ISOLATION AND CHARACTERIZATION OF A NOVEL GENE ENCODING KUNITZ-TYPE PROTEASE INHIBITOR FROM PIGEON PEA (CAJANUS CAJAN L.)Mukesh Kumar Vinay Kumar, Rekha Kansal, P.S. Srivastava and K.R. KoundalAbstract Protease Inhibitors (PI) have crucial functions in plant defense against pests and has been implicated in defense against insect pests. In the present investigation, total RNA was isolated from twelve days old developing seed of pigeonpea variety Pusa-33 and cDNA library was constructed from purified mRNA. The cDNA library was screened using heterologous probe of Tomato protease inhibitor (Tpi) cDNA clone and identified a total of six positive clones after tertiary screening and designated as (Pigeonpea Protease Inhibitor) PPI-1 to PPI-6. The positive clone PPI-6 was sequenced and produced 786 bp nucleotide sequences. The nucleotide sequence of PPI-6 produced open reading frame (ORF) of 696 bp having start and stop codon. The deduced amino acids showed homology with Kunitz-type trypsin inhibitors of chickpea, pigeonpea and soybean. The gene sequence was translated and deduced 230 amino acid sequences which belong to Soybean trypsin inhibitor (STI) super family. The nucleotide sequence was searched for homology using BLASTn search at NCBI revealed 100% homology with trypsin inhibitor mRNA of Cicer arietinum followed by 97% with protease inhibitor of Cajanus cajan. The novel PI gene has been submitted to the GenBank with Accession No. GU320336.1. The isolated novel PI gene from this valuable crop will broaden the gene pool of plant defense genes and could be exploited for developing transgenic plants resistant against insects and pathogens.
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