STUDIES ON ISOLATION AND CHARACTERIZATION OF TRYPSIN INHIBITOR (TI) GENE FROM Dolichos biflorus L. (Kulth)
| dc.contributor.advisor | NATH, A.K. | |
| dc.contributor.author | REENA KUMARI | |
| dc.date.accessioned | 2016-06-01T12:52:30Z | |
| dc.date.available | 2016-06-01T12:52:30Z | |
| dc.date.issued | 2013 | |
| dc.description.abstract | 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. | en_US |
| dc.identifier.uri | http://krishikosh.egranth.ac.in/handle/1/66538 | |
| dc.language.iso | en | en_US |
| dc.sub | Biotechnology | |
| dc.sub | Home Science | |
| dc.subject | enzymes, biochemical compounds, sowing, amino acids, genes, proteins, acidity, animal developmental stages, extraction, cloning | en_US |
| dc.subject | Protease, Trypsin Inhibitor | en_US |
| dc.these.type | M.Sc | |
| dc.title | STUDIES ON ISOLATION AND CHARACTERIZATION OF TRYPSIN INHIBITOR (TI) GENE FROM Dolichos biflorus L. (Kulth) | en_US |
| dc.type | Thesis | en_US |