VdMYB14 Regulates The Anthocyanin Synthesis In Vitis Davidii

Vitis davidii is one of the main wild grapes originated in China,and its berry contains rich anthocyanins and other polyphenols.V.davidii is commonly used in wine making and juice making,and has broad market prospects.Anthocyanins are a class of water-soluble flavonoids,which widely distribute in plant leaves,fruits,flowers and other organs.The content and compound variations of anthocyanin in grape skins resulte in the color change of the berries.The MYB transcription factor family play important regulatory roles in anthocyanin synthesis.The study of MYB transcription can further understand the regulation mechanism of anthocyanin synthesis.In our previous study,a candidate MYB gene related to anthocyanin synthesis was selected through transcriptome data analysis,which was named VdMYB14.In order to further explore the regulatory role of Vd MYB14 in anthocyanin synthesis of V.davidii,the following studies were carried out in this study,using Hunan Huitong black spine grapes as the test material.The variation of anthocyanins content and the expression pattern of Vd MYB14 gene in grape skins of six different developmental stages were analyzed;The CDS sequence of Vd MYB14 gene was cloned,and the physicochemical properties,phylogenetic analysis and subcellular localization analysis of the protein were performed by using the predicted amino acid sequence to preliminarily predict its function.To verify its regulatory role in anthocyanin synthesis by heterologous expression of VdMYB14 gene in tobacco.The main results are as follows:1.There were only a few anthocyanins in the skin before the veraison(80 days after flower).Anthocyanins began to accumulate in large quantities after veraison,and the content of anthocyanins in skins was 2.34 mg/g FW at mature stage(120 days after flower).However,the expression of VdMYB14 gene was higher before the veraison,and continued to decrease from veraison to the mature period.In the process of berry development,the expression pattern of VdMYB14 gene was contrary to the trend of anthocyanin accumulation in grape skins,suggesting that VdMYB14 might inhibit anthocyanins accumulation in the early growth stages of grape berry.2.The CDS region of VdMYB14 gene is 804 bp in length,encoding 267 amino acids in total,and has two MYB characteristic structural domains of R2 and R3 in the region of 3-153 amino acids.The protein encoded by the gene has a relative molecular weight of 29958.61 Da and an isoelectric point of6.51.Using tobacco transient expression to localize the location of VdMYB14 protein in cells,we found that the fluorescent signal of VdMYB14 fusion protein was mainly distributed in the nucleus,indicating that VdMYB14 mainly played a transcriptional regulatory role in the nucleus.Phylogenetic analysis showed that the VdMYB14 protein was clustered in the branch of MYB transcriptional repressors,and had high homology with apple MdMYB111.The result suggest that VdMYB14 might negatively regulate anthocyanin synthesis.3.After heterologous expression of VdMYB14 gene,the anthocyanins content in the corolla of transgenic tobacco was significantly lower than that of wild type,but the proanthocyanidins content was higher than that of wild type.VdMYB14 gene can down-regulate the expression of UFGT gene in anthocyanin synthesis pathway and up-regulate the expression of LAR and ANR genes inproanthocyanin pathway,resulting in the transfer of intermediate products of flavonoid pathway from anthocyanin pathway to proanthocyanin pathway,thus inhibiting anthocyanin synthesis of transgenic tobacco flowers and affecting the normal coloration of corolla.The results showed that VdMYB14 could effectively inhibit the anthocyanin synthesis,and the specific regulatory mechanism still needs further study to elucidate.