本文選題：煙曲霉 + 根癌農(nóng)桿菌�。� 參考：《吉林大學(xué)》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(wú)性繁殖,可產(chǎn)生大量的分生孢子。其孢子對(duì)環(huán)境適應(yīng)能力強(qiáng)、生存范圍廣,在腐爛的有機(jī)體、空氣粉塵、土壤中都含有大量的煙曲霉孢子。同時(shí),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(yán)重時(shí)可發(fā)生侵襲性曲霉病(invasive aspergillosis,IA)病死率高達(dá)70%-90%。近年來(lái),隨著惡性腫瘤、器官移植等需要進(jìn)行免疫抑制治療手術(shù)的不斷開(kāi)展,以及人們對(duì)抗生素的廣泛濫用,使得IA的發(fā)病率呈逐年上升趨勢(shì)。由于煙曲霉致病具有死亡率高、診療方法少等特點(diǎn),加之逐漸出現(xiàn)的耐藥菌株,因此對(duì)煙曲霉及其致病機(jī)制的研究迫在眉睫。煙曲霉在外界環(huán)境生長(zhǎng)中與其在宿主體內(nèi)感染時(shí)所面對(duì)的生長(zhǎng)壓力大不相同。為了應(yīng)對(duì)宿主體內(nèi)免疫系統(tǒng)的清除效應(yīng),煙曲霉細(xì)胞需要具有感知外界環(huán)境壓力、激活信號(hào)傳導(dǎo)、做出迅速適應(yīng)性反應(yīng)等能力,這對(duì)煙曲霉細(xì)胞在宿主體內(nèi)存活是十分必要的。同時(shí),有效的應(yīng)對(duì)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(xì)胞中,應(yīng)對(duì)環(huán)境壓力引起的應(yīng)答機(jī)制主要為總體翻譯抑制(global translational inhibition),通過(guò)抑制非必需蛋白的合成來(lái)保存代謝所需的能量,同時(shí)調(diào)整基因表達(dá)的有效性來(lái)應(yīng)對(duì)外界壓力。在面對(duì)環(huán)境壓力時(shí),真核細(xì)胞關(guān)閉部分轉(zhuǎn)錄進(jìn)程,同時(shí)將轉(zhuǎn)錄進(jìn)程中的相關(guān)組份聚集到應(yīng)激顆粒(stress granules,SGs)中。SGs在真核細(xì)胞中廣泛存在,在酵母和哺乳動(dòng)物細(xì)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(xì)胞的多種環(huán)境壓力應(yīng)答相關(guān)。通過(guò)序列分析我們發(fā)現(xiàn)在煙曲霉染色體當(dāng)中存在一個(gè)與米曲霉aopab1基因同源性較高的基因afpab1。因此,本研究應(yīng)用根癌農(nóng)桿菌(Agrobacterium tumefaciens)介導(dǎo)的遺傳轉(zhuǎn)化方法對(duì)煙曲霉IFM40808基因afpab1進(jìn)行了定向敲除,構(gòu)建了afpab1基因敲除株和回復(fù)株。希望以此來(lái)研究afpab1基因在煙曲霉應(yīng)對(duì)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(duì)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(yīng)激壓力(5、10、15 m M DTT)和營(yíng)養(yǎng)缺失壓力(氮源、碳源缺失)環(huán)境條件時(shí),Δafpab1與野生型煙曲霉的生長(zhǎng)并無(wú)差異,而在氧化應(yīng)激壓力(H2O2和甲萘醌)條件下,Δafpab1表現(xiàn)出顯著的敏感性,細(xì)胞表面發(fā)生嚴(yán)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(shí),在上述壓力條件下突變體細(xì)胞內(nèi)氧化應(yīng)激相關(guān)基因sod1和afyap1的m RNAs表達(dá)水平下降。以上結(jié)果表明在應(yīng)對(duì)外界環(huán)境壓力時(shí),afpab1基因與煙曲霉細(xì)胞應(yīng)對(duì)氧化應(yīng)激壓力相關(guān)。通過(guò)構(gòu)建免疫抑制小鼠模型檢測(cè)敲除株致病力情況時(shí)發(fā)現(xiàn),Δafpab1的致病力明顯低于野生型菌株和回復(fù)株。我們推測(cè),Δafpab1致病力的下降是由于細(xì)胞在應(yīng)對(duì)氧化應(yīng)激壓力所發(fā)生的改變導(dǎo)致。在煙曲霉基因功能研究當(dāng)中,根癌農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化技術(shù)已經(jīng)得到了廣泛的應(yīng)用。人們可以利用該技術(shù)對(duì)煙曲霉目的基因進(jìn)行敲除或干擾,從而研究目的基因功能。但該方法僅是對(duì)感興趣目的基因的預(yù)測(cè)功能進(jìn)行驗(yàn)證,而在煙曲霉基因組當(dāng)中仍存有大量的未知基因需要進(jìn)行功能解析。如能獲得具有一定規(guī)模的煙曲霉突變體庫(kù),并通過(guò)對(duì)突變體庫(kù)進(jìn)行有目的的篩選(如表型、性狀等),挖掘未知的基因功能,則會(huì)更加完善對(duì)煙曲霉基因功能的研究。本研究通過(guò)根癌農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化技術(shù)對(duì)煙曲霉IFM40808進(jìn)行T-DNA(Transfer DNA)隨機(jī)插入突變構(gòu)建,獲得了小規(guī)模的突變體庫(kù)(共計(jì)5712株)。并深入探討了轉(zhuǎn)化體系中的主要影響因素,該轉(zhuǎn)化體系為:以濃度為1×106個(gè)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(mén)-DNA供體,當(dāng)共孵育溫度為25℃、共孵育時(shí)間為48 h、潮霉素篩選濃度為200μg/m L時(shí),轉(zhuǎn)化效率可達(dá)到350個(gè)轉(zhuǎn)化子/106個(gè)分生孢子,且轉(zhuǎn)化子的陽(yáng)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(diǎn)隨機(jī)等特點(diǎn)。利用PCR、TAIL-PCR等分子生物學(xué)方法對(duì)煙曲霉T-DNA插入突變體進(jìn)行分析。通過(guò)提取突變體DNA,以潮霉素磷酸轉(zhuǎn)移酶(Hygromycin phosphotransferase,hph)為靶基因進(jìn)行PCR擴(kuò)增,可得到一條800 bp左右的目標(biāo)條帶,這表明T-DNA已成功插入到煙曲霉細(xì)胞的染色體當(dāng)中。同時(shí),應(yīng)用TAIL-PCR擴(kuò)增T-DNA插入位點(diǎn)的側(cè)翼序列,通過(guò)結(jié)合煙曲霉基因組信息可實(shí)現(xiàn)對(duì)其插入位點(diǎn)的精確定位。上述結(jié)果表明,應(yīng)用根癌農(nóng)桿菌介導(dǎo)的煙曲霉遺傳轉(zhuǎn)化方法是構(gòu)建突變庫(kù)的有效方法,是對(duì)煙曲霉進(jìn)行基因研究的有力工具。綜上,本研究通過(guò)對(duì)煙曲霉afpab1基因進(jìn)行敲除發(fā)現(xiàn),該基因與煙曲霉氧化應(yīng)激壓力相關(guān),并且該基因的缺失會(huì)導(dǎo)致煙曲霉致病力下降。同時(shí)通過(guò)建立小型煙曲霉T-DNA插入突變體庫(kù),可為今后挖掘煙曲霉未知基因功能、解析生物學(xué)性狀、探討致病機(jī)制奠定基礎(chǔ)。
[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(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R379

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9 曾榮;煙曲霉和念珠菌屬體外藥敏實(shí)驗(yàn)相關(guān)研究[D];北京協(xié)和醫(yī)學(xué)院;2014年

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