Characterization Of Genes Involved In Mannitol Metabolicof Pathway Ectocarpus Siliculosus And Saccharina Japonica

Mannitol and laminarin are the main forms of carbon storage substances inbrown algae. Exploration of pathways and ralative genes involved with mannitolmetabolism is important for objective breeding and genetic improvement ofSaccharina with high-content mannitol.Based on genomic data of Ectocarpus siliculosus, two genes encoding formannitol-1-phosphatase (named EsM1Pase1and EsM1Pase2) were isolated and theenzymatic function was verified subsequently. Through the amplification offull-length sequences, we analyzed the taxonomic distribution of EsM1Pase orthologsand spatial structure of EsM1Pase2. Recombinant EsM1Pase2was obtained via invitro expression in E. coli and purification by Ni2+affinity chromatography. It isshowed that EsM1Pase2was highly specific to hydrolyze mannitol-1-phosphate toproduce mannitol, and very sensitive to NaCl. Moreover, the kinetic parameters (Km,Vm etc.) were calculated with measurement of reaction velocity under various M1Pconcentrations. qPCR results showed that EsM1Pase2gene exhibited an analogousdiurnal rhythmicity with mannitol content. Our results demonstrated that M1Pase in E.siliculosus could be a new member of HAD super-family.Combined E. siliculosus genome data with transcriptomic analysis of Saccharinajaponica, the full-length sequence of gene encoding for mannitol-2-dehydrogenase(M2DH) was obtained. qPCR detection for juvenile sporophytes showed that withsalinities decreasing, M2DH gene expression levels increased; while it was barelytranscribed under high NaCl concentrations, indicating M2DH exhibited strongtolerance to low salinities. Treatments with H2O2(0.2-0.8mM) and short-perioddessication (<2h) could up-regulate M2DH gene transcriptional levels, indicating thatthe kelp could produce more mannitol for abiotic stresss tolerance. Compared todarkness treatment, Sj-M2DH showed relatively higher expression levels under white and blue light for2-6h. When immersed in darkness for8-10h, M2DH transcriptsexhibited slight up-regulation. It was deduced that M2DH gene could regulatereversible enzymatic reaction to adjust mannitol content. In addition, the detectedmannitol content varied with different strains, seasons and positions of the kelp.In conclusion, our study provided molecular data for deciphering the relationbetween mannitol pathway and its variations, and enriched genetic improvement ofthe kelp, which facilitated strains screening for large quantities of mannitol.