运用GLGI技术鉴定白念珠菌LongSAGE标签

发布时间：2018-03-25 09:58

本文选题：白念珠菌　切入点：基因表达序列分析　出处：《第三军医大学》2005年硕士论文

【摘要】： 白念珠菌是一种重要的条件性致病真菌,在各种真菌感染中占了首位。感染人的粘膜表面可引起鹅口疮、念珠菌性阴道炎疾病;在系统性疾病的病人和免疫缺陷的人群中,白念珠菌可以引起全身的播散性感染并导致死亡。白念珠菌的毒力因子和发病机理有关,主要包括促进白念珠菌粘附于宿主细胞的生物分子(黏附素),与侵入有关的酶SAP(分泌型天冬氨酸蛋白酶)和PL(磷脂酶),及其菌相转换(生长状态,可逆的单个酵母细胞和菌丝的转换)。并且,菌相转换和抗原表达的改变有关。为更全面的寻找白念珠菌酵母相和菌丝相致病相关基因及其毒力因子,本实验室前期应用LongSAGE技术,通过构建白念珠菌酵母相和菌丝相细胞LongSAGE标签库,来定性定量检测白念珠菌酵母相和菌丝相细胞表达基因的改变,并聚类分析表达基因功能,探讨酵母相和菌丝相细胞表达基因与菌相转换、菌株毒力的相关性。为了验证LongSAGE标签,我们利用SAGE标签产生长片段cDNA应用于基因识别技术(generation of longer fragments from serial analysis of gene espression(SAGE)tags for gene identification GLGI),将17bp的SAGE标签向3’端扩增到数百bp。17bp的LongSAGE标签作为正向引物,3’端使用的是单一碱基dA、dC或dG与oligo-dT结合的引物进行第1次PCR扩增,只有与LongSAGE标签相匹配的序列才能退火并得以扩增;在第2次循环中,只有5’端oligo-dA的前一碱基与锚定的oligo-dT相匹配的序列才能扩增,而单纯的oligo-dA与oligo-dT结合的片段因处于锚定状态则受抑制,因而最终得到的是含有LongSAGE标签的长片段DNA扩增产物,产物长度在200～1000bp之间。 GLGI技术提供了几种潜在的结果。首先,它为LongSAGE技术提供了发展策略;其次,结合应用LongSAGE-GLGI技术可以作为人或其它物种的基因的选择和表达;再次,它可以鉴定任何基因的3’端序列的外显子;最后,结合应用LongSAGE-GLGI技术可以确定人或其它物种的3’端在基因家族中所表达基因。 20个多重匹配标签中有15个获得稳定有效的扩增,长度在200～1000bp之间,获得的长片段进行序列分析后,有超过95%的碱基与已知基因相同,并且包括原来17bp的SAGE标签,表明获得的序列为此已知基因。其中酵母相多重匹配标签17是基因
[Abstract]:Candida albicans is an important opportunistic fungus, leading the way in fungal infections. Infected mucosal surfaces can cause thrush, candida vaginitis; in patients with systemic diseases and in people with immune deficiency, Candida albicans can cause systemic disseminated infections and lead to death. The virulence factor of Candida albicans is related to the pathogenesis of Candida albicans. These include biological molecules that promote Candida albicans adhesion to host cells (adhesin), invasion-related enzymes SAP (secretory aspartate protease) and PLL (phospholipase), and their phase transition (growth state). Reversible transformation of individual yeast cells and hyphae. In order to search for the pathogenicity genes and virulence factors of Candida albicans in yeast and hyphal phase more comprehensively, LongSAGE technique was used to construct the LongSAGE tagging library of Candida albicans yeast and hyphae cells. The expression genes in yeast and hyphal phase of Candida albicans were detected qualitatively and quantitatively, and the function of expressed genes was analyzed by cluster analysis, and the relationship between the expression genes of yeast phase and hyphae phase and the virulence of the strain was discussed. To verify the LongSAGE tag, We use SAGE tag to produce long fragment cDNA and apply it to gene recognition technique: generation of longer fragments from serial analysis of gene espression(SAGE)tags for gene identification GLGIN. 17bp SAGE tag is amplified to 3'end of 17bp and hundreds of bp.17bp LongSAGE tags are used as forward primer. The primers combined with oligo-dT were amplified by PCR for the first time. Only the sequence matching the LongSAGE tag can be annealed and amplified; in the second cycle, only the first base of the 5'terminal oligo-dA can be amplified by matching the anchored oligo-dT. However, the simple oligo-dA and oligo-dT binding fragments were inhibited because they were in the anchoring state, so the final product was the DNA amplification product containing the LongSAGE tag, and the length of the product was between 200~1000bp. GLGI technology offers several potential results. First, it provides a development strategy for LongSAGE technology; secondly, the combination of LongSAGE-GLGI technology can be used as gene selection and expression of human or other species. It can identify the exons of the 3'terminal sequence of any gene. Finally, the 3'terminal gene expressed in the gene family can be identified by using LongSAGE-GLGI technique. Fifteen of the 20 multiple matching tags were stably and effectively amplified, and the length of the fragments was between 200~1000bp. After sequence analysis, more than 95% of the bases were identical to the known genes and included the original 17bp SAGE tag. The obtained sequence is a known gene, and yeast phase multiple matching label 17 is a gene.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R379

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