Cloning CellobiohydrolaseⅠfrom Penicillium decumbens 114-2 with TAIL-PCR and comparing with its derepressed mutant JU-A10

Abstract:

[Objective] We studied the differences in gene sequence of cellobiohydrolaseⅠgene (cbh1) from Penicillium decumbens 114-2 and its derepressed mutant JU-A10. [Methods] We cloned cbh1 and its full-length cDNA from Penicillium decumbens 114-2 by the modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) and RT-PCR. [Results] The total length of cbh1 was 1500 bp. It contained 2 introns and encoded 453 amino acids (GenBank Accession No.EF397602). The upstream sequence (1.9 kb) of cbh1 gene was also cloned and sequenced. It contained two putative binding sites for the carbon catabolite repressor CREⅠand two putative binding sites for cellulases transcriptional regulator ACEⅠ. [Conclusion] The derepressed strain JU-A10 was a multiple mutant of the wild strain114-2. The mutant produced several times more cellobiohydrolase activity per ml of culture medium when compared with 114-2. The cbh1 gene sequence of the mutant was the same with the wild strain. While four single base mutations were detected on the upstream sequences (1.9 kb) of cbh1 gene.The result suggests that the evidently enhanced cellobiohydrolase activity of the mutant is not due to cbh1 protein-coded sequence . The true reason maybe refer to single base mutations of the upstream sequence that effect the transcription regulation of mutant JU-A10. As a result, the secretion of CBHⅠincreased.

Key words:Penicillium decumbens; TAIL-PCR; cbh1; derepression mutant