下,采用气调包装(4%O₂+2%CO₂+94%N₂、4%O₂+4%CO₂+92%N₂、4%O₂+6%CO₂+90%N₂)进行处理,采用L^*值、ΔE、褐变度、硬度、水分含量、呼吸强度、维生素C含量、总酚含量和菌落总数等指标评价气调包装对鲜切肉苁蓉褐变及品质的影响。结果表明,气调包装处理能有效抑制鲜切肉苁蓉在贮藏过程中褐变和品质劣变,且以4%O₂+6%CO₂+90%N₂气调包装保鲜效果最佳。该处理能有效抑制贮藏期间鲜切肉苁蓉多酚氧化酶(polyphenol oxidase, PPO)、过氧化物酶(peroxidase, POD)活性(P<0.05)、延缓其褐变过程。此外,该条件可显著抑制鲜切肉苁蓉呼吸强度,...     

橙汁腐败酵母菌的分子鉴定及其形态特征分析

目的：结合多种酵母菌鉴定方法,分析腐败橙汁中酵母菌的种类,为快速检测和控制商品橙汁中酵母菌污染奠定基础,也为酵母菌种类的快速鉴定提供参考。方法：以分离自腐败橙汁的8株酵母菌株为材料,观察菌落和细胞形态。用引物ITS1/ITS4扩增菌株5.8S rDNA 及其ITS间隔区(5.8S-ITS),对扩增产物用HhaⅠ、Hae Ⅲ 和 HinfⅠ进行限制性酶切片段多态性(RFLP)分析和测序;用引物NL1/NL4扩增26S rDNA D1/D2区并测序。结果：菌落特征观察将菌株初分为6组,细胞学观察粗分为3组,分子方法都分成4组。用限制性内切酶HhaⅠ、Hae Ⅲ和HinfⅠ对5.8S-ITS区产物进行 RFLP分析,观察到4种不同的图谱类型。5.8S-ITS 区和26S rDNA D1/D2区的序列分析结果相似,8株分离菌与GenBank中的4种酵母菌参考菌株序列一致性达99%以上。分离株与参考菌株以两区序列构建的NJ系统树都分成4枝：Y1与克鲁维毕赤酵母(Pichia kluyveri),Y12-3、Y18-1与发酵毕赤酵母(Pichia fermentans),Y22、Y23、Y26和Y56与Meyerozyma guilliermondii,Y47与Wickerhamomyces anomalus分别聚为一枝。结论：结合形态分析和核酸分析,将8株腐败橙汁分离菌鉴定为P. kluyveri var. kluyveri、P. fermentans、M. guilliermondii和W. anomalus 4个种,其中M. guilliermondii在腐败橙汁中首见报道;ITS- RFLP分析、26S rDNA D1/D2区测序与菌株形态特征结合能有效鉴定酵母菌,核糖体DNA分析可鉴定酵母到生物学种,菌落形态特征可反映种以下的遗传差异,因此,采用分子方法鉴定时不能忽视形态特征分析的重要性。     

大豆灰斑病菌生理小种PCR-RFLP分子检测

采用PCR-RFLP技术建立了大豆灰斑病菌生理小种分子检测体系。引物对ITS1和ITS4PCR扩增大豆灰斑病菌的16个生理小种的DNA,均得到600bp片段;继而用HinfⅠ、RsaⅠ和HaeⅢ三种限制性内切酶将扩增产物消化,得到15个多态性片段,可将16个生理小种区分开来。     

串珠镰孢菌来源的角质酶基因的克隆表达及其酶学性质

克隆一个来源于串珠镰孢菌(Fusarium moniliforme Sheld)的角质酶基因,该基因全长693 bp,编码231个成熟的氨基酸。克隆的角质酶基因构建到pPIC9K质粒,获得重组表达载体,转入毕赤酵母(Pichia pastoris Gs115)中进行高效表达。经甲醇诱导96 h,测得重组酶酶活为71.68 U/mL,经纯化获得比活力为2490.1 U/mg。对纯化后的角质酶进行酶学性质分析结果表明,其最适反应pH为9.0,在pH5.0~9.0范围内之间重组角质酶的酶活相对稳定;最适反应温度为35 ℃,在40 ℃条件下保温1 h酶活力保持70%以上。KCl、Triton X-100、MnCl₂、SDS对该酶活有促进作用,NaCl、BaCl₂、CuSO₄、Tween-20、FeSO₄、ZnSO₄、NiCl₂、EDTA、Tween-80对该酶活有抑制作用。     

限制性内切酶组合对阪崎克罗诺杆菌的脉冲场凝胶电泳基因分型效果的影响

目的利用多种限制性内切酶提高脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)技术对阪崎克罗诺杆菌(Cronobacter Sakazakii)的分型效果,找到更多的以PFGE为基础的阪崎克罗诺杆菌分子分型信息。方法本研究选用XbaⅠ、SpeⅠ、NheⅠ和BlnⅠ分别酶切34株克罗诺杆菌分离株的DNA,并经PFGE得到相应的基因图谱,通过束平均数、束菌株百分比、结与菌株比值与Simpson多样性指数对各限制内切酶以及结合酶的相对分辨率进行评价。结果 NheⅠ是PFGE单酶切分型中效果最好的一种限制性内切酶;4种限制性内切酶相结合的分型方法可提高PFGE阪崎克罗诺杆菌基因分型效果。结论运用PFGE技术对菌株进行亚分型时,选用的酶种类越多,则结果越可靠,使用XbaⅠ、SpeⅠ、NheⅠ、BlnⅠ4种内切酶结合分析,可以得到理想的菌株亚分型结果。     

保健食品建泽泻及其混淆品的PCR-RFLP分子鉴别

目的:寻找简易可重复的分子标记方法对保健食品建泽泻及其混淆品进行鉴定。方法:对建泽泻及其混淆品的rDNA ITS区进行PCR扩增、测序,运用ClustalX、Mega3.0、DNAMAN 4.0等软件对ITS区进行序列分析和PCR限制性酶切图谱分析(PCR-RFLP)。结果:依据建泽泻及其混品完整的rDNA ITS区片段长度,将慈菇和甘薯两种混淆品首先区分开;再通过PCR-RFLP分析,可将建泽泻从川泽泻、窄叶泽泻、小泽泻和芋等混淆品中成功鉴别出来。结论:rDNA ITS片段长度不足以用于鉴别保健食品建泽泻原料的真伪;PCR-RFLP可对保健食品建泽泻及其混淆品进行简便而快捷的分子鉴别。     

屎肠球菌源谷氨酸脱羧酶的制备及其酶学性质研究

为了开发谷氨酸脱羧酶(glutamate decarboxylase,GAD),以Enterococcus faecium为gadB基因供体、纤维素结合域(cellulose-binding domain,CBD)为亲和标签,利用内含肽DnaB自剪切作用分离GAD,对融合酶CBD-DnaB-GAD的构建、表达、GAD纯化及其酶学性质进行了探讨。结果表明:重组Escherichia coli GDMCC60446可高效表达CBD-DnaB-GAD,适宜自剪切液为0.2 mol/L Na₂HPO₄-NaH 2PO₄缓冲液(pH 6.5,含NaCl 0.5 mol/L、EDTA 1 mmol/L);经一步纯化和自剪切制备的GAD在SDS-PAGE电泳上呈单一条带,分子质量约为55.68 kDa,仅有1个亚基;GAD最适反应pH和温度分别为5.0和55℃,在pH 4.8~5.8和-25~55℃较稳定;5 mmol/L的NaCl、CaCl₂和乙二醇对GAD酶活力影响不大,而KCl、EDTA-2Na、ZnSO₄、CuSO₄、MnSO₄、MgSO₄、FeCl₂、FeCl₃、AlCl₃、AgNO 3和Pb(CH₃COO)₂对GAD酶活力具有不同程度的抑制作用;GAD仅催化L-谷氨酸发生脱羧反应,K m和V max分别为10.51 mmol/L和3.41μmol/(mL·min),催化反应无底物和产物抑制作用。GAD纯度和酶学性质优良,制备工艺简便,该技术有望应用于工业化生产。     

Molecular identification of yeast species associated with 'Hamei'--a traditional starter used for rice wine production in Manipur, India

In Manipur state of North-Eastern India, wine from glutinous rice using traditional solid state starter called 'Hamei' is particularly interesting because of its unique flavour. A total of 163 yeast isolates were obtained from fifty four 'Hamei' samples collected from household rice wine preparations in tribal villages of Manipur. Molecular identification of yeast species was carried out by analysis of the restriction digestion pattern generated from PCR amplified internal transcribed spacer region along with 5.8S rRNA gene (ITS1-5.8S-ITS2). Seventeen different restriction profiles were obtained from the size of PCR products and the restriction analysis with three endonucleases (Hae III, Cfo I and Hinf I). Nine groups were identified as S. cerevisiae, Pichia anomala, Trichosporon sp., Candida tropicalis, Pichia guilliermondi, Candida parapsilosis, Torulaspora delbrueckii, Pichia fabianii and Candida montana by comparing this ITS-RFLP profile with type strains of common wine yeasts, published data and insilico analysis of ITS sequence data available in CBS yeast database. ITS-RFLP profile of eight groups was not matching with available database of 288 common wine yeast species. The most frequent yeast species associated with 'Hamei' were S. cerevisiae (32.5%), P. anomala (41.7%) and Trichosporon sp. (8%). The identity of major groups was confirmed by additional restriction digestion of ITS region with Hind III, EcoRI, Dde I and Msp I. The genetic diversity of industrially important S. cerevisiae group was investigated using Pulsed Field Gel Electrophoresis (PFGE). Although most of the 53 strains of S. cerevisiae examined were exhibited a common species specific pattern, a distinct degree of chromosomal length polymorphism and variable number of chromosomal DNA fragments were observed with in the species. Cluster analysis showed seven major karyotypes (K1-K7) with more than 83% similarity. The karyotype pattern K1 was the most frequent (67.9%) among the strains from different samples. Other karyotypes K2-K7 were very unique with less than 80% similarity. Finally using mitochondrial DNA restriction analysis (mt-DNA RFLP), S. cerevisiae strains belonging to the major karyotype K1 were distinctly differentiated with highly polymorphic bands by Hinf I and Hae III endonucleases.     

利用12S rRNA基因的限制性酶切末端片段长度多态性鉴定动物种类

目的：建立一种精确可靠的鉴定常见的1 0 种动物( 猪、狗、牛、山羊、绵羊、马、鸡、鼠、三文鱼和鹿)的方法。方法：利用12S rRNA 基因的限制性酶切末端片段长度多态性(terminal restriction fragment lengthpolymorphism,T-RFLP)鉴别动物种类。将线粒体12S rRNA 基因通过引物的5＇端用FAM 荧光标记,从基因组DNA 中扩增450bp 的目的片段。引物对1(下游引物FAM标记,上游引物不标记)扩增的PCR 产物用限制性内切酶Alu Ⅰ酶切。引物对2(上游引物FAM标记,下游引物不标记)扩增的PCR 产物用限制性内切酶Tru9 Ⅰ酶切。得到的酶切产物分别在遗传分析仪ABI 3100 上进行毛细管电泳,片段大小用Peak Scanner 1.0 软件分析。结果：根据Alu Ⅰ酶切图谱能够区分鸡、马、猪和三文鱼,而鹿和牛、山羊和绵羊、鼠和狗因酶切图谱相同无法分开。根据Tru9 Ⅰ酶切图谱,能够进一步将鹿和牛、山羊和绵羊、鼠和狗分开。同一种动物不同个体的酶切图谱完全相同,结果具有可重复性。没有出现物种内多态的现象。大多数情况下,实际得到的末端片段长度与理论值非常接近,只存在2 ～5bp 的差异。结论：该方法操作简单、结果精确,适用于鉴定动物种类。     

Evaluation of ITS PCR and RFLP for Differentiation and Identification of Brewing Yeast and Brewery ‘Wild’ Yeast Contaminants

A reference library of ITS PCR/RFLP profiles was collated and augmented to evaluate its potential for routine identification of domestic brewing yeast and known ‘wild’ yeast contaminants associated with wort, beer and brewing processes. This library contains information on band sizes generated by restriction digestion of the ribosomal RNA‐encoding DNA (rDNA) internal transcribed spacer (ITS) region consisting of the 5.8 rRNA gene and two flanking regions (ITS1 and ITS2) with the endonucleases CfoI, HaeIII, HinfI and includes strains from 39 non‐Saccharomyces yeast species as well as for brewing and non‐brewing strains of Saccharomyces. The efficacy of the technique was assessed by isolation of 59 wild yeasts from industrial fermentation vessels and conditioning tanks and by matching their ITS amplicon sizes and RFLP profiles with those of the constructed library. Five separate, non‐introduced yeast taxa were putatively identified. These included Pichia species, which were associated with conditioning tanks and Saccharomyces species isolated from fermentation vessels. Strains of the lager yeast S. pastorianus could be reliably identified as belonging to either the Saaz or Frohberg hybrid group by restriction digestion of the ITS amplicon with the enzyme HaeIII. Frohberg group strains could be further sub‐grouped depending on restriction profiles generated with HinfI.     

基于内转录间隔区序列对云南12株鹅膏菌的分子分析

目的 基于内转录间隔区(internal transcribed spacer, ITS)序列对云南省12株鹅膏菌进行分类鉴定。方法 采用十六烷基三甲基溴化铵法(cetyltrimethylammonium Ammonium Bromide, CTAB)法提取12株鹅膏菌DNA, 以ITS4和ITS5为引物进行PCR扩增, 完成DNA序列测序, 对序列进行分析并对发育树进行构建。 结果 12个样品的ITS序列长度574~755 bp, GC含量39%~47%, 平均遗传距离为0.329, YN05、YN03与其他10株鹅膏亲缘关系较远。结论 ITS序列高度保守, 在真菌的科属种上能够初步实现对物种的鉴定和系统 发育分析, 为建立云南省鹅膏属分子数据库提供基础数据。     

Molecular study of Geotrichum strains isolated from Armada cheese

The aim of this work was the genetic characterization at the strain level of 39 presumed Geotrichum candidum isolates isolated throughout the artisanal manufacturing and ripening of Armada cheese and tentatively identified at genus and/or species level by phenotypic characteristics. The molecular identification of the strains included among others the amplification and sequencing of the D1/D2 domains of the 26S rRNA gene. A restriction fragment length polymorphism (RFLP) analysis with the ITS1-5.8S-ITS2 PCR amplicons and a randomly amplified polymorphic DNA (RAPD) analysis with five different primers were carried out. The bands pattern profile obtained through RFLP by enzymatic restriction with HinfI was the same for all the strains studied, which confirmed the classification of the strains at species level. A RAPD-PCR analysis with three different primers was applied to assess the intraspecific diversity, in this way 16 band profiles were obtained for the 39 strains studied by the combined use of primers Ari1 and Omt1. This study contributes to know the occurrence and genotypic biodiversity of G. candidum in Armada cheese.     

Use of molecular methods for the identification of yeast associated with table olives

A molecular approach is used for the identification of yeast isolated from table olives. Our results validate those obtained in the past by the classical biochemical methodology. Yeast were isolated from both aerobically and anaerobically processed black table olives and also from canned seasoned green table olives. Molecular identification methodology used included restriction pattern analysis of both PCR-amplified 5.8S rRNA gene and internal transcribed spacers ITS(1) and ITS(2). For some species, sequence analysis of the 26S rRNA gene was necessary. These techniques allowed the identification of three yeast species (Issatchenkia occidentalis, Geotrichum candidum and Hanseniaspora guilliermondii) which had not been described previously in table olives. Saccharomyces cerevisiae and Candida boidinii were the most frequent species in green seasoned olives and processed black olives, respectively. The molecular study of total DNA variability among the S. cerevisiae strains isolated indicates a quite heterogeneous population, with at least four different restriction patterns.     

Using ITS2 PCR-RFLP to generate molecular markers for authentication of Sophora flavescens Ait

BACKGROUND: Dried root of Sophora flavescens Ait. is a medicinal material occasionally misused or adulterated by other species similar in appearance. In this study the internal transcribed spacer (ITS) regions of DNA samples of S. flavescens Ait. collected from different areas of Taiwan were amplified by polymerase chain reaction (PCR) and compared. The effectiveness of using ITS2 PCR restriction fragment length polymorphism (RFLP)‐generated markers to differentiate S. flavescens Ait. from possible adulterants was also evaluated. RESULTS: The S. flavescens Ait. samples collected from different areas were extremely low in ITS sequence variability at species level. ITS2 PCR‐RFLP coupled with restriction enzymes Sac I, Sac II, Xho I or Pvu I produced specific fragments for all tested variants. ITS2 PCR‐RFLP coupled with Sac II was further performed to identify mixtures of DNA extracts of S. flavescens Ait. and Sophora tomentosa L. in various ratios. The developed ITS2 PCR‐RFLP markers could detect mixed DNA samples of S. flavescens Ait./S. tomentosa L. up to a ratio of 10:1. CONCLUSION: The present study demonstrates the usefulness of ITS2 PCR‐RFLP coupled with pre‐selected restriction enzymes for practical and accurate authentication of S. flavescens Ait. The technique is also suitable for analysing S. flavescens Ait. mixed with other adulterants. Copyright © 2011 Society of Chemical Industry     

Characterization of Pectinatus and Megasphaera Strains by Automated Ribotyping

A total of 32 Pectinatus and Megasphaera strains, isolated from spoiled beer or brewery environments and identified by conventional methods, were analysed by the automated RiboPrinter® System. One strain from each ribotype was further subjected to partial 16S rDNA sequencing to confirm the ribotyping results. The restriction enzyme EcoRI was used in ribotyping of Pectinatus strains. Eight strains, identified by conventional tests as P. cerevisiiphilus, generated five different ribotypes. The strains of three types were considered to be members of P. cerevisiiphilus, but the strains of two types were most probably members of a new species within the genus Pectinatus. The 24 strains identified by conventional tests as P. frisingenis generated nine different ribotypes. The similarity between the ribotypes was rather low, but all these strains obviously belonged to the same species. Thirteen Megasphaera cerevisiae strains were analysed with three restriction enzymes EcoRI, Pstl and PvuII and four, six and three different ribotypes, respectively, were generated resulting in seven different combinations. The best discrimination among these strains was obtained with Pstl. According to these results 12 of 13 brewery strains were considered to be M. cerevisiae, but one strain most probably represented a new species within the genus Megasphaera. During the work, 14 RiboPrint® patterns of Pectinatus, five of Megasphaera, two of Selenomonas and two of Zymophilus were created with EcoRI. In addition seven patterns of Megasphaera were created with Pstl and four with Pvull. All these identification patterns (genetic fingerprints) were saved at the database of VTT for future use.     

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.