The open reading frame (ORF) fragment of a Kunitz type protease inhibitor gene JcKTI was cloned by the RT-PCR and PCR technology, respectively, from the leaf cDNA and genomic DNA of Jatropha curcas. The genomic region corresponding to the open reading frame does not contain any introns. The open reading frame is of 540bp in length, encoding a mature peptide with 179 amino acid residues. The predictive protein contains the typical Kunitz structure exiting in the protein family members. Tissue specific expression study showed that the expression of JcKTI in root and stem was higher, and the expression was lower in leaf and seed. The prokaryotic expression vector pET32-JcKTI was constructed and expressed in E.coli BL21. The purified recombinant protein was obtained, which showed a certain degree of bovine trypsin inhibitory activity. The gene was over-expressed in the tobacco plantlets. The results of gelatin zymography and BAEE assays showed that the crude protein extracts from the transgenic plants exhibited stronger trypsin inhibitory activity than that of control. Further resistance experiment results showed that eating transgenic tobacco leaves led to the retardation larvae growth and reduced food intake. These results suggest that JcKTI gene may play a role in the roots and stems of Jatropha curcas response to pest attack.
