Interaction Of BvM14-Trx And BvM14-PrxR Genes In The Antioxidant Enzyme System Of Sugar Beet M14 Line Under Salt Stress

Under salt stress,the changes of the 7 main enzymes of antioxidant enzymes in the ROS scavenging mechanism of leaves and leaves of Sugarbeet M14 lines were: GO,Prx R,Trx,APX,MDAR,DHAR and CAT.In this experiment,the Trx and Prx R in the Prx R/Trx pathway were chosen for further study.This experiment is based on the research of resistance gene of sugar beet M14 on the Bv M14-Trx gene and Bv M14-Prx R gene as the research object,in-depth study by bioinformatics and molecular biology methods,and makes preliminary research on interaction of two gene.The main results are as follows:(1)The successful cloning of Bv M14-Trx gene,c DNA sugar beet M14 total 960 bp,maximum ORF included 558 bp,encoding 185 amino acids.Sequence analysis showed that the proteins are conserved domain of thioredoxin,the highest similarity of the protein and Suaeda Trx protein.The expression of Bv M14-Trx gene in sugar beet M14 flower,leaf,stem and root tissue using Real-Time PCR technology are analyzed,the results showed that Bv M14-Trx gene in sugar beet M14 flowers,leaves,stems and roots and widely expressed,the highest expression level in leaves,that of sugar beet M14 gene in leaves of Bv M14-Trx the highest expression level.These results suggest that the gene plays a physiological role in the process of plant antioxidation.The prokaryotic expression system of Bv M14-Trx gene was constructed.Under the condition of 0.5m M,IPTG and 37?induction,the expression of Bv M14-Trx protein was the largest,and the enzyme activity proved to have thioredoxin activity.These results indicate that Bv M14-Trx protein can be expressed normally in E.coli BL21(DE3)and has catalytic activity.This gene can be expressed in vitro,so it can be expressed in other plants.Bv M14-Trx gene expression vector was constructed,and wild plants and mutant plants of Arabidopsis thaliana were transformed respectively.The plants were observed and measured in Arabidopsis growth and root length,fresh weight,dry weight,H2O2 content,MDA content,GSH content and related gene expression at the transcriptional level,related protease activity and other physiological indexes,the results showed that Trx gene into Bv M14-Trx gene overexpression plants(T-OX)and Trx gene mutation recovery of plants(T-CO)compared with the control group,the plant growth under salt stress condition is good,the measured physiological indicators are better than the control group,indicating Bv M14-Trx transgenic plants to salt stress showed stronger tolerance.(2)The successful cloning of Bv M14-Prx R gene,c DNA sugar beet M14 total 911 bp,maximum ORF included 645 bp,encoding 214 amino acids.Sequence analysis showed that the proteins are conserved domain protein peroxide,the highest similarity of the protein and Prx R protein in spinach.The expression of Bv M14-Prx R gene in sugar beet M14 flower,leaf,stem and root tissue using Real Time-PCR technology are analyzed,the results showed that Bv M14-Prx R gene in sugar beet M14 flowers,leaves,stems and roots and widely expressed,the highest expression level in leaves,that of sugar beet M14 gene in leaves of Bv M14-Prx R the highest expression level.These results suggest that the gene plays a physiological role in the process of plant antioxidation.The prokaryotic expression system of Bv M14-Prx R gene was constructed.Under the condition of 0.5m M,IPTG and 37?induction,the expression of Bv M14-Prx R protein was the largest,and the enzyme activity proved to have the activity of peroxynitrite.These results indicate that Bv M14-Prx R protein can be expressed normally in E.coli BL21(DE3)and has catalytic activity.The results show that the gene can be expressed in vitro and can be expressed in other plants.Bv M14-Prx R gene expression vector was constructed,and wild plants and mutant plants of Arabidopsis thaliana were transformed respectively.The plants were observed and measured in Arabidopsis growth and root length,fresh weight,dry weight,H2O2 content,MDA content,GSH content and related gene expression at the transcriptional level,related protease activity and other physiological indexes,the results showed that Prx R gene into Bv M14-Prx R gene overexpression plants(P-OX)and Prx R gene mutation recovery of plants(P-CO)compared with the control group,the plant growth under salt stress condition is good,the measured physiological indicators are better than the control group,indicating Bv M14-Prx R transgenic plants to salt stress showed stronger tolerance.(3)The transfer of Bv M14-Trx gene or Bv M14-Prx R gene did not affect the content of MDA and GSH in plants,and the expression of APX gene,CAT gene and the activity of APX protein and CAT protein did not change significantly.The Bv M14-Trx gene and Bv M14-Prx R gene do not participate in MDA and GSH metabolic pathways,the Bv M14-Trx gene and the Bv M14-Prx R gene does not affect the APX pathway and CAT pathway.In plant antioxidant enzyme system,Prx R/Trx pathway has no obvious interaction with CAT pathway and APX pathway,and they are involved in the process of plant antioxidation.(4)The related genes of the above seven plants were detected at the level of gene transcription and protein expression.Get the sugar beet M14 Bv M14-Trx gene and Bv M14-Prx R gene in plants against abiotic stress especially Na Cl stress caused by oxidative stress,consistent with the change trend of two genes,the mutual influence mutual promotion.