Cloning and Characterization of Beta-1, 4 Glucosidase 2 (Bgl2) Gene from A High Producer Cellulolytic Enzyme Trichoderma Harzianum (T7)

In this study Trichoderma harzianum (T7) was selected as high producer of beta glucosidase enzyme in optimum conditions among 30 isolates of Trichoderma speices. Genomic DNA from a native Iranian isolate of Trichoderma harzianum (T7) that has been isolated from Kerman province was extracted by CTAB method and specific primers (TFbgl2 & TRbgl2) were used for bgl2 amplification. The amplified DNA fragment approximately 1.4 Kb was digested with appropriate enzymes and cloned into plasmid vector pUC19 and designated as pUCSA1. The sequence obtained from this construct is under accession number EF426298. For cDNA synthesis the mRNA of bgl2 was extracted from induced culture with 1% lactose and 1% CMC and used. This cDNA (approximately 1.3 kb) was isolated, digested, then confirmed with appropriate restriction enzymes and cloned into pUC19 plasmid vector and designated as pUCSA2. The DNA sequence of bgl2 from this construct is under accession number EF426299. Comparison of the genomic DNA with the cDNA sequences showed that bgl2 gene contains one short intron, 79 bp in length and its open reading frame (ORF) with approximately 1.3 kb encodes a polypeptide with 454 amino acids and estimated molecular mass of 51 kDa. Multiple alignment of the deduced amino acid sequence of Bgl2 with those from Hypocrea jecorina (BAA74959), T. viride (AAQ21384), Humicola grisea (BAA74958) and A. niger (AAF74209) showed 89.6%, 80.7%, 72.8% and 83.6% homology, respectively.