Cloning and Expression of Proteinase Inhibitor Gene from Vigna mungo (L.) Hepper and In-vitro Effectof Purified Protein on Second Stage Juveniles of Meloidogyne incognita

The plant proteinase inhibitors (PI’s) are one of the several defense mechanisms against plant parasitic nematodes. PI’s are the proteins which form stable complex with proteolytic enzymes and cause disruption in nematode feeding. The gene encoding PI from Black gram (Vignamungo) was amplified using Polymerase Chain Reaction (PCR). The size of gene was approximately 500 bp. The PCR product was cloned into pUC19 vector. The recombinant pUC19 plasmids were digested with NdeI and HindIII restriction enzyme. The purified product was sub-cloned in bacterial expression vector pET28a and recombinant rpET28a were transformed into Escherichia coli BL21(DE3) for over expression of recombinant protein. The whole protein was isolated, purified and loaded on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The approximately 25 kDa molecular weight pure protein was observed on SDS-PAGE. The effect of in-vitro feeding of purified protein was tested on second stage juveniles of root-knot nematode (RKN) Meloidogyne incognita.The protein was found effective and caused up to 55.50% mortality in RKN J2’s. Mortality was increased with increase in concentration of protein and time of exposure to protein. The results indicated that the protein has anti-nematode properties and may lead to new way of nematode management.