Isolation and characterization of a novel (S)-canadine synthase gene from Coptis chinensis

Background Berberine acts primarily as an important active ingredient in Coptis chinensis and has been traditionally applied in clinical treatment. Nevertheless, little information has been released about C. chinensis, as far as functional genes and biosynthetic pathway of berberine are concerned. H...

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Autores principales:	He,Yang, Hou,Pei, Fan,Gang, Li,Dongmei, Peng,Cheng
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2015
Materias:	Berberine biosynthesis Coptis chinensis Expression pattern
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000500009
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spelling	oai:scielo:S0717-345820150005000092016-03-21Isolation and characterization of a novel (S)-canadine synthase gene from Coptis chinensisHe,YangHou,PeiFan,GangLi,DongmeiPeng,Cheng Berberine biosynthesis Coptis chinensis Expression pattern Background Berberine acts primarily as an important active ingredient in Coptis chinensis and has been traditionally applied in clinical treatment. Nevertheless, little information has been released about C. chinensis, as far as functional genes and biosynthetic pathway of berberine are concerned. Here, we isolated a novel (S)-canadine synthase gene (designated as CAS-1) from C. chinensis by using RT-PCR and RACE techniques. Results Bioinformatics analysis showed that the cDNA is 1942 bp in length with a complete open reading frame (ORF) of 1476 bp, and the ORF encodes a polypeptide of 491 amino acids with a calculated molecular mass of 55.29 kDa and a pI value of 8.92. Real-time quantitative PCR analysis showed that CcCAS-1 was constitutively expressed in leaf, petiole and rhizome tissues, especially in the leaves of C. chinensis. However, the results of berberine content in different tissues by high-performance liquid chromatography with photodiode array detection (HPLC-DAD) method showed that the leaves and the petiole tissues have similar content of berberine. Conclusions We found that the berberine content in the rhizome was seven times (more or less) than that in the leaves and the petioles. In addition, the full length coding sequence of CcCAS-1 was inserted into pET-32a and was successfully expressed in Escherichia coli, laying a solid foundation for protein purification, activity assay and multi-clonal antibody preparation. Together, our data suggest that CcCAS-1 is a novel heme-thiolate enzyme essential for berberine biosynthesis in C. chinensis.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.18 n.5 20152015-09-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000500009en10.1016/j.ejbt.2015.08.004
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	Berberine biosynthesis Coptis chinensis Expression pattern
spellingShingle	Berberine biosynthesis Coptis chinensis Expression pattern He,Yang Hou,Pei Fan,Gang Li,Dongmei Peng,Cheng Isolation and characterization of a novel (S)-canadine synthase gene from Coptis chinensis
description	Background Berberine acts primarily as an important active ingredient in Coptis chinensis and has been traditionally applied in clinical treatment. Nevertheless, little information has been released about C. chinensis, as far as functional genes and biosynthetic pathway of berberine are concerned. Here, we isolated a novel (S)-canadine synthase gene (designated as CAS-1) from C. chinensis by using RT-PCR and RACE techniques. Results Bioinformatics analysis showed that the cDNA is 1942 bp in length with a complete open reading frame (ORF) of 1476 bp, and the ORF encodes a polypeptide of 491 amino acids with a calculated molecular mass of 55.29 kDa and a pI value of 8.92. Real-time quantitative PCR analysis showed that CcCAS-1 was constitutively expressed in leaf, petiole and rhizome tissues, especially in the leaves of C. chinensis. However, the results of berberine content in different tissues by high-performance liquid chromatography with photodiode array detection (HPLC-DAD) method showed that the leaves and the petiole tissues have similar content of berberine. Conclusions We found that the berberine content in the rhizome was seven times (more or less) than that in the leaves and the petioles. In addition, the full length coding sequence of CcCAS-1 was inserted into pET-32a and was successfully expressed in Escherichia coli, laying a solid foundation for protein purification, activity assay and multi-clonal antibody preparation. Together, our data suggest that CcCAS-1 is a novel heme-thiolate enzyme essential for berberine biosynthesis in C. chinensis.
author	He,Yang Hou,Pei Fan,Gang Li,Dongmei Peng,Cheng
author_facet	He,Yang Hou,Pei Fan,Gang Li,Dongmei Peng,Cheng
author_sort	He,Yang
title	Isolation and characterization of a novel (S)-canadine synthase gene from Coptis chinensis
title_short	Isolation and characterization of a novel (S)-canadine synthase gene from Coptis chinensis
title_full	Isolation and characterization of a novel (S)-canadine synthase gene from Coptis chinensis
title_fullStr	Isolation and characterization of a novel (S)-canadine synthase gene from Coptis chinensis
title_full_unstemmed	Isolation and characterization of a novel (S)-canadine synthase gene from Coptis chinensis
title_sort	isolation and characterization of a novel (s)-canadine synthase gene from coptis chinensis
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2015
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000500009
work_keys_str_mv	AT heyang isolationandcharacterizationofanovelscanadinesynthasegenefromcoptischinensis AT houpei isolationandcharacterizationofanovelscanadinesynthasegenefromcoptischinensis AT fangang isolationandcharacterizationofanovelscanadinesynthasegenefromcoptischinensis AT lidongmei isolationandcharacterizationofanovelscanadinesynthasegenefromcoptischinensis AT pengcheng isolationandcharacterizationofanovelscanadinesynthasegenefromcoptischinensis
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Isolation and characterization of a novel (S)-canadine synthase gene from Coptis chinensis

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