Using EGFP as a reporter to confirm the function of phytoene desaturase promoter in Duanliella bardawil

Abstract

Phytoene desaturase (PDS) is an upstream key enzyme of carotenoid metabolic pathway in Dunaliella bardawil. This research attempts to clone the Pds gene and its promoter and terminator from D. bardawil and identify the promoter activity. Using LA-PCR based genomic walking approach and semi-nested PCR method, the DbPds gene was cloned and its structure was analyzed. 5′ RACE technique was used to determine its transcriptional start site. The bioinformatics analysis hypothesized that DbPds is one of the isoenzyme genes in D. bardawil. The full length of DbPds gene isolated included 8113bp coding region (from ATG to TAA), 3010bp upstream sequence and 1555 bp downstream sequence. By constructing the DbPds promoter-EGFP-DbPds terminator exogenous gene expression cassette to drive EGFP expression in D. bardawil, the activities of promoter and terminator of DbPds upstream and downstream sequence separated in this experiment were confirmed. Online promoter analysis tool PlantPAN was utilized to analyze the potential related osmotic stress-regulated elements in DbPds promoter. The result suggested that DbPds promoter does not contain any related elements about salt regulation. Furthermore, through in vivo detection of the responsive transcriptional level of DbPds genes under different salt concentrations, it was found that salt stress could not apparently affect DbPds expression, indicating that DbPds genes does not participate in the response to salt stress.