Identification, isolation and cloning of cytochrome p450 from Thymus vulgaris

Thymol is one of the most widely used secondary metabolites in Thymus vulgaris, as a drug. One of the key genes which is involved in its synthesis is the cytochrome p450 gene. In order to isolate and cloning p450 gene, the extraction of RNA was carried out subsequently the cDNA synthesized. Amplification of cDNA was carried by using designed primers from conserve region in the presence of the HF enzyme that has the property of 3′ to 5′ exonuclease. In first stage cloning was done by T/A method in plasmid pTG19. Selection of recombinant colony was performed by PCR system and digestion with BamHI. The cloned fragments were sequenced for the final confirmation. In order to cloning in the plant expression vector pBI121GUS-9 , the fragments that were cut with the BamHI enzyme were cloned in homologous site of the pBI121GUS-9 vector under the promoter 35s and the Nos terminator. Confirmation of recombinant colonies was performed with colony PCR and digestion. This designed recombinant vector can be used for further research and over- expression of thymol.