Isolation and Sequencing of Chalcone synthase (CHS) in Calotropis procera

Aim: Chalcone synthase (CHS) is a key enzyme in the flavonoid biosynthesis pathway. This enzyme catalyses the first step of flavonoid biosynthesis pathway. The purpose of this study was to identify chalcone synthase gene in Calotropis procera.
Material and The genomic DNA was extracted from fresh leaves and used as template in PCR. Primers were designed according to the conserved regions of this gene in other plants. The PCR product was purified and sequenced. Multiple sequence alignment and sequence homology analyses were carried out with DNAMAN software using CHS sequences retrieved from Genbank. A neighbor-joining phylogenetic tree based on CpCHS and other CHS sequences were constructed using MEGA6 software.
PCR results indicated the existence of this gene and amplification of 570 nucleotides fragment that belong to exon2 of CHS gene. This gene was denominated as CpCHS and deposited at the GenBank accession KM878672. Comparison analysis based on nucleotide sequence alignment revealed that CpCHS shared a high degree of similarity to CHS from Arabidopsis thaliana (59%), Nicotiana tabacum (63%), Olea europaea (63%) and Petunia X hybrida (64%). Maximum similarity was observed between CpCHS and CHS from Catharanthus roseus (67%). Phylogenetic analysis showed that CpCHS was grouped into a branch with Catharanthus roseus.
These results suggest that CpCHS may play a similar role as other CHS in regulation of flavonoids biosynthesis pathway. Identification of this gene is an effective step which may lead to increase in the accumulation of useful medicinal extract in this plant.