Articles
ISOLATION OF A HYDROXYCINNAMOYLTRANSFERASE INVOLVED IN PHENYL-PROPANOID BIOSYNTHESIS IN CYNARA CARDUNCULUS L.
Article number
730_9
Pages
93 – 99
Language
English
Abstract
Most secondary metabolites found in plant tissues are of high pharmaceutical interest although they are usually present at low concentrations.
Among therapeutic molecules produced by plants the phenolic compounds are the most widespread.
In globe artichoke extracts the major phenolic compounds are di-caffeoylquinic acids (e.g. cynarin) which originate from the metabolism of phenylpropanoids.
In order to identify new ways of production, it is important to acquire new knowledge on the biosynthesis pathways of exploitable secondary metabolites.
The aim of our study was to identify genes encoding acyltransferases involved in acid chlorogenic synthesis. mRNAs were extracted from globe artichoke leaves and the cDNAs generated by reverse transcription.
Degenerate primers were designed on conserved regions of orthologous HCT (hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyltransferase) proteins to amplify in artichoke the HCT cDNA by PCR. Amplicons were sequenced, and a translated database search revealed high similarity with the tobacco HCT. After successful full length HCT cDNA isolation, the gene was cloned in plasmids and expressed in E. coli. Reaction products were identified by HPLC: the expressed enzyme catalyses the synthesis of p-coumaroyl-quinate, a chlorogenic acid precursor, from p-coumaroyl-CoA and quinic acid.
To evaluate the correlation between the expression of the isolated gene with caffeoyl quinic acids content in leaf tissues, northern blot assays were performed on Cynara cardunculus accessions.
Among therapeutic molecules produced by plants the phenolic compounds are the most widespread.
In globe artichoke extracts the major phenolic compounds are di-caffeoylquinic acids (e.g. cynarin) which originate from the metabolism of phenylpropanoids.
In order to identify new ways of production, it is important to acquire new knowledge on the biosynthesis pathways of exploitable secondary metabolites.
The aim of our study was to identify genes encoding acyltransferases involved in acid chlorogenic synthesis. mRNAs were extracted from globe artichoke leaves and the cDNAs generated by reverse transcription.
Degenerate primers were designed on conserved regions of orthologous HCT (hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyltransferase) proteins to amplify in artichoke the HCT cDNA by PCR. Amplicons were sequenced, and a translated database search revealed high similarity with the tobacco HCT. After successful full length HCT cDNA isolation, the gene was cloned in plasmids and expressed in E. coli. Reaction products were identified by HPLC: the expressed enzyme catalyses the synthesis of p-coumaroyl-quinate, a chlorogenic acid precursor, from p-coumaroyl-CoA and quinic acid.
To evaluate the correlation between the expression of the isolated gene with caffeoyl quinic acids content in leaf tissues, northern blot assays were performed on Cynara cardunculus accessions.
Authors
C. Comino, E. Portis, A. Acquadro, P. Pinelli, A. Hehn, F. Bourgaud, S. Lanteri
Keywords
Cynara cardunculus, acyltransferase isolation, HCT, phenylpropanoid biosynthesis
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