Cloning, expression and characterization of biotic stress inducible Ragi bifunctional inhibitor (RBI) gene from Eleusine coracana Gaertn.

Abstract

Ragi bifunctional inhibitor (RBI) of finger millet (Eleusine coracana Gaertn.) inhibits both α-amylase and trypsin simultaneously. It is for the first time the rbi gene was cloned from Eleusine coracana (GPU 28) seeds in pET22b(+) vector and sequenced (GenBank Accession No. DQ 494211). It was functionally expressed in E. coli Rosetta2(DE3) expression host and characterized in terms of enzyme inhibitory activity. The open reading frame (rbi) of 366 bp showing high GC content (68%) encoded 122 amino acids. The predicted amino acid sequence showed 92% identity with the seed purified RBI. Five disulfide bond containing RBI was expressed in the periplasmic space of Rosetta2(DE3). The enzyme inhibitory properties of the recombinant RBI were the same as those described for the seed RBI. The soluble recombinant protein of 0.9 mg/l was purified from the periplasmic fraction by His-bind resin column. Induction of the rbi transcript level in young finger millet leaves under Rhizopus oryzae and Curvularia lunata var aeria artificial infection were revealed by semi-quantitative RT-PCR analysis, that has advocated its involvement in the defense mechanism of finger millet.