Molecular isolation of cDNA encoding N-methylstylopine hydroxylase from greater celandine (Chelidonium majus L.) and its expression enhancement in response to salinity abiotic elicitor
2018
Zahra Soleimani | Sedigheh Fabriki | Soodabeh Mafakheri
<strong> </strong> The greater celandine (<em>Chelidonium majus</em> L.) contains important alkaloids such as Sanguinarine. Sanguinarine is an active alkaloid with potentially antimicrobial, anti-inflammatory and anti-tumor properties that is widely found in plants of Papaveraceae family. In this research, the isolation and sequencing of (s)-cis-N-methyl stylopine 14-hydroxylas (MSH) coding gene was performed as one of the key enzymes in Sanguinarine pathway from celandine. Then, the changes in <em>cmMSH</em> gene expression were investigated in roots, leave and stems at four salinity levels (0, 25, 50, and 100 mM) in a factorial experiment based on completely randomized design. In the present study, the cDNA encoding MSH enzyme was isolated from root and successfully integrated into PTG19-T plasmid and cloned at E. coli. After confirmation of recombinant clones by PCR, plasmids of recombinant bacteria were extracted and the gene segment sequenced. The sequenced segment had 784 nucleotides with an open reading frame of 261 amino acids which the derived protein with functional domains including helix K region, heme-binding region and aromatic region belonged to the Cytochrome-P450 protein family. In the phylogeny tree, the protein sequence of isolated <em>cmMSH</em> gene had the most similarity with methylstylopine 14-hydroxylase enzyme of poppy species. Mean comparison of gene relative expression showed that 50 mM salinity had maximal effect on increasing of <em>cmMSH</em> gene expression in root tissue. Also, the expression pattern showed that with increment the salinity up to 50 mM, the expression of <em>cmMSH</em> gene increased in leaves and roots, but with increment the salinity to 100 mM the gene expression decreased 35% in both tissues.
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