逆转座子激活对白念珠菌适应性形成的调控作用及其机制研究

发布时间：2018-04-02 20:23

本文选题：白念珠菌　切入点：逆转座子　出处：《第二军医大学》2011年硕士论文

【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(H_2O_2)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。H_2O_2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H_2O_2刺激激活逆转座子转录的机制,本课题在MCZ和H_2O_2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H_2O_2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、H_2O_2刺激所导致的细胞内ROS的产生积聚。提示MCZ和H_2O_2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;H_2O_2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。
[Abstract]:Candida albicans is one of the main pathogenic bacteria of opportunistic human fungal infection, many potential factors include immunosuppressive therapy, antibiotic therapy, human immunodeficiency virus infection and diabetes leads to increased incidence of infection of Candida albicans, Candida albicans and the environment itself highly adaptable more fungal infection situation of retrotransposons (retrotransposons). Is a movable gene fragment can be mediated by RNA occurred independent transposition of eukaryotes, widespread in multicellular, have important influence on the structure of eukaryotic genomic biology and genetic evolution. Recent studies about retrotansposons transposition activity mechanism and its influence on the expression of the host gene transcription is one of the hot topics in the field of life science research in the world. This paper aims to find the role of Candida albicans is involved in the regulation of reversal The possible mechanism of retrotransposon producing transcriptional transposition activity is preliminarily discussed, and the relationship between retrotransposon activation and its high adaptability in Candida albicans is explained from the perspective of genetic evolution.
Using the method of real-time RT-PCR, SC5314 for Candida albicans strains, study in damage repair pathway genes in a variety of conditions to stimulate the expression of retrotransposons, and further differences in expression of Candida albicans genome reversal ORFs encoding region protein transposon has transcriptional activity. In order to find a large number of literature that the activation of retrotansposons associated with DNA damage, so the first choice to direct effects on DNA molecules of carcinogen MMS stimulation, can cause in the damage repair genes, especially in homologous recombination repair gene expression increased, and can cause except TCA8 (orf19.6078 and orf19.6079) than most retrotransposon ORFs transcription in different degree of activation, suggesting that Candida albicans there is a retrotransposon activity. On this basis, we deeply study some important environmental stimuli (high permeability, oxygen Chemical stimulation, azoles) concentration reached the median lethal dose (IC50) on expression of transcription of Candida albicans encoding retrotransposon protein ORFs. The results showed that miconazole (MCZ) and hydrogen peroxide (H_2O_2) stimulation can cause DNA damage repair genes involved in extensive transcriptional activation, and in response to MCZ 6h, TCA3 (ORF19.2219), Zorro2 (ORF19.7274 and ORF19.7275) and TCA11 (ORF19.6469) occurred in the activation of.H_2O_2 transcription stimulated at 6 h can selectively activate TCA8 (ORF19.6078 and ORF19.6079) transcriptional activity, and at the time of repair gene prior to the retrotransposon activity. But resulting in high permeability stimulus conditions, whether it is NaCl or sorbital osmotic stimulation, were not found in retrotransposon activity, the expression of damage repair genes influence obviously. Therefore the environment expression activity regulation has selectivity to retrotransposons, which in turn Recording activation is closely related to the repair of cell damage.
For the further study of MCZ and H_2O_2 stimulation mechanism of activation of transcription of retrotransposons, this paper gives the antioxidant NAC in MCZ and H_2O_2 before stimulation, growth, joint action between the two strains of retrotransposons, participate in repair gene expression and intracellular levels of ROS the influence of three aspects. The results show that the bactericidal effect of NAC and MCZ combined stimulation can inhibit MCZ, effectively protect the yeast cells and the state, combined with H_2O_2, but the growth of strains had no protective effect; the combined effects of NAC and two can significantly reduce the expression of damage repair genes caused by stimulation, and stimulation activated retrotransposon expression activity will be weakened or disappeared active oxygen; fluorescence detection cells show that NAC can effectively inhibit MCZ production, accumulation of H_2O_2 caused by stimulation of ROS cells suggest that MCZ and H_2O_2 stimulated ROS accumulation so fine At the same time, the DNA damage repair pathway was initiated, and the expression of the retrotransposon was regulated.
Finally, we use Southern blotting technology in-depth analysis of all process whether these activated retrotransposons can be involved in the replication cycle, until the formation of a double stranded DNA molecule complete, further evaluation of the transcriptional activation of retrotransposons. Experimental first in the long time of the liquid culture medium environment pressure stimulation induced several strains of bacteria by retrotransposon probe hybridization after enzyme digestion of genomic and specific markers of color, found that MCZ induced bacteria copy of Zorro2 fragment was significantly increased, while TCA3 decreased H_2O_2 induced copy fragment; bacteria also showed that may occur in TCA8 copy fragments increased. Then using the broth dilution method and preliminary identification of spot assay phenotype, found after induction the increase of strain stimulus tolerance respectively. The results show that the stimulus will lead to some retrotransposons in the genome copies of the long-term environmental pressure , this genome remodeling may be through a variety of ways to change the host gene expression, regulation of the ability to adapt to environment. Therefore, we believe that the potential regulatory role of retrotransposons in the genome level is the inherent power of genetic evolution of Candida albicans, Candida albicans and high adaptability formation is likely related to regulation of retrotransposons are widely distributed and in the genome.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R379

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