Cloning Of Chalcone Synthase Gene From Dracaena Cambodiana And Functional Identification Of Its Promoter

Dragon’s blood is a red resin that forms in the stem of Dracaenas plants after they are wounded, which is widely used in traditional medicines. At present, the researches on dragon’s blood are mainly focused on the chemical composition and pharmacological activity. Currently, the molecular mechanism of dragon’s blood formation is still unknown. Flavonoids are the main compounds in dragon’s blood. Thus, understanding the biosynthesis and regulation of flavonoids in Dracaenas spp. is critically important in determining the mechanism of dragon’s blood formation. Chalcone synthase is the first key enzyme in biosynthesis of flavonoids. In this work, the cDNA (designated as DcCHS) encoding chalcone synthase was cloned from D. cambodiana. The expression profiles of DcCHS and the function of its promoter in D. cambodiana were also investigated. The present study perhaps contributes towards an understanding of the biosynthesis of flavonoids in D. cambodiana.DcCHS was isolated from D. cambodiana using rapid amplification of cDNA ends. DcCHS contains a1173bp open reading frame that encodes390amino acids. The deduced DcCHS protein was predicted to possess the conserved CHS_like domain, active sites and signal sequences. The amino acid sequence of DcCHS was aligned with those of the other plant chalcone synthase proteins that exhibit high sequence identities. There was a104bp intron in DcCHS. Prediction of intron functional elements showed that the intron contains promoter core sequence TATA-box and enhancer elements CAAT-box, as well as a bit of cis-acting elements which may regulate the transcription and stress respons of DcCHS.DcCHS expressed at different levels with the highest transcription in the flowers, and the lowest transcription occurring in stems. DcCHS transcription was up-regulated by6-BA and jasmonic acid treatment. The prokaryotic expression vector of Pet-28a-DcCHS has been successfully constructed and a45kD recombinant protein was expressed in E. coli.The promoter region of DcCHS was isolated by the PCR-based DNA walking method. TATA box and other core configurations were found in promoter. Several sequences similar to the eukaryotic cis regulatory elements were found in the promoter sequence of DcCHS. A promoter deletion analysis revealed that the DcCHS promoter drives specific expression of the GUS gene. The promoter was positively regulated expression by MeJA and6-BA.