Molecular cloning and expression analysis of manganese superoxide dismutase gene from Nelumbo nucifera embryonic axes

A manganese superoxide dismutase (Mn-SOD) gene, NnMSD1, was identified from embryonic axes of the sacred lotus (Nelumbo nucifera Gaertn). The NnMSD1 gene has an open reading frame of 699 bp, and encodes a deduced polypeptide of 233 amino acid residues with a predicted molecular mass 25.9 kDa and isoelectric point of 6.6. The protein contains all conserved residues of the Mn-SOD protein family, including four consensus metal binding domains and a signal peptide for mitochondrial targeting. Southern blot analysis suggests the existence of two Mn-SOD genes in sacred lotus. NnMSD1 was highly expressed in developing embryonic axes during seed development, but appeared in cotyledons only at the early stage of development and became undetectable in the cotyledons during late embryogenesis. The expression of NnMSD1 gene in germinating embryonic axes, in response to various stresses, such as heat shock, chilling, and exposure to stress-related chemicals, was also studied. Heat shock strongly inhibited the expression of the NnMSD1 gene whereas the NnMSD1 transcript level decreased at the early stage of chilling stress treatment, but was strongly expressed at the later stage. An increase in expression was also highly induced by H2O2 in germinating embryonic axes. The results suggest that the expression pattern of NnMSD1 gene differed between developing axes and cotyledons, and that NnMSD1 gene expression responds strongly to chilling and oxidative stress.