1河北农业大学农学院 / 华北作物种质资源研究与利用教育部重点实验室 / 河北省种质资源实验室,保定 071001; 2 河北易园生态农业科技有限公司,保定 071000
1College of Agronomy,Hebei Agricultural University/North China Key Laboratory for Crop Germplasm Resources of Education Ministry/Hebei Germplasm Resources Laboratory,Baoding 071001; 2 Hebei Yiyuan Ecological Agriculture Technology Co,Ltd,Baoding 071000
Key project of science and technology research in colleges and universities of the department of education in Hebei Province (ZD2019051)， Key project of science and technology research of modern seed industry of the department of S&T in Hebei Province(19226363D)
花生是重要的油料和经济作物，花生种皮色泽存在较大差异，具有白色、红色、紫色、粉色及花斑类型，花斑种皮花生是其中的独特成员。有关花斑花生种皮花青素合成的分子机制存在深入研究的必要性。本研究以花斑种皮花生VG-02为研究材料，采用液相色谱串联质谱（LC-MS/MS）法检测不同发育阶段种皮中花青素的相对含量变化，共检测到12种与种皮颜色相关的代谢物质。在花生种皮着色区（F）与非着色区（B）开花下针（DAF）45天的F2-B2比较组中差异代谢产物最多，结果表明矢车菊素3-O-半乳糖苷和矢车菊素O-丁香酸含量着色区低于非着色区，差异倍数分别为0.63和2.35；松香花青素O-己糖苷，原花青素A1、A2、B2、B3、矢车菊素，花翠素，花翠素3-O葡萄糖苷，矢车菊素3-O-半乳糖苷含量着色区高于非着色区，差异倍数1.05~11.55。花翠素和矢车菊素是导致着色区与非着色区颜色差异的主要代谢物。RNA-seq分析表明，1050个差异基因中筛选出与花斑种皮颜色形成高度相关的差异表达基因共27个，包括3个PAL，1个C4H，2个CHS,1个F3H，1个F3’H，2个DFR，2个LAR，2个IAA，4个bHLH和9个MYB，其中上调13个下调14个。KEGG(Kyoto Encyclopedia of Genes and Genomes)分析表明与种皮颜色合成相关所富集的代谢通路有苯丙氨酸代谢，苯丙醇生物合成，黄酮和黄酮醇生物合成，类黄酮生物合成，植物激素信号转导以及昼夜节律植物，其中类黄酮生物合成代谢途径是花生种皮花斑形成最直接的代谢途径。对20个差异基因进行qPCR验证，结果表明差异基因qPCR表达趋势与转录组测序结果显著一致。本研究结果对进一步揭示花生花斑种皮花青素合成调控机制具有一定的参考意义。
Peanut (Arachis hypogaea L.) is an important oil crop with economical interest. Peanut testa showed difference in colors, such as white, red, purple, pink and variegated. The molecular mechanism of the anthocyanin synthesis in variegated testa merit further in-depth research. In this study, the variegated testa peanut VG-02 was analyzed using Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis, in order to detect the relative changes in content of anthocyanins in variegated testa at different developmental stages. Twelve metabolites associating with the testa color were detected. In the comparison of F2-B2 at DAF45 (45 days after flowering and needling) stage the highest number of differential metabolites was revealed. The relative contents of cyanidin 3-O-galactoside and cyanidin O-syringic acid were lower by 0.63 and 2.35 fold in the pigmented area than that in the non-pigmented area, respectively. The relative contents of rosinidin O-hexoside, procyanidin A1, A2, B2, B3, cyanidin, delphinidin, delphinidin 3-O glucoside and cyanidin 3-O-galactoside were higher by 1.05 to 11.55 fold in the pigmented area than that in the non-pigmented area, respectively. Cyanidin and delphinidin were the main metabolites that caused the difference in color between the pigmented area and the non-pigmented area. RNA-seq analysis revealed 1,050 differentially-expressed genes (DEGs), which included 27 DEGs highly related to the synthesis of variegated testa color. Thirteen were up-regulated and 14 were down-regulated, including 3 PALs, 1 C4H, 2 CHSs, 1 F3H, 1 F3'H, 2 DFRs, 2 LARs, 2 IAAs, 4 bHLHs, and 9 MYBs. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis suggested that the metabolic pathways related to anthocyanin biosynthesis included phenylalanine metabolism, phenylpropanoid biosynthesis, flavone and flavonol biosynthesis, flavonoid biosynthesis, plant hormone signal transduction and circadian rhythm plant, among which flavonoid biosynthesis was proposed to be relevant with the testa variegation. Moreover, twenty DEGs were subjected for qPCR analysis. The similarity on transcriptional pattern revealed by qPCR and RNA-seq was observed. Collectively, the results of this study should help to further reveal the molecular regulatory mechanism of anthocyanin synthesis of the variegated testa in peanut.