| Laminariacea and Gracilariaceae are very important economic seaweeds, whichcan be widely used in food, medicine, chemical industry, aquaculture feed algae, frammanure fields cause of its rich polysaccharide. Trehalose and cellulose aredisaccharide and polysaccharide based on glucose molecules, act as two importantbranches of glucose metabolic pathway in all organism. Trehalose is a kind ofimportant resistant protective material in vivo. Cellucose is an important compositionelement cell wall in vascular plants and algae，the cell wall have the function ofsupport and protection. The research of genes related trehalose and cellucose pathwayin Saccharina japonica and Gracilaria chouae, not only will study and explore themechanism of glucose metabolism algae provide basic scientific basis, but also lay animportant foundation for the development and utilization of genetic resources.Combined with OneKP algae transcriptome plan, cDNA sequences wereobtained and the amino acids Bayes phylogenetic tree were constructed with thesequences of trehalose and cellulose related genes, with the results we found thateukaryotic algae UGPase may originate from endosymbiotic cyanobacteria in theprocess of endosymbiont. Red algae PGM, vasular plant plastic PGM and green algaePGM may originate from the bacteria, brown algae PGM and vasular plant cytosolPGM may originate from eukaryotic host. In addition to the original Galdieriasulphuraria containing3TPS sequences, the rest red algae are contained only1TPSsequence. Brown algae H TPS were origined from endosymbiotic red algae, class C,D, F, G were origined from the host involved in the second endosymbiont. In additionthere are multiple TPS sequences in brown algae and diatoms of the same species inclass F and G, which showed that TPS replication has appeared before the differentionof brown algae and diatoms. Red algae only exists CESA type, and clustered withoomycete. In brown algae exists CESA and CSL types of cellulose synathase, andCESA type gene duplication were token out in brown algae.In consideration of the important position of the trehalose synathesis andcellulose synathesis pathway, in this paper we selected the middle gene UGPase ofthe two pathway, the UGPase sequence is studied in Saccharina japonica and Gracilaria chouae. We got Gracilaria chouae and Saccharina japonica UGPasesequences by PCR amplification with the sequence length1488bp and1446bp,respectively. The similarity of the two sequences is21.6%, enconding495and481amino acid, protein molecular erights is54.97KDa and51.90KDa. The structureanalysis of the UGPase protein showed that both of the UGPase protein is acidicprotein, α-helix and random coil is higher in protain secondary structure, the proteintertiary structure is similar in the two protein. According to the studied of otherspecies UGPase, the nucleotide binding site (NB-LOOP) and substrate binding site(I-LOOP) are highly conserved in eukaryotes. We contrasted the prokaryoticexpression vector pET32a-hdUGPase and pET32a-cjlUGPase, screened theexpressing clone and purified the target UGPase protein, finally the purifiedSaccharina japonica UGPase is1.76mg/ml, the concentration of the Gracilariachouae UGPase protein is1.4mg/ml. Enzyme activity assay showed that theSaccharina japonica UGPase optimum reaction temperature is30-37℃, theoptimum pH is8.0, divalent metal ion Mg2+, Mn2+, Ca2+, Zn2+can promote theenzyme activity, while Pb2+, Cu2+inhibited the enzyme activity,0.5mM Mg2+showedthe greatest activity that is522.21U/g. Gracilaria chouae UGPase optimum reactiontemperature is40℃, the optimum pH is8.0, the effect of divalent metal ion issimilar with Saccharina japonica UGPase, but the greatest activity is780.49U/g inthe presence of0.5mM Mg2+. Saccharina japonica UGPase and Gracilaria chouaeUGPase protein Km value were4.33mol and1.67mol, both of the maximumreaction rate are1666.67mol/min/mg, the substrate affinity ability is better thanother reported species. |
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