Analysis Of Transcription Regulatory Sequence Of Genes Encoding 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 algaHaematococcus pluvialis accumulates the astaxanthin, with levels reaching up to 4%dry weight under environmental stress. The highlighted mass production ofastaxanthin using H. pluvialis remains problematic since the inhibition of cell divisionoccurs while astaxanthin is produced.Isopentenyl pyrophosphate (IPP) isomerase,β-carotene ketolase (BKT) andβ-carotene oxygenase (CRTO), with control being exhibited at the transcription level,play an important role in astaxanthin biosynthesis of H. pluvialis. This paper isintended to clone and character the cis regulatory sequences of genes of keybiosynthesis enzyme.Using adaptor-primer PCR method, we demonstrate the presence of two differentipi 5'-flanking regions (1.8kb and 2.5kb) carrying similar regulatory elements withsome of the known stress responsive genes in plants, suggesting that there might be analternative promoter for IPP isomerase to regulate ipi transcription in H. pluvialis.In addition, two crtO 5'-flanking upstream sequences (1kb and 2kb) are obtainedusing adaptor-primer PCR method. The transient expression of lacZ driven by crtO320bp 5'-flanking sequence shows that this sequence contains transcription regulatorysequences.Moreover, Electrophoresis mobility shift assay (EMSA) was used in H. pluvialis,to identify transcription factor binding sites within a 309 bp promoter region(-617/-309) of beta-carotene ketolase gene(bkt) and a 59bp sequence between-396 and-338bp was found to have a specific binding activity to the nuclear protein. Sequenceanalysis revealed that this functionally important region contains neither TATA norCAAT box but a G-box involved in the responsiveness of light, anaerobiosis,p-coumaric acid and hormone.According to cDNA sequences of ipi and crtO, a 2.3 kb ipi genomic sequenceand a 2.1kb crtO genomic sequence with six exons and five introns were cloned.Nearly all the exon-intron junctions conform closely to GU/AG consensus splicingrule. A maximum likelihood approach was employed to detect evidence of positiveselection in the evolution of ipi and crtO. Phylogenetic analysis was done on theprotein sequence of ipi and crtO.Based on above studies, further experiments are very necessary to explore theinteractions between these cis-acting elements and their relevant trans-acting factors.