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榛子正常发育与败育子房差异蛋白谱对比分析

程云清 齐名 赵永斌 邢继洋 刘剑锋

程云清, 齐名, 赵永斌, 邢继洋, 刘剑锋. 榛子正常发育与败育子房差异蛋白谱对比分析[J]. 北京林业大学学报, 2018, 40(3): 13-25. doi: 10.13332/j.1000-1522.20170352
引用本文: 程云清, 齐名, 赵永斌, 邢继洋, 刘剑锋. 榛子正常发育与败育子房差异蛋白谱对比分析[J]. 北京林业大学学报, 2018, 40(3): 13-25. doi: 10.13332/j.1000-1522.20170352
Cheng Yunqing, Qi Ming, Zhao Yongbin, Xing Jiyang, Liu Jianfeng. Comparative studies on differently expressed proteomes of developing and abortive ovary in hazelnut[J]. Journal of Beijing Forestry University, 2018, 40(3): 13-25. doi: 10.13332/j.1000-1522.20170352
Citation: Cheng Yunqing, Qi Ming, Zhao Yongbin, Xing Jiyang, Liu Jianfeng. Comparative studies on differently expressed proteomes of developing and abortive ovary in hazelnut[J]. Journal of Beijing Forestry University, 2018, 40(3): 13-25. doi: 10.13332/j.1000-1522.20170352

榛子正常发育与败育子房差异蛋白谱对比分析

doi: 10.13332/j.1000-1522.20170352
基金项目: 

国家自然科学基金项目 31670681

国家自然科学基金项目 31770723

国家自然科学基金项目 31370683

详细信息
    作者简介:

    程云清,博士,教授。主要研究方向:植物生物技术。Email:Chengyunqing1977@163.com 地址:136000吉林省四平市铁西区海丰大街1301号吉林师范大学生命科学学院

    责任作者:

    刘剑锋,博士,教授。主要研究方向:植物生物技术。Email:jianfengliu1976@163.com 地址:同上

  • 中图分类号: S722.3+7; Q943.1

Comparative studies on differently expressed proteomes of developing and abortive ovary in hazelnut

  • 摘要: 目的筛选参与调控榛子子房败育的候选蛋白,为榛子遗传改良研究提供科学依据。方法以平欧杂交榛‘达维’的正常发育与败育子房为材料,进行蛋白样品的同位素标记相对和绝对定量iTRAQ(isobaric tags for relative and absolute quantification)技术分析。对鉴定到的所有蛋白进行COG(Cluster of orthologous groups of proteins)功能分类,预测鉴定蛋白的功能。随后依据蛋白定量结果,筛选差异表达蛋白,进而开展GO(Gene ontology)功能富集与KEGG(Kyoto encyclopedia of genes and genomes)代谢路径富集分析以明确其分子功能和重要生物代谢路径。最后,主要从显著性富集路径中筛选可能参与子房败育调控的差异表达蛋白。结果蛋白鉴定共获得317068个二级谱图,特有多肽14267条,蛋白3538个。R、O、J、G和C类中的蛋白数量为最多,分别占有COG功能注释的蛋白总数的19.36%、9.97%、7.80%、7.67%和6.76%。共鉴定到249个差异表达蛋白,其中上调、下调表达蛋白分别为180和69个。GO富集分析结果表明,差异表达蛋白主要执行结合与催化分子功能。KEGG富集分析共找到11个显著性富集路径,最为显著的路径包括:苯丙素生物合成(ko00940),光合作用(ko00195),代谢路径(ko01100),光合作用-天线蛋白(ko00196),次生代谢产物生物合成(ko01110)。初步筛选获得可能参与调控榛子子房败育的候选蛋白37个。结论榛子败育子房的形成与光合作用、碳水化合物运输与代谢、能量合成与转换、花粉管生长与DNA甲基化等相关,本研究为深入解析榛子子房败育的分子机制提供了科学依据。

     

  • 图  1  蛋白鉴定结果汇总

    横坐标1、2、3、4、5、6分别代表总二级谱图、谱图、特有谱图、多肽、特有多肽、蛋白。

    Figure  1.  Protein identification overview

    1 to 6 in x-axis represent total spectra, spectra, unique spectra, peptide, unique peptide and protein, respectively.

    图  2  特异性谱图数目分布

    特异性肽段对应的谱图即为特异性谱图,x轴为特异性谱图数目,y轴为相应蛋白数目。

    Figure  2.  Unique spectrum number distribution

    Unique spectrum means spectrumof unique peptide. x-axis: unique spectrum number, y-axis: number of the corresponding proteins.

    图  3  特有多肽数量分布

    一个蛋白鉴定到的特异性肽段越多,相应蛋白可信度越高,x轴为特异性肽段数目,y轴为相应蛋白数目。

    Figure  3.  Unique peptide number distribution

    The more unique peptides identified in a protein implies its higher credibility. The x-axis: the unique peptide number of each protein, y-axis: the corresponding protein number.

    图  4  蛋白质分子量分布

    该图显示所有鉴定到蛋白质分子量分布情况,x轴为分子量,y轴为相应蛋白数目。

    Figure  4.  Protein molecular mass distribution

    Molecular weight distribution of all identified protein, x-axis: unique spectrum number, y-axis: number of the corresponding protein.

    图  5  鉴定蛋白的COG功能分布统计

    A.RNA加工与修饰;B.染色质的结构与动力学;C.能量产生与转换;D.细胞周期调控,细胞分裂,染色体分配;E.氨基酸运输与代谢;F.核酸运输与代谢;G.碳水化合物运输与代谢;H.辅酶运输与代谢;I.脂类运输与代谢;J.翻译、核糖体结构与生物合成;K.转录;L.复制、重组与修复;M.细胞壁/膜/包膜生物合成;N.细胞运动;O.翻译后修饰,蛋白质周转,伴侣;P.无机离子转运与代谢;Q.次级代谢产物的生物合成、运输和分解代谢;R.一般功能预测;S.功能未知;T.信号转导机制;U.胞内运输、分泌和囊泡运输;V.防御机制;Y.核结构;Z.细胞骨架。

    Figure  5.  COG functional histogram of all identified proteins

    A, RNA processing and modification; B, chromatin structure and dynamics; C, energy production and conversion; D, cell cycle control, cell division, chromosome partitioning; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation, ribosomal structure and biogenesis; K, transcription; L, replication, recombination and repair; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, posttranslational modification, protein turnover, chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; R, general function prediction only; S, function unknown; T, signal transduction mechanisms; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms; Y, nuclear structure; Z, cytoskeleton.

    图  6  正常与败育子房中蛋白表达差异倍数的对数分布

    TL=Log21.5=0.5849,TR= Log2(1/1.5)=-0.5849。红色、绿色与灰色三角分别表示上调、下调与非差异表达蛋白。

    Figure  6.  Logarithmic distribution plot of protein change folds of developing and abortive ovary

    Red, green and gray triangles represent up regulation, down regulation, and not differentially expressed proteins, respectively.

    图  7  差异表达蛋白的GO功能富集分析

    A.细胞; B.细胞组分; C.膜; D.膜外区域; E.大分子复合物; F.细胞器内部区域; G.细胞器; H.细胞器组分; I.抗氧化; J.结合; K.催化; L.电子载体; M.分子传感器; N.营养库; O.结构分子; P.翻译调节; Q.转运子; R.解剖结构形成; S.生物调控; T.细胞成分的生物合成; U.细胞成分的组织; V.细胞过程; W.死亡; X.发育过程; Y.定位建成; Z.生长; AA.免疫系统过程; AB.定位; AC.代谢过程; AD.多组织过程; AE.多细胞组织过程; AF.色素沉着; AG.生殖; AH.生殖过程; AI.刺激响应。

    Figure  7.  GO functional enrichment analysis of differently expressed proteins

    A, cell; B, cell part; C, envelope; D, extracellular region; E, macromolecular complex; F, membrane-enclosed lumen; G, organelle; H, organelle part; I, antioxidant; J, binding; K, catalytic; L, electron carrier; M, molecular transducer; N, nutrient reservoir; O, structural molecule; P, translation regulator; Q, transporter; R, anatomical structure formation; S, biological regulation; T, cellular component biogenesis; U, cellular component organization; V, cellular process; W, death; X, developmental process; Y, establishment of localization; Z, growth; AA, immune system process; AB, localization; AC, metabolic process; AD, multi\-organism process; AE, multicellular organismal process; AF, pigmentation; AG, reproduction; AH, reproductive process; AI, response to stimulus.

    表  1  差异表达蛋白的KEGG代谢通路富集分析

    Table  1.   KEGG pathway enrichment analysis of differently expressed proteins (DEPs)

    编号
    No.
    路径
    Pathway
    有路径注释的DEPs数量(比例
    )DEPs amount with pathway annotation (ratio)
    有路径注释的所有表达蛋白数量(比例)
    All expressed proteins with pathway annotation (ratio)
    P
    P-value
    路径号
    Pathway ID
    1 苯丙素生物合成Phenylpropanoid biosynthesis 23 (11.06%) 83 (2.76%) 2.82×10-9 ko00940
    2 光合作用Photosynthesis 10 (4.81%) 18 (0.6%) 5.37×10-8 ko00195
    3 代谢路径Metabolic pathways 86 (41.35%) 871 (28.92%) 4.73×10-5 ko01100
    4 光合作用-天线蛋白Photosynthesis-antenna proteins 4 (1.92%) 6 (0.2%) 2.97×10-4 ko00196
    5 次生代谢产物生物合成Biosynthesis of secondary metabolites 58 (27.88%) 555 (18.43%) 3.41×10-4 ko01110
    6 氮代谢Nitrogen metabolism 5 (2.4%) 13 (0.43%) 1.22×10-3 ko00910
    7 类黄酮生物合成Flavonoid biosynthesis 7 (3.37%) 28 (0.93%) 2.30×10-3 ko00941
    8 苯并恶嗪酮类物质生物合成Benzoxazinoid biosynthesis 2 (0.96%) 2 (0.07%) 4.75×10-3 ko00402
    9 植物MAPK信号转导通路MAPK signaling pathway-plant 8 (3.85%) 47 (1.56%) 1.37×10-2 ko04016
    10 同源重组Homologous recombination 4 (1.92%) 16 (0.53%) 2.08×10-2 ko03440
    11 DNA复制DNA replication 4 (1.92%) 16 (0.53%) 2.08×10-2 ko03030
    下载: 导出CSV

    表  2  可能参与子房败育调控的差异表达蛋白

    Table  2.   Differently expressed proteins which may be involved in the regulation

    蛋白路径
    Protein pathway
    蛋白质点编号
    Protein point No.
    蛋白编号
    Protein ID
    差异表达倍数
    Fold change
    分子量
    Molecular mass
    特有肽段数量
    Uniquepeptide number
    序列覆盖率
    Proteincoverage/%
    注释
    Annotation
    光合作用
    Photosynthesis
    2 213 Unigene25049_All 1.50 23 118 2 16.1 ATP合成酶β亚基ATP synthase beta subunit
    778 Unigene29018_All 1.62 20 944 2 13.6 ATP合成酶β亚基ATP synthase beta subunit
    456 Unigene41031_All 1.53 20 843 7 52.1 细胞色素f Cytochrome f
    462 Unigene20320_All 1.59 23 112 10 27.0 光系统Ⅰ反应中心亚基Ⅱ Photosystem Ⅰ reaction center subunit Ⅱ
    2 631 Unigene25432_All 1.71 49 003 9 23.2 光系统Ⅱ CP43叶绿素蛋白Photosystem Ⅱ CP43 chlorophyll apoprotein
    2 648 Unigene25729_All 2.73 18 844 1 14.9 光系统Ⅰ反应中心亚基PSI Photosystem Ⅰ reaction center subunit PSI
    892 CL10922.Contig2_All 2.14 13 964 1 26.7 细胞色素b6-f复合体铁硫蛋白亚基Cytochrome b6-f complex iron-sulfur subunit
    992 Unigene20387_All 1.85 28 922 7 25.5 光系统Ⅱ 22 kDa蛋白Photosystem Ⅱ 22 kDa protein
    1 033 Unigene20647_All 2.51 24 449 4 23.9 光系统Ⅰ反应中心亚基Ⅲ Photosystem Ⅰ reaction center subunit Ⅲ
    3 423 CL4056.Contig2_All 1.69 15 197 2 25.2 光系统Ⅰ反应中心亚基Ⅲ PSI reaction center subunit Ⅲ
    碳水化合物运输与代谢
    Carbohydrate transport and metabolism
    2 270 CL10718.Contig2_All 2.22 41 148 4 12.2 糖苷水解酶Glycoside hydrolase
    1 568 CL5678.Contig4_All 1.93 32 465 4 17.7 酸性几丁质内切酶Acidic endochitinase
    91 Unigene17825_All 1.90 59 673 6 11.9 α-L-呋喃糖苷酶1前体Alpha-L-arabinofuranosidase 1 precursor
    2 521 CL2311.Contig8_All 1.89 30 216 4 21.7 水通道蛋白PIP2 Aquaporin PIP2
    1 448 CL10219.Contig1_All 1.65 42 253 8 23.8 庚糖二磷酸酶Sedoheptulose-bisphosphatase
    2 360 CL9110.Contig3_All 1.64 30 890 5 29.9 水通道蛋白PIP1 Aquaporin PIP1
    3 324 Unigene27924_All 1.51 53 260 15 34.0 核酮糖1, 5 -二磷酸羧化酶/氧化酶大亚基(RbcL)Ribulose 1, 5-bisphosphate carboxylase/oxygenase large subunit
    1 281 Unigene9913_All 0.67 61 085 14 27.1 焦磷酸:果糖-6-磷酸1-磷酸转移酶β亚基(PFP)Pyrophosphate-fructose 6-phosphate 1-phosphotransferase subunit beta, PFP
    1 612 Unigene19879_All 0.65 53 563 1 12.2 丙酮酸激酶Pyruvate kinase
    3 253 CL1716.Contig1_All 1.55 22 841 3 18.3 类萌发素蛋白Germin-like protein
    能量合成与转换
    Energy production and conversion
    1 753 CL969.Contig2_All 2.13 20 122 9 51.1 Rubisco小亚基(RbcS)Rubisco small subunit
    2 852 Unigene20099_All 1.79 64 976 13 33.7 NADP-苹果酸酶(NADP-ME)NADP-dependent malic enzyme
    2 857 CL5660.Contig3_All 1.64 40 430 11 34.1 醛缩酶型TIM-barrel类家族蛋白亚型1 Aldolase-type TIM barrel family protein isoform 1
    160 Unigene17079_All 1.63 38 192 4 21.9 Perakine还原酶(PR)Perakinereductase-like
    3 133 CL5532.Contig7_All 1.59 53 235 5 11.0 醛脱氢酶(ALDH)Aldehyde dehydrogenase
    2 495 Unigene26012_All 2.14 41 113 10 32.7 线粒体甲酸脱氢酶(FDH) Formate dehydrogenase, mitochondrial
    967 Unigene15321_All 1.62 42 317 8 26.9 磷酸甘油酸脱氢酶(PGDH)Phosphoglycerate dehydrogenase
    2 807 CL6456.Contig2_All 1.66 33 779 4 58.0 2-亚甲基-呋喃-3-1还原酶2-methylene-furan-3-one reductase OS=Solanumlycoper-sicum
    1 138 CL3539.Contig1_All 1.52 32 347 7 30.0 NAD(P)H:醌氧化还原酶(NQO)NAD(P)H:quinoneoxidoreductase
    花粉管生长与DNA甲基化
    Pollen tube growth and DNA methylation
    2 248 CL7865.Contig2_All 4.09 15 766 2 23.0 花粉过敏原和伸展蛋白家族蛋白Ole e 1Pollen Ole e 1 allergen and extensin family protein
    1 218 Unigene909_All 1.77 17 902 3 25.3 主要花粉过敏原Bet v 1-A Major pollen allergen Bet v 1-A
    1 377 CL3839.Contig1_All 0.51 20 815 2 15.9 花粉特异蛋白SF3 Pollen-specific protein SF3
    1 129 Unigene5763_All 1.58 18 315 10 1.9 钙调素(CaM)Calmodulin
    571 CL2565.Contig2_All 1.88 40 363 6 19.4 咖啡酸-3-O-甲基转移酶(COMT)Caffeic acid 3-O-methyltransferase
    1 513 CL2173.Contig3_All 1.51 39 545 6 23.0 COMT
    1 624 CL4896.Contig1_All 0.56 55 950 7 33.3 磷酸乙醇胺N-甲基转移酶(PEAMT)Phosphoethanolamine N-methyltransferase
    2 474 CL10334.Contig1_All 0.63 38 914 6 22.8 甾醇C-24甲基转移酶(SMT)24-sterol C-methyltransferase
    下载: 导出CSV
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  • 收稿日期:  2017-10-23
  • 修回日期:  2018-01-23
  • 刊出日期:  2018-03-01

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