Cloning And Investigating Of Key Enzyme Gene Promoters And Their Salt Regulation Molecular Mechanisms In The Carotenogenic Metabolism From Dunaliella Bardawil

Dunaliella bardawil, one member of the genus Dunaliella (Chlorophyceae, Volvocales), ischaracterized by its two unique features:(1) it can survive in a wide range of NaClconcentrations ranging from about0.05mol/L to saturation (5.5mol/L), making it one of themost salt-tolerant plants on earth;(2) under high salt stress, it can massively accumulatecarotenoids such as β-carotene. The biosynthesis of β-carotene is fulfilled by several keycarotenogenic enzymes (CRTs) including phytoene synthase (PSY),phytoene desaturase (PDS),ζ-carotene desaturase (ZDS) and lycopene β-cyclase (LYC-B), etc.This paper attempts to clone the promoters of key carotenogenic genes (crts) from D.bardawil, analyze and identify related salt-regulated elements to investigate the salt toleranceregulation mechanisms of carotenogenesis metabolism in this algae. Using LA-PCR basedgenome walking approach and semi-nested PCR method, the gene structures of Dbpsy, Dbpds,Dbzds and DblycB1genes were cloned and analyzed. The promoters of Dbpsy, Dbpds, Dbzdsand DblycB1are3763bp,3010bp,3648bp and2621bp in length, respectively. Analysis ofthe DblycB1gene organization found that its first intron contains a GT-rich region which ispotentially involved in inducible expression by salt stress. The5’terminus of Dbpds gene is notin accordance with its template, thus5’ RACE technique was used to determine itstranscriptional start site, and bioinformatics analysis hypothesized that Dbpds is one of theisoenzyme genes in D. bardawil. To mine potential related salt-regulated elements, onlinepromoter analysis tool PlantPAN was utilized. Both Dbpsy and DblycB1promoters were foundto share a salt-regulated element (SRE) candidate; Dbzds promoter contains two potentialhypoosmolarity-regulated elements such as hypoosmolarity-responsive elements (HRE) andGBF5transcription factor binding site (GBF5BS); while Dbpds promoter does not contain anypotential related salt-regulated elements.In vivo detection of the responsive transcriptional level of Dbpsy, Dbpds, Dbzds, DblycB1and DblycB2genes under different salt concentrations found that Dbpsy, DblycB1and DblycB2genes are to some extent induced under high salt concentration. Dbpsy is the most obviousamong these gene, extreme high salt stress, could greatly promote Dbpsy expression (about18.479times of control); however, when salt concentration decreases, their expression dropsobviously with the exception of Dbzds, which is induced to upregulate. These results indicatedthat DbPSY, DbLYC-B1and DbLYC-B2are key enzymes in response to high salt stress,DbZDS is key enzyme in response to low salt stress. Salt stress could not apparently affectDbpds expression, DbPDS is not a key enzyme in response to salt stress. Under extreme low salt stress (0mol/L), Dbpsy maintains a very low expression level (only about0.069times ofcontrol) in spite of the fact that other crts still reach a certain level, indicating that as a rate-limitingenzyme,DbPSYexertsuniqueeffectonthecontrolofcarbonflowintocarotenogenesismetabolism. Isoenzymes DbLYC-B1and DbLYC-B2are in response to different salt stresses,respectively.Extremehighsaltstresscaninducebothenzymestranscriptionalexpression;underlow salt stress, DbLYC-B1keeps a certain level of transcriptional expression, while DbLYC-B2drops to lower level. We hypothesized that the GT-rich region in the first intron of DblycB2gene might be involved in this low salt stress expression.Deletion of the HRE element from Dbzds promoter found that hypoosmolarity-inducedexpression was lost in ble-egfp chimeric gene. Deletion of GBF5BS element, ble-egfp chimericgene expression resembled to that of wild type Dbzds promoter or that of in vivo Dbzds gene,hypoosmolarity-induced expression was not affected. Simultaneous deletion of the HRE andGBF5BS elements resembled to that of deletion of the HRE element, the hypoosmolarity-induced expression of ble-egfp chimeric gene was lost. Therefore, HRE is indispensable to thehypoosmolarity-induced expression of Dbzds; neither GBF5BS is hypoosmolarity-responsive,nor it can act in synergism with HRE to enhance Dbzds expression under hypoosmolarity, it’snot an authentic hypoosmolarity-regulated elements. Likewise, deletion of sense SRE in theDblycB1promoter led to loss of induced expression of DblycB1promoter in response to highsalt stress. SRE confer induced expression of DblycB1in response to high salt stress. Thedifferentiation of salt stress induced expression of CRTs correlates with differential promoterregulatory elements.