烟曲霉afpab1基因功能研究及突变体库的建立

发布时间：2018-06-10 01:07

本文选题：烟曲霉 + 根癌农杆菌　；参考：《吉林大学》2016年博士论文

【摘要】：烟曲霉(Aspergillus fumigatu)是一种典型的腐生性条件致病真菌,多为无性繁殖,可产生大量的分生孢子。其孢子对环境适应能力强、生存范围广,在腐烂的有机体、空气粉尘、土壤中都含有大量的烟曲霉孢子。同时,烟曲霉也是临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损人群引发真菌病(mycosis),严重时可发生侵袭性曲霉病(invasive aspergillosis,IA)病死率高达70%-90%。近年来,随着恶性肿瘤、器官移植等需要进行免疫抑制治疗手术的不断开展,以及人们对抗生素的广泛滥用,使得IA的发病率呈逐年上升趋势。由于烟曲霉致病具有死亡率高、诊疗方法少等特点,加之逐渐出现的耐药菌株,因此对烟曲霉及其致病机制的研究迫在眉睫。烟曲霉在外界环境生长中与其在宿主体内感染时所面对的生长压力大不相同。为了应对宿主体内免疫系统的清除效应,烟曲霉细胞需要具有感知外界环境压力、激活信号传导、做出迅速适应性反应等能力,这对烟曲霉细胞在宿主体内存活是十分必要的。同时,有效的应对外界环境压力也是烟曲霉完成侵袭性感染的必备条件。在真核细胞中,应对环境压力引起的应答机制主要为总体翻译抑制(global translational inhibition),通过抑制非必需蛋白的合成来保存代谢所需的能量,同时调整基因表达的有效性来应对外界压力。在面对环境压力时,真核细胞关闭部分转录进程,同时将转录进程中的相关组份聚集到应激颗粒(stress granules,SGs)中。SGs在真核细胞中广泛存在,在酵母和哺乳动物细胞中已有较为深入的研究。其组成受环境影响,主要包括m RNAs分子、小核糖体亚基和一些翻译起始因子。在米曲霉(Aspergillus.oryzae)中,aopab1基因所编码的蛋白是SGs的主要组成成份,该蛋白的功能与米曲霉细胞的多种环境压力应答相关。通过序列分析我们发现在烟曲霉染色体当中存在一个与米曲霉aopab1基因同源性较高的基因afpab1。因此,本研究应用根癌农杆菌(Agrobacterium tumefaciens)介导的遗传转化方法对烟曲霉IFM40808基因afpab1进行了定向敲除,构建了afpab1基因敲除株和回复株。希望以此来研究afpab1基因在烟曲霉应对外界环境压力和致病力方面所行使的功能。本研究发现,在面对温度压力(37℃、42℃和48℃)、渗透压压力(0.5、1、1.5 M山梨醇)、内质网应激压力(5、10、15 m M DTT)和营养缺失压力(氮源、碳源缺失)环境条件时,Δafpab1与野生型烟曲霉的生长并无差异,而在氧化应激压力(H2O2和甲萘醌)条件下,Δafpab1表现出显著的敏感性,细胞表面发生严重的改变(凹陷、光滑、形态不规则),并且其清除外界ROS的能力减弱。同时,在上述压力条件下突变体细胞内氧化应激相关基因sod1和afyap1的m RNAs表达水平下降。以上结果表明在应对外界环境压力时,afpab1基因与烟曲霉细胞应对氧化应激压力相关。通过构建免疫抑制小鼠模型检测敲除株致病力情况时发现,Δafpab1的致病力明显低于野生型菌株和回复株。我们推测,Δafpab1致病力的下降是由于细胞在应对氧化应激压力所发生的改变导致。在烟曲霉基因功能研究当中,根癌农杆菌介导的遗传转化技术已经得到了广泛的应用。人们可以利用该技术对烟曲霉目的基因进行敲除或干扰,从而研究目的基因功能。但该方法仅是对感兴趣目的基因的预测功能进行验证,而在烟曲霉基因组当中仍存有大量的未知基因需要进行功能解析。如能获得具有一定规模的烟曲霉突变体库,并通过对突变体库进行有目的的筛选(如表型、性状等),挖掘未知的基因功能,则会更加完善对烟曲霉基因功能的研究。本研究通过根癌农杆菌介导的遗传转化技术对烟曲霉IFM40808进行T-DNA(Transfer DNA)随机插入突变构建,获得了小规模的突变体库(共计5712株)。并深入探讨了转化体系中的主要影响因素,该转化体系为:以浓度为1×106个孢子/ml烟曲霉分生孢子为受体,以携带双元质粒载体p BHt1的根癌农杆菌AGL-1(OD600nm=0.8)为T-DNA供体,当共孵育温度为25℃、共孵育时间为48 h、潮霉素筛选浓度为200μg/m L时,转化效率可达到350个转化子/106个分生孢子,且转化子的阳性率可稳定在95%以上。这些突变体具有遗传背景清晰、有丝分裂稳定、插入位点随机等特点。利用PCR、TAIL-PCR等分子生物学方法对烟曲霉T-DNA插入突变体进行分析。通过提取突变体DNA,以潮霉素磷酸转移酶(Hygromycin phosphotransferase,hph)为靶基因进行PCR扩增,可得到一条800 bp左右的目标条带,这表明T-DNA已成功插入到烟曲霉细胞的染色体当中。同时,应用TAIL-PCR扩增T-DNA插入位点的侧翼序列,通过结合烟曲霉基因组信息可实现对其插入位点的精确定位。上述结果表明,应用根癌农杆菌介导的烟曲霉遗传转化方法是构建突变库的有效方法,是对烟曲霉进行基因研究的有力工具。综上,本研究通过对烟曲霉afpab1基因进行敲除发现,该基因与烟曲霉氧化应激压力相关,并且该基因的缺失会导致烟曲霉致病力下降。同时通过建立小型烟曲霉T-DNA插入突变体库,可为今后挖掘烟曲霉未知基因功能、解析生物学性状、探讨致病机制奠定基础。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R379

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