Molecular cloning and characterization of the gene encoding B-1, 3 - glucanase in Trichoderma spp.

Abstract

β–1,3–glucanase enzyme is widely distributed among bacteria, fungi and higher plants. Fungal β–1,3–glucanase produced by Trichoderma spp. is the key enzyme in the lysis of cell wall during their mycoparasitic action against several phytopathogenic fungi. This creates the potentiality of Trichoderma to be used as biocontrol agent. Biochemical characterization of the enzyme β–1,3–glucanase from different Trichoderma spp. has been reported. The gene encoding β–1,3–glucanase has been cloned and sequenced in several plants, bacteria and fungi. The study entitled “Molecular cloning and characterization of the gene encoding -1,3-glucanase in Trichoderma spp.” was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB) and Department of Plant Pathology, College of Horticulture, Vellanikkara during the period from 2006 to 2008. In the present study an attempt was made to determine the glucanase activity and to characterize β–1,3–glucanase gene of Trichoderma spp. Fifteen Trichoderma isolates, antagonistic to Phytophthora capsici, P. meadii, Pythium aphanidermatum and Ralstonia solanacearum were used for the present study. The enzyme activities of different isolates were found to be ranged from 1.3 U to 205.3 U per 50 ml culture filtrate for 10 min incubation. Among the fifteen isolates, the highest enzyme activity was found in T. viride–1 (205.3U) followed by T. harzianum-30 (157.8U), T. aureoviride (139.2U), T. viride-8 (121.9U) and these four were selected for gene isolation. The informations on glucanase gene sequences of different species of Trichoderma available in the public domain NCBI were collected and subjected to multiple sequence alignment to detect conserved boxes of the gene among species. Based on the data, three pairs of gene specific primer were designed for amplification of β–1,3–glucanase gene fragment of about 500 - 1400bp in Trichoderma spp. Genomic DNA was isolated from the four selected Trichoderma isolates. Amplification was obtained from two isolates by the primer combination of Glucan F1R1 and Glucan F2R2, whereas by Glucan F1R2, the amplification was obtained in all isolates. The amplicons obtained by Glucan F1R1 from T. aureoviride, by Glucan F2R2 from T. aureoviride and T. viride-8, by Glucan F1R2 from T. viride-1 and T. harzianum-30, were used for cloning. The amplicons were eluted, cloned in pGEM-T Easy Vector and transformed into E. coli JM 109 competent cells. High level of recombination was observed on blue-white screening. Recombination of the insert was confirmed by PCR of the plasmid, isolated from white colonies. The cloned fragments were sequenced to obtain the nucleotide sequence information. The sequences obtained after vector screening were named as GT-311, GT-322, BPT-822 and were subjected to Blast search. All the three sequences revealed significant levels of homology with glucanase genes of different Trichoderma spp. The sequences were also subjected to various theoretical sequence analysis using bioinformatics tools, which include ORF finder, GENSCAN, SOPMA, NEB cutter, Hydropathy plot, Interproscan, Motifscan, NASTATS and AASTATS tools of Biology Workbench. Sequences diversity of β–1,3–glucanase gene among different species of Trichoderma/Hypocrea was determined. The evolutionary distance of GT-322 from T. aureoviride and BPT-822 T. viride-8 was much lower and closely related to β-1,3-glucanase (glu) gene of T. viride having accession number EF176582.1. The two sequences cloned from T. aureoviride showed divergence, indicating the divergence of β–1,3–glucanase within the species. The sequence information obtained from the Trichoderma isolates during the present study can be further exploited for full length gene isolation to develop transgenic black pepper and ginger plants with resistance to Phytopthora capsici and Pythium aphanidermatum and can also be exploited to develop transgenic microorganism with high biocontrol potential.