Electromobility shift assay (EMSA) was used to scan 600 bp of 5′cis regulatory sequence of Aspergillus niger (A. niger ) T21 glucoamylase gene (glaA) for binding by partially fractionated T21 protein extracted from starch-induced mycelia. In this process, one protein, AngCP, was detected to bind specifically to three regions covering -374 to -344, -484 to -414 and -580 to -540 relative to the glaA translational start codon. UV-crosslinking of DNA-protein complex showed that MW of AngCP was 10 ku. DNaseⅠfootprinting analysis demonstrated that AngCP specifically binds to two CCAAT containing sequences within the regions between -374 and -344 and -484 and -414 bp. And the region between -580 and -540 bp contains CCAAT similar box, CCTAT. The results indicated that AngCP is probably one of the members of CCAAT-binding protein families, which are generally involved in enhancement of gene expression in filamentous fungi. These findings suggested that AngCP should be a transcription activator for high-level expression of glaA gene.
The molecular basis for increasing of the glucoamylase (GLA) production of an Aspergillus niger mutant T21 was investigated . Northern blot analysis showed that the amount of glaA specific mRNA of A . niger T21 was about 20 times higher than that of its start strain A . niger AS 3.795. The two glaA promoter fusions (PglaA)-uidAs were respectively introduced into A . niger. Analysis of GUS activity of the transformants revealed that the PglaA activity of the strain T21 is about 3 times stronger than that of the strain AS 3.795. It is considered to be one of the reasons for the increase of glaA transcriptional level in the strain T21. However, comparing with the 20 times increase in the amount of glaA mRNA the alteration of trans regulation should be the most important reason for that. The results of deletion analysis of 5′-cis region of A . niger T21 glaA gene indicated that the region from - 408 to - 513 bp upstream of ATG is responsible for the high level expression of glaA.
