| Transcription factors could identify and bind the cis-acting elements of promoter region specifically to activate or inhibit the transcript of genes. They play important roles in regulating varieties of physiological activities of organism in molecular level. At present, the transcription factors of terrestrial organism are well studied, but less in aquatic creatures, particularly in marine microorganisms. Aureochromes (AUREOs), the newly discovered transcription regulator from a xanthophycean alga Vaucheria, could be activated by blue light. All the AUREOs so far sequenced and/or isolated have always two functional domains, one bZIP domain and one LOV domain, at the same positions. The bZIP domain acts as transcription regulator, recognizing a consensus sequence of DNA, and the LOV domain acts as a photosensor, leading to a subsequent conformational change in the bZIP domain, which change its ability to bind to DNA under irradiation of blue light. Up to now, AUREOs have been found exclusively in stramenopile algaes, including Saccharina japonica (Phaeophyceae), Thalassiosira pseudonana (Bacillariophyceae), Ectocarpus siliculosu (Phaeophyceae), Ochromonas danica (Chrysophyceae), and so on, but their function are little known.Recently, we discovered another transcription factor AUREO from marine green microalgae Nannochloropsis gaditana, referring to as NgAUEROl. a full length cDNA of NgAUERO1 was isolated with rapid amplification of cDNA ends (RACE) method. It was 2086 bp in length, containing an open reading frame (ORF) of 1434 bp, a 5’-terminal untranslated region (UTR) of 162 bp, and a 3’-UTR of 490 bp. The ORF sequence was transformed into Saccharomyces cerevisiae INVScl by electroporation successfully. Positive transformants were cultured and viewed under optical microscopy continually. We discovered that the transgenic yeast cell size became bigger than wild yeast cell. The mean DNA and protein content of transgenic yeast were detected by flow cytometer. The result showed that the mean protein content increased from 3.75 to 6.28 and the mean DNA content increased from 16 to 18.3, which further indicated that the transgenic yeast cell volume has changed. The change of cell size is the direct result of cell-cycle regulatory network. To identify the potential regulation mechanism of NgAUREOl on yeast cell size, six cell-cycle-related genes (Pho85, Pho80, Pahl, Hsp12, Ies5 and Bck2), containing DNA binding sequence (TGACGT) of bZIP domain of NgAUREO1 in their 5’ promoter regions, were studied. The transcription of these genes in the transgenic and control yeast demonstrated that the transcript abundance of Pho85, Pho80, and Pahl were up-regulated, and Bck2 was repressed. We deduced that NgAUREO1 may affect the yeast cell size by regulating the cell cycle related genes.In addition, the fermentation characteristics of transgenic yeast were studied. The result revealed that transgenic yeast had higher ethanol fermentation ability than wild yeast, whose ethanol production increased from 9.68%to 11.59%(v/v) after fermenting 48h. The fermentation conditions and the components of culture medium for transgenic yeast were also optimized, the optimum fermentation conditions were 6% of inoculation proportion, initial pH of 6.0, speed of 220 r/min, fermentation temperature of 30℃ and fermentation time of 36h; the optimum carbon and nitrogen sources are glucose and the compound of peptone and yeast powder (quality ratio is 1:2), respectively.This study make us further understand the AUREOs functionality and lay the foundation for applying AUREOs in genetic modification to control gene expression in vivo. |
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