Studies On Cloing And Localizating Of VviBZR1 And Anthocyanin Regulator VviMYB5b In Wine Grapes And Corresponding Protein Interaction

Brassinosteriods(BRs)are steroid hormones with high-efficiency,broad-spectrum and non-toxic properties.They are a new type of plant hormone that regulates fruit ripening and can be high physiological effect at lower content.Grape berry treatment by exogenous BRs,anthocyanins and other phenolic substances of grape fruits increased significantly.BR fully play a regulatory effect,so that dephosphorylation of BZR1/BES1 induces downstream target gene expression in cells.VviBZR1 is a key transcription factor in BRs signal transduction and positively regulates BRs signaling pathway.However,in the process of BR signal transduction,the molecular mechanism of VviBZR1 gene regulating grape anthocyanin synthesis is still unclear.During the synthesis of anthocyanins from grape skin,VviMYB5 b is a regulatory gene for anthocyanin synthesis.VviMYB5 b is beneficial for the regulation of anthocyanin biosynthesis and the synthesis of CHS,F3’5’H,DFR,ANS and other structural genes in anthocyanin synthesis pathway.The previous study of the studying team found that the expression trends of VviBZR1 and VviMYB5 b genes were similar during the synthesis of anthocyanins in grape skin,and it was concluded that VviBZR1 and VviMYB5 b genes have a synergistic effect on regulating the biosynthesis of anthocyanins in grape skins.Therefore,the interaction relationship between VviBZR1 and VviMYB5 b transcription factors is of great significance for further study on the molecular mechanism of the interaction between VviBZR1 and VviMYB5 b in the regulation of anthocyanin synthesis in wine-grape skins.In this experiment,Vitis vinifera L.’Cabernet Sauvignon’ was used as material to clone the VviBZR1 and VviMYB5 b genes in grape skins for bioinformatics and subcellular localization analysis,using yeast two-hybrid and bimolecular fluorescence complementation test(Bi FC).To study the interaction between VviBZR1 and VviMYB5 b,and provide a theoretical basis for the regulation of the synthesis of anthocyanins in grape skin by VviBZR1 and VviMYB5 b.The main findings were as follows:(1)Mapping and analysis of VviBZR1 and VviMYB5 b.Subcellular localization results showed that VviBZR1-1,VviBZR1-2,VviBZR1-3,VviBZR1-4 and VviMYB5 b were all located in the nucleus and played a role as transcription factors;the full length of VviMYB5 b gene sequence was 1103 bp,which contains the basic domain of R2R3 MYB protein;the four homologous genes of VviBZR1 were named VviBZR1-1,VviBZR1-2,VviBZR1-3 and VviBZR1-4,and the total length of the gene sequences were 1368 bp,1235 bp,1071 bp and1820 bp,respectively;the similarity of the four homologous genes of VviBZR1 were 25.11%.The closest relationship with VviBZR1-4 was Arabidopsis At BZR1-4;VviMYB5 b was closely related to VviMYB5 a in grapes.(2)Analyze the interaction of VviBZR1 and VviMYB5 b proteins.The pGADT7-VviMYB5 b recombinant plasmid and the p GADT7 empty plasmid were separately transformed into Y2 H Gold yeast competent cells and coated with SD/-Leu plates.The results showed that the yeast colonies containing the transformed and untransformed plasmids had the same growth state,namely the bait recombinant plasmid has nontoxic to the yeast.The p GBKT7-VviBZR1 recombinant plasmid and the p GBKT7 empty plasmid were also transformed into Y2 H Gold yeast competent cells,respectively,and the SD/-Trp plate was coated.The results showed that the recombinant plasmid was not toxic to yeast;The p GADT7-VviMYB5 b plasmid was transformed into the competent state of Y2 H Gold yeast strain,and diluted at different concentrations and applied to SD/-Leu,SD/-Leu/-Trp,SD/-Leu/-Trp/X-α-Gal,SD/-Leu./-Trp/-His/-Ade/Ab A plate,also observed the growth of p GBKT7-VviBZR1 on SD/-Trp,SD/-Trp/X,SD/-Trp/A/X plates,all showed no self-activation.The yeast two-hybrid interaction assays showed that only VviBZR1-4 and VviMYB5 b proteins have an interaction relationship;the in vivo interaction between VviBZR1 and VviMYB5 b was analyzed by bimolecular fluorescence complementation assay(Bi FC).The fluorescence signal of the recombinant plasmid in Arabidopsis protoplasts was observed by laser scanning confocal microscope.It was found that only the VviBZR1-4 and VviMYB5 b plasmids were cotransduced in Arabidopsis protoplasts,and the yellow fluorescence signal appeared in the nucleus,thus verifying VviBZR1-4 and the interaction of VviMYBb protein,while VviBZR1-1,VviBZR1-2,VviBZR1-3 and VviMYB5b protein have no interaction relationship.