ABSTRACT
Genes encoding the cellobiohydrolase enzyme (CBHI), designated as cbhI, were isolated from the basidiomycetes Auricularia fuscosuccinea, Pleurotus giganteus, P. eryngii, P. ostreatus, and P. sajor-caju. Initially, the fungal genomic DNA was extracted using a modified cetyltrimethyl ammonium bromide (CTAB) protocol and used as a DNA template. The cbhI genes were then amplified and cloned using the pGEM-T Easy Vector Systems. The sizes of these PCR amplicons were between 700~800 bp. The DNA sequences obtained were similar showing high identity to the cbhI gene family. These cbhI genes were partial consisting of three coding regions and two introns. The deduced amino acid sequences exhibited significant similarity to those of fungal CBHI enzymes belonging to glycosyl hydrolase family 7.
Subject(s)
Humans , Amino Acid Sequence , Base Sequence , Basidiomycota , Bromides , Cellulase , Cellulose 1,4-beta-Cellobiosidase , Clinical Coding , Clone Cells , Cloning, Organism , DNA , Introns , Pleurotus , Polymerase Chain Reaction , Quaternary Ammonium Compounds , Sequence AnalysisABSTRACT
ACEI,ACEII and Xyr1 are transcriptional factors that regulate cellulase gene expression in Trichoderma koningii.In vitro experiments have shown that ACEI and Xyr1 bind to the cbh1 promoter fragment(-304 to-18) and regulate the gene transcription.However,whether ACEII binds to this 287bp fragment is still unclear.To further elucidate the regulatory mechanism of ACEII for cellulase,DNA-binding domains of ACEII from T.Koningii were expressed in E.coli.It could not show binding to the cbh1 promoter fragment(-304 to-18) by electrophoresis mobility shift assays,suggesting that it is Xyr1 but not ACEII binds playing an essential role during induction of cbh1 gene transcription.