| As a precursor of vitamin A,?-carotene has many functions,such as antioxidant,enhancing immunity,inhibiting cardiovascular and cerebrovascular diseases,preventing cancer and so on.It is widely used in medicine,health care,food and other industries.Dunaliella salina is a microalgae with high accumulation of ?-carotene and is an ideal model organism for carotenoid synthesis.It is of great research value to explore the molecular mechanism of accumulation of ?-carotene in Dunaliella salina from the perspective of molecular biology and improve the yield of ?-carotene by means of molecular genetic breeding.The main research contents and results are as follows:A single sterile Dunaliella salina cell was obtained by capillary separation,plate marking and antibiotic sterilization.Morphological and molecular biological identification of Dunaliella salina FACHB-435 and GY-H13 showed that the two strains belonged to Dunaliella.Prediction of the genome size of the two Dunaliella strains by flow cytometry was 385 Mb and 255 Mb,respectively.The second generation sequencing results showed that the genome size of Dunaliella salina FACHB-435 was about 368 Mb,which was similar to the predicted size.CDS sequences of LCYB and PSY genes of two Dunaliella strains were cloned by means of molecular biology,in which FACHB-435 and GY-H13 LCYB CDS were 1809 bp and 1794 bp respectively,and their similarity with FACHB-847 LCYB gene(KX218392.1)were 89% and 99%,respectively.The size of GY-H13 PSY CDS was 1275 bp,and the similarity with Db-847 PSY gene(EU328287.1)was 99%.The overexpression plasmid was constructed and transferred into two Dunaliella strains,and the successfully transformed strains were screened.The ?-carotene production of FACHB-435 LCYB overexpressing algae strain DFL11 increased by 14.06% compared with the original strain;The ?-carotene production of GY-H13 LCYB overexpressing algae strains DGL5,DGL7 and DGL10 increased by 37.16%?29.97%?98.55%(P<0.05)respectively,and PSY gene overexpressing strain DGP15,DGP28 increased by 67.2%?58.22%(P<0.05)respectively compared with the original strain.The effects of high salt,high light and nitrogen,phosphorus and sulfur deficiency on the accumulation of ?-carotene in Dunaliella salina were studied.The results showed that the content of ?-carotene in Dunaliella salina maximumly increased by 31.31%,30.29%,9.68%,50.84%,48.44% under high salt,hight light,-N,-P,-S conditions,respectively.Through ARTP mutagenesis breeding experiment,several mutant algae strains with fast growth rate and relatively high yield of ?-carotene were screened.The ?-carotene yield of mutant strains DF14 and DF15 were 2.06 and 2.28 times of the original strain,and the ?-carotene yield of DG15 and DG17 were 1.98 and 1.90 times of the original strain,respectively.The transcriptome sequencing technique was used to investigate the response of Dunaliella salina to high light stress.Clean reads were compared with reference Dunaliella salina genome,eggNOG,KEGG and KOG databases.The differentially expressed genes(AVsB)between two adjacent time points of stress experiment group and control group were compared.here were 6142 up-regulated genes,3949 down-regulated genes in control group on 5dVs13 d,8466 up-regulated genes and 2969 down-regulated genes in experimental group on 5d-HLVs13d-HL.Compared with the results of 5dVs13 d,the differentially expressed genes of 5d-HLVs13d-HL in carotenoid metabolism pathway were mostly up-regulated genes,and the content of ?-carotene in single cell was higher than that in control group.The results further showed that the high light stress promoted the synthesis of ?-carotene.Three differentially expressed genes were selected for RT-PCR verification,and the results were consistent with the prediction. |
PDF Full Text Request