【作者】 赵国庆；

【导师】 冉玉平；

【作者基本信息】 四川大学 ， 皮肤病与性病学， 2007， 博士

【摘要】 背景：近年来，马拉色菌越来越引起真菌学家和临床医学家的关注，它是一类可引起多种皮肤病的嗜脂性真菌，马拉色菌除可引起花斑糠疹、马拉色菌毛囊炎外尚可引起头皮糠疹、甲真菌病、龟头包皮炎、真菌血症、皮肤脓肿等。与诱发脂溢性皮炎、特应性皮炎、头皮糠疹的免疫机制密切相关，可能与融合性网状乳头瘤病相关，并与银屑病的发病和加重相关，在严重免疫缺陷及静脉营养患者尚可引起系统性感染。但马拉色菌致病分子机制至今尚未阐明，相关基因表达比较研究亦尚未见报道。马拉色菌最常导致的疾病为花斑糠疹，在花斑糠疹患者皮损刮取鳞屑直接镜检查见的马拉色菌大多呈菌丝态，而在健康人皮肤上则基本以酵母态形式存在，这种菌形态不同的原因目前还不清楚，推测可能与局部环境(温度、湿度、皮脂成分)不同有关。故糠秕马拉色菌在机体内外因素的作用下由酵母态转化成菌丝态很可能是临床致病的关键所在。在已公认的马拉色菌7个种中，糠秕马拉色菌较为常见，其菌丝态在试管内已被成功地诱导出，它也是目前唯一的一种可在体外培养出菌丝态的马拉色菌。通过比较糠秕马拉色菌酵母态和菌丝态的差异将对了解马拉色菌的致病机制具有重要意义。而随着微生物基因组序列的大量测定，通过比较基因表达差异分析致病微生物的分子致病机制已成为新近微生物基因组研究的热点。近年发展起来的抑制消减杂交技术为比较同源生物的基因表达差异提供了一条新的途径。目的：本研究试图应用抑制消减杂交技术比较糠秕马拉色菌的菌丝态和酵母态之间mRNA差异以分析糠秕马拉色菌的菌丝态和酵母态的基因表达差异，建立糠秕马拉色菌菌丝态和酵母态细胞的差异表达cDNA文库，获得其差异表达的基因片段，以了解其酵母态转化为菌丝态的相关分子机理，为进一步深入研究糠秕马拉色菌的致病机制奠定基础。方法：首先将糠秕马拉色菌(M.furfur CBS 1878)标准株分别接种于菌丝态和酵母态液体培养基，3天后分别提取糠秕马拉色菌菌丝态和酵母态细胞的总RNA，然后从中分离出mRNA，随后逆转录生成菌丝态和酵母态细胞双链cDNA，经RsaI酶切消化后，然后运用抑制消减杂交技术进行杂交，相同的片断将被消减掉，再经过2次抑制性PCR扩增消减后片断，获得菌丝态或酵母态细胞差异表达的cDNA片段并建立差异文库。再对差异cDNA片段进行克隆及斑点杂交鉴定，最后对阳性克隆进行测序、在GenBank上进行相似性对比和利用DNA序列软件进行序列分析。结果：1．成功培养出糠秕马拉色菌菌丝态。2．获得糠秕马拉色菌菌丝态和酵母态细胞的总RNA，并分离出其mRNA。3．建立了菌丝态及酵母态细胞差异表达的cDNA文库。4．经菌落PCR和斑点杂交鉴定差异文库，实际共得菌丝态特有片断克隆22个，酵母态特有表达片断克隆8个。5．将上述30个鉴定阳性克隆和4个可疑阳性克隆测序，用Blast程序在GeneBank中检索克隆片段基因序列的同源性，所有检索到与其他物种蛋白或序列有一定相似度的克隆可分为以下几类：①生化代谢类：A4与ATP合酶、A16与NADH脱氢酶Ⅰ、A23与细胞色素C氧化酶亚单位Ⅲ、A38与线粒体序列、A49与NADH脱氢酶Ⅱ等有一定同源度。B4与线粒体肌酸激酶有一定相似性，B7与ATP合酶的β亚基同源(A4与ATP合酶α亚基同源)，B9则与原海豹霉属的细胞色素b同源。②与转录有关：A8与剪接因子2或DNA结合蛋白相似，A13与可抑制白介素2表达的转录因子8及锌指蛋白具有高度同源，A14与核转录因子Y高度同源，与磷酸酯酶也具有一定同源性；A40与转录变体ACTN2高度同源。③与生长相关：A10与牛生长因子受体α多肽序列同源，但Blastx未找到同源蛋白。④细胞结构类：A12与细胞壁脂蛋白相关水解酶及脂蛋白具有一定同源性，A22与大肠杆菌嗜菌体衣壳蛋白有一定同源性，与米曲霉的基因组序列部分相同。⑤其他类：A15与肌联蛋白、A21与耻垢分支杆菌假设蛋白、A56与念珠棘虫属赤霉GTP结合蛋白有很高的相似度，B3与苏芸金芽胞杆菌质粒小部分序列相似，B10则有30％序列和玉蜀黍黑粉菌、绿藻门小部分序列具有相似性，B12与构巢曲霉的40S核糖体S7蛋白具有一定相似度。6．测序的21个序列中，经过Omiga 2.0等核酸序列分析软件进行正相三个和负相三个共六个相位的ORF搜索，发现所得序列均具有起始密码子和终止密码子，即为可能的ORF，翻译后的6相位的氨基酸序列均含有大量真菌蛋白质二级结构基序。表明所获得的片断均为编码蛋白质的基因片断。结论：采用消减杂交技术成功建立了糠秕马拉色菌的差异cDNA文库，筛选出一系列可能包含毒力相关基因的糠秕马拉色菌菌丝态和酵母态特有片段，片段部分同源则可能提示这16个片段与同源蛋白有某种功能或进化联系，分别与生化代谢酶、转录因子、生长因子、细胞结构或相关膜蛋白相似，提示这些基因片段可能与糠秕马拉色菌菌丝态和酵母态的代谢、分化、毒力或分子进化有关。这些结果对了解糠秕马拉色菌致病的分子机制提供了重要信息。更多还原

【Abstract】 Backgrounds The genus Malassezia has received considerable attentionin recent years from mycologists and clinicial doctors. This group oflipophilic yeasts, long known to be the pathogenic agents of pityriasis (tinea)versicolor and Malassezia folliculitis, is also increasingly being associatedwith the causation of balanitis, onychomycosis, papillomatosis, and invasivehuman infections, as well as potential immunogenic triggering of atopicdermatitis, seborrheic dermatitis, and dandruff. Otherwise, Malassezia canprobably irritate and deteriorate lesions in psoriasis; and inimmunocompromised patients and those of receiving intralipid therapy theycan induce severe systemic infection. Molecular pathogenic mechanism ofMalassezia is still unclear at present. Now genome project of Malasseziaisn’t broungt to consideration. Very few reports about gene experession andthe virulence-associated genes of the fungi appeared so far, which results inthe slow development in the study of pathogenic mechanism of Malassezia. Malassezia is regularly present in its yeast phase in the skin of healthyadults, produces the clinical lesions of pityriasis versicolor when developingits mycelial phase. The conditions that induce such transformation remainunclear, although the available data at present indicate that it could be causedby changes in the local conditions (e.g. temperature, humidity, sebumcomposition) on an idiosyncratic basis. The morphological transformation ofMalassezia from a yeast phase to a mycelial phase is the key pathogenicmechanism of Malassezia that be commonly received. Malassezia furfur(M.furfur) is the only one species of Malassesia that can be induced toproduce its mycelial phase in vitro. To compare the difference of geneexpression between mycelial phase and yeast phase of M.furfur can help us toknow its molecular pathogenic mechanism.Since 1990s, to compare the difference of gene expression betweenhomospecific microorganism has become a focus of attention inmicrobiologic society, accompanied with total genome in manymicroorganism have been sequenced. The recently developed method ofsuppression subtractive hybridization (SSH) provided a powerful new way tostudy molecular pathogenic mechanism of pathogenic microorganism bycomparison with the difference of microorganism mRNA.Objectives To compare the difference of gene expression betweenmycelial phase of M. furfur and its yeast phase, and we try to found twodifferential cDNA libraries. To understand the molecular pathogenicmechanism of the fungi and its morphological switching mechanism from ayeast phase of growth to a mycelial phase.Methods①To isolate the total RNA both in mycelial and yeast phase ofM. furfur, then to isolate mRNA from them severally.②Reverse them to ds-cDNA and then digest them by RsaI.③Hybridize with mycelial phase ofM.furfur as tester and its yeast phase as driver with the method of suppressionsubtractive hybridization (SSH), a reverse process also to be performed at thesame time.④The resulted PCR mixture was connected to T vector toconstruct the subtractive library.⑤Dot blot techniques were employed toindentify those positive clones.⑥That positive differential gene expressionclone to be sequenced, then search similary genes and proteins of thatacquired gene sequences by BLAST (basic local alignment search tool,BLAST) in GenBank to predict their function, and analyse them by DNAsequence analysis softwares.Results 1. Succeed in culturing mycelial phase of M. furfur. 2. Succeed ingaining total RNA both in mycelial and yeast phase of M. furfur and isolatingtheir mRNA. 3. Two differential cDNA libraries had been found with M.furfur in mycelial phase and its yeast phase. 4. Twenty-two positive cloneswere acquired by subtraction in positive direction. Eight positive clones wereacquired by subtraction in reverse direction. Positive clones in both directionsand four suspicious positive clones were sent to sequencing. 5. Thirty-fourfragments were compared through BLASTn and BLASTx in GenBank.Thirteen fragments were either repeated or belonging to vector sequences;fifteen fragments acquired by subtraction in positive direction and sixfragments from reverse direction weren’t repeated and also not belonging tovector sequences. 6. All of these 21 clones have more or less homologywith genes or proteins from other biological species. Sum up homologousgenes and proteins of these fragments, they can be clarified into fivecategories:①Fragments homologize to biochemical metabolic enzyme: A4,A16, A23, A38, A49, B4, B7, and B9.②Fragments homologize to transcription associated protein: A8, A13, A14, A40, and B 12.③Fragmentshomologize to growth factor: A10.④Fragments homologize to cell structureproteins: A12 and A22.⑤Others: A15, A21, B3, and B10.7. Open readingframeworks and lots of fungi motifs have been found existing in all of 21fragments by sequence analysis software. Initiation codons and terminationcodons exist in all of them too.Conclusions Succeed in founding two differential cDNA library with M.furfur in mycelial phase and yeast phase by suppressive subtractivehybridization techniques, and a series of fragments which possibly containedgenes related to virulence of M. furfur have been screened. These sequenceshomologize to genes of fungal virulence or molecular evolution such asmembrane protein, metabolic enzyme and cell structure, et cetra.更多还原