Isolation, characterization and expression analysis of 3-hydroxy-N-methylcoclaurine 4′–methyltransferase (4'OMT2) gene in Papaver somniferum L.

Poppy (Papaver somniferum L.) is one of the oldest medicinal plant in the world. It produces several important narcotic benzylisoquinoline alkaloids. In this study, 4’OMT2 (3′-hydroxy-Nmethylcoclaurine 4′-O-methyltransferase) gene was isolated, sequenced and characterized. After amplification of genomic and coding sequence of 4’OMT2 gene using PCR, the fragments were cloned into pTZ57R/T plasmid and sequenced. Sequencing results showed two fragments of 1074 and 1189bp for coding and genomic sequences, respectively. For the first time, results showed one intron of 115bp in the genomic sequence of 4’OMT2 in the species. Isolated gene from Iranian genotypes was similar to Papaver genus with high identity, and had 67% identity to Ranunculales plants. Further bioinformatics analyses revealed, 4’OMT2 gene produces a stable enzyme without signal peptide that localize in the cytoplasm. For gene expression analysis, five poppy RNA-seq (mRNA type) libraries (belonging to flower bud, leaf, developing fruit, stem and root tissues) were retrieved from SRA database and analysis of 4’OMT2 gene expression were done using IDEG6 software and statistical tests. Results showed that 4’OMT2 has differential expression among different tissues and has maximum expression in stem tissues. Identification and characterization of biosynthetic genes is the first step in genetic manipulation and metabolic engineering. In present study, for the first time, full length coding sequence of 4’OMT2 gene was isolated from poppy plant and presence of one intron in its sequence was determined.