Cloning Of Psy Flanking Regulatory Sequences And Analysis Of Their Environmental Stress Factor Regulatory Elements In Dunaliella Bardawil

Dunaliella bardawil is a halophilic unicelluar eukaryote with nude protoplast; it can accumulate massβ-carotene under stress, and has been utilized in commercial production and researches on carotenoid metabolic pathway and transgenic host. It can be expected that D. bardawil will be a perfect host for transgenic study due to its properties of simple culture conditions and effective genetic manipulation. At present, only a few genes have been cloned and studied with respect to regulatory mechanisms ofβ-carotene metabolism. Proper promoter and terminator are the key elements to determine and control the expression level of foreign gene in transgenic Dunaliella. Up to now, in Dunaliella some exogenous constitutive promoters and terminators have been characterized with reporter gene gus and gfp in transient expression pattern. Nevertheless, exogenous regulatory sequences, such as promoter and terminator, suffer from low expression efficiency and most even show transient expression. Consequently, the isolation of endogenic promoter and terminator to drive stable and effective expression of exogenous genes in Dunaliella is of great investigation meaning and practical value.Previous researches showed that phytoene synthase (PSY) from D. bardawil is the first key regulatory enzyme in carotenogenesis. This paper first obtained and validated the cDNA sequence of D. bardawil psy (Dbpsy) form total RNA. Using LA-PCR based genome walking method, we successfully obtained the psy promoter and terminator, respectively; and with bioinformatics tools, we analyzed and predicted the genomic sequence and protein structure of Dbpsy, correspondingly. The computed results showed that Dbpsy promoter possesses two regulatory elements which are induced by UV and salt, respectively, besides canonical TATA box, CCAAT box and GATA box, even BOXLCOREDCPAL,GT1CONSENSUS and GT1GMSCAM4. BOXLCOREDCPAL participates in up-regulation responses of DcPAL1 to UV-B irradiation mediated by DcMYB1, therefore, up-regulates expression of downstream gene; GT1CONSENSUS plays an intermediary role on interaction between TFIIA and GT-1-like factors to accumulateβ-carotene induced by light. GT1GMSCAM4 can up-regulate expression of downstream gene induced by light.Protein sequence analysis indicated a diverse N terminal along with a relative conserved C terminal in comparison with other species. We speculated the N terminal differentiation may underline the fine tune diversity of PSY between species.