Transcription activation of ß-carotene biosynthetic genes at the initial stage of stresses as an indicator of the increased ß-carotene accumulation in isolated Dunaliella salina strain GY-H13.

ß-carotene is an efficient antioxidant and its accumulation is an oxidative response to stressors. Dunaliella salina strain GY-H13 is rich in ß-carotene under environmental stresses, which was selected as material to understand the molecular mechanism underlying ß-carotene biosynthesis. Seven full length cDNA sequences in ß-carotene biosynthesis pathway were cloned, including geranylgeranyl pyrophosphate synthase (GGPS), phytoene synthase (PSY), phytoene desaturase (PDS), 15-cis-zeta-carotene isomerase (ZISO), zeta-carotene desaturase (ZDS), prolycopene isomerase (CRTISO), lycopene beta-cyclase (LCYb). The seven protein sequences from the strain GY-H13 showed the highest similarity with other D. salina strains. Especially, PSY, PDS and LCYb protein sequences shared 100 % identity. Phylogenetic analysis indicated all proteins from GY-H13 firstly clustered with those from other D. salina strains with a bootstrap of 100 %. Multiple alignment indicated several distinct conserved motifs such as aspartate-rich domain (ARD), dinucleotide binding domain (DBD), and carotene binding domain (CBD). These motifs are located near ligand-binding pocket, which may be required for the activity of enzyme. Expression levels of these genes and ß-carotene content were measured over 24-h cycle, showing clear daily dynamics. All genes were dramatically up-regulated in the morning but the highest accumulation of ß-carotene was observed at noon, suggesting a lag-effect between gene transcription and biological response. Furthermore, the accumulation of ß-carotene increased under nitrogen deficiency, Cd exposure and high light and decreased under high salinity in a time-dependent manner. No gene of ß-carotene biosynthesis was up-regulated by high salinity while most genes were activated by the other stresses at the beginning stage of exposure. Growth inhibition and oxidative damage were also observed under high salinity. Overall, transcription activation of ß-carotene biosynthetic genes at the initial stage of stress exposure is a determinant of the increased accumulation of ß-carotene in microalgae, which help their survive under harsh environments. The newly isolated D. salina strain GY-H13 would be a promising microalgae model for investigating the molecular mechanism of stress-induced ß-carotene biosynthesis.