Cloning Of Lycopene β-cyclase Gene And Activity Analysis Of Its Promoter From Dunaliella Bardawil

Dunaliella bardawil, a common unicellular green algae, so far it is the most haloduriceukaryotes in the world, it can grow in the extrme conditions such as the high saltconcentration, high light and high temperature and highly accumulate abundant of β-carotene.Lycopene β-cyclase (LycB) is one of the key enzymes for the synthesis of β-carotene, it cancatalysis lycopene to form β-carotene. The reason that the algae and plants highly accumulateβ-cyclase is directly related to the catalytic mechanisms of LycB and the molecular regulationof its gene. In this paper, a full-length DNA sequences and its upstream and downstreamsequence were obtained and analyzed from Dunaliella bardawil, respectively. Enhancedgreen fluorescent protein (EGFP) and Zeocin resistance gene (ble) was used as reporter gene,observed under the fluorescence microscope and through resistance screen, to detect thepromoter activities of LycB gene in the transgenic alga Dunaliella bardawil.Firstly, Dunaliella bardawil, as good material in our study, based on the full-lengthsequence of LycB cDNA in D. bardawil that our laboratory had coloned before, according tothe analysis of conserved regions of LycB gene from other species in database,5sets ofprimers was designed, we isolated the genomic sequence of LycB that was obtained by theapproach of PCR with genome walking techniques and analysed by us. Sequence resultsshowed that the nearly full-length of LycB genomic sequence was obtained, and it is5863bp,which included11exons and10introns.Secondly, two sets of primers was designed according to the LycB genomic sequence, andthe promoter sequence and terminator sequence of LycB was obtained using genome walkingtechniques. Then, the obtained promoter sequence was analysed by Plantpan software, and wefound that several promoter motifs such as GATABOX, SORLIP1A, SORLIP2AT whichwere related to light-induced, and GT1GMSCAM4that is related to salt-induced and so on.Thirdly, Promoter and terminator fragments of LycB were amplified and ligated to thereporter gene enhanced green fluorescent protein (EGFP) and Zeocin resistance gene (ble) toconstruct the expression vector. Then, the vector was introduced into Dunaliella bardawil todetect the promoter activities through the expression of EGFP and ble gene. We observed that green fluorescence appeared in the transgenic algae after48h and lasted to the sixth day underthe fluorescence microscope, at the same time, we observed that transgenic algae grow on thealgae agar medium contained the zecion. So, the research proved that that LycB promoterregion of Dunaliella bardawil had the activity.Forth，predicted salt-induced regulatory element was deleted by SOE PCR, the effects ofsalinity changes on exogenous gene expression were detected by real-time quantitative PCR.It initially illustrated that this predicted salt-induced regulatory element may have theregulatorty functions by salt.Cloning of promoter of LycB gene and analysis results of salt-induced and light-inducedelements, is helpful to us to further research the metabolic regulated mechanism of β-carotenesynthesis in Dunaliella bardawil. And it is of great significance to reveal the molecularmechanism of β-carotene accumulation in Dunaliella bardawil.