Proteomics study of mature rice seeds based on 4D label-free technology
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摘要:目的
从蛋白水平揭示水稻种子成熟的分子基础，探究调控水稻种子成熟的关键蛋白和代谢通路。
方法选用授粉后30 d的成熟水稻种子，利用4D label-free定量蛋白质组学进行质谱鉴定，通过生物信息学技术分析蛋白的亚细胞定位、结构域、GO注释和KEGG通路注释。
结果总共鉴定了3 484个种子成熟期的蛋白，相对分子质量大多在10 000~100 000之间，主要分布于细胞质、细胞核、叶绿体、线粒体和质膜上；结构域主要涉及蛋白质翻译的RNA识别基序和蛋白磷酸化修饰的蛋白激酶结构域；GO分析表明，成熟种子的蛋白主要参与了细胞过程和代谢过程，主要涉及催化活性和结合等功能，大多分布在细胞、细胞组分、细胞器和细胞膜等部位；KEGG分析发现，蛋白主要富集在核糖体、内质网中的蛋白质加工、氧化磷酸化和糖酵解等途径，推测蛋白质的翻译、加工和能量代谢是水稻种子成熟期的主要分子事件；进一步鉴定了脱落酸(Abscisic acid，ABA)信号和吲哚乙酸(Indoleacetic acid，IAA)代谢的相关蛋白，同时也发现了NAC(NAM、ATAF1/2 和 CUC2)家族的转录因子。
结论贮藏物质的积累和能量代谢是水稻种子成熟期的典型特征，ABA和IAA信号途径参与了种子成熟过程。
Abstract:ObjectiveTo reveal the molecular basis of rice seed maturation at the protein level, and explore the key proteins and metabolic pathways involved in regulating rice seed maturation.
MethodThe experiment used mature rice seeds at 30 days after pollination, and conducted mass spectrum identification using 4D label-free quantitative proteomics. Bioinformatics techniques were used to analyze the subcellular localization, domains, GO annotations, and KEGG pathway annotations of the proteins.
ResultA total of 3 484 seed maturation proteins were identified, with most having a relative molecular mass between 10 000 and 100 000. They were mainly distributed in the cytoplasm, nucleus, chloroplasts, mitochondria, and plasma membrane. The protein domain mainly involved the RNA recognition motifs of protein translation and the protein kinase domains of protein phosphorylation modification. GO analysis showed that the proteins of mature seeds were mainly involved in cellular and metabolic processes, mainly related to catalytic activity and binding functions, and were mostly distributed in cell, cellular component, organelle, and cytomembrane. KEGG analysis revealed that proteins were mainly enriched in pathways such as ribosome, protein processing in endoplasmic reticulum, oxidative phosphorylation, and glycolysis. It was speculated that protein translation, processing, and energy metabolism were the main molecular events of rice seed maturation. Furthermore, proteins related to abscisic acid (ABA) signaling and indoleacetic acid (IAA) metabolism were identified, and transcription factors of the NAM, ATAF1/2 and CUC2 (NAC) family were also discovered.
ConclusionThe accumulation of storage substances and energy metabolism are typical characteristics of rice seed maturation, and ABA and IAA signaling pathways are involved in the process of seed maturation.
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Keywords:
- Rice /
- Seed maturation /
- Metabolic pathway /
- Plant hormone
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图 1 不同发育阶段水稻种子的形态
图中比例尺长度为0.5 cm
Figure 1. Morphology of rice seeds at different development stages
The scale bars in figure are 0.5 cm
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图 2 总蛋白SDS-PAGE电泳图
M：蛋白Marker；1~3：蛋白样品
Figure 2. SDS-PAGE electrophoresis of total protein
M: Protein marker; 1−3: Protein samples
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图 5 亚细胞定位分析
括号中数据为占比
Figure 5. Subcellular localization analysis
The data in brackets are the proportion
表 1 成熟水稻种子中的植物激素和转录因子相关蛋白
Table 1 Proteins related to plant hormone and transcription factor in mature rice seeds
蛋白
Protein蛋白描述
Protein description基因
Gene平均强度
Average intensity分类
ClassificationA2ZAH5 ABA受体 ABA receptor OsRCAR10 415900 脱落酸 ABA Q7XQP4 丝氨酸/苏氨酸蛋白激酶
Serine/threonine-protein kinaseOsSAPK7 294850 脱落酸 ABA A2YDM7 ABA受体 ABA receptor OsRCAR7 75716 脱落酸 ABA Q8H3C8 IAA−氨基酸水解酶
IAA-amino acid hydrolaseOsILL8 296457 生长素 Auxin Q2QZU4 生长素抑制蛋白
Auxin-repressed proteinOsARP1 278910 生长素 Auxin Q84XG9 IAA−氨基酸水解酶
IAA-amino acid hydrolaseOsILL1 242487 生长素 Auxin Q01IX6 生长素氧化双加氧酶
Dioxygenase for auxin oxidationDAO 144843 生长素 Auxin P27934 α−淀粉酶 Alpha-amylase OsAmy3E 813397 赤霉素 GA B9EZ51 α−淀粉酶 Alpha-amylase OsAmy4A 604527 赤霉素 GA Q8H484 β−淀粉酶 Beta-amylase OsBmy7 204723 赤霉素 GA Q8RUI4 NAC转录因子
NAC transcription factorOs01g0938900 1154207 转录因子
Transcription factorA2Y3Z4 含有NAC-A/B结构域蛋白
NAC-A/B domain-containing proteinOs05g0373700 457820 转录因子
Transcription factorA2Y4P5 含NAC结构域蛋白
NAC domain-containing proteinOsNAC24 362233 转录因子
Transcription factorQ6H8A9 含NAC结构域蛋白
NAC domain-containing proteinOsNAC23 200103 转录因子
Transcription factorA2WJP3 含NAC结构域蛋白
NAC domain-containing proteinOsNAC20 132343 转录因子
Transcription factor
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