Molecular cloning, characterization and semi-quantitative expression of endochitinase gene from the mycoparasitic isolate of Trichoderma harzianum

Filamentous fungi from the genus Trichoderma are well known for their biocontrol potential and have been used as antagonistic agents as well as plant growth promoters. Chitinases released by Trichoderma spp. have been capable of hydrolyzing chitin by splitting their beta-1, 4-glucosidic bonds. The aim of the present study was to isolate and characterize an endochitinase gene from native Trichoderma harzianum isolate which is involved in mycoparasitism. In total, twelve Trichoderma isolates were screened for chitinolytic activity via dual plate method and greenhouse studies. T. harzianum isolate (SVPRT-THLi03) was selected as a target for isolation, cloning, characterization and expression profiling of an endochitinase gene due to its high chitinolytic activity recorded by the degradation of chitin substrates. The genomic DNA of Trichoderma isolates was amplified and cloned in pGEMT cloning vector. The recombinant clones were confirmed through colony PCR and restriction analysis. The sequenced 1223 bp clone nucleotide sequence of putative endochitinase gene, ChitTh showed 99% homology to T. harzianum chit-HAR2 endochitinase (AB041752.1) with 0.0 E-value. The complete nucleotide sequence of ChitTh contained a single ORF of 379 amino acids with 40.7 kDa molecular weight and theoretical pI 8.3. The precursor protein contained 22 amino acids long signal peptide at N terminus. Phylogenetic analysis showed that ChitTh protein was clustered into group V with other Trichoderma spp. Semi-quantitative endochitinase gene expression was analysed for different isolates viz. T. harzianum (SVPRT-THLi03 and SVPRT-47) and T. nigricans (SVPPP-7). Among the three isolates, higher expression was observed in SVPRT-THLi03 and SVPRT-47 whereas SVPPP-7 showed lesser gene expression with respect to the other isolates.