本文選題：白念珠菌 + 多銅氧化酶�。� 參考：《南開大學(xué)》2013年碩士論文

【摘要】：白念珠菌{Candida albicans)是念珠菌病原體中最常見的條件性致病菌,也是醫(yī)院獲得性真菌病的主要感染源之一。鐵離子獲得能力是白念珠菌在宿主中定居、存活和致病過程中的重要影響因素。多銅氧化酶(multicopper oxidase, MCO)基因是白念珠菌高親和性鐵獲得系統(tǒng)的關(guān)鍵組分。白念珠菌基因組中含有5個(gè)MCO編碼基因,研究這些基因的功能和表達(dá)調(diào)控關(guān)系,將有助于深入地了解白念珠菌在低鐵環(huán)境中的存活機(jī)制,為今后白念珠菌的感染治療和藥物開發(fā)提供重要的理論依據(jù)。 首先通過熒光定量PCR方法分析了鐵、銅離子對(duì)白念珠菌MCO基因的調(diào)控作用,結(jié)果發(fā)現(xiàn)：CaFET3、CaFET34和CaFET99是白念珠菌應(yīng)答低鐵條件的主要MCO基因；低銅條件只是對(duì)部分MCO基因的表達(dá)產(chǎn)生輕微影響。而當(dāng)鐵銅同時(shí)缺乏時(shí),所有MCO基因的表達(dá)均會(huì)明顯升高,表明鐵銅離子對(duì)MCO基因的表達(dá)具有重要的調(diào)控作用。 為了進(jìn)一步研究MCO基因在鐵代謝方面的作用,構(gòu)建了白念珠菌CaFET33和CaFET34基因釀酒酵母回補(bǔ)菌株,發(fā)現(xiàn)CaFET34可以有效地回補(bǔ)Scfet3△在低鐵條件下的缺陷表型。利用PCR介導(dǎo)的同源重組方法進(jìn)一步構(gòu)建了Cafet33△/△和Cafet34△/△缺失菌株,測(cè)定了其胞內(nèi)鐵含量和鐵吸收能力的變化。結(jié)果表明在缺鐵條件下,CaFET34的缺失會(huì)明顯降低細(xì)胞的鐵含量和鐵吸收能力,說明CaFET34在細(xì)胞鐵離子動(dòng)態(tài)平衡方面發(fā)揮著重要作用。對(duì)Cafet33△/△和Cafet34△/△缺失菌株在缺鐵條件下其它MCO基因表達(dá)進(jìn)行研究,結(jié)果表明當(dāng)白念珠菌中CaFET33/FET34基因缺失或者發(fā)生功能障礙時(shí),其它MCO基因會(huì)替代缺陷基因而發(fā)揮功能。 隨后我們觀察了Cafet33△/△和Cafet34△/△缺失菌株在菌絲誘導(dǎo)條件下的菌落形態(tài)。結(jié)果發(fā)現(xiàn)：在固體菌絲誘導(dǎo)培養(yǎng)基中,CaFET33和CaFET34,尤其是CaFET34的缺失會(huì)顯著影響白念珠菌菌落周邊菌絲的生長(zhǎng),而外源鐵的添加并不能回補(bǔ)此缺陷,表明CaFET33和CaFET34以非鐵依賴的方式參與白念珠菌菌絲發(fā)育。小鼠系統(tǒng)感染實(shí)驗(yàn)證實(shí)了CaFET34是一種重要的毒力因子,在白念珠菌的致病過程中起著一定作用。 構(gòu)建CaFET34啟動(dòng)子與報(bào)告基因LacZ融合質(zhì)粒,轉(zhuǎn)化野生型、aft2△/△和rin101△/△菌株,在不同pH、不同鐵離子濃度條件下測(cè)定p-半乳糖苷酶活性,表明Aft2p可能作為轉(zhuǎn)錄抑制子調(diào)控CaFET34基因的表達(dá)。而Rim101p可能作為轉(zhuǎn)錄激活子調(diào)控CaFET34基因的表達(dá)。 此外還構(gòu)建了fth1△/△單基因和fth1△/△fet33△/△雙基因缺失菌株,測(cè)定了其在缺鐵條件下的生長(zhǎng)情況、胞內(nèi)鐵含量、代謝轉(zhuǎn)換能力、菌絲以及菌落形態(tài)的變化,結(jié)果表明fth1△/△fet33△/△缺失菌株胞內(nèi)鐵含量明顯降低、代謝轉(zhuǎn)換受阻、菌落表面褶皺形成能力和菌絲聚集明顯增強(qiáng),表明CaFTH1和CaFET33基因產(chǎn)物可能在液泡膜上形成鐵轉(zhuǎn)運(yùn)復(fù)合物,參與胞內(nèi)鐵穩(wěn)態(tài)和液泡功能的維持。
[Abstract]:Candida albicans {Candida albicans is the most common opportunistic pathogen in Candida albicans, and it is also one of the main infection sources of nosocomial mycosis. Iron ion acquisition ability is an important factor in the host settlement, survival and pathogenicity of Candida albicans. The polycopper oxidase multicopper oxidase (MCO) gene is a key component in the high affinity iron extraction system of Candida albicans. The genome of Candida albicans contains five MCO coding genes. Studying the function and expression regulation of these genes will be helpful to understand the survival mechanism of Candida albicans in low iron environment. To provide an important theoretical basis for the treatment of Candida albicans infection and drug development in the future. The regulation of iron and copper ions on the MCO gene of Candida albicans was analyzed by fluorescence quantitative PCR method. The results showed that the major MCO genes of Candida albicans were CaFET34 and CaFET99 in response to low iron condition. The low copper condition had only a slight effect on the expression of some MCO genes. However, when iron and copper were deficient at the same time, the expression of all MCO genes increased significantly, indicating that iron and copper ions play an important role in regulating the expression of MCO gene. In order to further study the role of MCO gene in iron metabolism, CaFET33 and CaFET34 genes of Candida albicans Saccharomyces cerevisiae were constructed. It was found that CaFET34 could effectively complement the defective phenotypes of Scfet3 in low iron condition. Cafet33 / and Cafet34 / deficient strains were further constructed by PCR mediated homologous recombination method, and the changes of iron content and iron absorption capacity were measured. The results showed that the absence of CaFET34 could significantly reduce the iron content and iron absorption ability of the cells under iron deficiency, indicating that CaFET34 plays an important role in the dynamic equilibrium of iron ions in the cells. The expression of other MCO genes in Cafet33 / and Cafet34 / deficient strains under iron deficiency was studied. The results showed that when the CaFET33/FET34 gene in Candida albicans was absent or the function was impaired, other MCO genes would replace the defective genes and function. Then we observed the colony morphology of Cafet33 / and Cafet34 / deficient strains under hyphal induction. The results showed that the absence of CaFET33 and CaFET34, especially CaFET34, significantly affected the growth of mycelium around the colony of Candida albicans in solid hyphal induction medium, but the addition of exogenous iron could not compensate for the defect. The results showed that CaFET33 and CaFET34 participated in the development of Candida albicans hypha in a non-iron dependent manner. CaFET34 is an important virulence factor and plays a certain role in the pathogenic process of Candida albicans. The fusion plasmids of CaFET34 promoter and reporter gene LacZ were constructed and transformed into wild type CaFET34 / and rin101 / strains. The activity of pgalactosidase was determined at different pH and different iron ion concentration. It is suggested that Aft2p may act as a transcription suppressor to regulate the expression of CaFET34 gene. Rim101p may act as a transcriptional activator to regulate the expression of CaFET34 gene. In addition, fth1 / single gene and fth1 / fet33 / double gene deficient strains were constructed, and their growth, intracellular iron content, metabolic transformation ability, mycelium and colony morphology were determined. The results showed that the intracellular iron content of fth1 / fet33 / deficient strain was significantly decreased, the metabolic transition was blocked, and the fold forming ability and hyphae aggregation of colony surface were significantly enhanced. It is suggested that CaFTH1 and CaFET33 gene products may form iron transport complex on vacuolar membrane and participate in the maintenance of iron homeostasis and vacuolar function.
【學(xué)位授予單位】：南開大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R379.4;Q78

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本文編號(hào)：1928618