rRNA基因序列分析在阿萨希毛孢子菌鉴定中的应用

作者对近年有关毛孢子菌属rRNA分析的研究进行了综述,目前的研究结果表明。RNA的26S亚基的D1／D2区、ITS和IGS区的序列分析对毛孢子菌属的鉴定均有一定价值,其中ITS转录间区与18S、5．8S以及26S相比具有较高的特异性;IGS区序列分析优于ITS区序列分析;IGS1区优于IGS2区。     

陕西汉族人群12号染色体上7个STR基因座的遗传多态性分析

分析了中国汉族人群中12号染色体上7个短串联重复序列（short tandem repeat,STR）基因座的多态性。 采用荧光标记基因扫描对12号染色体上D12S1718、D12S1675、D12S358、D12S367、D12S1638、D12S1646和D12S1682基因座在80名陕西咸阳、榆林汉族人中的遗传多态性进行分析。结果在中国汉族人群中, D12S1718、D12S1675、D12S358、D12S367、D12S1638、D12S1646和D12S1682基因座分别检出7、10、8、8、6、9和11个等位基因,10、17、18、18、14、18和26个基因型,杂合度分别为44.28％、66.10％、78.89％、77.89％、73.69％、74.55％和82.39％。表明这7个STR基因座在中国人群中有较好的多态性,其基因型分布均符合Hard－Weinberg平衡（P>0.05）。     

斑马鱼肠道中一株酵母菌菌株的鉴定与系统发育分析

从斑马鱼肠道中分离到一株酵母菌,编号为ZF-5,进行了形态学观察、生理特征测定和26S rDNA D1/D2序列分析,并构建系统发育树。结果表明ZF-5菌株细胞呈卵圆形或杆状,为芽殖,有假菌丝;除乳糖外,能够发酵葡萄糖、蔗糖、麦芽糖等多种碳源;26S rDNA D1/D2区序列分析表明与季也蒙毕赤酵母Pichia guilliermondii的序列相似性最高,构建的系统发育进化树显示菌株ZF-5与Pichia guilliermondii模式菌株CBS 2030（= NRRL Y-2075）亲缘关系最近,     

基于26S rDNAD1/D2区序列分析的15株白地霉分子分类学研究

采用26S rRNA基因D1/D2区系统发育分析的方法对CICC(中国工业微生物菌种保藏管理中心)保藏的15株白地霉(Geotrichum candidum)菌种进行复核鉴定。系统发育分析结果表明15株白地霉属于地霉属的成员,且形成两个系统发育分支,系统发育上最接近Galactomyces geotrichum NRRLY-17569T,与其同源性为96.3%～98.3%。15株白地霉26S rRNA基因D1/D2区序列显著不同于地霉属的模式种及其它种,可能代表地霉属的两个新种,但这一结论尚需进一步的实验去证实。     

26S rDNA单链构象多态性分析在临床酵母菌菌种鉴定中的应用

摘要： 【目的】探讨酵母菌临床分离株26S rDNA D1/D2区序列种内相似性和种间差异性的快速检测方法,为临床酵母菌菌种鉴定方法的改进奠定基础。调查北京地区临床酵母菌的种群多样性,为国内酵母菌感染的流行病学研究提供新的基础数据。【方法】用5种常见临床酵母菌种的模式和权威菌株作为标准参考菌株,从北京四家综合性医院收集临床酵母菌260余株,PCR扩增其26S rDNA D1/D2区,对扩增产物进行单链构象多态性（Single-Strand Conformation Polymorphism,SSCP）分析和序列测定分析。【结果】常见病原酵母菌26S rDNA D1/D2区的SSCP图谱具有明显的种间差异性和种内相似性,可以通过该方法对菌株进行初步的菌种鉴定。D1/D2-SSCP和序列分析相结合,对260余株临床酵母菌进行了菌种鉴定,共鉴定有10个属20个种,优势属为念珠菌属(Candida),优势种及其所占比例分别是：C. albicans (57.7%), C. parapsilosis (10.0%), C. tropicalis (9.2%), C. glabrata (6.7%)和C. krusei (5.8%),并发现过去从未或很少报道致病的酵母菌种,愈来愈多地出现在临床分离菌株中。【结论】 26S rDNA D1/D2区的SSCP图谱分析为临床酵母菌株的快速鉴定提供了新的方法;北京地区酵母菌临床分离株呈种群多样性分布,C. albicans虽然仍占优势,但其它念珠菌种的比例已达42%。     

一株分离自酸梅酱的酵母菌的分离鉴定

目的:分离及鉴定来自于新疆塔城民间自制酸梅酱中的酵母菌。方法:用NL1/NL4引物对扩增酵母菌株的26S rDNAD1/D2区,测序结果进行序列分析,用Neighbour-joining(N-J)方法构建系统发育进化树,同时结合酵母的传统形态学鉴定对菌株进行鉴定。结果:26S rDNA D1/D2区序列分析表明菌株KKS与Metschnikowia aff.fructicola D3895相近,相似率为99.2%。在N-J法构建的系统发育进化树中,菌株KKS与Metschnikowia aff.fructicola聚类在同一分枝上。结论:从新疆塔城民间自制酸梅酱中分离得到一株酵母菌并将该菌株鉴定为Metschnikowia aff.fructicola。     

一株产虾青素酵母菌株的鉴定

从市售海水鱼内脏中分离得到一株产虾青素的酵母,编号为NZ- 01.采用传统形态学鉴定方法及rDNA序列分析法分别对从NZ- 01进行鉴定.形态学鉴定结果表明该菌为胶红酵母(Rhodotorula mucilaginosa),分别用特异性引物对rDNA 序列的18S rDNA D1/D2区、26S rDNA D1/D2区和ITS区进行扩增,PCR产物测序,将测序结果登录GenBank进行BLAST分析,结果均与形态学观察结果一致,NZ- 01为胶红酵母.     

DHPLC检测胃癌微卫星不稳定性

为探讨一种快速、简便、可靠的胃癌微卫星不稳定性（MSI）检测方法,变性聚丙烯酰胺凝胶电泳-银染法检测28例胃癌12个微卫星位点(D1S548、D1S552、D5S346、TP53、IGFIIR(G)8、IGFIIR(CT)5、TGFßRII(GT)3、TGFßRII(A)10、hMSH3(A)8、hMSH6(G)8、BAX(G)8和Bat26）,DHPLC柱温50℃检测Bat26位点。凝胶电泳发现MSI-H 2例（7.14%）,MSI-L胃癌15例（53.6%）,Bat26+2例均为MSI-H,Bat26改变和MSI-H表型一致（P<0.01,Fisher’s确切概率法）。DHPLC亦证实2例Bat26+胃癌,结果和凝胶电泳完全一致。结果表明,DHPLC检测Bat26位点是研究胃癌MSI-H的较好方法,有一定的临床应用价值。Abstact: To establish a fast, simple and solid method of studying microsatellite instability (MSI) in gastric cancer, a panel of 12 microsatellite sites,D1S548, D1S552, D5S346, TP53, IGFIIR(G)8, IGFIIR(CT)5, TGFßRII(GT)3, TGFßRII(A)10, hMSH3(A)8, hMSH6(G)8, BAX(G)8 and Bat26, were detected by denatured polyacrymide gel electrophoresis-silver stain in 28 gastric cancers. Bat26 was also analyzed by denatured high performance liquid chromatograph (DHPLC) at 50℃ in the DNASep Cartridge. Two MSI-H (7.14%) and 15 MSI-L cancers (53.6%) were identified in 28 gastric cancers. Bat26 was positive only in 2 MSI-H cancers. The alterations of Bat26 and MSI-H status were coincident (P<0.01). The two Bat26+ cancers were also confirmed by DHPLC. Results obtained from DHPLC and gel electrophoresis were completely consistent. Thus, DHPLC analysis of Bat26 site may be a favorable method of detecting MSI-H status in gastric cancer, and be of clinical importance.     

数据综合分析鉴定人类Auxilin基因

Auxilin蛋白诱导Hsp70c蛋白与笼形蛋白的结合,在真核细胞衣被小泡脱衣被的过程中扮演了重要的角色.通过对已有EST,STS等数据库的综合分析,我们将人类auxilin基因定位到1p31,D1S515和D1S198标记之间.26个EST构成的5个重叠群,占该基因中共约2.3 kb的部分cDNA序列,其中编码区长501 bp,得到的序列与牛的auxilin基因显示有极高的同源性.各EST数据显示,auxilin在人胚胎的多种组织中表达,在成人脑、表皮组织中也有表达.     

非小细胞肺癌6号染色体长臂的杂合性缺失的微卫星扫描分析

为探讨6号染色体长臂上与非小细胞肺癌发生相关的微卫星位点,应用多重PCR对41例非小细胞肺癌中6号染色体长臂上的36个微卫星位点进行扩增。PCR产物应用聚丙烯酰胶凝胶电泳分离,电泳结果用GeneScan^TM、Genotyper^TM软件进行分析。结果发现各个位点有明显不同的LOH频率,除D6S1579在41例非小细胞肺癌中未发现杂合性缺失外,其余35个位点均有至少一个位点发生突变,LOH频率从3.57%到75%不等,总LOH频率为78%（32/41）,其中D6S302位点的LOH频率最高（75%）。LOH频率超过20%的位点共有14个,主要分布在2个区域。其中有6个位点：D6S458（21.43%)、D6S1694（26.92%）、D6S1717（35.71%）、D6S1565（40%）、D6S302（75%）、D6S1706（36.36%）分布在6q21附近,具体区域是6q16.3-q21;有5个位点：D6S1550（38.4%）、D6S264（20%）、DS1585（25%）、D6S446（33.3%）、D6S281（30.77%）分布在6q27附近,具体区域是6q26-q27。因此6q21、q27附近可能存在与非小细胞肺癌发生相关的巳知或未知肿瘤抑制基因。     

Two new ballistoconidium-forming yeast species, Bullera melastomae and Bullera formosana, found in Taiwan

Two yeast strains, the cells of which contained xylose and Q-10 as the major ubiquinone, were isolated from a plant leaf collected in Taiwan. These yeasts were found to represent two new species of the genus Bullera in the Hymenomycetes. Identification was based on the sequence analysis of the 18S rDNA, the internal transcribed spacer (ITS) regions and the D1/D2 domain of 26S rDNA. The yeasts are named Bullera melastomae sp. nov. and Bullera formosana sp. nov. In the phylogenetic trees based on 18S rDNA and D1/D2 domain of 26S rDNA sequences, these two species constitute a cluster connected with Dioszegia cluster in the Cryptococcus luteolus lineage.     

<Emphasis Type="Italic">Metschnikowia viticola</Emphasis> sp. nov., a new yeast species from grape

Two yeast strains, producing needle-shaped ascospores under suitable conditions, were isolated from grapes grown in Hungary. Based on these two strains, Metschnikowia viticola (type strain NCAIM Y.01705, CBS 9950, JCM 12561) is proposed as a new yeast species. Considering its phenotypic features, the restriction fragment patterns of 18S rDNA and the sequence of the D1/D2 domain of 26S rDNA, the proposed new species is closely related to Candida kofuensis.     

Asterotremella gen. nov. albida, an anamorphic tremelloid yeast isolated from the agarics Asterophora lycoperdoides and Asterophora parasitica

Using a genotypic approach (PCR-fingerprinting, DNA/DNA reassociation, partial sequences of the 26S rDNA gene, complete sequences of the 18S rDNA gene, and sequences of the internal transcribed spacers) five tremelloid yeast isolates from the agarics Asterophora lycoperdoides and A. parasitica were shown to be conspecific with Cryptococcus ramirezgomezianus. It was not possible to distinguish the yeast strains from A. lycoperdoides and A. parasitica using sequences from the intergenic spacer (IGS1). Phylogeny based on the 26S (D1/D2-domain), ITS1-5.8S-ITS2 and complete 18S rDNA demonstrated that C. ramirezgomezianus is closely related to several additional Cryptococcus species (C. humicola, C. longus, C. musci, C. pseudolongus) within the Trichosporonales. A new genus, Asterotremella, and a new family, Asterotremellaceae were introduced for Cryptococcus species clustering within the Trichosporonales having a ubiquinone Q-9. Cryptococcus ramirezgomezianus is a synonym of Asterotremella albida.     

Changes of diversity and population of yeasts during the fermentations by pure and mixed inoculation of Saccharomyces cerevisiae strains

Mixed inoculation of Saccharomyces cerevisiae strains is used in winemaking for achieving high sensory quality of the wine. However, information on the diversity and population of yeasts during inoculated fermentation is very limited. In this study, we evaluated the effect of mixed inocula with different inoculation timing on the yeast community during fermentations of Cabernet Sauvignon. Grape must was inoculated with pure cultures of S. cerevisiae RC212 or S. cerevisiae R312, and simultaneous and sequential inoculation of both strains. Wallersterin Laboratory Nutrient (WLN) medium and sequence of the 26S rDNA D1/D2 domain were used to compare the diversity of yeast species. Five species, including Candida diversa, Hanseniaspora opuntiae, H. uvarum, Issatchenkia orientalis and I. terricola, were identified in the grape must, with Issatchenkia sp. being predominant (67.5 %). Three to four species were involved in each fermentation treatment. The fermentations by mixed inocula presented more yeast species than by pure inocula. Interdelta sequence typing was used to identify S. cerevisiae strains. Ten genotypes were identified among 322 isolated S. cerevisiae strains. Their distribution varied among different stages of fermentations and different inoculation treatments. The inoculated strains were not predominant, while indigenous genotypes I, III, and V showed strong competitiveness during fermentation. In general, this study provided information on the change of population structure and genetic diversity of yeasts in fermentations inoculated with pure and mixed S. cerevisiae strains.     

青海东部土壤中酵母物种多样性研究

从青海的互助、民和、门源等10个州县收集土样分离得到98株酵母菌, 利用26S rDNA D1/D2区域序列分析并结合形态学和生理生化特性对这些菌株进行了分类学研究, 探讨了青海东部土壤中酵母的物种多样性及其分布。共鉴定出10属13种(其中有两个疑似新种), 其中 Galactomyces geotrichum和Rhodotorula mucilaginosa为该地的优势种。     

Evaluation of yeast diversity during wine fermentations with direct inoculation and pied de cuve method at an industrial scale

The diversity and composition of yeast populations may greatly impact wine quality. This study investigated the yeast microbiota in two different types of wine fermentations: direct inoculation of a commercial starter versus pied de cuve method at an industrial scale. The pied de cuve fermentation entailed growth of the commercial inoculum used in the direct inoculation fermentation for further inoculation of additional fermentations. Yeast isolates were collected from different stages of wine fermentation and identified to the species level using Wallersterin Laboratory nutrient (WLN) agar followed by analysis of the 26S rDNA D1/D2 domain. Genetic characteristics of the Saccharomyces cerevisiae strains were assessed by a rapid PCR-based method, relying on the amplification of interdelta sequences. A total of 412 yeast colonies were obtained from all fermentations and eight different WL morphotypes were observed. Non-Saccharomyces yeast mainly appeared in the grape must and at the early stages of wine fermentation. S. cerevisiae was the dominant yeast species using both fermentation techniques. Seven distinguishing interdelta sequence patterns were found among S. cerevisiae strains, and the inoculated commercial starter, AWRI 796, dominated all stages in both direct inoculation and pied de cuve fermentations. This study revealed that S. cerevisiae was the dominant species and an inoculated starter could dominate fermentations with the pied de cuve method under controlled conditions.     

云南程海湖酵母菌多样性及应用

【目的】针对云南丽江永胜县境内程海湖环境的特殊性,研究高原湖泊环境中酵母菌的多样性,初步探索程海湖环境中酵母菌的利用价值。【方法】对程海湖的湖水和其周边土壤样品中的酵母菌进行分离;应用26S rDNA的D1/D2区域序列分析,并结合形态及生理生化指标对分离获得的酵母菌进行鉴定;采用筛选培养基对已鉴定酵母菌进行产酶定性实验,分析高原湖泊中酵母菌的多样性及可应用性。【结果】分离得到酵母菌64株,对其中63株进行鉴定,归属于9个属22个种(包括4个疑似新种或新变种);地霉属Geotrichum和隐球酵母属Cryptococcus是2种环境中的共有属;在产酶活性筛选中发现有9株产胞外酶活性的菌株,其中YM24373既产蛋白酶又可产淀粉酶。【结论】研究结果显示程海湖中酵母菌组成具有较为丰富的多样性,其应用价值值得进一步研究。     

Zygosaccharomyces kombuchaensis,a new ascosporogenous yeast from 'Kombucha tea'

A new ascosporogenous yeast, Zygosaccharomyces kombuchaensis sp. n. (type strain NRRL YB-4811, CBS 8849), is described; it was isolated from Kombucha tea, a popular fermented tea-based beverage. The four known strains of the new species have identical nucleotide sequences in domain D1/D2 of 26S rDNA. Phylogenetic analysis of D1/D2 and 18S rDNA sequences places Z. kombuchaensis near Zygosaccharomyces lentus. The two species are indistinguishable on standard physiological tests used for yeast identification, but can be recognized from differences in restriction fragment length polymorphism patterns obtained by digestion of 18S-ITS1 amplicons with the restriction enzymes DdeI and MboI.     

石河子不同树龄蟠桃土壤中可培养酵母多样性分析及功能酵母的筛选

为了探究新疆本土蟠桃园可培养酵母菌多样性,并挖掘功能酵母资源,本研究以新疆石河子蟠桃园3年、8年、15年树龄的根际和非根际土壤以及桃树叶片为材料,经过传统的分离培养方法获得可培养的酵母菌菌株,并进行形态学、生理生化以及26S r DNA的D1/D2区序列分析,共获得可培养酵母菌129株,从属于12个属17个种,其中子囊菌酵母为优势菌群,占分离属的88%,分布于威克汉姆酵母属(Wickerhamomyces),Vanrija属,Barnettozyma属和有孢圆酵母属(Torulaspora)等11个属的15个种。担子菌占分离属的12%,分布于隐球酵母属(Cryptococcus)的2个种。其中优势属威克汉姆酵母属,包括异常威克汉姆酵母(W. anomalus)和W. pijperi两个种,占总比例的33%,优势种异常威克汉姆酵母所占总株数比例为17%。从可培养酵母中共筛选出23株功能酵母,其中富硒酵母21株,优势种为白地霉(Galactomyces candidum),产蛋白酶酵母2株均属于隐球酵母属的Cryptococcus albidus。结果表明,新疆桃园中蕴含丰富的酵母菌资源,非根际土壤中的酵母多样性大于根际及叶片酵母多样性,且分离得到富硒酵母及产蛋白酶酵母。本研究挖掘了新疆本土可培养酵母菌资源,同时也为功能酵母的开发和利用提供理论指导。