Gene Cloning And Functional Identification Of Carotenoids Biosynthesis In Dunaliella And Mechanisms In Response To Light Stress

The eukaryotic unicellular green alga Dunaliella,which can grow in the Na Cl concentration of 0.5 ～ 5.5 M,is one of the most salt-tolerant eukaryotes found in the world.Dunaliella can produce large amounts of β-carotene under various environmental stresses.Due to these two features,Dunaliella has been considered as an excellent model organism to investigate the molecular mechanisms of low light stress responses and regulation of carotenoid metabolism.This thesis mainly focuses on genes involved in carotenoid biosynthesis metabolic pathways from Dunaliella and regulation mechanisms of carotenoid accumulation under low light stress at molecular biology level.It is of great significance for the production of β-carotene and the improvement of carotenoid content by utilizing Dunaliella cells.Dunaliella salina is the most salt-tolerant eukaryote and has the highest β-carotene content,but its carotenoid synthesis pathway is still unclear,especially the synthesis of lycopene,the upstream product of β-carotene.In this study,DsGGPS,DsPSY,DsPDS,DsZISO,DsZDS,DsCRTISO,and DsLYCB genes were cloned from D.salina and expressed in E.coli.A series of carotenoid engineering E.coli from phytoene to β-carotene were obtained.ZISO was first identified from chlorophyta,while CRTISO was first isolated from algae.It was found that DsZISO and DsCRTISO were essential for isomerization of carotenoids in photosynthetic organisms and could not be replaced by photoisomerization unlike some plants.DsZDS was found to have weak beta cyclization ability,and DsLYCB was able to catalyze 7,7’,9,9’-tetracis-lycopene to generate 7,7’,9,9’-tetra-cis-β-carotene,which had not been reported before.A new carotenoid 7,7’,9,9’-tetra-cis-β-carotene,the beta cyclization product of prolycopene,was discovered.Compared with bacterial-derived carotenoid synthesis pathway,there is a higher specificity and more efficiency of the carotenoid synthesis pathway in algae.This research experimentally confirmed that the synthesis of D.salina from phytoene to lycopene was similar to that of plants and different from bacteria,and provided a new possibility for the metabolic engineering of β-carotene.Then,In this study,it was proved that D.salina has the ketolase(DsBKT)of catalyzing the synthesis of astaxanthin,the downstream products of β-carotene.Therefore,the reason why D.salina does not synthesize astaxanthin is the purpose of this research.The enzymatic activity of DsBKT was detected by functional complementation assays in Escherichia coli,results showed that DsBKT had efficient ketolase activity towards β-carotene and zeaxanthin to produce astaxanthin,indicating that there were complete astaxanthin-producing genes in Dunaliella.Unlike the induced expression of Lycopene cyclase(catalyzing β-carotene synthesis)under salt stress,the expression of DsBKT was very low under both normal and stress conditions,which may be the main reason why D.salina cannot accumulate astaxanthin.On the contrary,with the astaxanthin-rich Haematococcus pluvialis as a control,its BKT gene was significantly up-regulated under salt stress.Further study showed that DsBKT promoter had strong promoter ability and could stably drive the expression of ble-egfp in D.salina.Obviously,DsBKT promoter is not the reason for DsBKT not being expressed which may be caused by Noncoding RNA.Finally,This research aimed to explore the response mechanism of carotenoid synthesis in Dunaliella bardawil to continued low light stress by analyzing carotenoid composition and its correlation with the carotenogenic genes(CRTs)differential expression,the promoter regulatory elements and transcription factors.Low light cis-elements,constructed ble-egfpexpressed D.bardawil strains driven by mutated DbZDS promoters with deletion of light regulatory elements were screened and identified.The relationship between DbZDS promoter and photosensitive transcription factors DbHY5,DbCO,and DbNF-YB was determined by yeast one-hybrid experiment to elucidate the mechanism of carotenoid accumulation in D.bardawil under low light stress.Lutein content reached the highest at 3000 lux and decreased by half at 500 lux,while β-carotene content reached the maximum 4.0 mg/g at 6000 lux and decreased to 0.27 mg/g at 500 lux.It was found that phytoene desaturase gene(DbPDS)and ζ-carotene desaturase gene(DbZDS)from D.bardawil were significantly up-regulated under low light conditions(≤1000 lux).Compared with other CRTs promoters,several light-responsive elements in DbZDS promoter,including ACE,ASF1,CCACA-box,and CCAAT-box were connected to low light regulation by gene mutation experiments.In addition,CONSTANS(DbCO)and nuclear transcription factor YB(DbNF-YB)from D.bardawil had a weak interaction and could bind to DbZDS gene promoter,whereas there was no interaction between DbZDS promoter and DbHY5.Consistent with DbZDS,DbCO gene was also highly expressed under low light conditions.These results show that DbCO and DbNF-YB form a complex that governs carotenoid accumulation responses by regulating DbZDS promoter in D.bardawil.