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Int. J. Mol. Sci. 2016, 17(1), 4; doi:10.3390/ijms17010004

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

1,2,†
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1,2,†
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1,2
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4,* and 1,2,*
1
College of Pharmacy, Jinan University, Guangzhou 510632, China
2
Guangdong Provincial Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, China
3
School of Life Sciences, Shenzhen University, Shenzhen 518060, China
4
State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China
These authors contributed equally to this work.
*
Authors to whom correspondence should be addressed.
Academic Editor: Hatem Rouached
Received: 15 September 2015 / Revised: 22 November 2015 / Accepted: 26 November 2015 / Published: 22 December 2015
(This article belongs to the Special Issue Plant Molecular Biology)
View Full-Text   |   Download PDF [2975 KB, uploaded 22 December 2015]   |  

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. View Full-Text
Keywords: Sonneratia alba; copper/zinc superoxide dismutase; protein expression; activity and stability; salt stress Sonneratia alba; copper/zinc superoxide dismutase; protein expression; activity and stability; salt stress
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MDPI and ACS Style

Yang, E.; Yi, S.; Bai, F.; Niu, D.; Zhong, J.; Wu, Q.; Chen, S.; Zhou, R.; Wang, F. Cloning, Characterization and Expression Pattern Analysis of a Cytosolic Copper/Zinc Superoxide Dismutase (SaCSD1) in a Highly Salt Tolerant Mangrove (Sonneratia alba). Int. J. Mol. Sci. 2016, 17, 4.

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