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A Kunitz trypsin inhibitor from chickpea (Cicer arietinum L.) that exerts anti-metabolic effect on podborer (Helicoverpa armigera) larvae

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Abstract

Chickpea (Cicer arietinum L.) seeds contain Bowman–Birk proteinase inhibitors, which are ineffective against the digestive proteinases of larvae of the insect pest Helicoverpa armigera. We have identified and purified a low expressing proteinase inhibitor (PI), distinct from the Bowman–Birk Inhibitors and active against H. armigera gut proteinases (HGP), from chickpea seeds. N-terminal sequencing of this HGP inhibitor revealed a sequence similar to reported pea (Pisum sativum) and chickpea α-l-fucosidases and also homologous to legume Kunitz inhibitors. The identity was confirmed by matrix assisted laser desorption ionization – time of flight analysis of tryptic peptides and isolation of DNA sequence coding for the mature protein. Available sequence data showed that this protein forms a distinct phylogenetic cluster with Kunitz inhibitors from Glycine max, Medicago truncatula, P. sativum and Canavalia lineata. The isolated coding sequence was cloned into a yeast expression vector and produced as a recombinant protein in Pichia pastoris. α-l-fucosidase activity was not detectable in purified or recombinant protein, by solution assays. The recombinant protein did not inhibit chymotrypsin or subtilisin activity but did exhibit stoichiometric inhibition of trypsin, comparable to soybean Kunitz trypsin inhibitor. The recombinant protein exhibited higher inhibition of total HGP activity as compared to soybean kunitz inhibitor, even though it preferentially inhibited HGP-trypsins. H. armigera larvae fed on inhibitor-incorporated artificial diet showed significant reduction in average larval weight after 18 days of feeding demonstrating potent antimetabolic activity. The over-expression of this gene in chickpea could act as an endogenous source of resistance to H. armigera.

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Abbreviations

BApNA:

Benzoyl-DL-Arginyl-para-Nitroanilide

BBI:

Bowman Birk type inhibitor

BSM:

Basal Salts Medium

CBB-R250:

Coomassie Brilliant Blue R-250

HGP:

Helicoverpa armigera Gut Proteinases

HGPI:

Helicoverpa armigera Gut Proteinases Inhibitor

HRP:

Horseradish Peroxidase

LB:

Luria Bertonii

LSLB:

Low Salt Luria Bertonii

TOF:

matrix assisted laser desorption ionization–time of flight

PAGE:

Polyacrylamide Gel Electrophoresis

PCR:

Polymerase Chain Reaction

PI:

Proteinase Inhibitor

PVDF:

Polyvinylene Difluoride

SAAAPLpNA:

Succinyl-Alanyl-Alanyl-Alanyl-Prolyl-Leucyl-para-Nitroanilide

SAAPFpNA:

Succinyl-Alanyl-Alanyl-Prolyl-Phenylalanyl-para-Nitroanilide

SDS:

Sodium Dodecyl Sulphate

SKTI:

Soybean Kunitz Trypsin Inhibitor

YPDS:

Yeast Extract Peptone Dextrose Sorbitol

YPG:

Yeast Extract Peptone Glycerol

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Author notes

  1. Abhay M. Harsulkar

    Present address: Interactive Research School for Health Affairs (IRSHA), Pune, 411043, India

Authors and Affiliations

  1. Plant Molecular Biology Group, Division of Biochemical Sciences, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India

    Ajay Srinivasan, Ashok P. Giri, Abhay M. Harsulkar & Vidya S. Gupta

  2. School of Biological Sciences, Science Laboratories, University of Durham, South Road, Durham, DH1 3LE, UK

    Ajay Srinivasan & John A. Gatehouse

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  1. Ajay Srinivasan
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  2. Ashok P. Giri
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Correspondence to Vidya S. Gupta.

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Srinivasan, A., Giri, A.P., Harsulkar, A.M. et al. A Kunitz trypsin inhibitor from chickpea (Cicer arietinum L.) that exerts anti-metabolic effect on podborer (Helicoverpa armigera) larvae. Plant Mol Biol 57, 359–374 (2005). https://doi.org/10.1007/s11103-004-7925-2

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