Function Analysis Of Salt Tolerance Of BvM14-MDHAR Gene In Sugarbeet M14 Line

Sugar beet M14 is the monosomic addition line by adding the 9th chromosome of Beta corolliflora Zoss.to the Beta vulgaris L.genome.It is identified that M14 has retained some excellent characters of B.corolliflora Zoss.,such as drought resistance,cold resistance,salt resistance and apomixis,possibly because the import of the 9th chromosome of B.corollilora Zoss.makes it obtain some good characters of wild species.M14 is a good material of exploring and using of quality gene of sugar beet.Monodehydroascorbate reductase(MDHAR)has two physiological functions in plants,the regeneration of the reduced state of ascorbic acid and participating in the process of electron transfer.MDHAR is the key enzyme in the regeneration of ascorbic acid,which can reduce monodehydroascorbate to ascorbic acid,thus maintaining the level of ascorbic acid in plant cells.This has important significance in the removal of reactive oxygen species,maintenance of plant cell reduction and resistance to salt stress.Our previous research showed that quantitative proteomics was applied to obtain the salt responsive proteins from the leaves and roots of M14 using iTRAQ LC-MS/MS technology under salt stress(0,200,400 mM NaCl).MDHAR was one of up regulated salt responsive proteins.According to the sequence of BvM14-MDHAR Unigene in sugar beeet RNAseq database obtained in our lab,the specific primers of BvM14-MDHAR gene were designed and the overall length of BvM14-MDHAR cDNA was obtained using PCR.The function of BvM14-MDHAR gene in response to salt stress was researched from bioinformatics,tissue-specific expression,prokaryote and plant expression systems.BvM14-MDHAR included a total of 1737bp sequence and contained the biggest ORF of 1059bp and coded 352aa.Multiple amino acid sequence alignment analysis showed that the similarity between BvM]4-MDHAR protein and MDHAR protein in Spinach was highest,reaching 92%.Hydrophilic predictive analysis found that hydrophilic amino acids were widely distributed in BvM14-MDHAR protein.Signal peptide prediction found that there was no signal peptide in BvM14-MDHAR protein.Tissue-specific expression of BvM14-MDHAR gene was analyzed in sugar beet M14 line using Real-Time PCR.BvM14-MDHAR gene in M14 line showed the highest level of expression in roots,followed by leaves,stems and flowers.The expression level of BvM14-MDHAR gene in the roots was 5.13 fold higher than that in the flowers.The prokaryotic expression vector pET28a-BvM14-MDHAR-His was constructed,and transformed into BL21(DE3).Western blot result showed that BvM14-MDHAR protein was confirmed to expressedin in BL21(DE3).In this study,transgenic Arabidopsis in response to salt stress were researched.The eukaryotic expression vector pCAMBIA 1305.1-BvM14-MDHAR was constructed and transformed into Agrabacterium tumefaciens EHA105 which was transformed into the wild-type plants(WT)and MDHAR mutation plants(KO)of Arabidopsis thaliana by inflorescence infestation method.The positive transgenic plants of the MDHAR gene overexpression plants(OX)and the MDHAR gene recovery type plants(CO)were selected.Many physiological indicators were compared in four types of plants(WT,KO,OX,CO):(1)At the phenotype level,fresh weight,root length of four types Arabidopsis plants were measured and compared.(2)At the physiology level,chlorophyll content,conductivity,ascorbic acid state,K+,Na+content were measured and compared.(3)At the transcription level,the DHAR(DHAR)gene expression level in leaves of four types of plants was measured.(4)At protein activity level,the monodehydroascorbate reductase(MDHAR)activity and dehydroascorbic acid reductase(DHAR)activity were compared.The results showed that compared to WT and KO plants,OX and CO plants in which the BvM14-MDHAR gene was transferred into WT and KO plants had a higher tollerance to salt stresse.It is clearly demonstrated that the BvM14-MDHAR gene could enhanc the salt tolerance ability of plants.