Isolation and Characterization of CPRgene Promoter from Artemisia annua by Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR)

摘要

Malaria, an infectious diseases, couses in almost 1 million deaths each year over the world. Artemisinin, a sesquiterpene lactone endoperoxide, is one of the most effective antimalarial drugs purified from Artemisia annua L. in China, 1970s. As the low content of this compound in plant many of studies have been focused on using elicitors affecting gene expression involved in artemisinin synthesis pathway. The main step to enhance artemisinin content in plant by using elicitors is characterization ofkey genes promoter. Cytochrome p450 Reductase (CPR) is one of the key enzymes that plays an important role in artemisinin synthesis pathway. Promoter sequence of key genes involving in artemisinin biosynthesis pathway included ADS, CYP71AV1 and DBR2 wasisolated except CPRgene. We used standard Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR) with some modification in thermal cycle numbers to isolate the unknown 5' flanking region of the CPRgene from Artemisia annua. Subsequent bioinformatics analysis to characterize functional cis-acting elements inside the promoter was performed. The 5' flanking sequence of CPR was cloned in pGEM-T Easy vector and sequenced. Subsequent sequence analysis for characterize functional motifs using bioinformatics software indicated a group of putative cis-acting elements such as TATA box, CAAT box, G box, W box and etc., inside the CPR promoter. This sequence was submitted in GenBank databases under the accession number KC243135. Present study demonstrated that characterization of cis-acting response elements can facilitate using elicitors to enhance artemisinin production in plant.