| The color of grape peel is one of the important appearance qualities of grape fruit,and it is also a key factor that determines the quality of wine.The color of peel is mainly determined by the content and composition of anthocyanins.The biosynthesis of anthocyanins is controlled by structural and regulatory genes.In this paper,yeast two-hybrid system,yeast one-hybrid system and virus induced gene silencing were used to analysis the mutual mechanism of VvmybAl,VvUFGT,VvDFR and the regulatory mechanism of the transcription factor mybA1 in the anthocyanin synthesis pathway.When mybAl was silenced,the expression of key enzymes in the anthocyanin synthesis pathway and the anthocyanin content were analyzed.The main results are as follows:1.Establishment of VIGS system for grape fruitUsing the Red Globe berries as a test material,the region of VvmybAl coding region 422-753 was inserted into pTRV2 vector in the reverse direction,and pTRV2-mybAl and pTRV1 were respectively transferred into Agrobacterium GV3101.’Red Globe’ berries were infected with suspensions of increasing bacterial densities(OD600=0.5,1;1.5,and 3),respectively.Next,the berries were artificially cultivated for 15 days at 26℃,under a 16-h light cycle,with 60%humidity.A significant difference in silencing phenotypes was observed at bacterial densities of OD600=1.2.Functional analysis of transcription factor mybAlThe bacterial cultures at densities of OD600=1 were used to infect ’JiZaoMi’ berries under the same conditions.After 10 days,the treatment group remained almost green,while the control group showed red color.The culture was continued for 5 days,the treatment group remained green.The results of anthocyanin assay showed that the content of anthocyanins in the control group was 3-4 times that of the treatment group,and there was a significant difference in the ’JiZaoMi’(P<0.05).The results of RT-PCR analysis showed that mybAl in the treated group was hardly expressed;the results of qPCR analysis showed that the gene expression levels of mybAl,mybA2,DFR,and UFGT in Red Globe berries decreased by 73%,47%,80%,and 8%,respectively(the expression levels of control were calculated as 100%);the gene expression levels of mybAl,mybA2,DFR,UFGT,LAR1,LAR2,ANR,and ANS in ’JiZaoMi’ were:97%,95%,72%,42%,45%,53%,73%,99%,respectively(control expression is calculated as 100%).Significant difference analysis showed that mybAl and DFR were significantly different(P<0.01)and mybA2 was significantly different(P<0.05)in Red Globe berries,and mybAl,mybA2,DFR,ANS were the difference was extremely significant(P<0.01)compared with the control in ’JiZaoMi’,and UFGT,LARI,LAR,and ANR were significantly different(P<0.05).3.Protein interaction analysisThe recombinant plasmids pGBKT7-mybAl,pGBKT7-DFR,pGBKT7-UFGT,and pGADT7-mybAl were constructed based on the VvmybAl,VvUFGT,VvDFR CDS sequences and pGBKT7 and pGADT7 polyclotase sites.Transcriptional activation analysis and toxicity test results showed that the protein encoded by VvmybAl has transcriptional activation function,and the proteins encoded by VvUFGT and VvDFR have no transcriptional activation function and are non-toxic and harmless.Yeast two-hybrid results showed that VvmybAl protein does not interact with UFGT and DFR protein at the yeast level.4.DNA-protein analysisThe promoter sequences of UFGT and DFR were obtained from the grape genome database.The PLANTCARE promoter was used to predict the promoter elements.MBS cis-acting elements bound to MYB were obtained.The MBS cis-acting elements were connected in tandem to obtain the bait element sequence.MBS cis-acting elements mutations were negative controls.pGADT7-mybA1 was co-transformed with pMBD(UFGT)-AbAi,pMBS(DFR)-AbAi and pGADT7-myhA1 to yeast Y1Hgold cells,respectively(test group);pGADT7-mybA1 was co-transformed with p[Mutant(UFGT)]-AbAi、p[Mutant(DFR)]-AbAi to yeast Y1Hgold cells,respectively(negative control)and p53-AbAi was co-transformed with pGAD-Rec-p53 to yeast Y1Hgold cells.Which were applied to SD/-Ura/-Leu,respectively On a 100 mM Aba solid plate.The results showed that both experimental and positive controls could grow on solid plates,while negative controls could not grow,demonstrating that the VvmybAl encoded protein could special recognit promoters of VvUFGT and VvDFR genes. |
