Cloning And Functional Analysis Of RtGRX And RtSOD In Rea Umuria Trigyna

Reaumuria trigyna,a highly xerophytic halophyte from the Eastern Alxa—Western Ordos area,has strong adaptability to the high saline and hyper-drought environment.According to the transcriptome sequencing database,we found that the antioxidant system of R.trigyna played a vital role in response to adversity stress.Superoxide dismutase SOD and Glutaredoxin GRX have important function to remove active oxygen and regulate redox status in cell.In present research,RtSOD and RtGRX genes were cloned from R.trigyna.Tissue specificity and expression pattern of genes under different stresses were analyzed.The function of the genes in transgenic Arabidopsis thaliana under abiotic stresses were detected.The results would lay the foundation for us to understand the antioxidant function of RtSOD and RtGRX to respond to different abiotic stresses.The main results are as follow:1.Glutaredoxin RtGRX and Superoxide dismutase RtSOD were isolated based on the transcriptome database of R.trigyna.The open reading frame(ORF)length of RtGRX is 807bp,encoding a protein with 268 amino acids(Molecular weight:66.98kDa,Isoelectric point:5.17).The open reading frame(ORF)length of RtSOD is 663bp,encoding a protein with 220 amino acids(Molecular weight:55.90kDa,Isoelectric point:5.11).Amino acid sequence alignment analysis showed that RtGRX belonged to the GRX family and RtSOD belonged to the Cu/Zn SOD family.Phylogenetic tree analysis indicated that the RtGRX was closely related to the GRX of Solanum pennellii and Nicotiana sylvestris,and RtSOD had the closest relationship with SOD of Tamarix hispida.2.Tissue specificity analysis showed that RtGRX and RtSOD were expressed in roots,stems and leaves,and the expression level in stems was significantly higher than those in roots and leaves.Expression specificity study indicated that under 400mM NaCl treatment,cold,drought,hydrogen peroxide,and ABA stress,the expression level of RtGRX and RtSOD were up-regulated;under different salt concentrations,the expression level of these genes reached the highest at 400mM NaCl.3.Overexpressing RtGRX and RtSOD in Arabidopsis,we found that under NaCl and drought stresses,the growth status(root length,fresh weight,chlorophyll content)of RtGRX and RtSOD transgenic lines was better than wild type,proline content and autioxidant enzyme activities(SOD,POD,CAT)increased significantly and the H2O2 and MDA content reduced dramatically in transgenic lines compared to the control,which incidated that overexpression of RtGRX and RtSOD in Arabidopsis could improve the antioxidant enzyme activity of transgenic plants,reduce the content of reactive oxygen species and alleviate the degree of membrane damage,thereby enhancing the resistance of transgenic plants.4.Under NaCl and drought stresses,the analysis of stress-related genes showed that the expression level of the genes related to antioxidant system,proline synthesis and the ion transport increased markedly in transgenic lines compared to the wild type.Thus,we draw the conclusion that the overexpression of RtGRX and RtSOD in transgenic lines could increase the gene activity involved in plant oxidation equilibrium,osmotic and ion balance,so as to enhance the tolerance of transgenic Arabidopsis thaliana to abiotic stresses.