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

Author:
He, Yang, Hou, Pei, Fan, Gang, Li, Dongmei, Peng, Cheng
Source:
Electronic Journal of Biotechnology 2015 v.18 pp. 376-380
ISSN:
0717-3458
Subject:
Coptis chinensis, Escherichia coli, active ingredients, amino acids, antibodies, berberine, biochemical pathways, biosynthesis, biotechnology, complementary DNA, gene expression, genes, high performance liquid chromatography, isoelectric point, molecular weight, open reading frames, petioles, polypeptides, quantitative polymerase chain reaction, reverse transcriptase polymerase chain reaction, rhizomes, tissues
Abstract:
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.Bioinformatics analysis showed that the cDNA is 1942bp in length with a complete open reading frame (ORF) of 1476bp, and the ORF encodes a polypeptide of 491 amino acids with a calculated molecular mass of 55.29kDa 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.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.
Agid:
5526758