Cloning, Characterization and Expression Pattern Analysis of a Cytosolic Copper/Zinc Superoxide Dismutase (SaCSD1) in a Highly Salt Tolerant Mangrove (Sonneratia alba)

Abstract

Mangroves are critical marine resources for their remarkable ability to tolerate seawater. Antioxidant enzymes play an especially significant role in eliminating reactive oxygen species and conferring abiotic stress tolerance. In this study, a cytosolic copper/zinc superoxide dismutase (SaCSD1) cDNA of Sonneratia alba, a mangrove species with high salt tolerance, was successfully cloned and then expressed in Escherichia coli Rosetta-gami (designated as SaCSD1). SaCSD1 comprised a complete open reading frame (ORF) of 459 bp which encoded a protein of 152 amino acids. Its mature protein is predicted to be 15.32 kDa and the deduced isoelectric point is 5.78. SaCSD1 has high sequence similarity (85%–90%) with the superoxide dismutase (CSD) of some other plant species. SaCSD1 was expressed with 30.6% yield regarding total protein content after being introduced into the pET-15b (Sma I) vector for expression in Rosetta-gami and being induced with IPTG. After affinity chromatography on Ni-NTA, recombinant SaCSD1 was obtained with 3.2-fold purification and a specific activity of 2200 U/mg. SaCSD1 showed good activity as well as stability in the ranges of pH between 3 and 7 and temperature between 25 and 55 °C. The activity of recombinant SaCSD1 was stable in 0.25 M NaCl, Dimethyl Sulphoxide (DMSO), glycerol, and chloroform, and was reduced to a great extent in β-mercaptoethanol, sodium dodecyl sulfate (SDS), H2O2, and phenol. Moreover, the SaCSD1 protein was very susceptive to pepsin digestion. Real-time Quantitative Polymerase Chain Reaction (PCR) assay demonstrated that SaCSD1 was expressed in leaf, stem, flower, and fruit organs, with the highest expression in fruits. Under 0.25 M and 0.5 M salt stress, the expression of SaCSD1 was down-regulated in roots, but up-regulated in leaves.

Molecular characterization and expression analysis of copper‐zinc superoxide dismutases from the freshwater alga Closterium ehrenbergii under metal stress

“…Generally, the CuZnSOD contains three conserved motifs defined as CuZnSOD signature motifs; these signature motifs KAVAVL, F(L)HL(I)HQ(A)F(L)GDTTNGC, and GNAGGRLACGVV(I)GL were also identified in the CeCSD1 and CeCSD2 proteins (Figures and ). These were in accordance with previous findings, indicating that these sites and motifs are essential for the structure and function of CuZnSOD and may be involved in stabilizing SOD conformation under harsh environmental conditions …”

“…Comparisons of CeCSDs with other CuZnSODs revealed that the C‐terminal was more conserved than the N‐terminal. These findings suggest that they may have functions similar to those of other SODs …”

Molecular adaptation to salinity fluctuation in tropical intertidal environments of a mangrove tree Sonneratia alba

“…It reaches an optimal growth in 5 to 50% seawater, indicating its capacity to tolerate high salinity and hypoxia [19]. It can synthesize organic solutes to adjust cellular osmotic potential and produce antioxidant enzymes for scavenging reactive oxygen species (ROS) against high salinity [20]. Therefore, it is an attractive ecological model to investigate genetic mechanisms underlying salt adaptation.…”

Chloroplast Genome Sequences and Comparative Analyses of Combretaceae Mangroves with Related Species

“…Mangroves are critical marine resources for their remarkable ability to tolerate seawater and are uniquely adapted to tropical and subtropical coasts [ 1 ]. Mangrove forests played an important role in ecosystem services and supported coastal livelihoods, yet they are relatively low in the number of species [ 2 ].…”