Comparative proteomics analysis of developing buds in Brassica napus L.
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摘要:
甘蓝型油菜（Brassica napus L.）是我国目前的主栽油菜类型，其花蕾发育包含一系列复杂的代谢和调控过程，尤其是小孢子成熟前的早期发育过程对油菜育性影响较大。本研究首先通过组织形态学观察，确定甘蓝型油菜花蕾大小在1~3 mm时期，对应于雄蕊发育的5~9期。利用高通量的HPLC-MS/MS质谱测序技术，对该发育时段的花蕾进行蛋白质组学分析，共鉴定到13 444 个蛋白，比较各时期鉴定到的蛋白筛选出613 个差异表达蛋白。对差异蛋白的功能进行分析，发现细胞壁建成、脂质代谢、细胞转运相关蛋白大量上调，这将有利于花蕾发育过程中的细胞建成和花粉发育；核酸相关蛋白表达与含量变化迅速，这与该时段细胞的急剧分裂与生长的代谢过程一致；而ABCDE发育模型相关蛋白的表达未发生显著变化，可能具有除诱导花蕾分化外的其他功能。研究结果说明油菜花蕾发育早期细胞代谢旺盛，大量代谢相关蛋白发生了显著变化，这将为进一步分析花蕾发育过程中的代谢及调控过程提供基础。
Abstract:Brassica napus L., the predominant variant of rapeseed cultivated in China, undergoes a series of complex metabolic and regulatory processes during flower bud development, especially the early developmental stages preceding microspore maturation, which significantly impacts fertility. Based on phenotypic and histological observations, flower bud sizes ranging from 1 mm to 3 mm in B. napus were found to correspond to the 5th–9th stages of angiosperm stamen development. Using high-throughput high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) sequencing technology, proteomics analysis was conducted on these flower buds. In total, 13 444 proteins were identified, including 613 differentially expressed proteins (DEPs). Functional analysis of the DEPs revealed a significant up-regulation in cell wall building, lipid metabolism, and cell transport-related proteins, crucial for cell development and pollen formation during the early stage of flower bud development. The expression of nucleic acid-related proteins markedly changed, consistent with rapid cell division and growth during this period. However, no significant changes were found in the expression of proteins related to the ABCDE development model, suggesting these proteins may have other functions beyond inducing flower bud differentiation. This study systematically studied the differences in tissue morphology and protein expression during the early stages of bud development in B. napus, paving the way for further research on the metabolism and regulation of flower bud development.
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Keywords:
- Brassica napus /
- Bud /
- Comparative Proteomics /
- Development
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图 1 甘蓝型油菜597花蕾发育形态观察
A：成熟花蕾的各组织；B：不同发育阶段的花蕾；C：不同发育阶段的花粉粒醋酸洋红染色检测。a~e：分别来自1~5 mm大小花蕾的花粉粒。
Figure 1. Morphology of rapeseed line 597
A: Tissues of mature flower buds; B: Buds at different developmental stages; C: Acetic acid magenta staining of pollen grains at different developmental stages. a–e: Pollen grains from 1–5 mm buds, respectively.
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图 2 油菜597不同发育阶段花蕾组织切片图
E：外表皮；En: 内表皮；T：绒毡层；Ms：花粉母细胞。
Figure 2. Histogram of flower buds of rapeseed line 597 at different developmental stages
E: Epidermis; En: Endothecium; T: Tapetum; Ms: Microsporocytes.
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图 3 不同发育时期597花蕾的蛋白质组学分析
A：597花蕾不同发育阶段各3次技术重复鉴定到的蛋白数量；B：鉴定蛋白的韦恩图分析；C：597花蕾不同发育阶段各3次技术重复鉴定到的蛋白主成分分析，X轴差异度为56.25%，Y轴差异度为20.86%；D：不同发育时期花蕾蛋白的差异数量分布。
Figure 3. Proteomics analysis of 597 buds at different developmental stages
A: Number of proteins identified in 597 buds with three technical replicates; B: Venn analysis of identified proteins; C: Principal component analysis of identified proteins; the Y and X axises present 56.25% and 20.86% difference, respectively; D: Distribution of DEPs at different developmental stages.
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图 4 油菜597不同大小花蕾的蛋白质组分析
A：差异蛋白的表达热图和HCL聚类分析；B：差异蛋白的K-mean分类，共分为4类（C1~C4）；C：差异蛋白的MapMan功能注释，上：每个MapMan功能分类中差异蛋白的数量，下：每个MapMan功能分类中的差异蛋白占C1~C4各类蛋白的比例。
Figure 4. Proteomics analysis of developing flower buds in rapeseed line 597
A: Heatmap and HCL clustering of all DEPs; B: K-mean clusters of all DEPs; C: MapMan annotation of all DEPs in four clusters, Up: number of DEPs in each cluster, Down: percentage of DEPs in each cluster.
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