【摘要】 【目的】克隆花青素合成关键基因VdF3′5′H2，分析其调控刺葡萄（Vitis davidii Fo?x）细胞花青素生物合成及组分含量比例的作用，为构建高产花青素的刺葡萄细胞和定向调控目标花青素组分生物合成提供技术支撑和理论依据。【方法】以刺葡萄细胞为研究材料，克隆VdF3′5′H2并构建植物表达载体，将其转化刺葡萄细胞，通过抗性筛选结合荧光观察和PCR鉴定阳性细胞系；分析转基因刺葡萄细胞的表型，测定其花青素、黄酮类化合物和原花青素含量，并利用UPLC-MS/MS检测以花青素为主的代谢物组分；使用实时荧光定量PCR(RT-qPCR)分析花青素合成相关基因的表达，并利用O2PLS法进行基因和差异代谢物的联合分析。【结果】VdF3′5′H2的ORF为1 527 bp，编码508个氨基酸残基；VdF3′5′H2蛋白与同科植物具有较高的同源性，均包含CYP75保守结构域、血红素结合位点和3个特征保守基序；系统进化分析发现F3′H与F3′5′H聚在同一大分支，显示F3′5′H从F3′H的进化关系，VdF3′5′H2在进化上比VdF3′5′H1更原始；VdF3′5′H2亚细胞定位于细胞质。在过表达VdF3′5′H2的2个转基因刺葡萄细胞中，花青素、黄酮类化合物和原花青素含量显著高于野生型对照。过表达VdF3′5′H2不仅使刺葡萄细胞中花青素组分的数量增加，而且显著促进了VdF3′5′Hs催化支路的花青素合成，2个转基因细胞的飞燕草素类花青素含量比例分别增加到7.82%(T6)和14.32%(T10)，矮牵牛素类分别增加到7.30%和10.16%，锦葵色素类分别增加到58.08%和42.30%，而野生型细胞中3类花青素的含量比例分别为1.92%、1.48%和8.49%；同时，2个转基因细胞中矢车菊素类和芍药花素类花青素含量比例大幅度下降。过表达VdF3′5′H2使刺葡萄细胞中PAL、CHS、CHI、F3H等基因表达量下调，而VdF3′5′H1、LDOX和UFGT表达量上调。基因表达与差异代谢物的联合分析表明，过表达VdF3′5′H2是通过调控花青素合成相关基因的表达进而调节不同种类花青素及其组分的合成与积累。【结论】过表达VdF3′5′H2显著促进刺葡萄细胞花青素生物合成与累积，通过调控基因表达改变花青素合成支路代谢流，进而调控刺葡萄细胞中花青素的组成和含量比例。更多还原

【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 Fo?x) 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.更多还原