Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (4): 802-818.doi: 10.3864/j.issn.0578-1752.2025.04.014

• RESEARCH NOTES • Previous Articles     Next Articles

Effect of VdF3′5′H2 Overexpression on the Accumulation of Anthocyanin Composition in Spine Grape Cells

GUO AoLin1,2(), LIN JunXuan1,2(), LAI GongTi2, HE LiYuan2, CHE JianMei3, PAN Ruo2, YANG FangXue1,2, HUANG YuJi1, CHEN GuiXin1(), LAI ChengChun2()   

  1. 1 College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002
    2 Institute of Food Science and Technology, Fujian Academy of Agricultural Sciences/Key Laboratory of Processing of Subtropical Characteristic Fruits, Vegetables and Edible Fungi, Ministry of Agriculture and Rural Affairs of China, Fuzhou 350003
    3 Institute of Resources, Environment and Soil Fertilizer, Fujian Academy of Agricultural Sciences, Fuzhou 350003
  • Received:2024-09-30 Accepted:2024-11-20 Online:2025-02-16 Published:2025-02-24
  • Contact: CHEN GuiXin, LAI ChengChun

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Abstract:

【Objective】This study aims to clone the key gene VdF3′5′H2 involved in anthocyanin biosynthesis and analyze its regulatory role of anthocyanin biosynthesis and component content in spine grape (Vitis davidii Foëx) cells, thereby providing technical support and a theoretical foundation for constructing spine grape cells with high-yield anthocyanin production and enabling targeted regulation of anthocyanin biosynthesis.【Method】Using spine grape cells as materials, the VdF3′5′H2 was cloned and a plant expression vector was constructed. This vector was subsequently transformed into spine grape cell, with positive cell lines were screened in antibiotic media and identified through fluorescence observation and PCR analysis. The phenotypic characteristics of the transgenic spine grape cells were analyzed, along with quantification of anthocyanin, flavonoid, and proanthocyanidin. Additionally, the metabolite profile of anthocyanins was detected by UPLC-MS/MS. The expression levels of genes associated with anthocyanin biosynthesis were assessed using real-time quantitative PCR (RT-qPCR), and a comprehensive analysis of gene expression and differential metabolites was carried out by the O2PLS method.【Result】The open reading frame (ORF) of VdF3′5′H2 gene is 1 527 bp, encoding a protein comprising 508 amino acid residues. The VdF3′5′H2 protein exhibits high homology with its counterparts in related plants within the same family, containing the CYP75 conserved domain, a heme-binding site, and three characteristic conserved motifs. Phylogenetic analysis revealed that F3′H and F3′5′H cluster within the same branch, implying an evolutionary relationship from F3′H to F3′5′H, with VdF3′5′H2 positioned at a more ancestral state compared to VdF3′5′H1. Furthermore, subcellular localization indicated that the VdF3′5′H2 protein resides in the cytoplasm. The contents of anthocyanins, flavonoids and proanthocyanidins in two transgenic spine grape cells overexpression VdF3′5′H2 showed a significant increase compared to the wild type control. The content ratio of delphinidin-anthocyanins in the two transgenic cells increased to 7.82% (T6) and 14.32% (T10), respectively, Petunidin-anthocyanins increaseed to 7.30% and 10.16%, respectively, while the content ratio of malvidin- anthocyanins increased to 58.08% and 42.30%, respectively. In contrast, the content ratios of the three types of anthocyanins in the wild-type cells were 1.92%, 1.48%, and 8.49%, respectively. Additionally, the content ratios of cyanidin- and paeonidin-anthocyanins in the two transgenic cells decreased significantly. Overexpression of VdF3′5′H2 led to the downregulation of PAL, CHS, CHI and F3H expression levels, while upregulating the expression levels of VdF3′5′H1, LDOX and UFGT genes. The comprehensive analysis integrating gene expression data with differential metabolites showed that the overexpression of VdF3′5′H2 modulated the transcriptional regulation of genes involved in anthocyanin biosynthesis, thereby influencing both the synthesis pathways and accumulation patterns of various types of anthocyanins.【Conclusion】The overexpression of VdF3′5′H2 gene significantly enhanced the biosynthesis and accumulation of anthocyanins in spine grape cells, by regulating gene expression and altering the metabolic flux of the anthocyanin biosynthesis pathway, there by modulating the composition and content ratio of antocyanins in spine grape cells.

Key words: spine grape, cell culture, flavonoids 3′5′-hydroxylase (F3′5′H), anthocyanins, overexpression

Table 1

Medium for spine grape cell culture and genetic transformation"

培养基种类 Type of medium 配方 Formulation
继代培养基Subculture medium MS+1.0 mg·L-1 2,4-D+6.0 g·L-1琼脂+30.0 g·L-1蔗糖（pH 5.8）
MS+1.0 mg·L-1 2,4-D+6.0 g·L-1 Agar+30.0 g·L-1 Sucrose (pH 5.8)
MS重悬液MS resuspension solution MS+20 µmol·L-1 AS (pH 5.8)
NAA重悬液NAA resuspension solution 10 mmol·L-1 MgCl2+100 mmol·L-1 MES (pH 5.7)+100 µmol·L-1 AS
液体LB培养基Liquid LB medium 10 g·L-1 胰蛋白胨+10 g·L-1 NaCl+5 g·L-1酵母浸粉（pH 7.0）
10 g·L-1 Tryptone+10 g·L-1 NaCl+5 g·L-1 Yeast infusion powder (pH 7.0)
共培培养基Co-culture medium MS+100 µmol·L-1 AS+7.0 g·L-1琼脂+30.0 g·L-1蔗糖（pH 5.8）
MS+100 µmol·L-1 AS+7.0 g·L-1 Agar+30.0 g·L-1 Sucrose (pH 5.8)
抗性筛选培养基Resistance screening medium MS+1.0 mg·L-1 2,4-D+50 mg·L-1 Kan+100 mg·L-1 Tim+7.0 g·L-1琼脂+30.0 g·L-1蔗糖（pH 5.8）
MS+1.0 mg·L-1 2,4-D+50 mg·L-1 Kan+100 mg·L-1 Tim+7.0 g·L-1 Agar+30.0 g·L-1 Sucrose (pH 5.8)

Fig. 1

Multiple sequence alignment and phylogenetic tree of F3′5′H proteins A: Multiple sequence alignment. The same residues in different sequences are denoted by a dark blue shading, while varying colors represent distinct levels of similarity. Ⅰ represents P450 motif “PPGP”, Ⅱ represents Ⅰ helical motif “AGTDT”, and Ⅲ represents heme-binding domain “FGAGRRICAG”. The black triangle represents the heme-binding site. B: Phylogenetic tree"

Fig. 2

Construction of plant expression vector, identification of transformed cell lines, and determination of subcellular localization A: Schematic diagram of the expression vector; B: GFP fluorescence assay of non-transgenic and transgenic spine grape cells; C: PCR amplification of VdF3′5′H2 from non-transgenic and transgenic spine grape cells; D: Subcellular localization of VdF3'5'H2. 35S::GFP and 35S:: VdF3'5'H2-GFP represents empty vector and recombinant vector, respectively. Bar =50 μm"

Fig. 3

Phenotypes of different spine grape cells and contents of anthocyanins, flavonoids and proanthocyanidins A: Non-transgenic (WT) and transgenic cells of spine grape (cultured for 0 and 35 days); B: Contents of anthocyanins, flavonoids and proanthocyanidins. Different lowercase letters indicate statistically significant differences (P<0.05). The same as below"

Fig. 4

Differential analysis of anthocyanin metabolites between non-transgenic and transgenic spine grape cells (WT vs T6 vs T10) A: Clustering heat map of metabolites; B: Content ratio of different anthocyanin components"

Fig. 5

Expression levels of key genes involved in anthocyanin biosynthesis between non-transgenic and transgenic spine grape cells"

Fig. 6

Comprehensive analysis of key gene expressions and differential anthocyanin metabolites A: Summary pathway diagram of changes in anthocyanin metabolit levels between non-transgenic and transgenic spine grape cells (The differences are expressed by the Log2 fold change value); B: Gene expression heatmap (The differences are expressed by the Log2 value); C: Load map associated with differential metabolites and gene expressions (WT vs T6); D: Load map of associated with differential metabolites and gene expressions (WT vs T10). Cy: Cyanidin; Dp: Delphinidin; Pn: Peonidin; Mv: Malvidin; Pt: Petunidin; Pg: Pelargonidin; digluc: Diglucoside; rhamn: Rhamnoside; gluc: Glucoside; sophor: Sophoroside; glucur: Glucuride; dima: Dimalonyl; coum: Coumaryl; galact: Galactoside; arabin: Arabinosidase; malon: Malonyl; arabin: Arabinoside; xyl: Xyloside; sambubi: Sambubioside; rutin: Rutinoside; acet: Acetyl; Trisuc: Trisuccinyl"

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