Characterization Of β-carotene Hydroxylase Gene From Dunaliella Salina And Its Transformation In Anabaena Sp.PCC7120

Zeaxanthin is a derivative of β-carotene found in nature. Scientists discovered that zeaxanthinis the most curcial portion of the macula and retina, helping protect the macular region of the eyefrom harmful form of light can cause photoxidative damage to the eye. Particularly the new-uncovered zeaxanthin-meso-zeaxanthin, it requires to maintain its structural density in macularregion to preserve age-related macular degeneration (ARMD). Meanwhile，zeaxanthin acts as anantioxidant which protects cell against the damaging effects of free redicals. Zeaxanthin is foundnaturally in the body, but is not made in the body and must be obtained from food or supplements.Zeaxanthin is rare in the nature and beyond the need of market.The biosynthesis of zeaxanthin isregulated by a series of enzymes, β-carotene hydroxylase (CHYB) is the rate-limiting enzymeduring the synthesis. It is a tendency to explore the zeaxanthin biosynthesis by transcriptionalregulation of β-carotene hydroxylase gene and gene engineering at the molecular level.The full-length cDNA of β-carotene hydroxylase (chyb) has been obtained from Dunaliellasalina based on RACE technology. The cDNA contains a969bp open reading frame (ORF)encoding322amino acids. It had four conserved histidine residue motifs and was homologouswith Volvox carteri f. Nagariensis (64%) and Chlamydomonas reinhardtii (58%). Sequencesanalysis showed that Dunaliella salina CHYB had four transmembrane domains and chloroplasttransit peptide, which further proved that β-carotene hydroxylase was located in the thylakoidmembrane. Phylogenetic tree demostrated that CHYB in Dunaliella salina and CHYBs from othergreen algae like Volvox carteri f. nagariensis and Chlamydomonas reinhardtii were grouped intoone clade. Upon the observation of chyb regulation expression, the expression of of Dschybincreased significantly after24h under high light treatment (P<0.01), and reached the peak of48htreatment (P<0.01). When darked-cultured cells were exposed to high light, sodium acetate andferrous sulfate, the Dschyb transcription obviously increased within the initial6h (P<0.01), butthen declined after12h of the treatment. HPLC results indicated that the content of zeaxanthin hadincreased by these treatment, especially under glucose treatment, the accumulation of zeaxanthinwas up53%compared to the control (P<0.01).The β-carotene hydroxylase gene (chyb) was inserted into the cargo plasmid pRL25C andtransferred into Anabaena sp. PCC7120by triparental mating. Through the selection of kanamycin, stable transgenic Anabaena sp. PCC7120was obtained. DNA molecule detection has intially beenconfirmed that chyb was integrated into the Anabaena sp. PCC7120. The zeaxanthin content oftrangenic and wild Anabaena sp. PCC7120were carried out by HPLC, the analysis indicated thatthe zeaxanthin of the transgenic had a59%increase from the wild. Under the highlight, thezeaxanthin of trangenic was approximate three times as much as the wild (P<0.01), and nearly fourtimes as much as the wild under moderate light (P<0.01). This provided a helpful message forproduction of zeaxanthin and established a good foundation to construction and widespread use ofmicroalgae bioreactor.