Isolation and Characterization of a Gene Coding for Chitinase in Developing Winged Bean Seed (Psophocarpus Tetragonolubus )

Chitinase, which catalyse the hydrolysis of the β-1,4-N-acetyl-D-glucosamine linkages of the fungal cell wall polymer chitin, is involved in inducible defences of plants. The aim of this research is to isolate a chitinase genes from seed of winged bean. In order to isolate genes encoding for chit...

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Main Author:	Sekeli, Rogayah
Format:	Thesis
Language:	English English
Published:	2000
Subjects:	Winged bean Chitinase Polymerase chain reaction
Online Access:	http://psasir.upm.edu.my/id/eprint/8421/1/FSMB_2000_11_IR.pdf
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id	my-upm-ir.8421
record_format	uketd_dc
spelling	my-upm-ir.84212024-01-15T03:50:34Z Isolation and Characterization of a Gene Coding for Chitinase in Developing Winged Bean Seed (Psophocarpus Tetragonolubus ) 2000-05 Sekeli, Rogayah Chitinase, which catalyse the hydrolysis of the β-1,4-N-acetyl-D-glucosamine linkages of the fungal cell wall polymer chitin, is involved in inducible defences of plants. The aim of this research is to isolate a chitinase genes from seed of winged bean. In order to isolate genes encoding for chitinases from a cDNA library of winged bean seed, two sets of degenerate primers were designed which corresponded to conserved regions of chitinases class I and class IV. These were then reverse transcribed and subsequently amplified using polymerase chain reactions (RT -PCR) using 4 weeks old seeds cDNA as template. A 1.1 kb fragment was cloned, and subjected to terminal sequence analysis to verify the presence of sequences encoding for chitinases. Nucleotide sequence analysis showed that the fragment appears to encode for a class I chitinase, and this fragment was then used as a probe to screen for a full length gene from the winged bean seed cDNA library. After library screening one clone, CHRZP was isolated and found to encode a chitinase gene. The complete nucleotide sequence of the winged bean chitinase (1324 bp) encoded a polypeptide of 289 amino acids that encodes for a basic chitinase with cysteine-rich domain at the N-terminal. A Comparison of amino acid sequence showed 88% similarity to a chitinase sequence isolated from Oryza sativa. RNA blot hybridisation revealed that mRNA that corresponded to CHRZP accumulates to high levels in leaves compared to seed, tuber and pod with a transcript size of 1.0 kb. Southern hybridisation analysis indicated that this gene was present as a single copy gene in the winged bean genome. Winged bean Chitinase Polymerase chain reaction 2000-05 Thesis http://psasir.upm.edu.my/id/eprint/8421/ http://psasir.upm.edu.my/id/eprint/8421/1/FSMB_2000_11_IR.pdf text en public masters Universiti Putra Malaysia Winged bean Chitinase Polymerase chain reaction Food Science and Technology Napis, Suhaimi English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
advisor	Napis, Suhaimi
topic	Winged bean Chitinase Polymerase chain reaction
spellingShingle	Winged bean Chitinase Polymerase chain reaction Sekeli, Rogayah Isolation and Characterization of a Gene Coding for Chitinase in Developing Winged Bean Seed (Psophocarpus Tetragonolubus )
description	Chitinase, which catalyse the hydrolysis of the β-1,4-N-acetyl-D-glucosamine linkages of the fungal cell wall polymer chitin, is involved in inducible defences of plants. The aim of this research is to isolate a chitinase genes from seed of winged bean. In order to isolate genes encoding for chitinases from a cDNA library of winged bean seed, two sets of degenerate primers were designed which corresponded to conserved regions of chitinases class I and class IV. These were then reverse transcribed and subsequently amplified using polymerase chain reactions (RT -PCR) using 4 weeks old seeds cDNA as template. A 1.1 kb fragment was cloned, and subjected to terminal sequence analysis to verify the presence of sequences encoding for chitinases. Nucleotide sequence analysis showed that the fragment appears to encode for a class I chitinase, and this fragment was then used as a probe to screen for a full length gene from the winged bean seed cDNA library. After library screening one clone, CHRZP was isolated and found to encode a chitinase gene. The complete nucleotide sequence of the winged bean chitinase (1324 bp) encoded a polypeptide of 289 amino acids that encodes for a basic chitinase with cysteine-rich domain at the N-terminal. A Comparison of amino acid sequence showed 88% similarity to a chitinase sequence isolated from Oryza sativa. RNA blot hybridisation revealed that mRNA that corresponded to CHRZP accumulates to high levels in leaves compared to seed, tuber and pod with a transcript size of 1.0 kb. Southern hybridisation analysis indicated that this gene was present as a single copy gene in the winged bean genome.
format	Thesis
qualification_level	Master's degree
author	Sekeli, Rogayah
author_facet	Sekeli, Rogayah
author_sort	Sekeli, Rogayah
title	Isolation and Characterization of a Gene Coding for Chitinase in Developing Winged Bean Seed (Psophocarpus Tetragonolubus )
title_short	Isolation and Characterization of a Gene Coding for Chitinase in Developing Winged Bean Seed (Psophocarpus Tetragonolubus )
title_full	Isolation and Characterization of a Gene Coding for Chitinase in Developing Winged Bean Seed (Psophocarpus Tetragonolubus )
title_fullStr	Isolation and Characterization of a Gene Coding for Chitinase in Developing Winged Bean Seed (Psophocarpus Tetragonolubus )
title_full_unstemmed	Isolation and Characterization of a Gene Coding for Chitinase in Developing Winged Bean Seed (Psophocarpus Tetragonolubus )
title_sort	isolation and characterization of a gene coding for chitinase in developing winged bean seed (psophocarpus tetragonolubus )
granting_institution	Universiti Putra Malaysia
granting_department	Food Science and Technology
publishDate	2000
url	http://psasir.upm.edu.my/id/eprint/8421/1/FSMB_2000_11_IR.pdf
_version_	1794018745923928064

Isolation and Characterization of a Gene Coding for Chitinase in Developing Winged Bean Seed (Psophocarpus Tetragonolubus )

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