Cloning and expression of the VvbHLH79 gene involved in grape tartaric acid biosynthesis

Abstract

Abstract [Objective] Tartaric acid not only affects the taste of grape berries but also determines the color, texture, microbial stability, and aging potential of the wine. It is of great value to explore the transcription factors and mechanisms that regulate tartaric acid biosynthesis, regulate the accumulation of tartaric acid in grapes through biotechnology or to cultivate wine grape varieties with high tartaric acid accumulation. [Methods] In this study, using ‘Traminer’ and ‘Marselan’ wine grapes as experimental materials, we cloned the tartaric acid related transcription factor VvbHLH79 by PCR. We performed bioinformatic and subcellular localization analyses of VvbHLH79. Furthermore, we measured the tartaric acid content and VvbHLH79 expression levels in grape berries at different developmental stages using HPLC and qRTPCR techniques. We also constructed a plant expression vector and the expression and tartaric acid content of VvbHLH79 were detected after transient transformation of young grape berries using Agrobacteriummediated fruiting pedicle infection, in order to clarify the role of the VvbHLH79 gene in regulating the content of tartaric acid in grape berries. [Results] (1) A grapevine VvbHLH79 gene was obtained by RNA-seq, with a CDS of 831 bp, amino acids of 277, molecular weight of 66.22 kD, and isoelectric point of 5.15. It belonged to the class of unstable hydrophilic proteins without signal peptides and transmembrane structures. Positions 147-232 of amino acid sequence contained the bHLH-SF conserved domain, which belonged to the bHLH transcription factor family. The protein was localized in the cell nucleus. (2) The grapevine VvbHLH79-encoded protein had the highest sequence similarity with the bHLH89-encoded protein in riverbank grape (accession number: XP_034699340.1), with a sequence identity of 99.64%. (3) There was a gradual decrease of VvbHLH79 expression during the development of ‘Traminer’ grape fruits, decreasing 67.25% from veraison to harvest. The accumulation of tartaric acids occurred mainly before color change of the grapes, with a rapid decrease after color change. (4) Transient expression of VvbHLH79 in low tartaric acid grapes resulted in a increase in tartaric acids, whereas silencing of VvbHLH79 in high tartaric acid grapes resulted in a decrease in tartaric acids, confirming that the VvbHLH79 gene positively regulated tartaric acid content. [Conclusion] The VvbHLH79 transcription factor may play a role in the tartaric acid biosynthesis in grape berries.

Keywords

• vitis vinifera
• vvbhlh79
• transcription factors
• tartaric acid
• gene cloning