title: 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.
authors.affiliation: College of Pharmacy, Jinan University, Guangzhou 510632, China. yangenze@foxmail.com.|Guangdong Provincial Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, China. yangenze@foxmail.com.
authors.forename: Enze
authors.initials: E
authors.lastname: Yang
authors.affiliation: College of Pharmacy, Jinan University, Guangzhou 510632, China. yishanze317@163.com.|Guangdong Provincial Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, China. yishanze317@163.com.
authors.forename: Shanze
authors.initials: S
authors.lastname: Yi
authors.affiliation: School of Life Sciences, Shenzhen University, Shenzhen 518060, China. 15016733519@163.com.
authors.forename: Fang
authors.initials: F
authors.lastname: Bai
authors.affiliation: College of Pharmacy, Jinan University, Guangzhou 510632, China. 18739977531@163.com.|Guangdong Provincial Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, China. 18739977531@163.com.
authors.forename: Dewei
authors.initials: D
authors.lastname: Niu
authors.affiliation: College of Pharmacy, Jinan University, Guangzhou 510632, China. 18825084106@163.com.|Guangdong Provincial Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, China. 18825084106@163.com.
authors.forename: Junjie
authors.initials: J
authors.lastname: Zhong
authors.affiliation: College of Pharmacy, Jinan University, Guangzhou 510632, China. qiuhong6506@126.com.|Guangdong Provincial Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, China. qiuhong6506@126.com.
authors.forename: Qiuhong
authors.initials: Q
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China. chsuf@mail.sysu.edu.cn.
authors.forename: Shufang
authors.initials: S
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China. zhrench@mail.sysu.edu.cn.
authors.forename: Renchao
authors.initials: R
authors.lastname: Zhou
authors.affiliation: College of Pharmacy, Jinan University, Guangzhou 510632, China. jnubiopharm@126.com.|Guangdong Provincial Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, China. jnubiopharm@126.com.
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D032407:Avicennia / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D017124:Conserved Sequence; D004795:Enzyme Stability; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055049:Salt Tolerance; D013379:Substrate Specificity; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D013482:Superoxide Dismutase
keywords: Sonneratia alba; activity and stability; copper/zinc superoxide dismutase; protein expression; salt stress