| Dunaliella salina is a kind of unicellular eukaryotes with extremely salt tolerance.Dunaliella salina can accumulate nutrients in high salt environment,such as β-carotene,glycerol,taurine,polyunsaturated fatty acids and other special substances with physiological activities.The most well-knowned is β-carotene,however,the content of lipids in Dunaliella salina is also very high,accounting for about 10-25% of the dry weight.It is found that Dunaliella salina has the potential as a substitute for fossil fuels.In order to cope with stress conditions such as high osmotic pressure and strong light,it can produce neutral lipids or triglycerides to store chemical energy.The synthesis of fatty acids requires a large supply of NADPH,which is considered to be the limiting factor of lipid synthesis in biology.Malic enzyme(ME)is one of the important sources of NADPH.It widely exists in nature.It is a key enzyme that plays an important role in fatty acid synthesis,organic acid metabolism and cell anaerobic growth.The ME genes of several species,such as P.tricornutum and C.pyrenoidosa,have been cloned and sequenced,but there are few reports on the ME genes closely related to oil accumulation in Dunaliella salina.In this paper,the sequences of Ds ME1 and Ds ME2 genes in Dunaliella salina were obtained by using the existing transcriptome data.Through bioinformatics analysis,the subcellular localization,conserved domain analysis and multiple sequence comparison of MEs in Dunaliella salina were studied,and the expression changes of Ds ME1 and Ds ME2 after salt stress were studied by fluorescence quantitative analysis.The promoters and terminators of Ds ME1 and Ds ME2 genes were successfully cloned,and the expression vectors p Ds ME1p-BEL and p Ds ME2p-BEL of Ds ME1 and Ds ME2 promoters were constructed by inserting EGFP gene and ble gene by homologous recombination method.The constructed promoter expression vector was transformed into Dunaliella salina cells by electric shock transformation for transient expression.After fluorescence microscope observation and ble resistance screening,a stable genetic transformation strain was successfully constructed and the promoter activity was verified.Then the promoter sequences of Ds ME1 and Ds ME2 genes were analyzed by bioinformatics tools.It was found that there were many cis-elements in the promoter sequences of Ds ME1 and Ds ME2,and there was a drought response element(MBS)in the promoter region of Ds ME2.The deletion mutation experiment after MBS knockout verified that the MBS gave the osmoregulation function of Ds ME2 promoter.Finally,taking Dunaliella salina as the starting strain,the overexpression vector was constructed with the cloned ME1 and ME2 genes,and transferred into Dunaliella salina cells by electric shock to construct the engineering algae with overexpression of ME.The biomass changes and oil content differences between wild algae and engineering algae under heterotrophic conditions were compared.Compared with wild algae,the oil yield of Ds ME1 OE cells was increased by30.49%,and that of Ds ME2 OE cells was increased by 36.32%.Two promising industrial microalgae strains were provided,which laid a foundation for the technological breakthrough of biodiesel production. |
