| As one of the most important secondary metabolites presented in red grape berries and wines, anthocyanins are responsible for the color and flavor of red grapes and wines. It has been widely accepted that most Vitis vinifera grape berries only contain anthocyanidin 3-0-glucosides, whereas non-V. vinifera and their hybrids contain both the anthocyanidin 3-O-glucosides and anthocyanidin 3,5-O-diglucosides.In this study, we analyzed the anthocyanin profile in Cabernet Sauvignon (V. vinifera L. cv) grape berries from different wine-producing regions in China, and detected the presence of anthocyanidin 3,5-O-diglucosides in the grape skins. We also confirmed the authenticity of the Cabernet Sauvignon grapevines by the help of SSR technology.To explain why anthocyanidin 3,5-O-diglucosides are detected in the Cabernet Sauvignon grapes, we designed the specific primers and cloned the full-length fragments of the four 5GT candidates according to previous report with bioinformatics methods. Multiple sequence alignment was performed, the potential biological functions were predicted and the phylogenetic tree was constructed. The recombination proteins of the target genes were obtained by using prokaryotic expression technique. An enzyme activity analysis revealed that among the recombination proteins of the four 5GT candidates, only Vv5GT3 recombinant protein could convert anthocyanidin 3-O-glucoside into anthocyanidin 3,5-O-dglucoside. A further analyze of substrate specificity and biochemical properties of the recombinant Vv5GT3 protein indicated that Vv5GT3 has the activities of both 3GT and 5GT, it could transfer the glycosyl moiety from UDP-glucose to both the 5-position and the 3-position of anthocyanidins and flavonols. Vv5GT3 had a higher affinity for anthocyanidin 3-O-glucosides and the highest catalytic activity of which was at pH 8.0 and around 35℃.To further understand the regulation of Vv5GT3 at a transcriptional level, we analyzed the relative expression of Vv5GT3 at the transcriptional level in the Cabernet Sauvignon grape berries of different developmental stages among different growing regions by using Real-Time PCR technique. In addition, the promoter of Vv5GT3 was PCR-amplified and the bioinformatic analysis of the promoter sequence was carried out. The results showed that the promoter of Vv5GT3 contained multiple cis-acting elements, could be activated in response to environmental factors. The promoter was then translationally fused to the luciferase reporter gene and subsequently transferred into Agrobacterium. Agrobacterium-infected leafs were cultured under different light conditions. The results revealed that activity of the Vv5GT3 gene promoter could be enhanced by strong light and weakened by shading treatment.The subcellular localizations of the proteins encoded by Vv5GT3 in plant cells were achieved by using transient expression technique. The result revealed that the protein encoded by Vv5GT3 was localized in the cytoplasm. To understand the biological function of Vv5GT3 in vivo, transgenic tobacco plants expressing Vv5GT3 were produced and analyzed. The relative expression of Vv5GT3 at the transcriptional level in different tissues of transgenic tobacco plants and the anthocyanin profiles of transgenic tobacco flowers were analyzed. The results showed that Vv5GT3 was transcriptionally expressed in flowers, leaves and stems of transgenic tobacco plants; anthocyanidin 3,5-O-diglucosides were detected in transgenic tobacco flowers while not detected in the control group, indicating that VvGT3 has indeed a functional activity of conversing anthocyanidin 3-O-glucosides into anthocyanidin 3,5-O-diglucosides in vivo.On the one hand, this finding brought a new understanding of the anthocyanins’ profile and their biosynthesis in V. vinifera; on the other hand, it answered whether there were anthocyanidin 3,5-O-diglucosides in V. vinifera or not and would be helpful for the identification of wine grapes in production practice. |
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