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 rhizomewas 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 andwas
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.
