Cloning And Functional Verification Of Key Enzyme Responsible For Carotenoid Biosynthesis In Dunaliella Salina

Dunaliella sallina is a kind of cell wall-free eukaryotic green algae that highly accumulates carotenoids in the extreme environments.Functional characterization of the genes involved in biosynthesis of carotenoids is crucial for directional breeding of the excellent germplasms of D.sallina having breakthrough traits.Such analysis could provide the scientific basis for developing the engineered algal strains rich in natural compounds with special biological functions and commercial production of high-valued algal products.Therefore,in this study,D.salina strain Ds-YC011 was used as the germplasm to isolate the target genes encoding Ds LYCB(lycopene ?-cyclase)and Ds LYCE(lycopene ?-cyclase),two enzymes in the carotenoid biosynthesis pathway of Dunaliella salina.The coding sequences of these two genes were cloned into the expression vectors,respectively.The target genes were separately introduced into the D.salina cells using electroporation method.The transient expressionins of the genes in tobacco leaves were obtained by Agrobacterium-mediated infiltration.Analysis on these transgenics was conducted to characterize the biological functions of the two enzyme genes,and subsequently,to develop the excellent algae strains with high yield of carotenoids.The main findings were described as the followings:1.The purified algae strain was isolated from the algae samples collected from Yuncheng Salt Lake.The algae strain was identified to be a new strain of D.sallina(namely Ds-YC011)by cellular morphology and 18 S r DNA sequence analysis.2.The expression vector contains the anti-hygromycin gene used as the selection marker.The sensitivity of D.salina cells to various concentrations of hygromycin was examined.The hygromycin dosage used for the positive transformant selection was identified to be 200 ?g/m L and 800 ?g/m L,in solid and liquid culture media,respectively.3.The coding sequences of Ds LYCB and Ds LYCE genes were successfully obtained from the algae by high-fidelity RT-PCR.Bioinformatics analysis showed that Ds LYCB is an unstable hydrophilic protein with no transmembrane structure.The ?-helix is the major form in the second structure which accounted for 39.9 %.Ds LYCE is an unstable hydrophilic protein with a transmembrane structure,showing that the irregular coil in the secondary structure is the major form whichaccounted for 43.27 %.The homologous sequence alignment and phylogentic analysis showed that Ds LYCB and Ds LYCE proteins of D.salina shared the highest similarity with Haematococcus pluvialis.4.The high-salt stress significantly up-regulated the expression of Ds LYCB and Ds LYCE genes in D.salina,leading to the highest expression level at 4 h after the stress.At this time point,the carotenoid content was up to the peak value.Notably,the expression levels of Ds LYCB and Ds LYCE genes were positively correlated with carotenoid enrichment in D.salina under the stress.5.In order to investigate whether Ds LYCB and Ds LYCE regulate the biosynthesis of carotenoids in higher plants,the two plant expression vectors p CAMBIA1303+Ds LYCB and p CAMBIA1303+Ds LYCE were constructed.Agrobacterium-mediated infiltration was employed to transfer these two genes into tobacco leaves,respectively,for their transient expression in the tissue.The biochemical analysis of tobacco leaves transiently-expressing the target gene showed that carotenoids and ?-carotenes increased by 24 % and 42.3 %,respectively,in the transgenic tobacco leaves compared to the wild type.In addition,the content of chlorophyll a and chlorophyll b in the transgenic leaves was also significantly higher than that in the wild-type tobacco leaves(P<0.05).This indicates that the Ds LYCB and Ds LYCE genes of D.salina can function normally,and promote the biosynthesis and accumulation of carotenoids in higher plants.6.An electroporation-based genetic transformation system of D.salina was established,and subsequently used for the transformation of Ds LYCB and Ds LYCE into D.salina cells to obtain the transgenic algal lines highly expressing the target genes.7.The examination of biomass and carotenoid content in the D.salina cells showed that the growth phenotypes were no difference between the transgenic and wild-type D.salina cells.The total amount of carotenoids and ?-carotene accumulated in the transgenic algae cells were significantly higher than those in the wild type(P<0.05).In conclusion,in this study,Ds LYCB and Ds LYCE genes encoding the enzymes having high catalytic activities were cloned fromthe D.salina strain.Moreover,an electroporation-based genetic transformation system of D.salina was successfully developed.All these data lay a foundation for the establishment of a highly-efficient genetic transformation system of D.salina in the future,and also provide the theoretical basis and technical support for developing an effective strategy to yield high-value-added products of D.salina and its industrial implications.