Interaction Of BvM14-DHAR3, BvM14-APX And BvM14-MDAR Genes In The AsA-GSH Cycle Under Salt Stress

Sugar beet monosomic addition line M14 was obtained by Professor Guo Dedong using the Beta vulgaris L.and hybridized with Beta corollifiora Zoss.The chromosome group in addition to 18 chromosomes,but also attached to the wild white sugar beet No.9 staining monomer.The identified beet M14 strain has some excellent traits such as drought resistance,salt tolerance,cold resistance and no fecundity,and is a good material for mining important genetic resources.A total of 76 differentially expressed proteins under different NaCl(0,200,400 mM)stress were identified by i TRAQ tandem LC-MS / MS.These amino acid sequences were matched to the Saccharomyces cerevisiae M14 transcripts of different NaCl(0,200,400 mM)obtained by Hiseq 2000 high throughput sequencing using NCBI localized t BLASTn software.BvM14-GO,BvM14-CAT,BvM14-Prx,BvM14-Trx,BvM14-APX,BvM14-MDAR and BvM14-DHAR3 genes were significantly different under different stress(P <0.05).Aspartic acid,BvM14-DHAR3,BvM14-APX and BvM14-MDAR play a key role in regulating plant stress response to oxidative stress.The three genes: BvM14-DHAR3,BvM14-APX and BvM14-MDAR play an important role in regulating plant stress response to oxidative stress.In this study,the full-length c DNAs of BvM14-DHAR3,BvM14-APX and BvM14-MDAR genes were obtained by PCR.The ORFs were found to be 807,763 and 732 bp,respectively,which were encoded by 268,250 and 243 aa respectively by bioinformatics analysis.It is predicted that the protein encoded by BvM14-DHAR3 is located in the chloroplast and belongs to the family of glutathione S-transferase,which has its N-terminal and C-terminal conserved domains.The protein encoded by BvM14-APX gene is located in chloroplast and mitochondria,The ascorbate peroxidase domain belongs to the plant peroxidase superfamily;the protein encoded by the BvM14-MDAR gene is located in the cytoplasm and has the pyridine disulfate nucleotide oxidoreductase domain.Three proteins were predicted to be hydrophilic.After the phylogenetic tree was constructed,the genetic relationship between the three genes and the same protein in spinach was the closest.Tissue-specific expression analysis of BvM14-DHAR3,BvM14-APX and BvM14-MDAR genes in the roots,stems,leaves and flowers of Sugar beet M14 lines showed that BvM14-DHAR3,BvM14-APX and BvM14-MDAR genes were expressed in leaves The highest expression level.This is because the three genes as the As A-GSH cycle of three key enzymes in the chloroplast expression,and participate in the plant light and the role of pathways,so the expression of the leaves in the most obvious.Construction of p ET28a-BvM14-DHAR3,p ET28a-BvM14-APX and p ET28a-BvM14-MDAR prokaryotic expression vector was transformed into E.coli BL21(DE3),and the expression of enzyme in supernatant was induced under the induction conditions of 0.05,0.1 and 0.03 mM IPTG,20,25 and 18 ? respectively.The pBI121-BvM14-DHAR3,pBI121-BvM14-APX and pBI121-BvM14-MDAR eukaryotic expression vectors were constructed.After transformation of Agrobacterium tumefaciens,the wild type and mutant of Arabidopsis thaliana were transformed into infestation,(CO)and gene overexpressing plants(OX).Three groups of plants(WT,KO,CO,OX)were treated with 0,150 mM NaCl stress.The contents of DHAR3,APX and MDAR and the relative expression of DHAR3,APX and MDAR genes in different plants were determined.The contents of DHAR3,APX and MDAR were measured.(1)The expression of DHAR3 gene was negatively correlated with the content of MDA and GSH accumulated in the plant,but the expression of MDA was not obvious when the expression of DHAR3 was inhibited,and the content of As A was positively correlated with the expression of DHAR3 relationship.This indicated that DHAR3 was the rate-limiting enzyme in As A-GSH cycle,and its expression level could affect its upstream and downstream substrate content.(2)The expression of APX was positively correlated with the content of MDA and negatively correlated with As A,but the expression of APX was not directly related to the content of GSH,and the content of GSH showed a decreasing trend.This indicates that when the APX activity of the active oxygen accumulated as a direct removal of the plant is changed,the accumulated ROS accumulated in the electron transport chain after the stress is reduced to varying degrees,which leads to the content of the upstream and downstream substrate Change.(3)The expression level of MDAR was positively correlated with the contents of GSH and As A,but negatively correlated with MDA content.This suggests that although MDA can produce an important substrate DHA that replenishes As A by nonenzymatic disproportionation,it is more efficient by MDAR catalyzed enzymatic reactions.(4)The level of enzyme activity or transcriptional expression of APX was positively correlated with the activity and expression of DHAR3 and MDAR.The level of enzyme activity or transcriptional expression of MDAR was negatively correlated with the activity and expression of DHAR3 and MDAR Relationship.The results showed that the three genes were the key enzymes in the As A-GSH cycle.When the plants were exposed to external salt stress,the three genes were involved in the regulation of the over-reduced electron transport chain,and the three enzymes But also the upstream enzyme product,so the changes in the expression of a single enzyme will lead to the other two kinds of enzyme activity occurred more obvious changes in the down and down,and DHAR3 as the cycle of regulation of dynamic equilibrium rate limiting enzyme,the changes in the expression of The other two enzymes caused by the change to be more obvious.