The Study On Gene Function Which Is Related To Growth And Propagation Of Aspergillus Fumigatus Based On T-DNA Inserted Mutant Library

Aspergillus fumigatus is one of the most important conditional pathogenic fungi and its morbidity is second behind Candida albicans in systematic fungal infections.In general,the Aspergillus fumigatus infection mainly caused by inhaled spores of Aspergillus fumigatus into alveolar.Spores can be survived in the surface of alveolar after germination,engraftment and ultimately causing systemic fungal infection.It is an important method by using T-DNA and transposon technology into chromosome DNA insertion mutants to research gene function in plants,fungi and other biological.During the early stage of the work,the Aspergillus fumigatus ATMT T-DNA insertion mutant library has been established preparing to be using for the study of the gene function in Aspergillus fumigatus.The main purpose of this study is to find out the genes which associated with Aspergillus fumigatus in growing,and make sure its relations with Aspergillus fumigatus virulence.Finally,we found 54 mutants changed in morphological and 3 of them obviously changed in phenotypic.In the procedure of this study,the key technologie is to determine the insertion site of the mutant.However,the existing methods shows many shortcomings such as complex operation,need restriction enzymes and T-A clone connection technology,low positive rate,nonspecific amplification,time consuming,cost higher and so on.This study combined with touch-down PCR,thermal asymmetric interlaced PCR and suppression-PCR technology,established a new type of high flux insertion site analysis technology.It has many advantages such as higher positive rate,simple and quick,saving time,manpower and material resources.It can be used to read flanking sequence length between 300-2500 bp,can used to the bioinformatics analysis of insertion site,can provide technical support on the high-throughput analysis insertion site in mutants.On this basis,we studied the insertional locus of T-DNA insertion mutant in Aspergillus fumigatus by using this technology.One of the mutant strains named AFM1006 which obviously albinism was studied in-depth gene function,we found that the interrupt Aspergillus chitin synthase gene for csm B genes,and it can influence of the phenotypic changes for stigma differentiation in Aspergillus fumigatus.These results can provide a scientific basis to find the pathways related to morphological differentiation and reveal the molecular mechanism in growing of Aspergillus fumigatus.1.Selecting the mutant which morphological changed observably from the mutant library.We obtained 54 T-DNA insertion mutants with phenotypic changes by screening Aspergillus fumigatus named IFM40808.Its phenotypic change covers the colony growth speed slow,bleaching,aerial hyphae increase,fold,etc.3 of them changed obviously on phenotypic.Strain AFM2690 bleaching,colony appear fold,hyphae bending,uneven thickness of one;Strain AFM3270,colony growth is very slow.Strain AFM1006 bleaching but no difference between the colony sizes.These mutants provide the further material support in the next analysis in the process of growth and differentiation of molecular mechanism in Aspergillus fumigatus.2.Building a new type of method can be used to high flux analysis insertional locus of the mutant.This study first use the principle of touch-down PCR,thermal asymmetric PCR and suppression-PCR to set up an analysis method that can be used for high-throughput found insert loci on mutation.The specific method is to design two consecutive semi nested fixed end primers,primer at the edge of the far distance requires higher annealing temperature,used for pre-amplification,on the edge of the insertion mutation area;Close distance from the edge of primers used in the second round of amplification.At the other end use in 5 ` side has specific conservative DNA fragments of degenerate primers random combine with template.Nonspecific fragments will be caused by the single reverse complementary reading backward at both ends and single cyclization,unable to further amplification.In the preliminary stage of amplification,through the pre-amplification-instantaneous cooling-slow warming-amplification way again,try to improve its amplification positive rate;In the second round of amplification,random primers replacement for human in the degenerate primers 5 ` side to join the specific fragment,make it into a simple PCR reaction,by touchdown PCR method,both the specificity and amplification efficiency.After two rounds of PCR amplification can amplify specific target band sequencing,only rubber cutting recycling without T-A cloning.By the experiment,we found that the integrity of template DNA is the key of the reaction can be completed correctly.This method only on the edge of the insertion site design two consecutive rounds of semi nested primers,and only the first round of primer needs higher annealing temperature,compared with the original successive rounds of semi nested primers,require a higher annealing temperature,greatly reduced the difficulty of primer design.Amplification products don't need T-A clone,A simple,easy to A single PCR A number of times to complete 96 samples,from genome response from the original extract to complete at least four days,reduce to three days to complete,cost of A single sample analysis by original within 10 dollars reduce to 4 dollars,it is suitable for high-throughput analysis of insertion mutation loci.The method has been applied successfully in our laboratory Aspergillus fumigatus,Aspergillus terreus,Sporothrix schenckii,Fusarium oxysporum ATMT T-DNA insertion mutant library insertion site analysis.Aspergillus fumigatus has been successfully identified more than 100 strains,and reveal the Aspergillus fumigatus molecular mechanism,to analyze the pathogenic mechanism of laid a solid foundation.3.The gene function study on albino mutant strains AFM1006 in Aspergillus fumigatus.By molecular analysis to determine the screening for Aspergillus fumigatus albino mutant strains AFM1006 break area for chitin synthase(putative)gene,its structure domain analysis finally confirmed it as a cigarette aspergillus V class chitin synthase csmB.By macroscopic observation,lactic acid phenol cotton blue stain fluorescent optical microscope observation,fluorescence staining white phase contrast microscope observation phase contrast microscope,scanning electron microscopy and fluorescence observation methods,such as the lack of the gene for Aspergillus fumigatus wire extension velocity,diameter,wall thickness,average branch distance and the average separation distance,sertoli cell differentiation,cell wall chitin content had no obvious change;Only affects the conidium top capsule on the differentiation process of produce small terrier,will affect the produce of the spores.By spectrophotometric method and Real-time PCR method for mutant physiological and biochemical analysis,determine the mutant strains of chitin content has no obvious change,always csmB gene expression while the rest of the chitin synthase have different degrees of compensatory sex expression.To analyze the sequence of amino acids on this gene,we found that it contains three domain structure,respectively as the myosin head structure domain,cytochrome b5 sample structure and structure of chitin synthase domains C.Accordingly,we put forward our hypothesis,Aspergillus fumigatus chitin synthase gene csmB only affects Aspergillus fumigatus conidium generated little terrier top capsule on the differentiation and its molecular mechanism for the gene expression after affected by actin finally mature in the top of the capsule membrane synthesis of cell wall chitin qualitative function into full play.At the same time also successfully established AFM1006 mutant strain response.The results can be further defined Aspergillus fumigatus growth and development of molecular mechanism,determination of Aspergillus fumigatus pathogenic mechanism to provide important technical data.In conclusion,this study screening of Aspergillus fumigatus form change mutant received a form difference of mutant strains of 54,3 strains of phenotypic changes obviously.We successfully established the touch-down PCR,thermal asymmetric PCR and suppression PCR insertion site analysis technology based on the land.High throughput,simple operation,this method only needs the PCR amplification without the need for restriction enzymes and T-A clone technology,from the genome only 3 days,extracted to complete within A single sample cost reduced to 25 yuan.Our laboratory has been successfully applied to a variety of T-DNA insertion mutant library insertional locus of analysis.This study has successfully identified more than one hundred strains of Aspergillus fumigatus T-DNA insertion mutant's insertion site.The albino strain AFM1006 further gene function analysis,ultimately determine its for csmB gene insertion mutations,affecting only Aspergillus fumigatus produce small terrier sac differentiation.The results of this study through reverse genetics method to analyze the pathogenic fungi pathogenic mechanism,resistance mechanism,such as foundation,at the same time,this study can also be for other pathogenic fungi,other insertion mutagenesis studies provide reference.