Construction Of CDNA Library Of Aspergillus Niger H1 And Screening Of The Phosphate-solubiliz-ing Related Gene

Phosphate-solubilizing microorganisms have remarkable benifits on several respects,such as transforming undissolved phosphorus into soluble phosphate in soil,increasing the availability of phosphate fertilizer and promoting crop growth.However,the application effect of the phosphate-solubilizing microorganisms may not stable due to many factors such as crop species,soil characteristic,climatic conditions and the inhabition of others microorganisms.Cloning the phosphate-solubilizing related genes was the first step toward the construction of high efficient and stable phosphate-solubilizing strains.In this study,a cDNA library of Aspergillus niger H1 was successfully constructed.The phosphate-solubilizing related genes were screened out on the solid medium with tricalcium phosphate as the sole phosphorus.The genes were analyzed with relative bioinformatic methods,and the functions of which were preliminary evaluated.A cDNA library of A.niger H1 was successfully constructed by SMART technique.Titer tests showed that the content of constructed A.niger HI cDNA library reached 5.65 ×106 cfu/mL,in which the percentage of recombinant clones was 99.15%.Sixty one clones were screened on the undissolved phosphorus solid medium.The clone H-54 and H-47 were selected for further study due to that their had more stable phosphate-solubilizing function than the others.Clone H-54 was designated as psgA,the full length cDNA of psgA was 839 bp.psgA had 99%of similarity with A.niger CBS 513.88 and 100%with A.niger contig An07c0100.Clone H-47 was designated as psgB,the full length cDNA of psgB was 625 bp.psgB had 99%of similarity with A.niger CBS 513.88 and A.niger contig An13c0090.The phosphate-solubilization mechanism of gene psgA was studied by analyzing the changes of the pH value,the soluble phosphate content and the production of organic acids in the insoluble phosphate liquid medium inoculated with the clones harboring psgA.The results showed that the expression of psgA in E.coli enhanced organic acids secretion and increased the kinds of organic acids.Formic acid and acetic acid were found in 12 h,malic acid and a-ketoglutarate were secreted in 24 h.The pH value of medium was decreased from 6.32 to 3.93 and soluble phosphate was released up to 0.105 mg/mL in 36 h.The recombinant plasmid of gene psgB was constructed.The phosphate-solubilizing capacity of the clone with the recombinant plasmid was identified by the same way with gene psgA.The results showed that the phosphate-solubilizing capacity was enhanced obviously by the recombinant clone.Formic acid,acetic acid,malic acid and citric acid were secreted,and the pH value of medium was decreased from 6.32 to 3.70 and soluble phosphate was released up to 0.1077 mg/mL in 24 h.