【作者】 朱春香；

【导师】 姜远英； 阎澜； 高平挥；

【作者基本信息】 第二军医大学 ， 药理学， 2011， 硕士

【摘要】 白念珠菌是人类真菌感染主要的机会致病菌之一,目前许多潜在因素包括免疫抑制剂治疗、抗生素治疗、人类免疫缺陷病毒感染以及糖尿病等导致白念珠菌感染发病率升高,而白念珠菌本身对环境高度的高适应能力更加重真菌感染的形势。逆转座子（retrotransposons）是一种可移动基因片段,可经RNA介导发生自主转座,广泛存在于多细胞的真核生物中,对真核生物的基因组结构以及遗传进化都具有重要的影响。近年来关于逆转座子的转座活性控制机制及其对宿主基因转录表达影响的研究是当前国际上生命科学领域研究的热点课题之一。本课题旨在发现白念珠菌中参与调控作用的逆转座子,并初步探讨逆转座子产生转录转座活性的可能机制,从遗传进化的角度解释白念珠菌中的逆转座子激活与其高适应性形成的关系。实验采用实时定量RT-PCR的方法,以白念珠菌SC5314为供试菌,考察参与损伤修复通路的基因在各种刺激条件下表达情况,并进一步比较白念珠菌基因组中逆转座子蛋白编码区ORFs的表达差异以寻找具有转录活性的逆转座子。大量文献提示逆转座子的激活与DNA损伤相关,因此首先选择直接作用于DNA分子的致癌剂MMS刺激,可引起参与损伤修复的基因,特别是参与同源重组修复的基因表达升高,并且可引起除TCA8（orf19.6078 and orf19.6079）以外的多数逆转座子ORFs发生程度不同的转录激活,提示白念珠菌中存在着有活性的逆转座子。在此基础上,我们深入地研究一些重要的环境刺激因素（高渗透、氧化刺激、唑类药物）浓度达到半数致死剂量（IC50）时对白念珠菌逆转座子蛋白编码区ORFs的转录表达影响。结果表明咪康唑（MCZ）和双氧水（H₂O₂）刺激均可引起参与DNA损伤修复的基因广泛地转录激活,并且在MCZ刺激6h时,TCA3 （ORF19.2219）、Zorro2 （ORF19.7274和ORF19.7275）以及TCA11 （ORF19.6469）发生转录激活。H₂O₂刺激6 h时能选择性地激活TCA8 （ORF19.6078和ORF19.6079）的转录活性,且在时间上修复基因优先于逆转座子产生活性。但在高渗透刺激条件下,无论是NaCl还是sorbital高渗透刺激,均未发现有活性的逆转座子,对损伤修复基因的表达影响无上调作用。由此推测环境对逆转座子表达活性的调节具有选择性,其转录激活与细胞的损伤修复密切相关。为进一步探讨MCZ和H₂O₂刺激激活逆转座子转录的机制,本课题在MCZ和H₂O₂刺激前给予抗氧化剂NAC,比较二者联合作用对菌株的生长、逆转座子、参与损伤修复基因的表达以及细胞内ROS水平三方面的影响。研究表明NAC与MCZ刺激联合时可抑制MCZ的杀菌作用,有效地保护酵母态细胞,而与H₂O₂联用却对菌株的生长情况无保护作用;NAC与二者联合作用均可显著地降低刺激所导致的损伤修复基因表达上调,并且刺激所激活的逆转座子表达活性也会相应的减弱或消失;荧光检测细胞内活性氧显示NAC可有效地抑制MCZ、H₂O₂刺激所导致的细胞内ROS的产生积聚。提示MCZ和H₂O₂刺激后的ROS积聚使得细胞间接发生氧化损伤,同时启动DNA损伤修复通路,调控逆转座子的表达。最后,我们采用southern blotting技术深入分析这些激活的逆转座子是否能够参与复制周期的所有过程,直至形成完整的双链DNA分子,进一步评估转录活化的逆转座子功能。实验首先在液体培养基中长时间的环境加压刺激诱导若干株菌,通过酶切后的基因组与特定标记的逆转座子探针杂交显色,发现MCZ诱导菌中Zorro2的拷贝片段明显增加,而TCA3的拷贝片段反而减少;H₂O₂诱导菌也显示可能发生TCA8拷贝片段的增加。随后采用微量液基稀释法及spot assay实验初步鉴定表型,发现诱导后的菌株对各自的刺激源耐受性增加。结果表明环境长期的加压刺激会导致某些逆转座子在基因组中的拷贝变化,这种基因组重塑可能通过多种方式改变宿主基因的表达,调控对环境的适应能力。因此,我们认为逆转座子在基因组水平的潜在调节作用是白念珠菌遗传进化的内在动力,而白念珠菌高适应性的形成也很有可能与其基因组中广泛分布的逆转座子的调控作用相关。更多还原

【Abstract】 Candida albicans, the major human fungal pathogen, is capable of causing superficial to life-threatening infections. Recently, many predisposing factors, including immunosuppressive therapy, antibiotic therapy, human immunodeficiency virus infection and diabetes, cause the high rate of C.albicans infection; however the high adaptability to environment change for C. albicans cells leads to current critical infection situation. Retrotransposons are mobile genetic elements capable of autonomous transposition via RNA intermediates. They are widespread in multicellular eukaryotes, and have an important effect on the structure of eukaryotic genomic and genetic evolution. In recent years, researches about retrotansposons transposition activity mechanism and their impact on the host gene transcription are one of the hot topics on the life science in current international research. The aim for this study is to discover active retrotansposons involving in regulating host genes in C. albicans, preliminarily to investigate the possible mechanism for retrotansposons transcription and transposition activity, and finally to explain the relationship between activated retrotansposons and the high adaptive ability in C. albicans from the perspective of genetic evolution.In this study, real-time quantitative RT-PCR for C. albicans SC5314 was adopted to calculate the relative express quantity for genes involving in DNA damage repair parthway, meanwhile, according to the relative expression variation for retrotansposons protein encoded regions （represented by ORFs） in C. albicans genome in response to stresses, the transcriptionally active retrotansposons could be found. Based on the previous studies,it is considered that the activation of retrotansposons is associated with DNA damage. So carcinogenic agent MMS, which directly impacted on the DNA molecule, was first selected. Results showed that most of the genes participating in gene damage reparation, especially in homologous recombination （HR） progress, were transcriptionally activated, and most of the seleted retrotransposons ORFs were activated except TCA8 （ORF19.6078 and orf19.6079）. Further studies were carried out to investigate the relative expression for retrotansposons protein encoded regions in response to kinds of environmental stresses such as （Hyperosmotic、H₂O₂、azole drug ）. The concentration of all these stresses reached half of lethal dose （IC50）. It is showed that genes involving in DNA damage reparation were widely upregulated in the response to miconzole （MCZ） and H₂O₂ stress, and TCA3 （ORF19.2219）、TCA11 （ORF19.6469） and Zorro2 （ORF19.7274 and ORF19.7275） were transcriptionally activated under the treatment with MCZ for 6 h; And TCA8 was selectively activated under the treatment with H₂O₂ for 6 h. Moreover, the activated repair genes showed priority to retrotansposons activity in time. However, hyperosmotic stress, neither NaCl hyperosmotic nor sorbital hyperosmotic, couldn’t increase the retrotansposons expression, and no up-regulated genes involving DNA-damage repair were found in response to hyperosmotic. It is suggested that the regulation function for retrotansposons activity is condition-selective, and closely related with cell gene damage repair pathway.To further explore the possible mechanism for retrotansposons transcriptionally activity induced by MCZ and H₂O₂ stress, anti-oxidant NAC was added before the two kinds of stresses. The combinated effects on the strains growth, the relayive expression changes of retrotansposons and damage repair related genes, and the cellular ROS level were acquired to evaluate the combination function between NAC and MCZ, H₂O₂ stresses. Results showed that NAC could inhibit the killing cells caused by MCZ and effectively protect the yeast state cells, while no function was found under H₂O₂ stress; The up- regulated retrotansposons induced by MCZ and H₂O₂ stress were decreased and almost all the activity of damage repair genes were significantly reduced with the function of NAC; Besides, the generated intracellular ROS caused by MCZ and H₂O₂ stress could be completely inhibited with the combination function of NAC according to fluorenscence intensity detection. We proposed that accumulation of ROS by MCZ and H₂O₂ stress could indirectly cause cell oxidative damage, start the DNA damage repair parthway, and finally regulate the expression of retrotansposons.In the last part, to evaluate the function of activated retrotransposon, southern blotting technique was adopted to in-depth analysis of those active retrotansposons element whether capable of performing all the steps of the replication cycle, up to and including the generation of full-length double-stranded DNA molecules. Several strains were selected under long time pressure in the liquid medium （YPD）. The digested genome of parental and derivative strains, were hybridized to particular probe for retrotansposons elements. The results revealed that more copy numbers of Zorro2 were found in the MCZ induced strains, while decrease of TCA3 copy numbers in derivative strains. Besides, TCA8 copy number was also seemed to increase in H₂O₂ induced strains. Using broth microdilution method and spot assay, all the derivative strains represented increased tolerance to the matching stress respectively. It is indicated that stress could change the copy numbers of retrotansposons, and the genomic remodeling due to retrotransopsoms may change the host gene expression through a number of different ways, and be capable to improve the ability to adapt to environment.Therefore, we propsose that the potential regulation function for the genome in C.albicans would become internal power for genetic evolution, and the high adaptive ability of C. albicans may closely related to the regulation function of widespread retrotansposons in C. albicans genome.更多还原