Cloning and expression analysis of β-carotene hydroxylase gene(chyb) from Dunaliella salina.

Zeaxanthin is a derivative of β-carotene found in nature and it is the most curcial portion of the macula and retina ,helping protect the macular region of the eye from harmful form of light can cause photoxidative damage to the eye. The biosynthesis of zeaxanthin is regulated by a series if enzymes, β-carotene hydroxylase(HYB) is the ratelimiting enzyme during the synthesis. To investigate the changes of Dschyb expression and content of zeaxanthin in Dunaliella salina under some stress, the full-length cDNA of chyb (GenBank accession No. JN118489) had been obtained from D.salina based on RACE technology, and four factors influencing Dschyb and zeaxanthin were analyzed. The cDNA was comprised of 1 433 bp containing a 969 bp open reading fragment, which encoded a polypeptide of 322 amio acids with a predicted molecular mass of 35.5 kDa and theoretical pI of 9.01. It had four conserved histidine residue motifs and was homologous with Volvox carteri f. nagariensis (64%) and Chlamydomonas reinhardtii(58%). Sequence analysis showed that D. salina CHYB had four transmembrane domains and chloroplast transit peptide, which further proved that β-carotene hydroxylase was located in the thylakoid membrane, no signal peptide was predicted in DsCHYB. Phylogenetic tree demostrated CHYB in D.salina and CHYBs from other green algae like Volvox carteri f. nagariensis and Chlamydomonas reinhardtii were grouped into one clade. Upon the observation of chyb regulation expression, the Dschyb expression level had little fluctuation when treated with intensive light at the treatment groups of 6 and 12 h, but with a significant rise from 24 h (P0.01), and at the peak of 48 h (P0.01). When darkedcultured cells were exposed to high light, sodium acetate and ferrous sulfate, the Dschyb transcription obviously increased within the initial 6 h (P0.01), but then declined after 12 h of the treatment. When D. sallina was exposed to glucose, the expression level of Dschyb reached a peak after 1.5 h. In contrast to the control group, there was no change observed in the group of 6 h. Most strikingly, glucose induction had not been observed after treatment with RNA polymerase inhibitor actinomycin D, indicating that actinomycin D might have a strong inhibitory effect on glucose. Dschyb mRNA was expressed significantly less than that in the control group (P0.01), especially in groups of 1.5 and 3 h. Zeaxanthin was identified according to their chromatograms on HPLC and UV-vis absorption spectra. The retention time of the standard was 26.742 min while the sample was 29.008 min. The content of zeaxanthin increased due to sodium acetate, ferrous sulfate, high light and glucose treatment. The content of zeaxanthin had increased 16% (P0.05), 28%(P0.05) and 53%(P0.01) compared with control group, respectively. These results support that high light and glucose play critical roles in Dschyb expression and provide a helpful message for production of zeaxanthin.