基于转录组测序的宁夏枸杞不同品种果实活性成分合成差异表达基因分析

Comparative analysis of differentially expressed genes for biosynthesis of active ingredients in fruits of different cultivars of Lycium barbarum L. based on transcriptome sequencing

摘要:

为探究不同品种宁夏枸杞果实活性成分生物合成相关基因的表达水平，筛选关键差异表达基因（differentially expressed genes，DEGs），揭示宁夏枸杞品种间活性成分含量差异的分子机制，本研究采用Illumina NovaSeq 6000高通量测序技术，对宁夏枸杞‘宁杞1号’和‘宁杞7号’青果期、转色期及成熟期果实进行转录组测序，比较2个品种果实不同发育期相关基因表达谱的变化。结果显示：转录组测序共获得811 818 178条clean reads，有121.76 Gb有效数据。‘宁杞1号’和‘宁杞7号’在青果期、转色期和成熟期差异表达基因分别有2 827、2 552和2 311个；分别有2 153、2 050和1 825个差异基因在基因本体论（gene ontology，GO）、京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）富集分析和同源蛋白簇（clusters of orthologous groups of proteins，KOG）分析等6个数据库中被成功注释。青果期、转色期和成熟期果实的差异表达基因，在GO数据库分别有1 307、865和624个被富集到生物学过程、细胞组分及分子功能3个部分中；KEGG通路富集结果均集中在代谢途径、次生代谢物生物合成和植物-病原互作过程；在KOG数据库，3个发育期分别注释了1 775、1 751和1 541个差异表达基因。对注释的基因进行PubMed数据库检索，在青果期、转色期和成熟期分别筛选到与枸杞活性成分合成相关的差异表达基因18、26和24个，这些基因主要参与类胡萝卜素、类黄酮、萜类、生物碱和维生素等代谢途径。选取7个差异表达基因进行RT-qPCR验证，结果与转录组测序数据表达趋势一致。本研究从转录水平为不同品种宁夏枸杞活性成分含量差异提供了初步证据，为进一步挖掘枸杞活性成分生物合成的关键基因及解析其表达调控机制提供了研究基础。

Abstract:

To explore the differentially expressed genes (DEGs) related to biosynthesis of active ingredients in wolfberry fruits of different varieties of Lycium barbarum L. and reveal the molecular mechanism of the differences of active ingredients, we utilized Illumina NovaSeq 6000 high-throughput sequencing technology to conduct transcriptome sequencing on the fruits of ‘Ningqi No.1’ and ‘Ningqi No.7’ during the green fruit stage, color turning stage and maturity stage. Subsequently, we compared the profiles of related gene expression in the fruits of the two varieties at different development stages. The results showed that a total of 811 818 178 clean reads were obtained, resulting in 121.76 Gb of valid data. There were 2 827, 2 552 and 2 311 DEGs obtained during the green fruit stage, color turning stage and maturity stage of ‘Ningqi No. 1’ and ‘Ningqi No. 7’, respectively, among which 2 153, 2 050 and 1 825 genes were annotated in six databases, including gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG) and clusters of orthologous groups of proteins (KOG). In GO database, 1 307, 865 and 624 DEGs of green fruit stage, color turning stage and maturity stage were found to be enriched in biological processes, cell components and molecular functions, respectively. In the KEGG database, the DEGs at three developmental stages were mainly concentrated in metabolic pathways, biosynthesis of secondary metabolites and plant-pathogen interaction. In KOG database, 1 775, 1 751 and 1 541 DEGs were annotated at three developmental stages, respectively. Searching the annotated genes against the PubMed database revealed 18, 26 and 24 DEGs related to the synthesis of active ingredients were mined at the green fruit stage, color turning stage and maturity stage, respectively. These genes are involved in carotenoid, flavonoid, terpenoid, alkaloid, vitamin metabolic pathways, etc. Seven DEGs were verified by RT-qPCR, which showed consistent results with transcriptome sequencing. This study provides preliminary evidences for the differences in the content of active ingredients in different Lycium barbarum L. varieties from the transcriptional level. These evidences may facilitate further exploring the key genes for active ingredients biosynthesis in Lycium barbarum L. and analyzing their expression regulation mechanism.