Functional analysis of Vitis davidii R2R3-MYB transcription factor Vd-MYB14 in the regulation of flavonoid biosynthesis

Abstract:【Objective】Flavonoids are important secondary metabolites in grapes and are associated with the sensory characteristics of red wine such as color and astringency. The flavonoid pathway is reg- ulated by the MBW transcription complex at the transcriptional level. In this complex, the MYB tran- scription factor is a decisive regulator that positively or negatively regulates various structural genes in the flavonoid pathway to maintain the balance in flavonoid content in different plant organs. Clustering analysis and functional enrichment analysis of differentially expressed genes were carried out based on the transcriptome data of grapes and the MYB gene VdMYB14 was selected for participation in the regu- lation of anthocyanin synthesis. In this study, we analyzed the structure and function of the MYB tran- scription factor to further elucidate its role in regulating anthocyanin synthesis in grapes. This analysis coul contribute to in-depth understanding of the mechanism of flavonoid synthesis and improving fruit quality.【Methods】Berries skins of huitong black spine grapes (Vitis davidii‘1338’) in 6 developmen- tal stages after flowering were used as experimental materials. qRT-PCR were used to analyze the ex- pression of the VdMYB14. The plant total RNA rapid extraction kit was used to extract the total RNA from the samples. The PrimeScriptTM RT kit with DNAase was used to remove contaminated genomic DNA and to carry out reverse transcription for cDNA synthesis. The plant anthocyanin content measure- ment kit (Solarbio) and plant proanthocyanidin content measurement kit (Solarbio) were used to mea- sure anthocyanin and proanthocyanidin content, respectively. The MEGA software was used to analyze the phylogenetic relationship between the VdMYB14 protein and the other flavonoid-related MYB. Ho- mologous recombination was used to insert the VdMYB14 gene in the PBI121 vector and tobacco leaves were transfected. The expression position of the VdMYB14 protein in the cells was observed. Heterolo- gous expression was carried out in the tobacco to validate whether gene VdMYB14 could regulate antho- cyanin synthesis.【Results】qRT-PCR results showed that the gene VdMYB14 expression level increased in the early stage and reached its peak 40 days after flowering. Subsequently, its expression level de- creased, which wass consistent with the previous transcriptome results. The subcellular localization re- sults showed that the VdMYB14 was mainly localized in the nucleus and only a few VdMYB14 were lo- calized in the cytoplasm. The phylogenetic tree results showed that the protein VdMYB14 was highly homologous to the VvMYB14 and the VvMYB15 from grapes (Vitis vinifera L.). Furthermore, these three genes and the MdMYB111 from apples were located at the same branch, indicating that the Vd- MYB14 and theMdMYB111 had a similar function. In the identification of the petal color of tobacco, the degree of petal color of the transgenic lines was significantly lighter than that of the wild type, and there were differences between different transgenic lines. Measurements of anthocyanin content in the transgenic tobacco showed that anthocyanin content in the petals of transgenic tobacco was significantly lower than that in the wild-type plants. However, the proanthocyanidin measurement results showed that the proanthocyanidin content in the petals of the transgenic tobacco was higher than that in the wild- type. The overexpression of the gene VdMYB14 in tobacco affected the expression of the structural genes in the flavonoid pathway. Compared with the wild-type, the expression levels of the NtCHI, NtD- FR, NtLAR and NtANR in the VdMYB14-overexpressing tobacco petals were significantly upregulated. Among them, the difference in the expression levels of NtLAR and NtANR was the highest although there were no significant changes in the expression levels of the NtCHI, NtCHS and NtF3H. However, the NtUFGT expression in the transgenic tobacco petals was lower than that in the wild-type petals. However, in the VdMYB14-overexpressing tobacco, the expression levels of the NtANR and NtLAR were significantly upregulated, and the expression level of the structural gene NtUFGT of the anthocyanin synthesis pathway decreased. This would cause more leucoanthocyanidin and anthocyanidin to enter the proanthocyanidin pathway, thereby inhibiting the synthesis of anthocyanin. This showed that the Vd- MYB14 gene might regulate the transfer of the anthocyanin pathway to proanthocyanidin pathway, there- by inhibiting anthocyanin synthesis and affecting the the petal color of tobacco. In this study, the expression level of the VdMYB14 was high in the early stage of grape berry development. As the fruit entered the veraison, its expression gradually decreased. This indicated that the gene VdMYB14 might inhibit the accumulation of anthocyanins in the early stage of berry development【. Conclusion】This study demon- strated the effect of the grape VdMYB14 in regulating anthocyanin and proanthocyanidin synthesis. Vd- MYB14 overexpression in tobacco significantly affected the expression of structural genes in the flavo- noid pathway, particularly key structural genes in the proanthocyanidin pathway and anthocyanin path- way, causing intermediate products from the anthocyanin pathway to enter the proanthocyanidin pathway.