title: Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean.
abstract: This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327 bp encoding 109 amino acids was cloned from rice bean seeds using degenerate primer set. BlastP search revealed that the RbTI encoded amino acid of approx 13.0 kDa and shared 99% homology each with BBI from Phaseolus parvulus, Vigna trilobata and Vigna vexilata. Phylogenetic tree also showed close relationship of RbTI with BBI from other members of Leguminaceae family. RbTI gene was further confirmed as intronless (GenBank accession no. KJ159908). The secondary and 3D-structural models for the RbTI were predicted with homology modeling. qRT-PCR studies revealed the highest RbTI expression in the seeds nearing maturity, whereas the low expression of the gene was noticed in young leaves. The isolated RbTI was successfully expressed in Escherichiacoli and the highest expression was recorded after 5.5h of induction. Study on the inhibitory activity of expressed protein against the gut proteases of Hessian fly larvae revealed 87% inhibition. The novel RbTI gene will further broaden the pool of plant defense genes and could be an ideal choice for developing transgenic crops resistant to insect pests with high economic value. In addition, it has the potential to be used as a probe for selection of insect- and pathogen-resistant genotypes.
authors.affiliation: Biochemistry Laboratory, Department of Crop Improvement, CSKHPKV, Palampur, HP 176062, India. Electronic address: rajankatoch@yahoo.com.
authors.forename: Rajan
authors.initials: R
authors.lastname: Katoch
authors.affiliation: Department of Biotechnology, Institute of Himalayan Bioresource Technology, Palampur 176062, India.
authors.forename: Sunil Kumar
authors.initials: SK
authors.lastname: Singh
authors.affiliation: Department of Zoology, PAU, Ludhiana 141004, India.
authors.forename: Neelam
authors.initials: N
authors.lastname: Thakur
authors.affiliation: Department of Biotechnology, Institute of Himalayan Bioresource Technology, Palampur 176062, India.
authors.forename: Som
authors.initials: S
authors.lastname: Dutt
authors.affiliation: Department of Biotechnology, Institute of Himalayan Bioresource Technology, Palampur 176062, India.
authors.forename: Sudesh Kumar
authors.initials: SK
authors.lastname: Yadav
authors.affiliation: Department of Entomology, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Rich
authors.initials: R
authors.lastname: Shukle
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007887:Fabaceae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors
keywords: Bowman–Birk protease inhibitor; Gene expression; Hessian fly; Insect resistance; Rice bean