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作物学报 ›› 2020, Vol. 46 ›› Issue (01): 31-39.doi: 10.3724/SP.J.1006.2020.94036

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

利用酵母双杂交系统筛选大豆结瘤因子受体NFR1α的互作蛋白

柯丹霞(),彭昆鹏   

  1. 信阳师范学院生命科学学院/大别山农业生物资源保护与利用研究院, 河南信阳 464000
  • 收稿日期:2019-03-07 接受日期:2019-08-09 出版日期:2020-01-12 网络出版日期:2019-09-02
  • 通讯作者: 柯丹霞
  • 基金资助:
    本研究由国家自然科学基金项目(31400213);河南省科技攻关计划项目(182102110448);信阳师范学院“南湖学者奖励计划”青年项目(2016)资助

Screening of NFR1α-interactive proteins in soybean using yeast two hybrid system

KE Dan-Xia(),PENG Kun-Peng   

  1. College of Life Sciences, Xinyang Normal University/Institute for Conservation and Utilization of Agro-bioresources in Dabie Mountains, Xinyang 464000, Henan, China
  • Received:2019-03-07 Accepted:2019-08-09 Published:2020-01-12 Published online:2019-09-02
  • Contact: Dan-Xia KE
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31400213);Science and Technology Research Projects of Henan Province(182102110448);Nanhu Scholars Program for Young Scholars of XYNU

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摘要：

大豆是重要的植物蛋白作物和粮豆间作作物, 挖掘大豆的生物固氮潜力, 对推动生态农业的可持续发展具有深远意义。大豆结瘤因子受体蛋白GmNFR1α (Nod Factor Receptor)对结瘤至关重要, 但其具体调控机制尚不明确。本研究以大豆mRNA为模板, 利用RT-PCR方法扩增到GmNFR1α蛋白的激酶结构域(GmNFR1α-pk), 构建了pGBKT7-GmNFR1α-pk诱饵表达载体, 通过酵母双杂交技术, 筛选大豆根瘤AD-cDNA文库, 从文库中分离到与GmNFR1α-pk相互作用的71个阳性克隆, 经测序和同源性分析筛选到12种与GmNFR1α-pk互作的蛋白, 包括钙离子结合手性蛋白、豆血红蛋白、结瘤素Nod44等蛋白。以大豆豆血红蛋白GmLbc2为例, 回转酵母以及烟草体内BiFC验证其与诱饵蛋白的相互作用, 对其进行同源蛋白比对及系统进化树分析, 并利用百脉根毛根转化技术鉴定GmLbc2在结瘤过程中的生物学功能。研究结果进一步补充和完善了GmNFR1α介导的结瘤信号传递途径, 为大豆与根瘤菌共生互作机制提供了新的分子证据。

关键词: 大豆, 共生固氮, 结瘤因子受体蛋白, 酵母双杂交, 豆血红蛋白

Abstract:

Soybean is the important plant protein crop and grain-bean intercropping crop. Exploring the biological nitrogen fixation potential of soybean is of far-reaching significance to promote the sustainable development of ecological agriculture. GmNFR1α, a soybean nod factor receptor protein, is very important for nodulation, but its specific regulatory mechanism is still unclear. Soybean mRNA was used as template to amplify the kinase domain of GmNFR1α protein (GmNFR1α-pk) by RT-PCR method and the pGBKT7-GmNFR1α-pk bait plasmid was constructed. Seventy-two positive clones interacted with GmNFR1α-pk were isolated through yeast two hybrid screening of soybean nodule AD-cDNA library from the library. Among them, 12 proteins including calcium ion binding chiral protein, hemoglobin, nodulin Nod44 and other proteins interacted with GmNFR1α-pk were screened by sequencing and homology analysis. The interaction between soybean hemoglobin and bait protein was verified by re-transforming in yeast and BiFC in tobacco. Comparison of homologous proteins and phylogenetic tree analysis were also done at the same time. The important function of GmLbc2 in nodulation process was clarified by hairy root transformation technology in Lotus japonicus. The results further complement and improve the signal transduction pathway mediated by GmNFR1α, and provide new molecular evidence for the symbiotic interaction mechanism between soybean and rhizobia.

Key words: soybean, symbiotic nitrogen fixation, nod factor receptor protein, yeast two-hybrid, soybean hemoglobin

图1

诱饵质粒pGBKT7-GmNFR1α-pk的构建及自激活检测 (A) M: trans 2K Plus II DNA marker; 1: GmNFR1α-pk目的片段扩增; 2: pGBKT7-GmNFR1α-pk诱饵质粒双酶切; 3: pGBKT7空载体双酶切。(B)阳性对照(pGBKT7-53和pGADT7-T)。(C)阴性对照(pGBKT7-lam和pGADT7-T)。(D) pGBKT7-GmNFR1α-pk与pGADT7空载体。"

图2

酵母双杂交 cDNA 文库的筛选 (A) QDO/X/A平板上的阳性克隆; (B) PCR 检测酵母双杂交cDNA文库插入片段大小。"

图3

GmNFR1α与GmLbc2相互作用的验证 (A)酵母双杂交系统验证GmNFR1α-pk与GmLbc2的相互作用。1: 阳性对照(pGBKT7-53和pGADT7-T); 2: 阴性对照(pGBKT7-lam和pGADT7-T); 3: pGBKT7-GmNFR1α-pk与pGADT7-GmLbc2。(B)双分子荧光互补技术验证GmNFR1α全长与GmLbc2的相互作用。"

图4

大豆GmLbc2与其他植物同源蛋白的序列比对分析 Gm: 栽培大豆, Glycine max; Gs: 野生大豆, Glycine soja; Cc: 木豆, Cajanus cajan; Va: 赤豆, Vigna angularis; Vr: 绿豆, Vigna radiata; Mt: 蒺藜苜蓿, Medicago truncatula; Lj: 百脉根, Lotus japonicus; As: 紫云英, Astragalus sinicus。"

图5

大豆GmLbc2蛋白与同系物的系统进化分析 标尺代表遗传相似性, 表明不同物种间同系物进化关系的远近。缩写同图4。"

图6

过表达GmLbc2对百脉根阳性毛根共生结瘤的影响 (A)空载体对照复合体植株的结瘤表型。(B)超表达GmLbc2 (GmLbc2-OX)复合体植株的结瘤表型; 接种根瘤菌30 d 后照相, Bars = 5 mm。(C)平均每个植株的结瘤数目, **表示P<0.01。(D) qRT-PCR检测GmLbc2、NIN、Enod40-1和Enod40-2 的表达水平。"

[1] Giles E D, Oldroyd G E, Downie J A . Calcium, kinases and nodulation signalling in legumes. Nature, 2004,5:566-576.
doi: 10.7554/eLife.33506 pmid: 29957177
[2] Oldroyd G E, Downie J A . Coordinating nodule morphogenesis with rhizobial infection in legumes. Annu Rev Plant Biol, 2008,59:519-546.
doi: 10.1146/annurev.arplant.59.032607.092839 pmid: 18444906
[3] Oldroyd G E . Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants. Nat Rev Microbiol, 2013,11:252-263.
doi: 10.1038/nrmicro2990
[4] Radutoiu S, Madsen L H, Madsen E B, Felle H H, Umehara Y, Grønlund M, Sato S, Nakamura Y, Tabata S, Sandal N, Stougarrd J . Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature, 2003,425:569-570.
doi: 10.1038/425569a pmid: 14534570
[5] Masdsen E B, Madsen L H, Radutoiu S . A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals. Nature, 2003,425:637-640.
doi: 10.1038/nature02045 pmid: 14534591
[6] Limpens E, Franken C, Smit P, Willemse J, Bisseling T, Geurts R . LysM domain receptor kinases regulating rhizobial Nod factor-induced infection. Science, 2003,302:630-633.
doi: 10.1126/science.1090074 pmid: 12947035
[7] Arrighi J F, Barre A, Ben Amor B, Bersoult A, Soriano L C, Mirabella R, de Carvalho-Niebel F, Journet E P, Ghérardi M, Huguet T, Geurts R, Dénarié J, Rougé P, Gough C . The Medicago truncatula lysin motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes. Plant Physiol, 2006,142:265-279.
doi: 10.1104/pp.106.084657 pmid: 16844829
[8] Smit P, Limpens E, Geurts R, Fedorova E, Dolgikh E, Gough C, Bisseling T . Medicago LYK3, an entry receptor in rhizobial nodulation factor signaling. Plant Physiol, 2007,145:183-191.
doi: 10.1104/pp.107.100495 pmid: 17586690
[9] Madsen E B, Antolin-Llovera M, Grossmann C, Ye J, Vieweg S, Broghammer A, Krusell L, Radutoiu S, Jensen O N, Stougaard J, Parniske M . Autophosphorylation is essential for the in vivo function of theLotus japonicus Nod factor receptor 1 and receptor-mediated signalling in cooperation with Nod factor receptor 5. Plant J, 2011,65:404-417.
doi: 10.1111/j.1365-313X.2010.04431.x
[10] Broghammer A, Krusell L, Blaise M, Sauer J, Sullivan J T, Maolanon N, Vinther M, Lorentzen A, Madsen E B, Jensen K J, Roepstorff P, Thirup S, Ronson C W, Thygesen M B, Stougaard J . Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding. Proc Natl Acad Sci USA, 2012,109:13859-13864.
doi: 10.1073/pnas.1205171109 pmid: 22859506
[11] Sørensen K K, Simonsen J B, Maolanon N N, Stougaard J, Jensen K J . Chemically synthesized 58-mer lysM domain binds lipochitin oligosaccharide. Chembiochem, 2014,15:2097-2105.
doi: 10.1002/cbic.201402125
[12] Mbengue M, Camut S, de Carvalho-Niebel F, Deslandes L, Froidure S, Klaus-Heisen D, Moreau S, Rivas S, Timmers T, Hervé C, Cullimore J, Lefebvre B . The Medicago truncatula E3 ubiquitin ligase PUB1 interacts with the LYK3 symbiotic receptor and negatively regulates infection and nodulation. Plant Cell, 2010,22:474-488.
doi: 10.1104/pp.15.01694 pmid: 26839127
[13] Tsikou D, Ramirez E E, Psarrakou I S, Wong J E, Jensen D B, Isono E, Radutoiu S, Papadopoulou K K . ALotus japonicus E3 ligase interacts with the Nod Factor Receptor 5 and positively regulates nodulation. BMC Plant Biol, 2018,18:217.
doi: 10.1186/s12870-018-1425-z pmid: 30285618
[14] Lefebvre B, Timmers T, Mbengue M, Moreau S, Hervé C, Tóth K, Bittencourt-Silvestre J, Klaus D, Deslandes L, Godiard L, Murray J D, Udvardi M K, Raffaele S, Mongrand S, Cullimore J, Gamas P, Niebel A, Ott T . A remorin protein interacts with symbiotic receptors and regulates bacterial infection. Proc Natl Acad Sci USA, 2010,107:2343-2348.
doi: 10.1073/pnas.0913320107 pmid: 20133878
[15] Tóth K, Stratil T F, Madsen E B, Ye J, Popp C, Antolín-Llovera M, Grossmann C, Jensen O N, Schüssler A, Parniske M, Ott T . Functional domain analysis of the Remorin protein LjSYMREM1 inLotus japonicus. PLoS One, 2012,7:e30817.
doi: 10.1371/journal.pone.0030817 pmid: 22292047
[16] Ke D X, Fang Q, Chen C F, Zhu H, Chen T, Chang X J, Yuan S L, Kang H, Ma L, Hong Z L, Zhang Z M . The small GTPase Rop6 interacts with NFR5 and is involved in nodule formation inLotus japonicus. Plant Physiol, 2012,159:131-143.
doi: 10.1104/pp.112.197269
[17] 柯丹霞, 李祥永 . 结瘤信号途径中相关调控蛋白的研究进展. 信阳师范学院学报(自然科学版), 2015,28:621-626.
Ke D X, Li X Y . Research progress of key regulatory proteins in nodulation pathway. J Xinyang Nor Univ (Nat Sci Edn), 2015,28:621-626 (in Chinese with English abstract).
[18] Duan L J, Pei J Q, Ren Y P, Li H, Zhou X Z, Zhu H, Duanmu D Q, Wen J Q, Mysore K S, Cao Y R, Zhang Z M . A dihydroflavonol-4-reductase-like protein interacts with NFR5 and regulates rhizobial infection inLotus japonicus. Mol Plant Microbe Int, 2018,32:401-412.
doi: 10.1094/MPMI-04-18-0104-R pmid: 30295579
[19] Indrasumunar A, Searle I, Lin M H, Kereszt A, Men A, Carroll B J, Gresshoff P M . Nodulation factor receptor kinase 1α controls nodule organ number in soybean (Glycine max L. Merr). Plant J, 2011,65:39-50.
doi: 10.1111/j.1365-313X.2010.04398.x
[20] 柯丹霞, 熊文真, 彭昆鹏, 李祥永 . 抗盐基因Gm01g04890大豆子叶节遗传转化研究. 信阳师范学院学报(自然科学版), 2017,30:46-51.
Ke D X, Xiong W Z, Peng K P, Li X Y . Study on genetic transformation of salt resistant gene Gm01g04890 in soybean. J Xinyang Nor Univ (Nat Sci Edn), 2017,30:46-51 (in Chinese with English abstract).
[21] Choudhury S R, Pandey S . Specific subunits of heterotrimeric G proteins play important roles during nodulation in soybean. Plant Physiol, 2013,162:522-533.
doi: 10.1104/pp.113.215400
[22] Choudhury S R, Pandey S . Phosphorylation-dependent regulation of G-protein cycle during nodule formation in soybean. Plant Cell, 2015,27:3260-3276.
doi: 10.1105/tpc.15.00517 pmid: 26498905
[23] Yin Y, Vafeados D, Tao Y . A new class of transcription factors mediates brassino steroid-regulated gene expression inArabidopsis. Cell, 2005,120:249-259.
doi: 10.1016/j.cell.2004.11.044 pmid: 15680330
[24] Navascués J, Pérez-Rontomé C, Gay M, Marcos M, Yang F, Walker F A, Desbois A, Abián J, Becana M . Leghemoglobin green derivatives with nitrated hemes evidence production of highly reactive nitrogen species during aging of legume nodules. Proc Natl Acad Sci USA, 2012,109:2660-2665.
doi: 10.1073/pnas.1116559109 pmid: 22308405
[25] Sainz M, Calvo-Begueria L, Pérez-Rontomé C, Wienkoop S, Abián J, Staudinger C, Bartesaghi S, Radi R, Becana M . Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/peroxide-dependent mechanism. Plant J, 2015,81:723-735.
doi: 10.1111/tpj.12762 pmid: 25603991
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