本文選題：馬爾尼菲青霉菌 + 基因敲除�。� 參考：《廣西醫(yī)科大學》2017年碩士論文

【摘要】：馬爾尼菲青霉真菌(Penicillium marneffei PM)是青霉真菌屬中唯一的一種溫度依賴性的雙相型條件致病真菌,它是馬爾尼菲青霉真菌病(Penicilliosis marneffei PSM)的病原菌,25℃時以菌絲相生長,在37℃體外培養(yǎng)或者感染人體后轉換為致病性酵母相生長;主要流行區(qū)域在東南亞地區(qū),PM對免疫功能低下的患者感染性強,對感染者造成致命性的全身性感染。目前,馬爾尼菲青霉真菌雙相轉換和致病性的分子機制尚不清楚。細胞轉錄因子是細胞感受外界信號后調控基因表達的關鍵分子,許多轉錄因子在調控基因表達的同時其基因自身的表達會受到調控。深入研究轉錄因子及其調控基因功能探討馬爾尼菲青霉雙相轉換與致病性分子機制,可為馬爾尼菲青霉病防治和藥物開發(fā)提供科學依據[1-6]。目的:1、本課題組前期進行的馬爾尼菲青霉菌鏈特異性轉錄組的測序分析發(fā)現,編碼含亮氨酸拉鏈(bZIP)的轉錄因子基因Atf21和編碼含有同源異型盒(homeobox)結構域的轉錄因子基因Phx1在雙相轉換過程中差異表達顯著,本課題通過構建馬爾尼菲青霉菌Atf21和Phx1基因缺失突變體,對轉錄因子基因Atf21和Phx1的功能進行了初步研究,探討馬爾尼菲青霉雙相轉換與致病性分子機制,可為馬爾尼菲青霉病防治和藥物開發(fā)提供科學依據。2、本課題組前期研究發(fā)現編碼角質酶轉錄因子β亞基基因(Ctf1β)及其調控的角質酶基因在酵母相表達顯著升高。本課題擬通過克隆馬爾尼菲青霉菌角質酶的基因,構建角質酶原核表達重組菌,進行角質酶的活性鑒定與分析,獲得重組蛋白為進一步制備馬爾尼菲青霉菌角質酶抗體和探討該抗體在防治馬爾尼菲青霉病中的應用奠定基礎。方法:1、用實時熒光定量方法分析馬爾尼菲青霉菌株gxff在雙相轉換過程中基因表達量和基因表達特征。2、利用同源重組方法,構建目的基因敲除突變株,然后與spm4野生菌株進行對比研究,初步研究和分析突變缺失基因的功能。3、通過引物設計、pcr擴增馬爾尼菲青霉菌角質酶基因序列,構建誘導型表達載體,并對重組質粒進行pcr鑒定和質粒的測序;用堿式滴定法測定酶活性;提取蛋白對角質酶進行表達鑒定。結果:1、atf21和phx1基因在馬爾尼菲青霉菌雙相轉換不同時相的表達進行分析結果表明,在馬爾尼菲青霉菌株gxff中atf21和phx1基因表達在菌絲相生長狀態(tài)顯著高于在酵母相生長狀態(tài),與在馬爾尼菲青霉菌株frr2161中轉錄組測序分析atf21基因表達結果一致;atf21在雙相轉換早期基因表達明顯改變,在雙向轉化過程中phx1基因表達出現明顯改變時間較晚。2、成功構建了atf21和phx1基因敲除菌株,與對照菌spm4對比,atf21突變菌株對cu更加敏感,phx1突變菌株對cu的敏感性無影響。3、成功克隆了馬爾尼菲青霉菌角質酶基因,構建了誘導型表達載體并進行測序驗證;構建了馬爾尼菲青霉菌角質酶基因大腸桿菌原核表達重組菌株;用堿式滴定法測定重組菌株酶活性約為對照菌1.7倍,粗酶液的酶活性可達21.8u/ml。結論:1、馬爾尼菲青霉菌atf21和phx1基因在雙相轉換不同時相表達差異顯著,atf21和phx1基因表達在菌絲相生長狀態(tài)顯著高于在酵母相生長狀態(tài);atf21在雙相轉換早期基因表達明顯改變,在雙向轉化過程中phx1基因表達出現明顯改變時間較晚。2、成功構建了atf21和phx1基因敲除菌株,創(chuàng)新性地發(fā)現馬爾尼菲青霉菌轉錄因子基因atf21具有調控銅離子代謝功能,為深入探討馬爾尼菲青霉雙相轉換與致病性分子機制奠定了基礎。3、成功克隆了馬爾尼菲青霉菌角質酶基因,構建了馬爾尼菲青霉菌角質酶基因大腸桿菌原核表達重組菌株,為進一步獲得重組蛋白制備馬爾尼菲青霉菌角質酶抗體和探討該抗體在防治馬爾尼菲青霉病中的應用奠定基礎。
[Abstract]:Penicillium marneffei PM is the only temperature dependent biphasic pathogenic fungus in the genus Penicillium. It is the pathogen of Penicilliosis marneffei PSM (Penicilliosis marneffei PSM). At 25 C, it is grown in mycelium and converted to pathogenic yeast at 37 C in vitro or infected with human body. The main epidemic area is in Southeast Asia, PM is highly infectious to patients with low immune function and causes fatal systemic infection to infected people. At present, the molecular mechanism of biphasic conversion and pathogenicity of Penicillium marneffy fungi is not clear. On the other hand, many transcriptional factors will be regulated while regulating gene expression. Further study on the function of transcription factors and their regulatory genes to study the biphasic transformation and pathogenicity of Penicillium marneffei can provide scientific basis for the prevention and control of Penicillium marneffei and the development of drug [1-6].. 1. The sequence analysis of Penicillium marneffei chain specific transcriptome found that the transcriptional factor gene Atf21 containing leucine zipper (bZIP) and the transcriptional factor gene Phx1 containing the homologous type box (homeobox) domain were expressed differently in the biphasic transformation process. The subject was constructed by the construction of Penicillium marneffei Atf21 and the Atf21 of Penicillium marneffei. The function of Phx1 gene deletion mutant and the function of the transcription factor gene Atf21 and Phx1 were preliminarily studied. The biphasic conversion and pathogenic molecular mechanism of Penicillium marneffei could provide a scientific basis for the prevention and control of Penicillium marneffei and the development of drug,.2. In the previous study, the keratinocyte transcription factor beta subunit gene (Ctf1 beta) was developed. The expression of the keratinase gene and its regulated genes in the yeast phase increased significantly. This topic is to clone the keratinase gene of Penicillium marneffy, construct the recombinant bacteria of the keratinocyte, identify and analyze the activity of the keratinase, and obtain the recombinant protein for the further preparation of the antibody to the Penicillium marnffy keratinase and to discuss the antibody against this antibody. The basis for the application of the treatment of Penicillium marneffei was established. Method: 1, the gene expression and gene expression of Penicillium marneffei strain gxff were analyzed by real time fluorescence quantitative method (.2). The target gene knockout mutant was constructed by homologous recombination method, and then compared with the wild strain of spm4, the preliminary study was carried out. And analyze the function.3 of the mutant deletion gene. Through the primer design, PCR amplification of the Penicillium marneffy keratinase gene sequence, the inducible expression vector was constructed, the recombinant plasmid was identified by PCR and the plasmid was sequenced. The enzyme activity was determined by the alkaline titration, and the expression of the protein diagonal enzyme was identified. The results were as follows: 1, atf21 and phx1 genes were found. The results showed that the expression of atf21 and phx1 gene expression in the mycelial phase of Penicillium marneffei gxff was significantly higher than that in the yeast phase, which was consistent with the atf21 gene expression results in the transcriptional sequence analysis in Penicillium marneffei frr2161; atf21 was in the form of atf21 gene expression. The expression of gene expression in the early stage of biphasic transformation was obviously changed. In the process of bi-directional transformation, the expression of phx1 gene was obviously changed later.2. The atf21 and phx1 gene knockout strains were successfully constructed. Compared with the control bacteria spm4, the atf21 mutant strain was more sensitive to Cu, and the sensitivity of phx1 mutant to Cu was not affected.3, and the Penicillium marneffei was cloned successfully. The inducible expression vector was constructed and sequenced, and the recombinant strain of Escherichia coli was constructed. The enzyme activity of the recombinant strain was about 1.7 times that of the control bacteria by alkaline titration, and the enzyme activity of the crude enzyme could reach 21.8u/ml. conclusion: 1, Penicillium marneffei atf21 and phx1 The expression of atf21 and phx1 gene expression in the mycelial phase was significantly higher than that in the yeast phase, and the expression of atf21 in the early stage of biphasic transformation was significantly changed, and the expression of phx1 gene in the process of biphasic transformation was significantly changed during the bi-directional transformation, and the atf21 and phx1 gene knockout were successfully constructed. It has been found that the Penicillium marneffei transcriptional factor gene atf21 has the function of regulating copper ion metabolism, which lays a foundation for the in-depth study of the biphasic transformation and pathogenic molecular mechanism of Penicillium marneffei, which has successfully cloned the Penicillium marneffei keratinase gene and constructed the coliform pole of the Penicillium marneffei keratinase gene. The recombinant strain expressed the recombinant strain to prepare the recombinant protein of Penicillium marneffei and explore the application of the antibody in the prevention and control of Penicillium marneffei.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R379

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