本文選題：煙曲霉 + 根癌農(nóng)桿菌��； 參考：《吉林大學》2016年博士論文

【摘要】：煙曲霉(Aspergillus fumigatu)是一種典型的腐生性條件致病真菌,多為無性繁殖,可產(chǎn)生大量的分生孢子。其孢子對環(huán)境適應能力強、生存范圍廣,在腐爛的有機體、空氣粉塵、土壤中都含有大量的煙曲霉孢子。同時,煙曲霉也是臨床中最主要的致病性曲霉菌(占全部曲霉感染的90%左右),多感染免疫功能缺陷或受損人群引發(fā)真菌病(mycosis),嚴重時可發(fā)生侵襲性曲霉病(invasive aspergillosis,IA)病死率高達70%-90%。近年來,隨著惡性腫瘤、器官移植等需要進行免疫抑制治療手術的不斷開展,以及人們對抗生素的廣泛濫用,使得IA的發(fā)病率呈逐年上升趨勢。由于煙曲霉致病具有死亡率高、診療方法少等特點,加之逐漸出現(xiàn)的耐藥菌株,因此對煙曲霉及其致病機制的研究迫在眉睫。煙曲霉在外界環(huán)境生長中與其在宿主體內(nèi)感染時所面對的生長壓力大不相同。為了應對宿主體內(nèi)免疫系統(tǒng)的清除效應,煙曲霉細胞需要具有感知外界環(huán)境壓力、激活信號傳導、做出迅速適應性反應等能力,這對煙曲霉細胞在宿主體內(nèi)存活是十分必要的。同時,有效的應對外界環(huán)境壓力也是煙曲霉完成侵襲性感染的必備條件。在真核細胞中,應對環(huán)境壓力引起的應答機制主要為總體翻譯抑制(global translational inhibition),通過抑制非必需蛋白的合成來保存代謝所需的能量,同時調(diào)整基因表達的有效性來應對外界壓力。在面對環(huán)境壓力時,真核細胞關閉部分轉錄進程,同時將轉錄進程中的相關組份聚集到應激顆粒(stress granules,SGs)中。SGs在真核細胞中廣泛存在,在酵母和哺乳動物細胞中已有較為深入的研究。其組成受環(huán)境影響,主要包括m RNAs分子、小核糖體亞基和一些翻譯起始因子。在米曲霉(Aspergillus.oryzae)中,aopab1基因所編碼的蛋白是SGs的主要組成成份,該蛋白的功能與米曲霉細胞的多種環(huán)境壓力應答相關。通過序列分析我們發(fā)現(xiàn)在煙曲霉染色體當中存在一個與米曲霉aopab1基因同源性較高的基因afpab1。因此,本研究應用根癌農(nóng)桿菌(Agrobacterium tumefaciens)介導的遺傳轉化方法對煙曲霉IFM40808基因afpab1進行了定向敲除,構建了afpab1基因敲除株和回復株。希望以此來研究afpab1基因在煙曲霉應對外界環(huán)境壓力和致病力方面所行使的功能。本研究發(fā)現(xiàn),在面對溫度壓力(37℃、42℃和48℃)、滲透壓壓力(0.5、1、1.5 M山梨醇)、內(nèi)質(zhì)網(wǎng)應激壓力(5、10、15 m M DTT)和營養(yǎng)缺失壓力(氮源、碳源缺失)環(huán)境條件時,Δafpab1與野生型煙曲霉的生長并無差異,而在氧化應激壓力(H2O2和甲萘醌)條件下,Δafpab1表現(xiàn)出顯著的敏感性,細胞表面發(fā)生嚴重的改變(凹陷、光滑、形態(tài)不規(guī)則),并且其清除外界ROS的能力減弱。同時,在上述壓力條件下突變體細胞內(nèi)氧化應激相關基因sod1和afyap1的m RNAs表達水平下降。以上結果表明在應對外界環(huán)境壓力時,afpab1基因與煙曲霉細胞應對氧化應激壓力相關。通過構建免疫抑制小鼠模型檢測敲除株致病力情況時發(fā)現(xiàn),Δafpab1的致病力明顯低于野生型菌株和回復株。我們推測,Δafpab1致病力的下降是由于細胞在應對氧化應激壓力所發(fā)生的改變導致。在煙曲霉基因功能研究當中,根癌農(nóng)桿菌介導的遺傳轉化技術已經(jīng)得到了廣泛的應用。人們可以利用該技術對煙曲霉目的基因進行敲除或干擾,從而研究目的基因功能。但該方法僅是對感興趣目的基因的預測功能進行驗證,而在煙曲霉基因組當中仍存有大量的未知基因需要進行功能解析。如能獲得具有一定規(guī)模的煙曲霉突變體庫,并通過對突變體庫進行有目的的篩選(如表型、性狀等),挖掘未知的基因功能,則會更加完善對煙曲霉基因功能的研究。本研究通過根癌農(nóng)桿菌介導的遺傳轉化技術對煙曲霉IFM40808進行T-DNA(Transfer DNA)隨機插入突變構建,獲得了小規(guī)模的突變體庫(共計5712株)。并深入探討了轉化體系中的主要影響因素,該轉化體系為:以濃度為1×106個孢子/ml煙曲霉分生孢子為受體,以攜帶雙元質(zhì)粒載體p BHt1的根癌農(nóng)桿菌AGL-1(OD600nm=0.8)為T-DNA供體,當共孵育溫度為25℃、共孵育時間為48 h、潮霉素篩選濃度為200μg/m L時,轉化效率可達到350個轉化子/106個分生孢子,且轉化子的陽性率可穩(wěn)定在95%以上。這些突變體具有遺傳背景清晰、有絲分裂穩(wěn)定、插入位點隨機等特點。利用PCR、TAIL-PCR等分子生物學方法對煙曲霉T-DNA插入突變體進行分析。通過提取突變體DNA,以潮霉素磷酸轉移酶(Hygromycin phosphotransferase,hph)為靶基因進行PCR擴增,可得到一條800 bp左右的目標條帶,這表明T-DNA已成功插入到煙曲霉細胞的染色體當中。同時,應用TAIL-PCR擴增T-DNA插入位點的側翼序列,通過結合煙曲霉基因組信息可實現(xiàn)對其插入位點的精確定位。上述結果表明,應用根癌農(nóng)桿菌介導的煙曲霉遺傳轉化方法是構建突變庫的有效方法,是對煙曲霉進行基因研究的有力工具。綜上,本研究通過對煙曲霉afpab1基因進行敲除發(fā)現(xiàn),該基因與煙曲霉氧化應激壓力相關,并且該基因的缺失會導致煙曲霉致病力下降。同時通過建立小型煙曲霉T-DNA插入突變體庫,可為今后挖掘煙曲霉未知基因功能、解析生物學性狀、探討致病機制奠定基礎。
[Abstract]:Aspergillus fumigatus (Aspergillus fumigatu) is a typical saprophytic pathogenic fungus, which is mostly asexual reproduction and produces a large number of conidia. The spores have strong adaptability to the environment and a wide range of survival. In the rotting organisms, air dust and soil, there are plenty of Aspergillus fumigatus spores. Meanwhile, Aspergillus fumigatus is the most important in the clinic. Pathogenic Aspergillus (about 90% of all Aspergillus infection), multiple infection of immune function defects or impaired people cause fungal disease (mycosis), the fatality rate of invasive aspergillosis (invasive aspergillosis, IA) is higher than 70%-90%. in recent years. With malignant tumor and organ transplantation, the operation of immunosuppressive therapy needs to be continuously opened. The incidence of IA is increasing year by year, as well as the widespread abuse of antibiotics, which has the characteristics of high mortality and less diagnosis and treatment, as well as the gradual emergence of drug-resistant strains. Therefore, the study of Aspergillus fumigatus and its pathogenesis is imminent. In order to cope with the immune system clearance in the host, the Aspergillus fumigatus cells need to have the ability to perceive the pressure of the external environment, activate the signal conduction, and make the rapid adaptive response. This is very necessary for the Aspergillus fumigatus cells in the host memory. At the same time, it is effective to deal with the outer ring. The environmental pressure is also a prerequisite for the invasive infection of Aspergillus fumigatus. In eukaryotic cells, the response mechanism caused by environmental pressure is mainly global translational inhibition. By inhibiting the synthesis of non essential proteins to preserve the energy needed for metabolism, and to adjust the effectiveness of gene expression to respond to the outside world. Stress. In the face of environmental pressure, eukaryotic cells close some of the transcriptional processes, while the related components in the transcriptional process are aggregated to stress particles (stress granules, SGs), and.SGs exists widely in eukaryotic cells. It has been deeply studied in yeast and mammalian cells. Its composition is affected by the environment, mainly including m RNAs. Micronucleus subunits and some translation initiation factors. In Aspergillus.oryzae, the protein encoded by the aopab1 gene is the main component of SGs. The function of the protein is related to various environmental pressure responses of Aspergillus oryzae. By sequence analysis, we found that there is a Aspergillus oryzae aopab1 in the Aspergillus fumigatus chromosome. Gene homologous gene afpab1., therefore, this study uses Agrobacterium tumefaciens mediated genetic transformation method to targeted knockout of IFM40808 gene afpab1 of Aspergillus fumigatus, and constructs a afpab1 gene knockout strain and a response strain. In the hope of studying the afpab1 gene in Aspergillus fumigatus to cope with the pressure of the environment and the pressure of the external environment. The study found that when the temperature pressure (37, 42 and 48), the osmotic pressure (0.5,1,1.5 M sorbitol), the stress pressure of the endoplasmic reticulum (5,10,15 m M DTT) and the nutrient loss pressure (nitrogen source and carbon source), there was no difference between the growth of the delta afpab1 and the wild type Aspergillus fumigatus, but in the oxidative stress pressure Under the conditions of force (H2O2 and naphthoquinone), the delta afpab1 showed significant sensitivity, and the cell surface had serious changes (sunken, smooth, irregular), and its ability to remove the external ROS was weakened. At the same time, the expression level of M RNAs expression of the oxidative stress related gene SOD1 and afyap1 in the mutant cells decreased. The above results were found. Afpab1 gene and Aspergillus fumigatus were related to oxidative stress when coping with environmental pressure. The pathogenicity of delta afpab1 was significantly lower than that of wild type and response strain by building an immunosuppressive mouse model to detect the pathogenicity of the knockout strain. We speculated that the decrease of the virulence of delta afpab1 was due to the response of the cells to the response. Changes in oxidative stress cause changes in stress stress. In the study of Aspergillus fumigatus gene function, genetic transformation technology mediated by Agrobacterium tumefaciens has been widely used. This technique can be used to knock out or interfere with the target gene of Aspergillus fumigatus to study the function of the target gene, but this method is only for the interested gene. A large number of unknown genes still exist in the genome of Aspergillus fumigatus, which needs functional analysis. For example, a library of Aspergillus fumigatus with a certain scale can be obtained, and the unknown gene function can be improved by digging out the unknown gene function through the screening of the mutant library (such as phenotypes, traits and so on). In this study, a random insertion mutation of T-DNA (Transfer DNA) of Aspergillus fumigatus IFM40808 was constructed by Agrobacterium tumefaciens mediated genetic transformation technology, and a small scale mutant library (total 5712 strains) was obtained. The main factors in the transformation system were discussed in depth. The transformation system was a concentration of 1 x 106 spores /ml. The conidia of Aspergillus fumigatus is a receptor, which carries the AGL-1 (OD600nm=0.8) of Agrobacterium tumefaciens AGL-1 (OD600nm=0.8), which carries the dual plasmid vector p BHt1. When the incubation temperature is 25, the incubation time is 48 h, when the concentration of the hygromycin is 200 mu g/m L, the conversion efficiency can reach 350 transformants / 106 conidium, and the positive rate of the transformant can be stabilized at 95%. These mutants have the characteristics of clear genetic background, stable mitosis and random insertion sites. Using PCR, TAIL-PCR and other molecular biological methods to analyze the T-DNA insertion mutant of Aspergillus fumigatus. By extracting the mutant DNA, the Hygromycin phosphotransferase, HPH as the target gene for PCR amplification can be carried out. A target band of about 800 BP is obtained, which indicates that T-DNA has been successfully inserted into the chromosomes of Aspergillus fumigatus. At the same time, the TAIL-PCR amplification of the flanking sequence of the T-DNA insertion site and the precise location of the insertion site can be achieved by combining the genomic information of Aspergillus fumigatus. The results show that the application of Agrobacterium tumefaciens mediated smoke The genetic transformation method of Aspergillus is an effective method to construct the mutant library and is a powerful tool for the gene study of Aspergillus fumigatus. In this study, the gene was knocked out by the knockout of the afpab1 gene of Aspergillus fumigatus, and the gene was related to the oxidative stress pressure of Aspergillus fumigatus, and the deletion of the gene could lead to the decline of the pathogenicity of Aspergillus fumigatus. The T-DNA insertional mutant library of Aspergillus fumigatus can lay a foundation for digging out the unknown gene function of Aspergillus fumigatus, analyzing biological characteristics and exploring pathogenic mechanism.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R379

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