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STUDIES ON ISOLATION AND CHARACTERIZATION OF TRYPSIN INHIBITOR (TI) GENE FROM Dolichos biflorus L. (Kulth)

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Title STUDIES ON ISOLATION AND CHARACTERIZATION OF TRYPSIN INHIBITOR (TI) GENE FROM Dolichos biflorus L. (Kulth)
 
Creator REENA KUMARI
 
Contributor NATH, A.K.
 
Subject enzymes, biochemical compounds, sowing, amino acids, genes, proteins, acidity, animal developmental stages, extraction, cloning
Protease, Trypsin Inhibitor
 
Description ABSTRACT
Protease inhibitors are one of the most promising agents that confer resistance in plants
against insect pests by inhibiting larval midgut proteases. Maximum extraction of trypsin inhibitor
protein from seed flour of Dolichos biflorus L. was in 0.1 M phosphate buffer (pH 7.6) after four
hours of extraction. Screening of Dolichos biflorus L. cultivars for trypsin inhibitor activity
revealed maximum activity in HPK4 cultivar and further studies were conducted in this cultivar.
Crude trypsin inhibitor of all cultivars inhibited midgut protease of P. brassicae larvae. Inhibitor
activity was detected at early stages of seed development (3 days after flowering (DAF)) and it
increased progressively with seed development (21 DAF to 60 DAF). Trypsin inhibitor activity
decreased during seed germination as compared to dry seeds. Crude trypsin inhibitor extracted
from developing and germinating seeds also inhibited larval midgut protease of S. littoralis.
Neonate larvae of P. brassicae fed on cabbage leaf discs coated with 0.025-2.50 mg crude trypsin
inhibitor caused 10–80 % larval mortality. The calculated LC50 value was 1.05 mg crude trypsin
inhibitor and for 2.5 mg crude trypsin inhibitor the calculated LT50 value was 3.2 days. Leaf area
eaten and faecal matter produced by treated larvae were significantly lower as compared to
untreated controls. Larvae fed on leaf discs coated with 2.5 mg crude trypsin inhibitor for 5 days
had significantly less total soluble protein in faecal matter and midgut trypsin activity as
compared to untreated control. Significant reduction in egg hatching (75%) was observed in egg
mass treated with 5.3mg of crude trypsin inhibitor of mature seeds. Trypsin inhibitor gene (309
bp) was amplified from cDNA synthesized from mature seeds of Dolichos biflorus L. HPK4
cultivar using designed primers. The amplified PCR product was cloned and sequenced. Sequence
of Dolichos biflorus L. HPK4 cultivar trypsin inhibitor (DbTI) gene has been submitted to NCBI
with Accession No. JQ259858. DbTI gene and its deduced amino acid sequence showed
homology with Bowman-Birk inhibitors of Dolichos spp., Phaeolus spp., Vigna spp. and Glycine
spp. The predicted molecular weight of deduced amino acid sequence was ~11.5 KDa and it had
N terminal signal peptide of 19 amino acid residues. The secondary structure of deduced amino
acid sequence of DbTI showed dominance of coils and sheets over alpha helix. Homology
modelling was employed to predict the three dimensional structure of DbTI. Docking of trypsin
enzyme and DbTI showed the inhibitor to be of non- competitive type.
 
Date 2016-06-01T12:52:30Z
2016-06-01T12:52:30Z
2013
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/66538
 
Language en
 
Format application/pdf