Identification, Cloning and Structural Analysis of Major Genes from Portulaca oleracea L. Hairy Roots that Involved in the Biosynthesis of Dopamine

Abstract

Dopamine is one of the important medications of Portulaca oleracea L. To optimize the production of dopamine, one of the methods is the identification and engineering of metabolite pathways. To investigate the tyrosine decarboxylase (TDC) and tyrosinase, which seem to be the most important genes in dopamine synthesis pathway, hairy roots were produced from Portulaca oleracea using Agrobacterium rhizogenes and total RNA was extracted from hairy roots. A cDNA library was synthesized using RT-PCR. Then, the twogenes were amplified, isolated and cloned in a pTG 19-T vector. Bioinformatics' databases were used to predict the details of the structural, functional and biological characteristic of these genes. Nucleotide sequence analysis revealed that the cloned cDNAs expressed TDC and tyrosinase, and contained a single open reading frame of 1800 bp and 1750 bp, respectively. TDC has the most similarity with TDC of Arabidopsis thaliana (L.) Heynh.,but tyrosinase has 98% similarity withtyrosinase of Agaricus bisporus. Because of More negatively charged amino acids the TDC has hydrophobic properties, therefore affinity and hydrophilic chromatography can be used for purification of TDC. But tyrosinase has hydrophilic properties and hydrophobicity chromatography can be used for its purification. There were two peroxisomal signal peptide (KLAKEFEQL) and (KIEGRPLHL) in the TDC and tyrosinase, respectively. Therefore, they are biologically active in the peroxisomes, and included in biosynthesis dopamine through the transformation of L-lysine to L-dopa and finally to the dopamine. In conclusion, increasing the expression of TDC and tyrosinase through the genetic engineering can increase dopamine production in the Portolaca.