|
|
|
| Expression and Characterization of ThMSD from Thellungiella halophila in Escherichia coli |
| XU Xiao-jing, AN Hui-ling, CHEN Ning-mei, YANG Jing, ZHOU Yi-jun |
| College of Life and Environmental Sciences, Minzu University of China, Beijing 100081, China |
|
|
|
Abstract Superoxide dismutases (SODs) are ubiquitous antioxidative enzymes found in eukaryotic and differ by the named metal cofactor, Cu/Zn, Mn or Fe. Plant mitochondria Mn-SOD play a vital role in fight aginst stresses. ThMSD, a gene of Mn-SOD was cloned from Thellungiella halophila with dramatic stresses tolerance in earlier research. ThMSD was ligated to a prokaryotic expression vector pET30a(+) and transferred to E. coli BL21 to get recombinant BL21(pET30a-ThMSD). SDS-PAGE analysis showed an induced expression product band,with a molecular weight of about 32 kDa,which was consistent with the expected value, in all recombinant strains.Western blotting confirmed this band with anti-His monoclonal antibody. The results of SOD activity assay of soluble fractions of total protein from 3 recombinant strains showed that much higher SOD activity than control. The strain with highest expression level of ThMSD was selected and was cultured in large scale and target protein was purified using a Ni2+ Chelating column. The effect of temperature on the enzyme activity of ThMSD was tested, the result showed that ThMSD enzyme still had more than 50% residual activities at 55℃. The thermal stability of purified ThMSD was examined at 42℃, the result showed that SOD activity of ThMSD reduced with the time elapse, but it retained more than 60% residual activities after 40 min. In assay of NaCl tolerance of BL21(pET30a-ThMSD), the recombinant strain can grow faster than control in higher concentration of NaCl. From above, the recombinant ThMSD protein had high SOD activity and high thermal stability, and BL21 transformed with ThMSD have increased NaCl tolerance. It was concluded that ThMSD may connect with extreme stresses tolerance of Thellungiella halophila.
|
|
Received: 12 November 2012
Published: 25 April 2013
|
|
|
|
|
[1] Juarez J C, Manuia M, Burnett M E, et al. Superoxide dismutase 1 (SOD1) is essential for H2O2-mediated oxidation and inactivation of phosphatases in growth factor signaling. Proc Natl Acad Sci USA, 2008, 105: 7147-7152.
[2] Poage M, Le Martret B, Jansen M A, et al. Modification of reactive oxygen species scavenging capacity of chloroplasts through plastid transformation. Plant Mol Biol, 2011, 76(3-5): 371-384.
[3] Kliebenstein D J, Monde R A, Last R L. Superoxide dismutase in Arabidopsis: an eclectic enzyme family with disparate regulation and protein localization. Plant Physiol, 1998, 118: 637-650.
[4] Gutteridge J M, Halliwell B. Free radicals and antioxidants in the year 2000: a historical look to the future. Ann NY Acad Sci, 2000, 899: 136-147.
[5] Wang Y C, Qu G Z, Li H Y, et al. Enhanced salt tolerance of transgenic poplar plants expressing a manganese superoxide dismutase from Tamarix androssowii. Mol Biol Rep, 2010, 37(2): 1119-1124.
[6] Gonzalez A, Steffen K L, Lynch J P. Light and excess manganese. Implications for oxidative stress in common bean. Plant Physiol, 1998, 118(2): 493-504.
[7] Lee H, Xiong L, Ishitani M, et al. Cold regulated gene expression and freezing tolerance in an Arabidopsis thaliana mutant. The Plant Journal, 1999, 17: 301-308.
[8] Wang F Z, Wang Q B, Kwon S Y, et al. Enhanced drought tolerance of transgenic rice plants expressing a pea manganese superoxide dismutase. J Plant Physiol, 2005, 162(4): 465-472.
[9] Samis K, Bowley S, McKersie B. Pyramiding Mn-superoxide dismutase transgenes to improve persistence and biomass production in alfalfa. J Exp Bot, 2002, 53(372): 1343-1350.
[10] Wang F Z, Wang Q B, Kwon S Y, et al. Enhanced drought tolerance of transgenic rice plants expressing a pea manganese superoxide dismutase. J Plant Physiol, 2005, 162(4): 465-472.
[11] Zhu J. Plant salt tolerance. Trends Plant Sci, 2001, 6: 66-71.
[12] Taji T, Seki M, Satou M, et al. Comparative genomics in salt tolerance between Arabidopsis and Arabidopsis-related halophyte salt cress using Arabidopsis microarray. Plant Physiol, 2004, 135(3), 1697-1709.
[13] Inan G, Zhang Q, Li P H, et al. Salt cress. A halophyte and cryophyte Arabidopsis relative model system and its applicability to molecular genetic analyses of growth and development of extremophiles. Plant Physiol, 2004, 135: 1718-1737.
[14] Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc Nat Acad Sci USA, 1979, 76: 4350-4354.
[15] Stewart R R C, Bewley J D. Lipid peroxidation associated with accelerate aging of soybean axes. Plant Physiol, 1980, 65: 245-248.
[16] Moran J F, James E K, Rubio M C, et al. Functional characterization and expression of a cytosolic iron-superoxide dismutase from cowpea root nodules. Plant Physiol, 2003, 133: 773-782.
[17] 李晓泽, 姜静, 李德斌, 等. 重组大肠杆菌BL21(PET28a-e1F5A)的抗逆性分析. 生物技术, 2006, 16(3): 25-28. Li X Z, Jiang J, Li D B, et al. Stress tolerance assay of recombinant BL21(PET28a-e1F5A). Biotechnology, 2006,16(3):25-28.
[18] 石金磊, 徐建遥, 蒋昌华, 等. 月季eIF-5A基因的表达增强宿主大肠杆菌对高温和低温的耐性. 中国生物工程杂志, 2008, 28(1): 18-24. Shi J L, Xu J Y, Jiang C H, et al. Increasing the thermotolerance of E.coli with expression of eIF-5A from Rosa chinensis. China Biotechnology, 2008,28(1):18-24. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
