CHARACTERIZATION OF THE FULL-LENGTH GENOMIC SEQUENCE OF THREE PUTATIVE CHS GENES IN OLEA EUROPAEA

Chalcone synthase (CHS) is a type III polyketide synthase (PKS) that represents the first committed enzyme of the flavonoid metabolic pathway. CHS catalyzes the condensation of p- coumaroyl and three malonyl-CoA molecules to form the naringenin chalcone. The CHS genetic structure is strictly conserved and consists in two exons separated by one intron, with the exception of the Antirrhinum majus gene showing the presence of three exons and two introns (Sommer and Saedler, 1986). Two partial putative sequences of CHS paralogues have been previously identified in Olea europaea in our laboratory (Scialpi et al., 2005). These sequences, named CHSA and CHSB respectively 2002 and 1466 bp long, included the 5’ upstream sequence, the exon 1, the intron 1 and part of the exon 2. In this work, CHSA and CHSB complete nucleotide sequences were isolated using a degenerate primer strategy. Moreover the PCR approach allowed the identification of a third putative paralogue of the gene named CHSC The computational analysis of the PCR products showed that all the CHS putative paralogues showed the same gene structure with three exons and two introns, the second intron showing different length in CHSA and CHSB but the same conserved position at Leu290. In contrast, the CHSC intron 2 showed the same position at Gly163 of the intron 2 of Antirrhinum majus. FastA (EMB-EBI) similarity search showed high identity among O. europaea CHSA, B and C and other orthologous CHS genes. In particular, the in silico translation of the O. europaea genes evidenced high identity with different orthologous CHS proteins and the presence of conserved amino acids important for the geometry of the active site (Cys164, Phe215, His303, and Asn336), for coumaroyl-binding protein (Ser133, Glu192, Thr194, Thr197, and Ser338) and for the cyclization pocket (Thr132, Met137, Phe215, Ile254, Gly256, Phe265, and Pro375) (Yasuyo et al., 2005). Finally, the NJ phylogenetic tree based on different CHS proteins placed all O. europaea putative CHS in the same cluster of other Lamiales. These results suggested that the three putative CHS genes could be three paralogues of the O. europaea CHS gene family. These results also showed that in O. europaea as in Antirrhinum majus, there is an important structural exception to the conserved CHS gene structure.