Molecular cloning of endochitinase 33 (ECH33) gene from Trichoderma harzanium

Abstract

This study was conducted to screen for the presence of ech33 gene in 80 isolates of Trichoderma. Furthermore, using gene specific primers, ech33 gene were cloned into pTZ57R/T from T. harzanium IABT1068. The clone was confirmed through PCR amplification and restriction analysis. The  clones were sequenced and analyzed for homology at nucleotide and protein level to find out conserved domain of protein. Gene encoding endochitinase from both species have 96 and 95%  homology with reported sequence both at nucleotide and protein level. The cloned ech33 has a size of 1159 bp, of which 9 bp corresponds to the 5' untranslated region, with a 650 bp open reading frame. The amino acid sequence of gene has signal peptide sequence ranges from 1 to 19. The nucleotide sequence analysis using GENETOOL software revealed presence of three exon and four introns, and has unique restriction sites for HindIII, BamHI and SalI, at 881, 308 and 485 positions, respectively.

Key words: Trichoderma harzanium, ech33, signal peptide.