Molecular Coloning And Expression Analysis Of Important Antioxidant Genes In Nelumbo Nucifera

Nelumbo nucifera is an ancient species and native of China. All parts of N. nucifera had been used as the traditional food and herbs in our country. Present researches were mainly focus on its taxology, morphology, physiology, molecular biology and value in medicine. As an important economic aquatic crop, few researches about molecular cloning and transgenic engineering of N. nucifera were reported. Antioxidant enzymes (MnSOD, CuZnSOD, APX and CAT) could protect cells from the damage caused by reactive oxygen species (ROS), delay senescence and improve the resistance of organism. In order to provide excellent genes with tolerance against environmental stress for transgenic plants, it was necessary to clone the complete sequences of important antioxidant genes and analyses the characters of their expression in N. nucifera. Our researches were mainly focus on four aspects as follow:1 Molecular cloning and expression analysis of MnSOD geneSODs could catalyze the dismutation of the O2-. to molecular O2 and H2O2, three styles of SODs were classified according to their metal cofactor and their subcellular localization:MnSOD, CuZnSOD and FeSOD. The obtained full-length cDNA of N. nucifera MnSOD was 926 bp, the active sites and structure of N. nucifera MnSOD showed the common characters of MnSOD family. The recombinant MnSOD showed high thermal stability and remained more than 90% activity even after 30 min treatment at 30-50℃. The MnSOD mRNAs were differentially expressed in various parts, and significantly increased in short-term mechanical wounding and temperature stress. 2 Molecular cloning and expression analysis of CuZnSOD geneTwo novel CuZnSOD cDNAs were achieved in N. nucifera; the full-length cDNAs of CuZnSOD1 and CuZnSOD2 were 723 bp and 713 bp respectively. The active sites and structure of N. nucifera CuZnSOD showed the common characters of CuZnSOD family. According to homology modeling, we speculated the isforms had similar activity, and this result was proved by the assay of activity. So it probably provided the tool for analysis the enzyme activity by bioinformatic analysis. The recombinant CuZnSOD showed high thermal stability and remained more than 90% activity even after 30 min treatment at 30-65℃. GFP fluorescence visualized by fluorescence microscopy indicated that the CuZnSOD-GFP accumulated in cytosol and nucleus. This was the first research on the differences in the three styles of CuZnSOD (chlCuZnSOD, cytCuZnSOD, ecCuZnSOD). The CuZnSOD mRNAs were differentially expressed in various parts, and significantly increased in short-term mechanical wounding and temperature stress. We concluded the expression of CuZnSOD and MnSOD had some similar characters in aquatic botany.3 Molecular cloning and expression analysis of APX geneAPX utilized ascorbate as its specific electron donor to reduce H2O2 to H2O with the concomitant generation of monodehydroascorbate. The obtained full-length cDNA of N. nucifera APX was 1379 bp. The phylogenetic analysis revealed APX of N. nucifera grouped together with chloroplastic APX of high plants. Enzymatic analysis showed a recombinant enzyme with APX activity (0.04 mM ascorbate min-1 mg-1 protein). The APX mRNAs were differentially expressed in various parts, and significantly increased in short-term mechanical wounding.4 Molecular cloning and expression analysis of CAT geneThe obtained full-length cDNA of N. nucifera CAT was 1666 bp. The phylogenetic analysis revealed CAT of N. nucifera grouped together with CAT of high plants. The CAT mRNAs were differentially expressed in various parts, and synergistic expression of N. nucifera MnSOD, CuZnSOD, APX and CAT mRNA were indicated in short-time mechanical wounding.