| Aspergillus fumigatus is a typical saprophytic opportunistic pathogen, which produce a large number of asexual conidia. The spores can survive in different conditions and are widely spread in soil, rotted organisms and air dust. The A. fumigatus is also an important clinial pathogen, accounting for 90% Aspergillus infection. It mainly infect immuno-deficient or immunosupressed subjects, causing mycosis or even severe invasive aspergillosis and the mortality rates is as high as 70%-90%.In rencent dacades, with the widely development of immunosuppressive therapy for malignancy and organ transplantation, the incidence of invasive aspergillosis is increasing year by year. Due to the high mortality, limited diagnositc methods and occurance of drug resistant strains, study on the pathogenic mechanism of A. fumigatus is urgent.A. fumigatus face hugely different stress when living in vivo compared with in external enviroment. In order to resist the scavenging effect of host immune system, A. fumigatus cells need to sense environmental stimulation, activate signal transduction and make rapid adaptive response, which is necessary for survival in the host. The ability to cope with external enviromental stress effectively is also essential for establishing a successful infection. In eukaryotic cells, the main response mechanism to environmental stress is golbal translational inhibition, which suppress the synthesis of non-essential proteins to save metabolic energy and regulate the effectivity of gene expression to respond to external stress. Eukaryotic cells shut down part of the transcription process when encountering environmental stress and aggregate related compotents in stress granules(SGs). The composition of SGs is affected by the enviroment, mainly including m RNAs molecules, small ribosomal subunits and some translation initiation factors.SGs exists widely in Eukaryotic cells and have been investigated deeply in both yeast and mammalianis cells. In Aspergillus oryzae cells, the protein encoded by aopab1 gene is the main compotents of SGs and is associated with various stress response in the cells. We found there is a gene in A. fumigatus called afpab1 which is highly homologous with aopab1 gene in A. oryzae cells by sequence analysis. So in this research, Agrobacterium-mediated transformation was emplyed to knock out IFM40808 gene afpab1 targetedly in A. fumigatus cells, constructing deleted strain and complementary mutant strain of afpab1. These strains may help to study the function of afpab1 in A. fumigatus cells when encountering stress from outside.In our research, no difference was found between ?afpab1 and wild A. fumigatus cells when living in high temperature(37, 42, 48 ?)?hyperosmolarity(0.5, 1, 1.5 M sorbitol), endoplasmic reticulum stress(5, 10, 15 m M DTT)?malnutrition(nitrogen/carbon deficiency). However, ?afpab1 showed high sensitivity to oxidative stress(H2O2 and menadione) and serious change was found on the surface of mutant cells(depression?smoothness?irregular figure) and the ability to clear ROS decreased. The results suggested that afpab1 gene was associated with the response of A. fumigatus to oxidative stress. By constructing immunosuppressed mice model to test the pathogenicity of ?afpab1, we found the pathogenicity of ?afpab1 was significantly weaker than wild strains and reverse mutant strains. It is estimated that the increase of sensitivity to oxidative stress may lead to the decrease of pathogenicity of mutant strain.In the exploration of gene function in A. fumigatus cells, Agrobacteriummediated transformation is widely applied. People can use this technology to knock down specific gene or disturb their expression and then explore their function. However, this technology can only confirm the possible function of known genes while there are numerous novel genes in A. fumigatus cells to be analyzed. If we can construct a mutant library with certain scale and screen genes purposely, the function of more genes will be known and the study of A. fumigatus will be further improved.In this study, a small-scale library of 5712 mutant strains was obtained by using Agrobacterium-mediated random insertion mutagenesis of IFM40808. The main influencing factors of this transformation system was further explored: when the receptor is A. fumigatus concentration is 1 × 106 spores/ml, the donor is Agrobacterium tumefaciens AGL-1 carrying dual plasmid vector p BHt1 concentration is OD600 nm =0.8, the coincubation temperature is 25 ?, the coincubation time is 48 h, the selection concentration of hygromycin is 200 ?g/m L, the maximum transformation efficiency 350 transformants/106 conidium will be obtained and the positive rate can be kept at 95%. These mutants have clear genetic backgrounds, stable mitosis and random insertion sites, making them perfect for studying gene function.Molecular biological methods like PCR?TAIL-PCR were applied to analyze the mutants. Hygromycin phosphotransferase was obtained as a 800 bp target bands through PCR amplification, which confirm the successful insertion of T-DNA to the A. fumigatus gene. At the same time, flanking sequences of T-DNA insertions was obtained through TAIL-PCR and the precise location of insertion site was analized. These results suggest that Agrobacterium-mediated genetic transformation is an effective method to construct mutant library and explore gene function of A. fumigatus.In conclusion, by knocking down afpab1 gene of A. fumigatus, our research suggested that afpab1 gene was associated with oxidative stress and the deletion of it lead to the increase of pathogenicity. The construction of small-scale T-DNA inserted mutant library lay a foundation for exploring the function of unknown genes, analyzing biological characteristics, and investigating pathogenic mechanism. |
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