本文選題：白念珠菌　切入點(diǎn)：逆轉(zhuǎn)座子　出處：《第二軍醫(yī)大學(xué)》2011年碩士論文

【摘要】：白念珠菌是人類真菌感染主要的機(jī)會致病菌之一,目前許多潛在因素包括免疫抑制劑治療、抗生素治療、人類免疫缺陷病毒感染以及糖尿病等導(dǎo)致白念珠菌感染發(fā)病率升高,而白念珠菌本身對環(huán)境高度的高適應(yīng)能力更加重真菌感染的形勢。逆轉(zhuǎn)座子(retrotransposons)是一種可移動基因片段,可經(jīng)RNA介導(dǎo)發(fā)生自主轉(zhuǎn)座,廣泛存在于多細(xì)胞的真核生物中,對真核生物的基因組結(jié)構(gòu)以及遺傳進(jìn)化都具有重要的影響。近年來關(guān)于逆轉(zhuǎn)座子的轉(zhuǎn)座活性控制機(jī)制及其對宿主基因轉(zhuǎn)錄表達(dá)影響的研究是當(dāng)前國際上生命科學(xué)領(lǐng)域研究的熱點(diǎn)課題之一。本課題旨在發(fā)現(xiàn)白念珠菌中參與調(diào)控作用的逆轉(zhuǎn)座子,并初步探討逆轉(zhuǎn)座子產(chǎn)生轉(zhuǎn)錄轉(zhuǎn)座活性的可能機(jī)制,從遺傳進(jìn)化的角度解釋白念珠菌中的逆轉(zhuǎn)座子激活與其高適應(yīng)性形成的關(guān)系。 實(shí)驗(yàn)采用實(shí)時定量RT-PCR的方法,以白念珠菌SC5314為供試菌,考察參與損傷修復(fù)通路的基因在各種刺激條件下表達(dá)情況,并進(jìn)一步比較白念珠菌基因組中逆轉(zhuǎn)座子蛋白編碼區(qū)ORFs的表達(dá)差異以尋找具有轉(zhuǎn)錄活性的逆轉(zhuǎn)座子。大量文獻(xiàn)提示逆轉(zhuǎn)座子的激活與DNA損傷相關(guān),因此首先選擇直接作用于DNA分子的致癌劑MMS刺激,可引起參與損傷修復(fù)的基因,特別是參與同源重組修復(fù)的基因表達(dá)升高,并且可引起除TCA8(orf19.6078 and orf19.6079)以外的多數(shù)逆轉(zhuǎn)座子ORFs發(fā)生程度不同的轉(zhuǎn)錄激活,提示白念珠菌中存在著有活性的逆轉(zhuǎn)座子。在此基礎(chǔ)上,我們深入地研究一些重要的環(huán)境刺激因素(高滲透、氧化刺激、唑類藥物)濃度達(dá)到半數(shù)致死劑量(IC50)時對白念珠菌逆轉(zhuǎn)座子蛋白編碼區(qū)ORFs的轉(zhuǎn)錄表達(dá)影響。結(jié)果表明咪康唑(MCZ)和雙氧水(H_2O_2)刺激均可引起參與DNA損傷修復(fù)的基因廣泛地轉(zhuǎn)錄激活,并且在MCZ刺激6h時,TCA3 (ORF19.2219)、Zorro2 (ORF19.7274和ORF19.7275)以及TCA11 (ORF19.6469)發(fā)生轉(zhuǎn)錄激活。H_2O_2刺激6 h時能選擇性地激活TCA8 (ORF19.6078和ORF19.6079)的轉(zhuǎn)錄活性,且在時間上修復(fù)基因優(yōu)先于逆轉(zhuǎn)座子產(chǎn)生活性。但在高滲透刺激條件下,無論是NaCl還是sorbital高滲透刺激,均未發(fā)現(xiàn)有活性的逆轉(zhuǎn)座子,對損傷修復(fù)基因的表達(dá)影響無上調(diào)作用。由此推測環(huán)境對逆轉(zhuǎn)座子表達(dá)活性的調(diào)節(jié)具有選擇性,其轉(zhuǎn)錄激活與細(xì)胞的損傷修復(fù)密切相關(guān)。 為進(jìn)一步探討MCZ和H_2O_2刺激激活逆轉(zhuǎn)座子轉(zhuǎn)錄的機(jī)制,本課題在MCZ和H_2O_2刺激前給予抗氧化劑NAC,比較二者聯(lián)合作用對菌株的生長、逆轉(zhuǎn)座子、參與損傷修復(fù)基因的表達(dá)以及細(xì)胞內(nèi)ROS水平三方面的影響。研究表明NAC與MCZ刺激聯(lián)合時可抑制MCZ的殺菌作用,有效地保護(hù)酵母態(tài)細(xì)胞,而與H_2O_2聯(lián)用卻對菌株的生長情況無保護(hù)作用;NAC與二者聯(lián)合作用均可顯著地降低刺激所導(dǎo)致的損傷修復(fù)基因表達(dá)上調(diào),并且刺激所激活的逆轉(zhuǎn)座子表達(dá)活性也會相應(yīng)的減弱或消失;熒光檢測細(xì)胞內(nèi)活性氧顯示NAC可有效地抑制MCZ、H_2O_2刺激所導(dǎo)致的細(xì)胞內(nèi)ROS的產(chǎn)生積聚。提示MCZ和H_2O_2刺激后的ROS積聚使得細(xì)胞間接發(fā)生氧化損傷,同時啟動DNA損傷修復(fù)通路,調(diào)控逆轉(zhuǎn)座子的表達(dá)。 最后,我們采用southern blotting技術(shù)深入分析這些激活的逆轉(zhuǎn)座子是否能夠參與復(fù)制周期的所有過程,直至形成完整的雙鏈DNA分子,進(jìn)一步評估轉(zhuǎn)錄活化的逆轉(zhuǎn)座子功能。實(shí)驗(yàn)首先在液體培養(yǎng)基中長時間的環(huán)境加壓刺激誘導(dǎo)若干株菌,通過酶切后的基因組與特定標(biāo)記的逆轉(zhuǎn)座子探針雜交顯色,發(fā)現(xiàn)MCZ誘導(dǎo)菌中Zorro2的拷貝片段明顯增加,而TCA3的拷貝片段反而減少;H_2O_2誘導(dǎo)菌也顯示可能發(fā)生TCA8拷貝片段的增加。隨后采用微量液基稀釋法及spot assay實(shí)驗(yàn)初步鑒定表型,發(fā)現(xiàn)誘導(dǎo)后的菌株對各自的刺激源耐受性增加。結(jié)果表明環(huán)境長期的加壓刺激會導(dǎo)致某些逆轉(zhuǎn)座子在基因組中的拷貝變化,這種基因組重塑可能通過多種方式改變宿主基因的表達(dá),調(diào)控對環(huán)境的適應(yīng)能力。因此,我們認(rèn)為逆轉(zhuǎn)座子在基因組水平的潛在調(diào)節(jié)作用是白念珠菌遺傳進(jìn)化的內(nèi)在動力,而白念珠菌高適應(yīng)性的形成也很有可能與其基因組中廣泛分布的逆轉(zhuǎn)座子的調(diào)控作用相關(guān)。
[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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R379

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 徐來祥,張知彬,宋銘晶;逆轉(zhuǎn)座子與生物遺傳多樣性[J];動物學(xué)雜志;2002年06期

2 孫偉瑋;高平揮;王英;姜遠(yuǎn)英;;ABC轉(zhuǎn)運(yùn)蛋白與臨床白念珠菌耐藥[J];第二軍醫(yī)大學(xué)學(xué)報;2008年09期

3 張勝利;許桂芳;李東方;李學(xué)斌;吳大付;;逆轉(zhuǎn)座子對真核生物基因組及宿主基因表達(dá)的影響[J];核農(nóng)學(xué)報;2010年02期

4 吳紹熙,廖萬清,郭寧如,李春陽,毛玲娥,張宏,曾凡欽,李錫儒,封紹奎,李若瑜,石玉秀,鄭岳臣,冉玉平,王家俊,喻楠,譚升順,江致德;中國致病真菌10年動態(tài)流行病學(xué)研究[J];臨床皮膚科雜志;1999年01期

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