STUDIES ON CHARACTERIZATION AND CLONING OF BOWMAN-BIRK TRYPSIN INHIBITOR GENE FROM A LOCAL BEAN (Phaseolus vulgaris L.) CULTIVAR

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dc.contributor.advisorNATH, AMARJIT K.
dc.contributor.authorSUBHASH, CHAND
dc.date.accessioned2017-04-25T09:15:08Z
dc.date.available2017-04-25T09:15:08Z
dc.date.issued2017
dc.description.abstractABSTRACT Legumes are rich source of proteases inhibitors (PIs) and the particular are known as most promising weapons that confer resistance against insects by inhibiting proteases present in gut of insect larvae. In the present study, before selecting a cultivar for isolation of trypsin inhibitor gene, the local bean cultivars were characterized using EST-SSR markers. Out of 25 EST-SSR primers used for characterization, 19 primers were selected to analyze polymorphism present among bean cultivars. Total number of bands amplified was 26, out of which 24 (92.31%) were polymorphic and 2 (7.69%) were monomorphic. The numbers of unique bands obtained were 2 and the size of amplified fragments ranged from 70 bp to 800 bp. Dendrogram constructed using UPGMA method grouped the bean cultivars into two major clusters. Similarity matrix was constructed using Jaccard’s coefficients and the value of similarity coefficient ranged from 0.17 to 0.90. Maximum similarity coefficient (0.90) was obtained between cultivar Capsule and Kaju and minimum (0.17) between Chitra and Luxmi. Screening of Phaselous vulgaris L. cultivars for total and biological inhibitor activity revealed maximum activity in Baspa cultivar and further studies were conducted in this cultivar. Total trypsin inhibitor activity was detected at early stage of seed development, 10 days after flowering (DAF) and it increased progressively with seed development (10-60 DAF). The inhibitor activity decreased during seed germination as compared to dry seeds. Crude trypsin inhibitor extracted from developing and germinating seeds also inhibited larval midgut proteases of Pieries brassicae, Helicoverpa armigera and Spodoptera littoralis. Significant reduction in egg hatching was observed in egg masses treated with crude trypsin inhibitor protein extracted from mature seeds. The inhibitor protein was purified by ammonium sulfate precipitation, gel filtration chromatography and ion exchange chromatography on DEAE-Sephadex column. The purified inhibitor protein showed antimicrobial activity against fungus (viz., Alternaria sp., Fusarium oxysporium, Phytophtora nicotiana, Pythium sp. and Rhizoctonia solani) and bacteria (viz., Providencia stuartii, Bacillus subtilis and Pseudomonas aeroginosa). The trypsin inhibitor gene (372 bp) was amplified from cDNA synthesized from developing seeds (40 DAF) of Baspa cultivar using designed primers. Amplified PCR product was cloned and sequenced. The isolated gene was also integrated into plant and bacterial expression vector pBI121 and pET 23a, respectively. Sequence of Phaseolus vulgaris L. Baspa cultivar trypsin inhibitor gene has been submitted to NCBI with accession no. KX703026. Trypsin inhibitor gene and its deduced amino acid sequence showed homology with Bowman-Birk inhibitors of legumes crops. The predicted molecular weight of deduced amino acid sequence was ~ 13.4 kDa and it had N terminal signal peptide of 29 amino acid residues. The secondary structure of deduced amino acid sequence of trypsin inhibitor gene showed dominance of coils and sheets over alpha helix. Homology modeling was employed to predict the three dimensional structure of protein. Ramachandran plot for Phaseolus vulgaris L. cultivar Baspa trypsin inhibitor showed that the model generated by GENO3D2software had most residues in favorable region and had overall good quality.en_US
dc.identifier.urihttp://krishikosh.egranth.ac.in/handle/1/5810010071
dc.keywordsBEAN,CULTIVARS,TRYPSIN INHIBITOR GENEen_US
dc.language.isoenen_US
dc.publisherUHF,NAUNIen_US
dc.subBiotechnologyen_US
dc.subjectnullen_US
dc.themeBEAN,CULTIVARS,TRYPSIN INHIBITOR GENEen_US
dc.these.typePh.Den_US
dc.titleSTUDIES ON CHARACTERIZATION AND CLONING OF BOWMAN-BIRK TRYPSIN INHIBITOR GENE FROM A LOCAL BEAN (Phaseolus vulgaris L.) CULTIVARen_US
dc.typeThesisen_US
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