Cloning And Functional Identification Of The Promoter Of Dunaliella Bardawil’s ζ-carotene Desaturase Gene

Dunaliella, a genus of unicellular and halophilic green bifagellate microalga without arigid cell wall structure, is the most halo-tolerant eukaryotic organism known so far, whichcan grow in50mM to5M NaCl solution, and has been reached as model organism aboutsalt-tolerant mechanism in plant and alga in recent years. As one the common Dulaliella, D.bardawil can massively accumulate two major stereoisomers: all-trans and9-cis β–carotene,when exposed to stress conditions such as high salt concentration, extreme temperatures ornitrate deficiency, and so on, in order to protect cells against photo-damage. In differentinduction conditions, the β–carotene content in the cells of Dunaliella can reach up to14%ofthe cell’s dry weight. The accumulation of β–carotene in D. bardawil is mainly regulated bydifferent enzymes in the carotenoid biosynthetic pathway; on the other way, these enzymesare also regulated by different genes in genome of D. bardawil. Many motifs exist in thepromoter of these genes and regulate expression amount of enzymes according to differentinvironment elements.ζ-carotene desaturase (ZDS) is one of the key enzymes in the carotenoid biosyntheticpathway. According to the full-length sequence of the ZDS cDNA in D. bardawil that ourlaboratory has obtained before, we isolated the coding region sequence of ZDS by theapproach of PCR. And then, we amplified flanking sequence of the coding region by themethod of genome walking, including promoter and terminator of ZDS. The length of thewhole cDNA is2209bp, including33bp5’-untranslated region (UTR),1749bp open readingframe (ORF) and411bp3’-UTR. In this experiment, the whole sequence of ZDS that we gotis11,896bp. it contains6435bp coding region (from ATG to TAA),4091bp upstream of thecoding region, and1370downstream of the coding region. Blastn the coding region and ORFof ZDS, we find that there are12exons and11introns in coding region. The total length ofthe introns is4086bp, about2.68fold of the total length of exons (1749bp). Each intron startswith GT and end with AG, belonging to the most common style of exon. According to the information of transcription start site (TSS) analyzed by bioinformaticstools, we constructed an expression cassete-“pZDS-EGFP-tZDS” by taking2899bpnucleotides upstream the coding region of ZDS as promoter,1217bp nucleotides downstreamthe coding redion as termitor, and enhanced green fluorescent protein (EGFP).Therecombinant plasmid PCR2.1containing the expression cassete is8741bp long andtransformed D. bardawil by Electroporation. At last, the EGFP was expressed successfully inthe cells of D. bardawil and we conclude that promter and the terminator is available.