| Saccharina japonica is one of the important commercial brown seaweeds,which has significant ecological and economic values.Alginate is the major carbon metabolite in S.japonica,which is widely used in food,pharmaceuticals,chemical industry,and other applications.Therefore the analysis on the alginate synthesis pathways and involved genes is significant both in theory and application.Based on the kelp genome and transcriptome data,we predicted the alginate biosynthetic pathway in S.japonica.In total three mannose-6-phosphate isomerase(MPI)genes,two phosphomannomutase(PMM)genes,three GDP-mannose 6-dehydrogenase(GMD)genes,one glycosyltransferases 2(GT2)gene and 125 mannuronate C5-epimerase(MC5E)genes were annotated.It was found that the transcription of these genes exhibits a decreasing trend at different developmental stages.The result is consistent with the fact that the kelp cells grow more rapidly at the early stages than the later stages when the organic substance was synthesized and accumulated rapidly.Comparing with the top part of S.japonica,the cells in the base part of kelp have stronger ability to devide,so the transcription levels of GMD1?GT2 and MC5E2 genes were significantly higher than those in the cells of the top part.While for MPI3 and PMM1 genes,which were also involved in other carbohydrates synthesis pathways,the transcription levels increased from the base part to the top part of the kelp.We analyzed the structure and function of the highly expressed phosphomannomutase/phosphoglucomutase(PMM/PGM)gene in S.japonica.The PMM/PGM gene(Sjpmm/pgm1)was cloned,encoding 252 amino acids which belonged to the haloacid dehydrogenase(HAD)superfamily with four conserved motifs.We also conducted the expression and function analysis of PMM/PGM from S.japonica(Sj PMM/PGM1),and found that Sj PMM/PGM1 showed both PMM and PGM activities with cofactor Mg2+.The optimal temperature for both PMM and PGM activities was 30?,and the optimal p H values were 7.5 and 7.0,respectively.The Km for the substrates G1 P and M1 P were 0.08 m M and 1.15 m M,respectively.In addition,it was found that with high temperature(25?)and highlight(55±5 ?mol/m2/s)treatments,the transcription levels of Sjpmm/pgm1 increased within a short time,and then gradually returned to the initial levels,which indicated that the synthesis of alginate might be involved in the acclimation of S.japonica to temperature and light stress.The trend analysis was conducted on the 49 highly expressed MC5 E genes,and the result showed that the transcription of most MC5 E genes decreased gradually at different developmental stages,which was closely related with the higher M/G ratio of alginate in the base part at the late development stage of S.japonica.From the base part to the top part,the transcription of most MC5 E genes exhibited a decreasing trend,and only a few genes,such as MC5E16 showed an increasing trend.In the study of cloning and expression of MC5E2 gene,we obtained a sequence with the full-length of 1458 bp,which encoded 485 amino acids.It was predicted that the MC5E2 has a typical right-handed ?-helix structure,and belonged to the ?-helix superfamily.We successfully expressed the soluble eukaryotic MC5E2 in the prokaryotic expression system,and confirmed its mannuronate C5-epimerase activity by the 1H-NMR spectrum analysis.Based on omics data,we studied the transcription and function of genes invloved in the alginate synthesis in S.japonica.Our data were not only positive to analyze the metabolic pathway of alginate synthesis,but also provided reference for improving the physicochemical property of alginate by biological modification,and further for controlling the structure of alginate precisely in its industrial production. |
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