Functional Identification Of Genes Involved In Carotenoids Biosynthesis And Degradation In Dunaliella Bardawil And Mechanisms In Response To Salt Stress

The eukaryotic unicellular green alga Dunaliella bardawil,which can grow in the NaCl concentration of 0.5 ~ 5.5 mol/L,is one of the most salt-tolerant eukaryotes found in the world.D.bardawil can produce large amounts of ?-carotene under various environmental stresses,such as high light and/or high salinity.Due to these two features,D.bardawil has been considered as an excellent model organism to investigate the molecular mechanisms of salt stress responses and regulation of carotenoid metabolism.This thesis mainly focuses on genes involved in carotenoid biosynthesis and degradation metabolic pathways from D.bardawil and regulation mechanisms of carotenoid accumulation under salt stress at molecular biology level.It is of great significance for the production of ?-carotene and the improvement of carotenoid content by utilizing Dunaliella cells.Firstly,with the help of transcriptome sequencing and rapid amplification of cDNA ends(RACE)technology,the full-length coding sequences(CDS)of carotenogenic genes GGPS,ZISO,CRTISO,LcyE,BCH,CYP97s(CYP97A,CYP97 B,and CYP97C)and degradation related genes CCDs(CCD1A,CCD1 B,and CCD8)can be cloned form D.bardawil.The carotenoid metabolic pathways of D.bardawil were similar to that of other green algae,higher plants and cyanobacteria,and were significantly different from bacteria and fungi.The protein sequences of carotenogenic genes in D.bardawil had high homologies with other green algae and higher plants,and had low homologies with bacteria and fungi.The protein sequences of CCDs genes related to carotenoid degradation in D.bardawil had high homologies with other green algae,and were significantly different from bacteria and fungi.The CCDs genes in D.bardawil were not as complex as those in higher plants.Then by using plasmids(pACCRT-EIB,pACCAR16?crtX and pACCAR25?crtX)carrying a gene cluster from Erwinia uredovora capable of carotenoid biosynthesis,the functional activities of GGPS,BCH,and CCDs from D.bardawil can be identified in Escherichia coli by complementary experiment.GGPS,PSY,and LcyB genes from D.bardawil coupled with crtI gene from E.uredovora can result in the accumulation of ?-carotene in E.coli.This provides the feasibility of constructing the carotenoid metabolic pathway of D.bardawil in E.coli.DbBCH can catalyze the conversion of ?-carotene to zeaxanthin.Unlike Haematococcus pluvialis,D.bardawil lacks ?-carotene ketolase,and cannot synthesize astaxanthin.DbCCDs has functional activities to cleave ?-carotene into ?-ionone by preliminary identification.No corresponding cleavage products of lycopene can be detected.It seemed that DbCCDs may participate in degrading upstream substrates of lycopene or produce other unusual cleavage products of lycopene.These remain to be determined.The treatment of triethylamine in D.bardawil led to a reduction in the biomass of algal cells,but it can significantly improve the accumulation of lycopene.Maybe it was because the upstream genes(GGPS,PSY,PDS,and ZDS)of the biosynthesis of lycopene were induced,and lycopene cyclases genes(LcyB and LcyE)and downstream hydroxylase gene(BCH)were down-regulated,and then the carotenoid metabolic pathway in D.bardawil was altered,and led to the accumulation of lycopene.This method may be valuable for the development of lycopene production.Finally,by genome walking technique,the promoters of carotenogenic genes(GGPS,CRTISO,and BCH)were cloned from D.bardawil.By using online website analysis,it was found that the promoters of carotenogenic genes from D.bardawil contained several stressresponsive cis-acting element.Promoters of several genes(GGPS,PSY,CRTISO,LcyB,and BCH)contained salt-regulated element(GT1GMSCAM4),which conferred a salt induction pattern in carotenogenesis of D.bardawil.Using quantitative Real-Time PCR(qRT-PCR)technique,it was showed that salt stress upregulated both carotenogenic genes(GGPS,PSY,ZISO,CRTISO,LcyB,LcyE,and BCH)and four WRKY transcription factors genes in D.bardawil.All these may cause the accumulation of carotenoids under salt stress in D.bardawil.