Cloning Of Chalcone Isomerase Gene From Dracaena Cambodiana And Funcitional Identification Of Its Promoter

Dragon's blood is a red resin mainly extracted from Dracaena plants,and has been widely used as a traditional medicine in East and Southeast Asia.The chemical constituents and pharmacological activities of dragon's blood have been extensively studied,but research on its biosynthetic mechanism remains unknown.Dragon's blood is the base of the plant produced by the red resin extracted.Dragon's blood of the base plant is the genus of Dracaena Vand.ex L.,Daemonorops Bl.,Euphorbiaceae Croton L.and Pterocarpus indicus Willd.D.cambodiana is is the genus of Dracaena Vand.ex L..In this study,we cloned a DcCHI and its promoter,which is one of the key genes on flavonoid biosynthetic pathway,aim to understanding the biosynthesis of flavonoids in D.cambodiana.The c DNA encoding a chalcone isomerase,designated as DcCHI was isolated from D.cambodiana.DcCHI was containing a 642 bp open reading frame encoding a putative protein of 213 amino acids,The molecular formula is C1081H1684N270O322S6,the relative molecular mass is 23.8 KD,the PI value is 4.97,the protein instability coefficient was 46.98,the fat coefficient was 91.60,and the hydrophilic coefficient was-0.053.Most Glu content of all the amino acids,Cys content at least.The deduced amino acid sequence shared over 70%amino acid homology compared with CHI from Narcissus tazetta var.chinensis and Ornithogalum longebracteatum.The phylogenetic tree analysis of DcCHI protein and other species showed that DcCHI belongs to Type ?.The prokaryotic expression vector of p ET-28-DcCHI was constructed,and then transferred into E.Coli.The recombinant protein was produced.In vitro DcCHI could catalyze naringenin chlorenone into naringenin by HPLC(High Performance Liquid Chromatography).This is the first time found that the Type ? isomerase chalcone have activity.The promoter region of DcCHI was isolated by the PCR-based DNA walking method.Several sequences similar to the eukaryotic cis regulatory elements were found in the promoter sequence of DcCHI.The promoter was positively regulated expression by Me JA and6-BA.Yeast one hybrid analysis indicated that Dcb HLH1 and Dcb HLH5 were able to specifically recognize and interacted with the promoter of DcCHI.