Constitutive expression of protease inhibitor gene isolated from black gram (Vigna mungo L.) confers resistance to Spodoptera litura in transgenic tobacco plants

Abstract

Plant insecticidal proteins like protease inhibitors have been reported to confer insect resistance in transgenic plants. We have cloned a protease inhibitor gene of black gram (<em>Vigna</em> <em>mungo </em>L.) and developed transgenic tobacco plants expressing the protease inhibitor constitutively under the 35S promoter by <em>Agrobacterium tumefaciens </em>mediated genetic transformation. A full-length open reading frame (ORF) of 545 nucleotides codes for a serine protease inhibitor protein with a deduced sequence of 180 amino acids contributing to a predicted molecular mass of 20.2 kDa. The <em>Vigna mungo</em> protease inhibitor (<em>VmPI</em>) is basically a Bowman-Birk type proteinase inhibitor (BBI) of double headed type; family of plant serine protease inhibitors that block trypsin or chymotrypsin. Comparison for conserved domain in the protein by National Centre for Biotechnology Information (NCBI) conserved domain (CD) search showed that the regions from 113-168 amino acid residues have the conserved domain of which 9 residues compose reactive loops on the conserved domain of BBI. The transgenic tobacco plants developed were confirmed by Southern, northern hybridization, polymerase chain reaction (PCR) and reverse transcriptase-PCR (RT-RCR) analysis. When transgenic T₁ tobacco plants were analyzed for insecticidal bioassays, the larval growth of <em>Spodoptera litura</em>, the tobacco cutworm was found severely retarded as compared to their growth on control plants. Therefore, black gram protease inhibitor could be used as an alternative individually or in combination with other insecticidal genes for the development of insect-resistant transgenic crop plants.