Cloning, Expression and Antifungal Activity of an Endochitinase Gene Derived from Barley (Hordeum Vulgare)

Fungal phytopathogens pose serious problems worldwide in the cultivation of economically important plants, especially in the tropical and subtropical regions. Plants respond to fungal pathogens by producing various pathogenesis-related (PR) proteins, such as chitinases, which restrict the spread of the pathogen in incompatible interactions and allow for systemic acquired resistance. In this study, chitinase gene from barley (bec) was cloned and over-expressed in E. coli. Sequence analysis indicated that the bec gene consists of an open reading frame of 798 nucleotides, which encodes a protein of 266 amino acid residues. Amino acid sequence analysis revealed that BEC has 95% and 92% similarity with a class II chitinase from wheat (Triticum aestivum) and a seed chitinase from rye (Secale cereale), respectively. The recombinant bec (29kDa protein) was produced as insoluble inclusion bodies and was released into active soluble form under alkaline pH and mild denaturing condition. The purified chitinase was assayed against the three economically important fungal pests, Alternaria alternate, the causative agent of leaf spot, Fusarium oxysporium, and Fusarium solani, the causative agents of leaf wilting and damping off diseases. BEC inhibits the growth of the three fungi at 100 ^g- and 200 ^g ml-1. Data obtained from this study suggest that bec-coding sequence can be introduced into the genome of different crop plants to enhance its fungal-resistance property.