[Cloning cellobiohydrolase I from Penicillium decumbens 114-2 with TAIL-PCR and comparing with its derepressed mutant JU-A10].
OBJECTIVE
We studied the differences in gene sequence of cellobiohydrolase I gene (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 CRE I and two putative binding sites for cellulases transcriptional regulator ACE I.


CONCLUSIONS
The derepressed strain JU-A10 was a multiple mutant of the wild strain 114-2. 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 CBH I increased.
Liu
Ziyong
Z
State key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China. liuziyong1010@126.com
Sun
Xianyun
X
Qu
Yinbo
Y
D001665:Binding Sites; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D050505:Mutant Proteins; D043366:Cellulose 1,4-beta-Cellobiosidase