Cloning Of β-1, 3-glucanase Gene From Trichoderma Virid LTR-2 And Its Expression In Pichia Pastoris

Trichoderma viride, a soil-borne filamentous fungus,exists widly in the nature. It is capable of parasitizing several plant pathogenic fungi, playing an important role in biocontrol of plant pathogens. Many strains of T. viride produce cell-wall degrading enzymes in whichβ-1,3-glucanase and chitinase are the most important ones and these enzymes degrade fungal cell wall during mycoparasitim .β-1,3-glucanases catalyze the hydrolysation ofβ-1,3-glucan. Most of plant pathogenic fungi attacked by T. harzianum have cell walls that consist of chitin as a structural back- bone and laminarin (β-1,3-glucan) as a filling material.β-1,3-glucanase is thus considered to be the most effective enzyme in the degradation of plant pathogenic fungal cell walls.Theβ-1,3-glucanase encoding gene glu was obtained by PCR amplification using Trichoderma viride LTR-2 cDNA as the template, cloned into plasmid pMD18-T. The recombinant plasmid pMD18-T-glu was transformed into E.coli Top10 F′, and the gene fragment was sequenced and analyzed.Results revealed that an Open Reading Frame of 2289 nucleotides that encoded a protein of 762 amino acids. The sequence showed high homology to threeβ-1,3-glucanase genes reported from Trichoderma.The nucleotide sequence was submitted to NCBI GenBank and the Accession Number is EF176582.The gene glu from recombinant plasmid pMD18-T-glu was cut and cloned into Pichia pastoris shuttle vector pPIC9K.The recombinant plasmid pPIC9K-glu was constructed and transformed into Pichia pastoris KM71 by electroporation. Recombinants were screened on sorbitol selective medium. PCR amplification showed that glu was inserted in the chromosome DNA of Pichia pastoris. SDS-PAGE and enzymatic kinetic analysis indicated thatβ-1,3-glucanase was secreted effectively and expressed highly. The enzyme activity in culture liquid amounted to 889U/mL which was 3.76 times more than that of LTR-2.The recombinantβ-1,3-glucanase showed similar biological activity and characteristics to natural ones.