Cloning And Analysis Of Cu/ZnSOD Gene From Galega Orientalis L.

Galega orientalis is a kind of excellent legumious forage. SOD is an important enzyme existing in eukaryote extensively and played an essential role in stress-tolerance of plants. In order to study the effect of SOD genes on resisting oxidative damage and improving stress-tolerance in Galega orientalis, using rapid amplification of cDNA ends (RACE) method, and based on an EST sequence which was achieved from a SSH cDNA library induced by salt stress from Galega orientalis, a cDNA of Cu/ZnSOD gene was cloned and analyzed perliminarily.The full-length of cDNA sequence was 935 bp and included a 600 bp open reading frame which encoded a 199-amino-acid polypeptide. The molecular weight of Cu/ZnSOD protein was 20.35 KDa, and the theoretical isoelectric point was 5.88. Named as GoCu/ZnSOD, accession number: HM777020.Amino acid of GoCu/ZnSOD protein analyses were conducted using the online programs of protein blast and ScanProsite. The residues required for coordinating copper ion were His-91, 93, 108 and 165, and for zinc ion were His-108, 116, 125 and Asp-128. The cysteine (C-91) in GNAGGRIACGVV(183-194 bp) could form a single disulfide bond.The results of GoCu/ZnSOD gene expression by Real-Time PCR showed that the gene expression level was highest in leaves, moderate in stems, and least in roots. NaCl and PEG could up-regulated the expression of GoCu/ZnSOD gene. Low temperature had no significant effect on the expression of GoCu/ZnSOD gene . And the exogenous hormone ABA down-regulated the expression of GoCu/ZnSOD gene.The overexpression vector pCAMBIA1302-GoCu/ZnSOD has been constructed. Based on the characteristic of the GFP protein could sending out green fluorescent under laser light, subcellular localization of the GoCu/ZnSOD protein was conducted using tobacco infection method. The result of observation using laser confocal microscope indicated that the GoCu/ZnSOD protein located in the chloroplasts.