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GST-pull down技术筛选毛白杨天冬氨酸蛋白酶PtoAED3互作蛋白

赵杰 王兵 骆梅 莫黎杰 李慧 刘迪 陆海

赵杰, 王兵, 骆梅, 莫黎杰, 李慧, 刘迪, 陆海. GST-pull down技术筛选毛白杨天冬氨酸蛋白酶PtoAED3互作蛋白[J]. 北京林业大学学报, 2021, 43(5): 64-74. doi: 10.12171/j.1000-1522.20200365
引用本文: 赵杰, 王兵, 骆梅, 莫黎杰, 李慧, 刘迪, 陆海. GST-pull down技术筛选毛白杨天冬氨酸蛋白酶PtoAED3互作蛋白[J]. 北京林业大学学报, 2021, 43(5): 64-74. doi: 10.12171/j.1000-1522.20200365
Zhao Jie, Wang Bing, Luo Mei, Mo Lijie, Li Hui, Liu Di, Lu Hai. Identification of aspartic acid protease PtoAED3-interacting proteins through GST pull-down assays in Populus tomentosa[J]. Journal of Beijing Forestry University, 2021, 43(5): 64-74. doi: 10.12171/j.1000-1522.20200365
Citation: Zhao Jie, Wang Bing, Luo Mei, Mo Lijie, Li Hui, Liu Di, Lu Hai. Identification of aspartic acid protease PtoAED3-interacting proteins through GST pull-down assays in Populus tomentosa[J]. Journal of Beijing Forestry University, 2021, 43(5): 64-74. doi: 10.12171/j.1000-1522.20200365

GST-pull down技术筛选毛白杨天冬氨酸蛋白酶PtoAED3互作蛋白

doi: 10.12171/j.1000-1522.20200365
基金项目: 国家自然科学基金面上项目(31971618)
详细信息
    作者简介:

    赵杰。主要研究方向:树木分子生物学。Email:isaaczhao1994@163.com 地址:100083北京市海淀区清华东路35号北京林业大学生物科学与技术学院

    责任作者:

    刘迪,博士,副教授。主要研究方向:树木分子生物学。Email:liudi@bjfu.edu.cn 地址:同上

  • 中图分类号: S792.117;Q556.9

Identification of aspartic acid protease PtoAED3-interacting proteins through GST pull-down assays in Populus tomentosa

  • 摘要:   目的   天冬氨酸蛋白酶属于蛋白水解酶家族,为了解析毛白杨天冬氨酸蛋白酶PtoAED3在植物生长发育中的分子调节机制,利用GST-pull down联合质谱技术,对PtoAED3的互作蛋白进行鉴定和分析。   方法   通过同源克隆获得了毛白杨PtoAED3的CDS序列,构建含GST标签的原核表达载体pGEX-4T-PtoAED3。使用IPTG诱导GST-PtoAED3融合蛋白表达后,利用GST标签对PtoAED3蛋白进行纯化,纯化后的PtoAED3蛋白与毛白杨植株总蛋白共孵育,应用GST-pull down技术获得毛白杨总蛋白中与PtoAED3蛋白相互作用的候选蛋白,随后通过质谱技术对筛选到的候选蛋白进行鉴定与分析。   结果   通过质谱技术鉴定这些蛋白的氨基酸序列,共筛选出128 个与PtoAED3特异性结合的候选互作蛋白,这些互作蛋白涉及到细胞进程、代谢过程、应激反应、生物调节、发育过程等多个生物学过程。   结论   通过GST-pull down实验联合质谱技术,筛选出毛白杨中与PtoAED3互作的候选蛋白,为研究毛白杨PtoAED3与底物或复合物的互作及其影响毛白杨生长发育的分子调节机制提供了初步方向。

     

  • 图  1  毛白杨PtoAED3基因的克隆

    A. 毛白杨的总RNA;B. 毛白杨PtoAED3 PCR结果;C. 毛白杨PtoAED3核酸和PtoAED3蛋白序列信息;D. 毛白杨 PtoAED3 信号肽预测。A, total RNA extraction result of Populus tomentosa; B, PCR result of PtoAED3 cDNA in Populus tomentosa; C, sequence information of PtoAED3 nucleic acid and PtoAED3 protein; D, signal peptide prediction of PtoAED3 protein.

    Figure  1.  Results of PtoAED3 gene cloning in P. tomentosa

    图  2  pGEX-4T-PtoAED3原核表达载体双酶切电泳图

    M. 2 000 bp DNA Marker;泳道1. pGEX-4T-1空载体双酶切电泳图;泳道2. pGEX-4T-PtoAED3双酶切电泳图。Lane 1, electrophoresis result of pGEX-4T-1 empty vector after BamHⅠ/XhoⅠ digestion; lane 2, electrophoresis result of pGEX-4T-PtoAED3 after BamHⅠ/XhoⅠ digestion.

    Figure  2.  Double enzyme digestion of pGEX-4T-PtoAED3 vector from P. tomentosa

    图  3  毛白杨PtoAED3基因表达和纯化

    A. 毛白杨PtoAED3基因原核表达SDS-PAGE电泳图;泳道1. IPTG未诱导的pGEX-4T-1上清溶液; 泳道2. IPTG未诱导的pGEX-4T-PtoAED3上清溶液; 泳道3. IPTG诱导的pGEX-4T-1蛋白表达上清溶液; 泳道4.IPTG诱导的pGEX-4T-PtoAED3蛋白表达上清溶液; 泳道5. IPTG诱导的pGEX-4T-1菌体沉淀; 泳道6. IPTG诱导的pGEX-4T-PtoAED3菌体沉淀。B. 利用GST标签蛋白纯化GST-PtoAED3蛋白的SDS-PAGE电泳图;泳道1 ~ 3分别为洗脱5、3、1次后,谷胱甘肽琼脂糖凝胶偶联物通过SDS-PAGE检测。C. 抗体anti-PtoAED3与纯化后的原核表达蛋白免疫杂交;泳道1. 抗体与IPTG诱导的pGEX-4T-1原核表达蛋白杂交; 泳道2. 抗体与IPTG诱导的pGEX-4T-PtoAED3原核表达蛋白杂交。红色箭头表示原核表达的目的蛋白。A, SDS-PAGE electrophoresis of PtoAED3 prokaryotic expression; lane 1, non-IPTG induced pGEX-4T-1 protein expression in supernatant; lane 2, non-IPTG induced pGEX-4T-PtoAED3 protein expression in supernatant; lane 3, IPTG induced pGEX-4T-1 protein expression in supernatant; lane 4, IPTG induced pGEX-4T-PtoAED3 protein expression in supernatant; lane 5, IPTG induced pGEX-4T-1 protein expression in precipitation; lane 6, IPTG induced pGEX-4T-PtoAED3 protein expression in precipitation. B, SDS-PAGE electrophoresis results using GST-tagged protein purification for GST-PtoAED3 protein; lane 1−3, the glutathione-sepharose eluted 5,3,1 times are detected by SDS-PAGE. The red arrow indicates the interested proteins expressed in E.coli. C, western blotting of anti-PtoAED3 antibody with the purified prokaryotic expression protein; lane 1, western blotting of anti-PtoAED3 antibody with the IPTG-induced pGEX-4T-1 prokaryotic expression protein; lane 2, western blotting of anti-PtoAED3 antibody with the IPTG pGEX-4T-PtoAED3 protein hybridization. The red arrow indicates the protein of interest expressed prokaryotically.

    Figure  3.  Expression and purification of PtoAED3 in P. tomentosa

    图  4  利用GST-pull down在毛白杨中筛选与GST-PtoAED3互作的蛋白

    A. 毛白杨植株全蛋白电泳图; B. 还原型谷胱甘肽洗脱与GST-PtoAED3互作的蛋白。泳道1和2为未洗脱的植株全蛋白,泳道3和4分别为利用还原型谷胱甘肽洗脱3和5次收集的蛋白。A, electrophoresis of whole protein of P. tomentosa; B, GST-PtoAED3 interacting protein eluted with reduced glutathione. Lane 1 and 2, uninteracting proteins; lane 3 and 4, proteins with reduced glutathione detected by SDS-PAGE for 3 and 5 times.

    Figure  4.  Screening of proteins interacting with GST-PtoAED3 in P. tomentosa by GST-pull down

    图  5  PtoAED3互作蛋白GO功能富集

    Cellular processes 生物学途径包括细胞进程; Metabolism process 代谢过程; Response to stimulus 应激反应; Biological regulation 生物调节; Multicellular organismal process 多细胞器官进程; Regulation of biological process 生物进程调节; Development process 发育过程; Cellular component organization or biogenesis 细胞组分组成或发育; Localization 定位; Reproductive process 繁殖过程; Positive regulation of biological process 生物过程正调节; Multi-organism process 多器官进程; Signaling 信号途径; Negative regulation of biological process 生物过程负调节; Rhythmic process 节律进程; Growth 生长; Immune system process 免疫体统进程; Pigmentation 色素沉积; Biological adhesion 生物固定; Carbon utilization 碳利用; Binding 分子功能包括结合; Catalytic activity 催化活性; Transporter activity 转运活性; Transcription regulator activity 转录调节因子活性; Structural molecular activity 结构分子活性; Antioxidant activity 抗氧化活性; Cell part 细胞组分则包括细胞部分; Cell 细胞; Organelle 细胞器; Membrane 膜; Membrane part 膜组分; Organelle part 细胞器组分; Protein-containing complex 蛋白复合体; Membrane-enclosed lumen 膜封闭腔; Cell junction 细胞连接; Symplast 共质体; Extracellular region 胞外域; Supramolecular complex 超分子复合体; Synapse 突触; Synaptic part and extracellular region part 突触局部及胞外域局部

    Figure  5.  GO function enrichment of PtoAED3IPs

    表  1  GST-pull down筛选的PtoAED3互作蛋白

    Table  1.   PtoAED3 interacting proteins screened by GST-pull down

    项目
    Item
    蛋白编号
    Protein No.
    肽段
    Peptide segment
    蛋白注释
    Protein annotation
    肽段得分
    Peptide score
    覆盖率
    Coverage/%
    高丰度蛋白
    High abundance
    protein
    Q8LPG9 K.EDVEALWLK.K/
    R.SQAIIK.L
    Protein IQ-DOMAIN 14
    蛋白IQ结构域
    20.84/21.91 3.31
    Q56XU4 R.MKM*DR.E Zinc finger CCCH domain-containing protein 6
    锌指结构域
    26.92 1.15
    C6E2H7 K.KLEWYEK.F GTPase Era
    GTP 水解酶
    27.89 1.99
    F4I0N3 R.ELQQLK.E WEB family protein
    WEB 转录因子
    39.89 2.91
    O04590 R.VAAEALIR.L Pentatricopeptide repeat-containing protein
    三角状五肽
    23.91 4.88
    蛋白质加工降解
    Protein processing
    and degradation
    Q9D832 K.NPVNK.K DnaJ homolog subfamily B member 4
    分子伴侣
    21.05 1.58
    P43297 K.EGAVAEVK.D Cysteine proteinase RD21A
    半胱氨酸蛋白酶
    20.47 1.72
    Q9C5D2 R.MQENISR.F F-box/LRR-repeat protein 4
    SCF复合体亚基
    25.39 1.94
    Q9FZK1 K.ILGGKATWATTK.C F-box only protein 6
    SCF 复合体亚基
    21.78 3.22
    Q94AH6 R.LLFDK.S Cullin-1 SCF 复合体亚基 37.5 1.22
    Q94F30 K.FTLSR.V Ubiquitin-like-specific protease ESD4
    泛素特异蛋白酶
    22.41 0.97
    Q9JIG7 R.QNLGKAK.L Coiled-coil domain-containing protein 22
    螺旋卷曲结构域蛋白
    40.1 1.67
    O22993 R.FTLSR.S Probable inactive ATP-dependent zinc
    metalloprotease FTSHI 1
    金属蛋白酶
    22.41 0.62
    胁迫响应相关
    Stress response
    correlation
    P43297 K.EGAVAEVK.D Cysteine proteinase RD21A
    半胱氨酸蛋白酶
    20.47 1.72
    O80396 M.AGLEELK.K Mitogen-activated protein kinase kinase 3
    分裂原活化蛋白激酶
    27.02 1.33
    O04203 R.LPELR.K Nematode resistance protein-like HSPRO2
    类线虫抗性蛋白
    24.23 5.38
    Q9LII8 R.IDSELR.H Protein KINESIN LIGHT CHAIN-RELATED 2
    驱动蛋白轻链
    30.55 3.61
    O81825 K.LTELR.K Probable disease resistance protein At4g27220
    抗病蛋白
    26.75 0.83
    Q9LFP7 R.NVGLK.T Probable serine/threonine-protein kinase PIX7
    丝氨酸/苏氨酸蛋白激酶
    21.76 1.13
    Q7XA40 R.SLDELK.R Putative disease resistance protein RGA3
    抗病蛋白
    28.92 7.69
    Q9LRR4 R.LLEIR.A Putative disease resistance RPP13-like protein 1
    抗病蛋白
    28.42 1.79
    Q9LX93 K.LPRSR.M E3 ubiquitin-protein ligase RING1
    E3 泛素连接酶
    22.21 17.86
    Q7VG78 K.AGPLSARKPASK.Q Probable GMP synthase [glutamine-hydrolyzing]
    GMP合成酶
    22.29 23.53
    Q9C5D2 R.MQENISR.F F-box/LRR-repeat protein 4
    SCF 复合体亚基
    25.39 1.94
    Q0WQF4 K.ITELR.E Vacuolar protein sorting-associated protein 53 A
    液泡蛋白分类相关蛋白
    26.75 1.48
    O22769 R.LLEIR.Q NADH dehydrogenase [ubiquinone] flavoprotein 2
    NADH 脱氢酶[泛醌]黄素蛋白
    28.42 1.97
    Q9UI10 K.IIIADK.V Translation initiation factor eIF-2B subunit delta
    翻译起始因子亚基
    25.12 0.94
    O65202 R.ILELR.L Peroxisomal acyl-coenzyme A oxidase 1
    过氧化物酶体酰基辅酶A氧化酶
    28.42 0.75
    F4I9E1 R.LKM*IPR.T Protein NUCLEAR FUSION DEFECTIVE 4
    核融合缺陷蛋白
    25.57 1.06
    P47140 R.EQSNVIAR.Q Altered inheritance rate of mitochondria protein 25
    线粒体蛋白
    21.26 2.31
    O43099 R.LPELR.K Peroxiredoxin Asp f3
    过氧化物酶
    24.23 4.13
    Q9FYA2 K.ENYHLVPR.T Probable WRKY transcription factor 75
    WRKY转录因子
    32.11 6.35
    Q84X53 R.SLDELK.E Transcription termination factor MTEF1, chloroplastic
    转录终止因子
    28.92 2.48
    发育过程相关
    Developmental
    process
    Q9UI10 K.IIIADK.V Translation initiation factor eIF-2B subunit delta
    翻译起始因子亚基
    25.12 0.94
    Q84JG2 K.ILRFEELDLQM*EK.E SWI/SNF complex subunit SWI3B
    SWI/SNF复合体亚基
    23.07 2.60
    Q12955 K.AGMTPLDLATNEEIR.L Ankyrin-3 锚蛋白 24.76 8.72
    Q8GWQ6 K.AYALDK.K UPF0235 protein At5g63440 UPF蛋白 21.38 6.25
    Q84X53 R.SLDELK.E Transcription termination factor MTEF1, chloroplastic
    转录终止因子
    28.92 2.48
    F4JRR5 R.VDTFR.I TITAN-like protein
    TITAN蛋白
    21.33 3.03
    Q94AH6 R.LLFDK.S Cullin-1
    SCF 复合体亚基
    37.5 1.22
    O22993 R.FTLSR.S Probable inactive ATP-dependent zinc metalloprotease FTSHI 1
    金属蛋白酶
    22.41 0.62
    Q9LII8 R.IDSELR.H Protein KINESIN LIGHT CHAIN-RELATED 2
    驱动蛋白轻链
    30.55 3.61
    Q0D4T4 K.VELKR.I MADS-box transcription factor 18
    MADS转录因子
    23.86 2.53
    Q9ZVP8 K.VLEQR.A NAC domain-containing protein 35
    NAC转录因子
    25.43 1.98
    Q94F30 K.FTLSR.V Ubiquitin-like-specific protease ESD4
    泛素特异蛋白酶
    22.41 0.97
    Q9FYA2 K.ENYHLVPR.T Probable WRKY transcription factor 75
    WRKY转录因子
    32.11 6.35
    Q9FII5 R.LGGMIPAK.T Leucine-rich repeat receptor-like protein kinase TDR
    受体蛋白激酶
    23.78 5.88
    Q9LHP4 R.IPELR.T Receptor-like protein kinase 2
    受体蛋白激酶
    24.23 3.57
    Q9SN39 R.ILFDK.Y Pentatricopeptide repeat-containing protein DOT4
    三角状五肽蛋白
    37.5 2.53
    F4I9E1 R.LKM*IPR.T Protein NUCLEAR FUSION DEFECTIVE 4
    核融合缺陷蛋白
    25.57 1.06
    Q8LEZ4 R.AGVERK.A Protein NUCLEAR FUSION DEFECTIVE 5
    核融合缺陷蛋白
    25.82 0.84
    Q9FZK1 K.ILGGKATWATTK.C F-box only protein 6
    SCF 复合体亚基
    21.78 3.22
    Q9M811 K.EAPQVPASPR.E Rop guanine nucleotide exchange factor 11
    鸟苷酸交换因子
    21.47 7.41
    Q2QDF6 .LSEVR.G Steroid 5-alpha-reductase DET2
    类固醇还原酶
    22.75 6.33
    下载: 导出CSV
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出版历程
  • 收稿日期:  2020-11-21
  • 修回日期:  2021-02-07
  • 网络出版日期:  2021-04-21
  • 刊出日期:  2021-05-27

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