College of Agriculture, Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu, 030801, Shanxi, China.
Shanxi Key Laboratory of Germplasm Innovation and Molecular Breeding of Minor Crop, Taigu, 030801, Shanxi, China.
BMC Plant Biol. 2022 Mar 8;22(1):105. doi: 10.1186/s12870-022-03467-2.
The grains of foxtail millet are enriched in carotenoids, which endow this plant with a yellow color and extremely high nutritional value. However, the underlying molecular regulation mechanism and gene coexpression network remain unclear.
The carotenoid species and content were detected by HPLC for two foxtail millet varieties at three panicle development stages. Based on a homologous sequence BLAST analysis, these genes related to carotenoid metabolism were identified from the foxtail millet genome database. The conserved protein domains, chromosome locations, gene structures and phylogenetic trees were analyzed using bioinformatics tools. RNA-seq was performed for these samples to identify differentially expressed genes (DEGs). A Pearson correlation analysis was performed between the expression of genes related to carotenoid metabolism and the content of carotenoid metabolites. Furthermore, the expression levels of the key DEGs were verified by qRT-PCR. The gene coexpression network was constructed by a weighted gene coexpression network analysis (WGCNA).
The major carotenoid metabolites in the panicles of DHD and JG21 were lutein and β-carotene. These carotenoid metabolite contents sharply decreased during the panicle development stage. The lutein and β-carotene contents were highest at the S1 stage of DHD, with values of 11.474 μg /100 mg and 12.524 μg /100 mg, respectively. Fifty-four genes related to carotenoid metabolism were identified in the foxtail millet genome. Cis-acting element analysis showed that these gene promoters mainly contain 'plant hormone', 'drought stress resistance', 'MYB binding site', 'endosperm specific' and 'seed specific' cis-acting elements and especially the 'light-responsive' and 'ABA-responsive' elements. In the carotenoid metabolic pathways, SiHDS, SiHMGS3, SiPDS and SiNCED1 were more highly expressed in the panicle of foxtail millet. The expression of SiCMT, SiAACT3, SiPSY1, SiZEP1/2, and SiCCD8c/8d was significantly correlated with the lutein content. The expression of SiCMT, SiHDR, SiIDI2, SiAACT3, SiPSY1, and SiZEP1/2 was significantly correlated with the content of β-carotene. WGCNA showed that the coral module was highly correlated with lutein and β-carotene, and 13 structural genes from the carotenoid biosynthetic pathway were identified. Network visualization revealed 25 intramodular hub genes that putatively control carotenoid metabolism.
Based on the integrative analysis of the transcriptomics and carotenoid metabonomics, we found that DEGs related to carotenoid metabolism had a stronger correlation with the key carotenoid metabolite content. The correlation analysis and WGCNA identified and predicted the gene regulation network related to carotenoid metabolism. These results lay the foundation for exploring the key target genes regulating carotenoid metabolism flux in the panicle of foxtail millet. We hope that these target genes could be used to genetically modify millet to enhance the carotenoid content in the future.
黍稷的籽粒富含类胡萝卜素，使黍稷呈现黄色，营养价值极高。然而，其分子调控机制和基因共表达网络尚不清楚。
利用高效液相色谱法（HPLC）检测两个黍稷品种三个穗发育阶段的类胡萝卜素种类和含量。基于同源序列 BLAST 分析，从黍稷基因组数据库中鉴定出与类胡萝卜素代谢相关的基因。利用生物信息学工具分析保守蛋白结构域、染色体位置、基因结构和系统发育树。对这些样品进行 RNA-seq 以鉴定差异表达基因（DEGs）。通过 Pearson 相关性分析，对类胡萝卜素代谢相关基因的表达与类胡萝卜素代谢物含量进行相关性分析。此外，通过 qRT-PCR 验证关键 DEGs 的表达水平。通过加权基因共表达网络分析（WGCNA）构建基因共表达网络。
DHD 和 JG21 穗中的主要类胡萝卜素代谢物为叶黄素和β-胡萝卜素。这些类胡萝卜素代谢物含量在穗发育阶段急剧下降。DHD 的 S1 期叶黄素和β-胡萝卜素含量最高，分别为 11.474μg/100mg 和 12.524μg/100mg。在黍稷基因组中鉴定出 54 个与类胡萝卜素代谢相关的基因。顺式作用元件分析表明，这些基因启动子主要含有“植物激素”、“抗旱”、“MYB 结合位点”、“胚乳特异性”和“种子特异性”顺式作用元件，特别是“光响应”和“ABA 响应”元件。在类胡萝卜素代谢途径中，SiHDS、SiHMGS3、SiPDS 和 SiNCED1 在黍稷穗中表达较高。SiCMT、SiAACT3、SiPSY1、SiZEP1/2 和 SiCCD8c/8d 的表达与叶黄素含量显著相关。SiCMT、SiHDR、SiIDI2、SiAACT3、SiPSY1 和 SiZEP1/2 的表达与β-胡萝卜素含量显著相关。WGCNA 显示珊瑚模块与叶黄素和β-胡萝卜素高度相关，鉴定出类胡萝卜素生物合成途径中的 13 个结构基因。网络可视化显示 25 个具有潜在功能的基因，它们可能控制着类胡萝卜素代谢。
通过转录组学和类胡萝卜素代谢组学的综合分析，我们发现与类胡萝卜素代谢相关的差异表达基因与关键类胡萝卜素代谢物含量的相关性更强。相关性分析和 WGCNA 鉴定并预测了与类胡萝卜素代谢相关的基因调控网络。这些结果为探索黍稷穗中调控类胡萝卜素代谢通量的关键靶基因奠定了基础。我们希望这些靶基因可用于未来对谷子进行遗传改良，以提高类胡萝卜素含量。
