Cloning and characterization of trichome-specific promoter of cpr71av1 gene involved in artemisinin biosynthesis in Artemisia annua L.

Abstract

Artemisinin, a sesquiterpene lactone endoperoxide derived from Artemisia annua L.(Asteraceae), is the most effective antimalarial drug. We used two methods: genome walking and thermal asymmetric interlaced polymerase chain reaction, to isolate the unknown 5′-flanking sequence of the cyp71av1 gene. The subsequent sequence analysis using bio informatics software revealed that there are several cis-acting elements inside the cyp71av1 promoter. The 5′-rapid amplification of the cDNA ends method was used to determine the transcription start site of the cyp71av1 gene. We then mapped it at the 18 base upstream of the ATG initiation codon. For simple functional characterization, we built fusion vectors between the 5′-deletion promoter and the gus reporter gene. The expression levels of the transferred vectors into A. annua L. were analyzed by the transient expression way. The β-glucuronidase assay results indicated that deletion of the region to −1551 bp did not lead to much damage in the GUS activity, whereas further deletion, to −1155 bp, resulted in a 5.5-fold reduction of GUS activity. In stabilized transgenic A. annua L. seedlings we observed that GUS expression was restricted to trichomes, which means that the promoter of the cyp71av1 gene is trichome-specific. Compared with the constitutive CaMV 35S promoter, which can express genes throughout the plant, influence on the trichome system through the trichome-specific expression promoter merely imperils plant growth. In addition, the promoter of the cyp71av1 gene contains several binding sites for transcription factors, which implies that the cyp71av1 promoter responds to more than one form of stimulation.