Submitted to: Gene
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/23/2014
Publication Date: 8/10/2014
Citation: Katoch, R., Singh, S.K., Thakur, N., Dutt, S., Yadav, S.K., Shukle, R.H. 2014. Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean. Gene. 546:342-351. Interpretive Summary: Insect pests of crop plants, such as the Hessian fly, cause significant yield loss in agro-ecosystems. One way to protect crops from such insect attack is through the use of transgenes for insect resistance. One class of toxic proteins that has potential for resistance to insect pests are the protease inhibitors. We have cloned, characterized and conducted inhibitor studies with a novel protease inhibitor from a little known, little studied, and little exploited wild legume called rice bean that is endogenous to Indo-China (i.e. Nepal). The expressed Rice Bean protease inhibitor strongly blocked the digestive proteases of Hessian fly larvae, suggesting its use as a resistance gene to this pest in genetically engineered wheat. Knowledge from these studies will help scientists ensure resistance in wheat to Hessian fly and will benefit wheat producers and consumers with increased yield and quality without increased cost.
Technical 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 327bp 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 13.0kDa 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 Escherichia coli 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.