Cloning and characterization of full-length cDNA clones encoding chalcone synthase from the orchid Bromheadia finlaysoniana

Abstract

cDNA clones encoding chalcone synthase (CHS) (EC 2.3.1.74), a key enzyme involved in flavonoid and anthocyanin biosynthesis were isolated from a cDNA library constructed from flowers of the orchid, Bromheadia finlaysoniana (Lindl.) Rchb.f using a homologous probe generated via PCR. The complete nucleotide sequences of the 3 clones, OCHS3, OCHS4 and OCHS8 were determined. The lengths of OCHS3, OCHS4 and OCHS8 are 1 445, 1 382 and 1 439 bp, respectively. All the cDNAs contained a single open reading frame of 1 185 bp, encoding a polypeptide of 394 amino acids with a calculated molecular mass of 42.9 kDa. The nucleotide sequences of OCHS3, OCHS4 and OCHS8 showed that they were not identical but exhibited a high degree of similarity (> 97%). The deduced amino acid sequence showed 76-82% homology, whereas the nucleotide sequence showed 59-68% homology to CHS of other plants. Southern blot analysis indicated that CHS is possibly encoded by a small multigene family in the genome of B. finlaysoniana. Northern blot analysis revealed that CHS was expressed at very high levels in young leaves which are flushed with anthocyanin, whereas it was expressed at much lower levels in flowers which are faintly coloured and completely undetectable in other organs. Within different floral organs, CHS was found to be expressed at the highest levels in sepals, followed by stalks and columns whereas it was hardly detectable in lips and petalS. However, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, a significantly more sensitive method compared to northern hybridization, demonstrated that CHS transcripts could be amplified from all parts of the plant and all floral organs examined.