Analysis Of The Function Of The Promoter Of Glyoxalase Ⅰ Gene In Sugar Beet M14 Strain And Analysis Of Its Transcription Factor Isolation

The sugar beet M14 line is a sugar beet monosomic addition line obtained by crossing and backcrossing a diploid cultivated sugar beet(Beta vulgaris L.)with a tetraploid wild white flower sugar beet.Related research has been confirmed: The sugar beet M14 has the excellent characteristics of wild white flower beet,such as drought resistance,cold resistance,salt tolerance and no fused reproduction.A comparative proteomic analysis of sugar beet M14 strains under salt stress was conducted in the laboratory,76 differential proteins were obtained.Among them,glyoxalase I is up-regulated by salt stress.The full-length BvM14-glyoxalase I gene was obtained from the beet M14 strain using electronic cloning and RACE technology,and gene function was studied.The results showed that overexpression of BvM14-glyoxalase I gene could increase the resistance of tobacco to salt stress.In order to further understand the salt-tolerance mechanism of this gene from the level of transcriptional regulation,this study analyzed the function of the promoter of the BvM14-glyoxalase I gene of the sugar beet M14 strain and isolated and analyzed the transcription factors bound to it.(1)Using the Plant CARE software,functional components of the 1830 bp promoter sequence(P0)upstream of the BvM14-glyoxalase I gene obtained in the laboratory were analyzed.The promoters were found to contain basic components such as TATA-box and CAAT,and were also associated with endosperm formation.Components: GCN4＿motif,Skn-1 motif;regulatory elements related to photoreaction:ACE,Box-4,GAG-motif;elements related to stress signals: TC-rich repeats,MBS;elements that respond to hormones: CGTCA-motif.(2)In order to determine the BvM14-glyoxalase I gene promoter in response to the core region of salt stress,the 5’ end deletion was performed according to the position of the TATA-box and the response stressor in the promoter,and amplification was designed by designing primers to obtain P1(1483 bp),P2.(1063 bp),P3(809 bp),P4(714 bp),P5(567 bp)promoter deletion fragment.In order to detect the activity of each promoter fragment of BvM14-glyoxalase I gene,a plant expression vector containing different lengths of BvM14-glyoxalase I gene promoter was constructed.The transient expression of tobacco was used to detect the GUS staining results of tobacco before and after salt stress.The promoter activity of each deletion fragment of the promoter and its ability to respond to salt stress were rapidly identified.In order to further verify the transient expression of tobacco,and to stabilize the expression of Arabidopsis thaliana,the GUS activity and GUS staining results of Arabidopsis thaliana positive plants before and after salt stress were detected.The P4 fragment had the strongest priming activity by transient expression and stable expression of GUS activity and GUS staining results;After salt stress,the GUS activity of the P1-P5 promoter fragment driving GUS expression increased compared with the control,and the color of GUS staining was deeper than that of the control.The P4 fragment drove the most increased GUS activity and the color of GUS stained the most.The P4 fragment was the strongest in response to salt stress,and the P4 fragment was determined to be the core sequence of the gene promoter in response to salt stress.(3)The cDNA library of beet M14 strain treated with 0 mM and 200 mM NaCl was constructed,and the P4 fragment of BvM14-glyoxalase I gene promoter was used as a bait.The cDNA library treated with 0 mM and 200 mM NaCl was screened by yeast one-hybrid assay.Clone and sequence.The sequencing results were analyzed by NCBI and compared with the cultivated sugar beet transcription factor database to determine the transcription factor sequence of BvM14-glyoxalase I gene regulated by 0mM and 200 mM NaCl,which was regulated under treatment with 200 mM NaCl.The cDNA sequence of seven unique transcription factor genes of the BvM14-glyoxalase I gene.(4)Bioinformatics analysis of 7 unique transcription factor gene cDNA sequences under treatment with 200 mM NaCl.Using the transcriptome data of the leaves and roots of the sugar beet M14 strain obtained in the early stage of the laboratory under the treatment of 200 mM and 400 mM NaCl,the cDNA sequences of the seven transcription factor genes obtained were analyzed under salt stress,and the results showed that they all responded to salt stress.(5)Aligning 7 transcription factor sequences with the Arabidopsis database to find the protein sequence of the homologous gene,using online predictive protein interaction string website.A molecular network was obtained which interacts with these seven transcription factors and BvM14-glyoxalase I.