Cloning, Analysis And Prokaryotic Expression Of SAMS Gene In Undaria Pinnatifida

Total RNA was extracted from the gametophytes of Undaria pinnatifida with improved SDS method. S-adenosylmethionine synthetase gene (UpSAMS) had been isolated from the economic seaweed U. pinnatifida by using degenerate primers. The cDNA sequence was 1,491 bp in length with an ORF of 1,194 nucleotides, encoding a deduced 397 amino acid residues, and had a predicted molecular weight of 43.2 kDa and was Iso-electronic point of 5.244. The sequence began with the presumed initial ATG codon and stop TAA codon,with the 5′UTR and 3′UTR 92 bp and 205 bp in length, respectively. UpSams comprised 42 strongly basic, 57 strongly acidic, 132 hydrophobic, and 93 polar amino acids. The amino acid sequence included two typical motifs, namely, signature motif-1 GAGDQG (at position 127–132) and signature motif-2 GGGAFSgKD (at position 274–282). BLASTx result revealed the Sams sequence of U. pinnatifida (UpSams) shared 68%–96% identity with the previously published Sams sequences of other species. U. pinnatifida was clustered with L. digitata and E. siliculosus of brown algae; with green and red algae; with diatoms and dinoflagellate; and with terrestrial plants. Phylogenetic analysis indicated that the phylogenetic relationship of UpSams with other seaweeds was closer than those from higher plants. Hydrophobicity analysis showed that the strongest hydrophilic amino acid was glycine and the most hydrophobic amino acid was lysine. Phosphorylation analysis showed that there were 10 serine,4 threonine and 3 possible tyrosine.Under different stress conditions, relative mRNA expression levels of UpSAMS were measured by real-time fluorescence quantitative PCR and the results demonstrated that the UpSAMS was related to stress tolerance. UpSAMS was transformed into E.coli BL21. UpSAMS expression in the original, SDS-PAGE showed UpSAMS had been combined in pGEX4T-1 expression vector, and was the formation of the fusion protein.Gene cloning, analysis and expression of UpSAMS had laid an important foundation for further study to the resistant mechanism.