Isolation and characterization of GA 20-oxidase gene from sago palm (Metroxylan sagu)

GA 20-oxidase is one of the enzymes that are involved in the production of gibberellins. Gibberellins are plant hormones that are involved in controlling seed germination, stem elongation, leaf expansion, flower induction and development and growth of seed and fruit. Based on previously published c...

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Main Author:	Bala, Jamel
Format:	Thesis
Language:	English
Published:	2013
Subjects:	Q Science (General)
Online Access:	http://ir.unimas.my/id/eprint/9392/1/Bala%20anak%20Jamel%20ft.pdf
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id	my-unimas-ir.9392
record_format	uketd_dc
spelling	my-unimas-ir.93922023-04-12T04:56:33Z Isolation and characterization of GA 20-oxidase gene from sago palm (Metroxylan sagu) 2013 Bala, Jamel Q Science (General) GA 20-oxidase is one of the enzymes that are involved in the production of gibberellins. Gibberellins are plant hormones that are involved in controlling seed germination, stem elongation, leaf expansion, flower induction and development and growth of seed and fruit. Based on previously published conserved amino acid sequences of the plant GA 20- oxidase cDNA clones, oligonucleotide primers were used to amplify GA 20-oxidase gene from genomic DNA of sago palm. PCR amplification using this primer produced a single band with the estimated size of 500 bp in length. BLAST analysis has shown that this fragment was homologous with GA 20-oxidase gene. Based on the sequence obtained, several sets of gene specific primers were constructed and used to amplify the full-length gene both from genomic DNA and cDNA. The total size of GA20-oxidase gene obtained from genomic DNA and cDNA was 1332 and 1161 bp respectively. Comparison between genomic and cDNA sequence showed that, the GA 20-oxidase gene is comprised of two introns and three exons. In order to study the heterologous expression, GA 20-oxidase gene was cloned into pPCR-Script Amp SK (+) vector in sense direction and in correct reading frame, and was then transformed into Escherichia coli (E. coli). Protein was then extracted from E. coli and the fusion protein was analysed using western blot analysis. Heterologous expression was compared with endogenous GA 20-oxidase of sago palm. Expression activity was detected with polyclonal antibody which was induced and produced in a rabbit using short peptide sequence of sago GA 20-oxidase. Western hybridization analysis showed the presence of an intact 32 kDa band, for both crude protein samples obtained from sago tissue and E. coli containing sense GA 20-oxidase gene. No band was observed for protein extracted from negative control sample. This study indicated that the endogenous and heterologous products might be encoded by the same gene, GA 20-oxidase. University Malaysia Sarawak, UNIMAS 2013 Thesis http://ir.unimas.my/id/eprint/9392/ http://ir.unimas.my/id/eprint/9392/1/Bala%20anak%20Jamel%20ft.pdf text en validuser phd doctoral University Malaysia Sarawak, UNIMAS Faculty of Resource Science and Technology
institution	Universiti Malaysia Sarawak
collection	UNIMAS Institutional Repository
language	English
topic	Q Science (General)
spellingShingle	Q Science (General) Bala, Jamel Isolation and characterization of GA 20-oxidase gene from sago palm (Metroxylan sagu)
description	GA 20-oxidase is one of the enzymes that are involved in the production of gibberellins. Gibberellins are plant hormones that are involved in controlling seed germination, stem elongation, leaf expansion, flower induction and development and growth of seed and fruit. Based on previously published conserved amino acid sequences of the plant GA 20- oxidase cDNA clones, oligonucleotide primers were used to amplify GA 20-oxidase gene from genomic DNA of sago palm. PCR amplification using this primer produced a single band with the estimated size of 500 bp in length. BLAST analysis has shown that this fragment was homologous with GA 20-oxidase gene. Based on the sequence obtained, several sets of gene specific primers were constructed and used to amplify the full-length gene both from genomic DNA and cDNA. The total size of GA20-oxidase gene obtained from genomic DNA and cDNA was 1332 and 1161 bp respectively. Comparison between genomic and cDNA sequence showed that, the GA 20-oxidase gene is comprised of two introns and three exons. In order to study the heterologous expression, GA 20-oxidase gene was cloned into pPCR-Script Amp SK (+) vector in sense direction and in correct reading frame, and was then transformed into Escherichia coli (E. coli). Protein was then extracted from E. coli and the fusion protein was analysed using western blot analysis. Heterologous expression was compared with endogenous GA 20-oxidase of sago palm. Expression activity was detected with polyclonal antibody which was induced and produced in a rabbit using short peptide sequence of sago GA 20-oxidase. Western hybridization analysis showed the presence of an intact 32 kDa band, for both crude protein samples obtained from sago tissue and E. coli containing sense GA 20-oxidase gene. No band was observed for protein extracted from negative control sample. This study indicated that the endogenous and heterologous products might be encoded by the same gene, GA 20-oxidase.
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Bala, Jamel
author_facet	Bala, Jamel
author_sort	Bala, Jamel
title	Isolation and characterization of GA 20-oxidase gene from sago palm (Metroxylan sagu)
title_short	Isolation and characterization of GA 20-oxidase gene from sago palm (Metroxylan sagu)
title_full	Isolation and characterization of GA 20-oxidase gene from sago palm (Metroxylan sagu)
title_fullStr	Isolation and characterization of GA 20-oxidase gene from sago palm (Metroxylan sagu)
title_full_unstemmed	Isolation and characterization of GA 20-oxidase gene from sago palm (Metroxylan sagu)
title_sort	isolation and characterization of ga 20-oxidase gene from sago palm (metroxylan sagu)
granting_institution	University Malaysia Sarawak, UNIMAS
granting_department	Faculty of Resource Science and Technology
publishDate	2013
url	http://ir.unimas.my/id/eprint/9392/1/Bala%20anak%20Jamel%20ft.pdf
_version_	1783728055610507264

Isolation and characterization of GA 20-oxidase gene from sago palm (Metroxylan sagu)

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