Characterization and Expression Analyses of Chalcone Synthase (CHS) and Anthocyanidin Synthase (ANS) Genes in Clivia miniata

Abstract

Chalcone synthase (CHS) and Anthocyanidin synthase (ANS) are amongst the key enzymes responsible for the production of anthocyanins in plants. They are generally encoded by multi-gene families with some members of these families contributing to colour pigmentation. The study examined CHS and ANS genes in Clivia miniata; whose flowering tissues undergo colour changes. The RNA to cDNA was isolated from floral tissues and three unigene(s) (CmiCHS 11996, CmiCHS 43839 and CmiANS) of short sequence lengths were initially obtained using next-generation sequencing technique. Gene-specific primers were designed from the unpublished initial short sequences of CHS and ANS. The length of the amplified cDNA after PCR were CmiCHS 11996 (933 bp), CmiCHS 43839 (951 bp) and CmiANS (983 bp). The start translated ORF frame corresponding to the predicted protein of 390 amino acid deduced protein (AEN04070) for CHS genes, and predicted 355 amino acid in respect to ANS gene (AGD99672). In silico analysis revealed the calculated molecular weight and theoretical isoelectric point (pI) CmiCHS 11996 and CmiCHS43839 were 31.0 kDa - 6.95 and 34.6 kDa - 7.54 respectively. The important motifs of the product binding site and active site were successfully identified from the deduced amino acid sequences. Multiple sequence alignment showed that the CmiCHS and CmiANS sequences were highly conserved and shared high sequence identity (>83%) with chalcone synthases from other plants. However, another assay was performed to determine the expression profiles of these genes in different tissues as well as the tepals (orange and yellow flower) using the real-time quantitative PCR. The expression levels of CmiCHS and CmiANS were higher in tepals compared to other tissues (leaves, style and stigma and scape). These expression patterns of the genes in the tissues corresponded to the accumulation of anthocyanin, suggesting that the orange and yellow colour pigments was certainly related to the regulation of chalcone synthase and anthocyanin synthase.