Isolation And Function Characterization Of Transcription Regulatory Sequence Of Genes Encoding Two Key Biosynthesis Enzymes Of Astaxanthin In Haematococcus Pluvialis

Astaxanthin with high antioxidant activity is of great application value. Haematococcus pluvialis has a rich accumulate astaxanthin content up to 4% dry weight, ranking the best producer of astaxantion. The highlighted mass production of astaxanthin using H. pluvialis remains problematic since the inhibition of cell division occurs while astaxanthin produced. It has been proved that biosynthesis of astaxanthin is regulated, at least partially at level transcription. This paper is intended to clone and character the cis regulatory sequences of genes of key biosynthesis enzyme. Based on cDNA sequences of (3-carotene ketolase(cr/ff) and p-carotene hydroxylase(c/-fZ), a 1.6 kb crtW genomic sequence with six introns, and a 3kb crtZ genomic sequence with five introns were clone, respectively. All the exon-intron junctions conform closely to GU-AG consensus splicing rule. Two crtW 5'-flanking upstream sequences (795bp and 695bp, respectively) and one crtZ 5'-flanking upstream sequence (302bp) are obtained using adaptor-primer PCR method. The transient expression of lacZ driven by crtW 306bp S'-flanking sequence and crtZ 302bp S'-flanking sequence shows that these two sequences habour transcription regulatory sequences.