Identification and cloning of chalcone synthase gene family in milk thistle (Silybum marianum L.) plant

Silymarin is a flavonoid compound derived from milk thistle plant (Silybum marianum) seeds comprising several pharmacological applications. chalcone synthase (CHS) is a key enzyme in the biosynthesis of flavonoids, thereby identification of CHS gene in milk thistle plant can be of great importance. Seeking that, the available sequences of CHS genes from different plants collected, aligned and the conserved regions detected. Then, 4 degenerate primers designed based on the CHS consensus sequences. The fragments of CHS genes from Borazjan's native genotype and Majar's modified genotype were amplified by polymerase chain reaction and then, cloned and sequenced. Analysis of the resultant nucleotide and deduced amino acid sequences lead to identification of two different members of CHS gene family from Silybum marianum. 6 specific primers were designed based on the diverged regions of each member for amplification of related cDNA from majar's total RNA in the RACE system. Amplified fragment of cDNA in 3'RACE and 5'RACE were cloned and sequenced. Full length cDNA was identified by overlapping the 3'RACE and 5'RACE sequences of member 1 whose open reading frame contains 1239 bp including exon 1 (190 bp) and exon 2 (1049 bp) encoding 63 and 349 amino acid residues, respectively. Altogether, analysis of the resulting nucleotide and deduced amino acid sequences lead to identification of three different members of chalcone synthase from Silybum marianum containing the conserved chalcone synthase C-terminal and N-terminal domains.