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Molecular Cloning and Expression Analysis of an Mn-SOD Gene from Nelumbo nucifera

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Abstract

A rapid amplification cDNA end (RACE) assay was established to achieve the complete sequence of mitochondrial manganese-superoxide dismutase (Mn-SOD) cDNA in Nelumbo nucifera. The obtained full-length cDNA of Mn-SOD was 926 bp and contained a 699-bp open reading frame encoding an Mn-SOD precursor of 233 amino acids. The recombinant of Mn-SOD expressed by PET-32a vector in Escherichia coli BL21 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting assays. A 3D structural model of the Mn-SOD was constructed by homology modeling. Real-time polymerase chain reaction analysis revealed that Mn-SOD mRNA was expressed in young leaves, blossom, stems, and terminal buds during reproductive stage but with the highest expression in young leaves. This significant difference demonstrated the differential expression of Mn-SOD in various organs of N. nucifera.

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Abbreviations

Mn-SOD:

mitochondrial manganese-superoxide dismutase

SOD:

superoxide dismutase

ROS:

reactive oxygen species

RACE:

rapid amplification cDNA end

IPTG:

isopropylthio-b-d-galactoside

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Acknowledgments

The authors are greatly indebted to Prof. Yunchun Song and Ying Diao for critical reading of this manuscript, and doctor Hui Liu for his assistance in the laboratory work. We are especially appreciative of the reviewers for their helpful comments and detailed suggestions. This work was financially supported by the Key grant Project of Chinese Ministry of Education (no. 307018), National Science and Technology Pillar Program (no. 2007BAD37B06), and Natural Science Foundation of Hubei Provience (no. 2007ABD003)

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Authors and Affiliations

  1. Key Lab of the Ministry of Education for Plant Developmental Biology, College of Life Science, Wuhan University, Wuhan, 430072, China

    Chen Dong, Guolin Li, Zhuoqi Li, Honglin Zhu & Zhongli Hu

  2. Lotus Center, Wuhan University, Wuhan, 430072, China

    Mingquan Zhou

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  1. Chen Dong
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  2. Guolin Li
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  3. Zhuoqi Li
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  4. Honglin Zhu
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  6. Zhongli Hu
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Correspondence to Zhongli Hu.

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Dong, C., Li, G., Li, Z. et al. Molecular Cloning and Expression Analysis of an Mn-SOD Gene from Nelumbo nucifera . Appl Biochem Biotechnol 158, 605–614 (2009). https://doi.org/10.1007/s12010-008-8410-1

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  • DOI: https://doi.org/10.1007/s12010-008-8410-1

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