Key Lab of the Ministry of Education for Plant Developmental Biology, College of Life Science, Wuhan University, Wuhan 430072, China.
Appl Biochem Biotechnol. 2009 Sep;158(3):605-14. doi: 10.1007/s12010-008-8410-1. Epub 2008 Nov 19.
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.
建立了一种快速扩增 cDNA 末端（RACE）技术，以获得荷花线粒体锰超氧化物歧化酶（Mn-SOD）cDNA 的全长序列。获得的 Mn-SOD 全长 cDNA 为 926bp，包含一个编码 233 个氨基酸的 Mn-SOD 前体的 699bp 开放阅读框。通过 SDS-PAGE 和 Western blot 实验证实了 Mn-SOD 在大肠杆菌 BL21 中由 PET-32a 载体表达的重组体。通过同源建模构建了 Mn-SOD 的 3D 结构模型。实时聚合酶链反应分析显示，Mn-SOD mRNA 在生殖期的幼叶、花朵、茎和顶芽中表达，但在幼叶中表达最高。这一显著差异表明 Mn-SOD 在荷花不同器官中的表达存在差异。
