Differential expression analysis of flowering-related genes in stem apex tissues of flowering Chinese cabbage based on transcriptome sequencing
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摘要: 【目的】基于转录组测序数据分析鉴定调控菜心抽薹开花相关的候选基因及信号通路，为今后阐明菜心抽薹开花调控分子机制及选育不同熟性菜心品种提供理论参考。【方法】对四九-19号（早熟型）和80天油绿（晚熟型）菜心的七叶期和现蕾期茎尖组织进行转录组测序，比较不同熟性菜心品种间及不同生长时期间的差异表达基因（DEGs），并对其进行GO功能注释及KEGG信号通路富集分析，结合FLOR-ID和UniProt数据库筛选出不同熟性菜心参与调控抽薹开花时间的候选基因及信号通路，通过实时荧光定量PCR验证转录组数据的可靠性。【结果】菜心茎尖组织转录组测序数据为81.93 Gb，经质控过滤后得到536679570条Clean reads，GC含量为47.24%~47.63%，Q20为97.11%~98.00%，Q30为91.97%~94.11%，平均87.98%的Clean reads比对上参考基因组。四九-19号菜心的七叶期和现蕾期间有1965个DEGs，80天油绿菜心的七叶期和现蕾期有6007个DEGs。GO功能注释结果显示，涉及分子功能的DEGs最多，主要注释为蛋白质二聚化活性、血红素结合和四吡咯结合等；其次是生物学过程，主要注释为小分子代谢过程、脂质代谢过程和离子运输等过程；细胞成分类别中，DEGs主要注释为核糖体、细胞器部分、染色体、核小体、蛋白质－DNA复合物和DNA包装复合体。KEGG信号通路富集结果显示，四九-19号菜心七叶期和现蕾期间的DEGs显著（Padj<0.05，下同）富集在光合生物中的碳固定及角质、木栓质和蜡生物合成等通路；80天油绿菜心七叶期和现蕾期间的DEGs显著富集在苯丙素生物合成、DNA复制和植物激素信号转导等通路。四九-19号和80天油绿菜心品种间有750个DEGs，其中16个为开花相关基因，涉及光周期途径、自主途径、赤霉素途径、春化途径、温度途径和年龄途径等，部分开花基因在2个菜心品种中表达量存在明显差异。实时荧光定量PCR检测结果与转录组测序结果相关性较强。【结论】菜心抽薹开花过程受到光周期途径、自主途径和赤霉素途径等关键途径调控，同时受到春化途径、温度途径和年龄途径的影响。虽然2个不同熟性菜心品种开花调控所涉及的途径基本相同，但开花调控途径中的基因响应不一致，导致二者开花时间存在差异。
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关键词:
- 菜心 /
- 茎尖组织 /
- 转录组 /
- 差异表达基因(DEGs) /
- 开花调控
Abstract: 【Objective】The purpose of the study was to analyze and identify candidate genes and signal pathways related to the regulation of bolting and flowering of flowering Chinese cabbage based on transcriptome sequencing data， in order to explore the regulatory molecular mechanism of bolting and flowering of flowering Chinese cabbage and provide theoretical reference for the selection and breeding of flowering Chinese cabbage varieties with different maturity properties in the future. 【Method】Transcriptome sequencing was performed on stem apex tissues at both seven-leaf stage and budding stage of two flowering Chinese cabbage varieties， Siju-19 （early-maturing type） and 80 Tian Youlü （latematuring type）. Differentially expressed genes （DEGs） between different maturity properties of flowering Chinese cabbage and between different growth stages were compared. GO functional annotation and KEGG signal pathway enrichment analysis were conducted to analyze DEGs. Candidate genes and signal pathways involved in the regulation of bolting and flowering time of flowering Chinese cabbage with different maturity properties were selected by integrating FLOR-ID and UniProt databases. The reliability of transcriptome data was verified through real-time fluorescence quantitative PCR. 【Result】Transcriptome sequencing data of flowering Chinese cabbage stem apex tissues was 81.93 Gb， and 536679570 clean reads were obtained after quality control filtering. The GC content ranged from 47.24% to 47.63%， with Q20 between 97.11% and 98.00%， and Q30 between 91.97% and 94.11%. On average， 87.98% of the data were aligned to the reference genome. A total of 1965 and 6007 DEGs were identified in Siju-19 and 80 Tian Youlü during the seven-leaf stage and the budding stage， respectively. GO functional annotation results revealed that the majority of the DEGs were associated with molecular function， mainly annotated as protein dimerization activity， heme binding and tetrapyrrole binding. Followed by biological process， which was mainly annotated as small molecule metabolic process， lipid metabolic process and ion transport. In the cellular component category， the DEGs were mainly annotated as ribosomes， organelles part， chromosomes， nuclesome， protein-DNA complexes and DNA packaging complexes. The results of KEGG signal pathway enrichment analysis showed that DEGs of Sijiu-19 flowering Chinese cabbage were significantly enriched （adj<0.05， the same below） in photosynthetic organisms pathways such ascarbon fixation ， cutin， suberin and wax biosynthesis. DEGs of 80 Tian Youlü flowering Chinese cabbage were significantly enriched in pathways such as phenylpropanoid biosynthesis， DNA replication and plant hormone signal transduction during the seven-leaf stage and the budding stage. There were 750 DEGs between Sijiu-19 and 80 Tian Youlü， 16 of which were flowering-related genes， involving the photoperiod pathway， autonomous pathway， gibberellin pathway， vernalization pathway， temperature pathway and age pathway. Some flowering-related genes showed obvious differences in expression between the two flowering Chinese cabbage varieties. The results of real-time fluorescence quantitative PCR were strongly correlated with those of transcriptome sequencing. 【Conclusion】The bolting and flowering process of flowering Chinese cabbage is regulated by key pathways such as the photoperiodic pathway， autonomous pathway and gibberellin pathway， while it is also affected by vernalization pathway， temperature pathway and age pathway. The pathways involved in the flowering regulation of two flowering Chinese cabbage varieties of different maturity are fundamentally consistent. However， the inconsistent gene responses in the flowering regulatory pathway leads to the differences in flowering time between the two flowering Chinese cabbage varieties. -
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