Molecular cloning and characterization of transcripts encoding terpene synthase genes from Aquilaria malaccensis Lam

Aquilaria malaccensis Lam. is an endangered tropical tree that produces agarwood, a natural product well-known for its fragrance and medicinal properties, in response to external stimuli. Agarwood contains a wide variety of terpenes and phenylethyl chromone derivatives. However, sesquiterpene was th...

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Main Author:	Wong, Mun Theng
Format:	Thesis
Language:	English
Published:	2017
Subjects:	Aquilaria malaccensis - Conservation Biosynthesis Aquilaria malaccensis
Online Access:	http://psasir.upm.edu.my/id/eprint/68452/1/FH%202018%207%20IR.pdf
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id	my-upm-ir.68452
record_format	uketd_dc
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
topic	Aquilaria malaccensis - Conservation Biosynthesis Aquilaria malaccensis
spellingShingle	Aquilaria malaccensis - Conservation Biosynthesis Aquilaria malaccensis Wong, Mun Theng Molecular cloning and characterization of transcripts encoding terpene synthase genes from Aquilaria malaccensis Lam
description	Aquilaria malaccensis Lam. is an endangered tropical tree that produces agarwood, a natural product well-known for its fragrance and medicinal properties, in response to external stimuli. Agarwood contains a wide variety of terpenes and phenylethyl chromone derivatives. However, sesquiterpene was the only terpene synthase (TPS) gene that was being studied in Aquilaria. Therefore, both monoterpene and diterpene synthase genes which are equally important as sesquiterpene in agarwood formation from A. malaccensis were identified in this study. The aim of this study described here was to determine candidate TPS transcripts from A. malaccensis stems, to analyze the transcript expression profile in different tissues of A. malaccensis and then use this information to express terpene synthase in an Escherichia coli expression system. Using an in-house transcriptome assembled from A. malaccensis RNA sequencing project, specific primers targeted for TPS genes were designed. Three TPS candidate genes were selected based on their sequence similarities to other known plant TPSs and named AmTPS01, AmTPS02 and AmTPS03. The partial-length and full-length cDNAs were amplified from stem tissues using the RACE approach and cloned into the pSTBlue-1 vector. Only AmTPS03 is a full-length cDNA (3027 nucleotides), while the AmTPS01 and AmTPS02 sequences included only the 3’-ends (2014 and 1601 nucleotides, respectively). Gene annotation analysis indicates that the deduced peptide sequences of AmTPS01, AmTPS02 and AmTPS03 had 603, 421 and 786 amino acids, respectively. BLAST searches against the GenBank revealed that AmTPS01 had 72% similarity to myrcene synthase from Morus notabilis, AmTPS02 to 3R-linalool synthase from Theobroma cacao (62%) and AmTPS03 to ent-kaurene synthase from Castanea mollissima (81%). From phylogenetic analyzes, the three TPSs are found clustered within the functional homologs of the terpene synthase subfamilies. AmTPS01 and AmTPS02 are clustered with the monoterpene Tps-b subfamily, while AmTPS03 with the diterpene Tps-e subfamily. For the transcript expression analysis, qPCR experiments were carried out using different tissues and treatments. In tissue culture plantlets of A. malaccensis, AmTPS01 and AmTPS02 transcripts were highly expressed in the roots and stems, respectively, compared to leaves. The preference of expression in wood tissues is supported from the experiment using nursery-grown tree, where AmTPS02 also showed high expression in both stem (450-fold) and roots (100-fold) compared to leaves, while AmTPS01 showed the highest expression in stem (10-folds). AmTPS01 and AmTPS02 in calli responded to methyl jasmonate (MJ), a known trigger to production of fragrance constituents in agarwood with the highest expression detected 6 hours after treatment. Drill-wounding and application of a liquid inducer were carried out on plantation trees in a time-course experiment (0, 2, 4, 6, 12 and 24 hours). The liquid inducer apparently induced expression of AmTPS01 (4 hours) and AmTPS03 (6 hours) to the highest level (10-30-folds) compared to drill-wounding. AmTPS03 transcript was abundant in induced-plantation trees but was lowly expressed in MJ-treated calluses, tissue culture plantlets and nursery-grown trees, suggesting its expression is related to the tree’s physiological age and a strong inducer. To functionally characterize the three genes, the ORFs were ligated into pET-28a vector and expressed in the Escherichia coli BL21 (DE3) strain. Expression of the three His-tag fused proteins, AmTPS01 (70.90 kDA), AmTPS02 (50.29 kDa) and AmTPS03 (90.55 kDa) in bacteria resulted in the accumulation of the protein in insoluble forms. In conclusion, the cloned A. malaccensis TPS genes are wood-specific and respond differently to triggers of agarwood induction. In future, studies should be conducted to understand their actual roles in transforming basic isoprene building blocks into terpene compounds. This study provides a foundation for further elucidating the role of TPS genes in the biosynthesis of agarwood compounds in A. malaccensis.
format	Thesis
qualification_level	Doctorate
author	Wong, Mun Theng
author_facet	Wong, Mun Theng
author_sort	Wong, Mun Theng
title	Molecular cloning and characterization of transcripts encoding terpene synthase genes from Aquilaria malaccensis Lam
title_short	Molecular cloning and characterization of transcripts encoding terpene synthase genes from Aquilaria malaccensis Lam
title_full	Molecular cloning and characterization of transcripts encoding terpene synthase genes from Aquilaria malaccensis Lam
title_fullStr	Molecular cloning and characterization of transcripts encoding terpene synthase genes from Aquilaria malaccensis Lam
title_full_unstemmed	Molecular cloning and characterization of transcripts encoding terpene synthase genes from Aquilaria malaccensis Lam
title_sort	molecular cloning and characterization of transcripts encoding terpene synthase genes from aquilaria malaccensis lam
granting_institution	Universiti Putra Malaysia
publishDate	2017
url	http://psasir.upm.edu.my/id/eprint/68452/1/FH%202018%207%20IR.pdf
_version_	1747812577988050944
spelling	my-upm-ir.684522019-05-14T01:28:16Z Molecular cloning and characterization of transcripts encoding terpene synthase genes from Aquilaria malaccensis Lam 2017-08 Wong, Mun Theng Aquilaria malaccensis Lam. is an endangered tropical tree that produces agarwood, a natural product well-known for its fragrance and medicinal properties, in response to external stimuli. Agarwood contains a wide variety of terpenes and phenylethyl chromone derivatives. However, sesquiterpene was the only terpene synthase (TPS) gene that was being studied in Aquilaria. Therefore, both monoterpene and diterpene synthase genes which are equally important as sesquiterpene in agarwood formation from A. malaccensis were identified in this study. The aim of this study described here was to determine candidate TPS transcripts from A. malaccensis stems, to analyze the transcript expression profile in different tissues of A. malaccensis and then use this information to express terpene synthase in an Escherichia coli expression system. Using an in-house transcriptome assembled from A. malaccensis RNA sequencing project, specific primers targeted for TPS genes were designed. Three TPS candidate genes were selected based on their sequence similarities to other known plant TPSs and named AmTPS01, AmTPS02 and AmTPS03. The partial-length and full-length cDNAs were amplified from stem tissues using the RACE approach and cloned into the pSTBlue-1 vector. Only AmTPS03 is a full-length cDNA (3027 nucleotides), while the AmTPS01 and AmTPS02 sequences included only the 3’-ends (2014 and 1601 nucleotides, respectively). Gene annotation analysis indicates that the deduced peptide sequences of AmTPS01, AmTPS02 and AmTPS03 had 603, 421 and 786 amino acids, respectively. BLAST searches against the GenBank revealed that AmTPS01 had 72% similarity to myrcene synthase from Morus notabilis, AmTPS02 to 3R-linalool synthase from Theobroma cacao (62%) and AmTPS03 to ent-kaurene synthase from Castanea mollissima (81%). From phylogenetic analyzes, the three TPSs are found clustered within the functional homologs of the terpene synthase subfamilies. AmTPS01 and AmTPS02 are clustered with the monoterpene Tps-b subfamily, while AmTPS03 with the diterpene Tps-e subfamily. For the transcript expression analysis, qPCR experiments were carried out using different tissues and treatments. In tissue culture plantlets of A. malaccensis, AmTPS01 and AmTPS02 transcripts were highly expressed in the roots and stems, respectively, compared to leaves. The preference of expression in wood tissues is supported from the experiment using nursery-grown tree, where AmTPS02 also showed high expression in both stem (450-fold) and roots (100-fold) compared to leaves, while AmTPS01 showed the highest expression in stem (10-folds). AmTPS01 and AmTPS02 in calli responded to methyl jasmonate (MJ), a known trigger to production of fragrance constituents in agarwood with the highest expression detected 6 hours after treatment. Drill-wounding and application of a liquid inducer were carried out on plantation trees in a time-course experiment (0, 2, 4, 6, 12 and 24 hours). The liquid inducer apparently induced expression of AmTPS01 (4 hours) and AmTPS03 (6 hours) to the highest level (10-30-folds) compared to drill-wounding. AmTPS03 transcript was abundant in induced-plantation trees but was lowly expressed in MJ-treated calluses, tissue culture plantlets and nursery-grown trees, suggesting its expression is related to the tree’s physiological age and a strong inducer. To functionally characterize the three genes, the ORFs were ligated into pET-28a vector and expressed in the Escherichia coli BL21 (DE3) strain. Expression of the three His-tag fused proteins, AmTPS01 (70.90 kDA), AmTPS02 (50.29 kDa) and AmTPS03 (90.55 kDa) in bacteria resulted in the accumulation of the protein in insoluble forms. In conclusion, the cloned A. malaccensis TPS genes are wood-specific and respond differently to triggers of agarwood induction. In future, studies should be conducted to understand their actual roles in transforming basic isoprene building blocks into terpene compounds. This study provides a foundation for further elucidating the role of TPS genes in the biosynthesis of agarwood compounds in A. malaccensis. Aquilaria malaccensis - Conservation Biosynthesis Aquilaria malaccensis 2017-08 Thesis http://psasir.upm.edu.my/id/eprint/68452/ http://psasir.upm.edu.my/id/eprint/68452/1/FH%202018%207%20IR.pdf text en public doctoral Universiti Putra Malaysia Aquilaria malaccensis - Conservation Biosynthesis Aquilaria malaccensis

Molecular cloning and characterization of transcripts encoding terpene synthase genes from Aquilaria malaccensis Lam

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