[Construction of cDNA library of Aspergillus niger H1 and screening of phosphate-dissolving related gene].
OBJECTIVE
To obtain phosphate-dissolving genes from cDNA library of Aspergillus niger H1.


METHODS
The double-stranded cDNA was synthesized using switching mechanism at 5'end of RNA transcript technique and ligated to the vector pBluescript II SK (+). We transformed recombinant plasmid into E. coli HST08, resulting in a primary cDNA library. We screened clones with phosphate-dissolving activities on the insoluble phosphate medium and blasted the sequence in National Center for Biotechnology Information (NCBI). To study the phosphate dissolving mechanisms of the cloned gene, we analyzed 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 the phosphate-dissolving gene.


RESULTS
A cDNA library of A. niger H1 was successfully constructed. Titer tests showed that the content of constructed A. niger H1 cDNA library reached 5.65 x 10(6) cfu/mL, in which the percentage of recombinant clones was 99.15%. We screened 61 clones with phosphate-dissolving activities on the solid medium with insoluble phosphate. The corresponding gene in one of these clones was identified. The full length cDNA of clone H-54 was 839 bp, encoding a predicted protein with 180 amino acid residues. The expression of phosphate-dissolving gene in E. coli enhanced organic acids secretion and improved the phosphate solubilizing activity. Formic acid and acetic acid were found in 12 h, and malic acid and alpha-ketoglutarate were secreted in 24 h. The clone H-54 decreased the pH value of medium from 6.32 to 3.93 and released soluble phosphate up to 0.105 mg/mL in 36 h.


CONCLUSIONS
We had obtained a phosphate-dissolving gene designated psgA from Aspergillus niger H1.
College of Life Sciences, Nanjing Agricultural University, Key Laboratory for Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Nanjing 210095, China. tangchaoxi240@163.com
Chaoxi
C
Tang
Mingbo
M
Gong
Shunpeng
S
Li
Changxiong
C
Zhu
D001234:Aspergillus niger / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D007656:Ketoglutaric Acids / Q000378:metabolism; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism*
D005656:Fungal Proteins; D007656:Ketoglutaric Acids; D010710:Phosphates