Characterization Of Cis-acting Elements Of Genes Encoding Two Key Enzymes In Astaxanthin Biosynthesis In Haematococcus Pluvialis

Astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione), a type of red ketocarotenoid, is of great commercial interest because of its high price. The unicellular green alga Haematococcus pluvialis accumulates the astaxanthin, with levels reaching up to 4% dry weight under environmental stress. β-carotene ketolase (BKT) and β-carotene hydroxylase (CRTR-B), with control being exhibited at the transcription level, play an important role in astaxanthin biosynthesis of H. pluvialis. Recently, transient expression of lacZ driven by bkt 306bp 5'-flanking sequence and crtR-B 302bp 5'-flanking sequence showed that these two sequences harbor transcription regulatory sequences. Based on studies above, this paper is continually intended to clone and character the complete 5'-flanking regions of two key genes in astaxanthin biosynthesis. Based on previous studies, we demonstrate the presence of a 900bp 5'-flanking region of crtR-B including a promoter-like region (-378/-22) downstream from the ATG start codon. Results of the site-directed mutagenesis of a C-repeat/DRE, an ABRE and a MBS motifs in a promoter-like region (-378/-22) suggested that ABRE motif might have an influence on the crtR-B transcription. Results of 5'-deletions constructs and transient β-galactosidase expression assays demonstrate that there may be negative regulatory elements governing expression in the shorter promoter at -919/-378. Based on previous studies, we demonstrate the presence of two separate 5'-flanking regions (1.5kb and 2kb) of bkt (bkt1 and bkt2), which possess regulatory elements similar to known stress responsive genes in plants. Moreover, results of the site-directed mutagenesis of a C-repeat/DRE and two APE-like motifs in a promoter-like region (–630/–308) suggested that two APE-like motifs might be essential for transcriptional regulation of the bkt gene. Furthermore, with the suggestion that there may be at least two kinds of regulatory patterns controlling bkt astaxanthin biosynthesis in H. pluvialis, results of 5'-deletions constructs and transient β-galactosidase expression assays demonstrate possible positive elements and negative elements governing expression of bkt. Furthermore, our present studies reveal that the first intron (+371/+497) downstream from the 1.5kb 5'-UTR of bkt1 may function as a negative regulatory element to regulate its own promoter. Based on above studies, further experiments are very necessary to explore the interactions between these cis-acting elements and their relevant trans-acting factors.