拟南芥CKI1基因上游转录调控因子筛选及鉴定
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摘要
拟南芥CKI1(cytokininindependent 1)是双组分信号系统中一个组氨酸激酶蛋白,通过作用于下游组氨酸磷酸转移蛋白激活双组分信号通路,在调控胚囊中央细胞命运分化和发育过程中具有重要作用。然而目前对于CKI1基因上游转录调控因子还知之甚少。本研究分析了不同长度的CKI1启动子在拟南芥胚囊中的活性,并利用酵母单杂交技术对CKI1上游转录调控因子进行了筛选和鉴定。结果表明,位于内含子区域中的F5/R2片段表现出与CKI1启动子全长相一致的表达活性。进一步选取3个串联重复的F5/R2片段用于构建诱饵表达载体,同时,选取拟南芥雌蕊构建cDNA文库,通过酵母单杂交筛选获得226个阳性克隆。去除低质量及冗余重复的序列后共获得66条可读序列,其中8条序列对应的基因编码具有DNA结合功能的蛋白。研究结果为进一步揭示CKI1基因的转录调控机制提供了重要参考信息。
Arabidopsis CKI1(cytokinin independent 1) is a histidine kinase protein involved in the two-component system, which can activate two-component signaling via the downstream histidine phospho-transfer proteins, playing the essential roles in central cell fate determination and development regulation in embryo sacs. However, studies on CKI1 upstream transcription regulators are still limited. In the present study, promoter activities with varying fragments were investigated, and CKI1 upstream transcription regulators were screened and identified by the yeast-one hybrid technique.Results indicated F5/R2 fragments located in the intron region showed promoter activities in embryo sacs, which is consistent with CKI1 full-length promoters. Then three tandem repeats of F5/R2 fragments were used to construct the bait expression vector, and Arabidopsis pistils were collected for cDNA library construction. Totally, 226 positive clones were screened by the yeast-one hybrid technique, 66 readable sequences were retrieved after removing sequences with low quality and redundant repeats, among which eight proteins could act as DNA-binding proteins. These results provided some important clues to study the molecular function of CKI1 in the transcription regulation network.
Arabidopsis CKI1(cytokinin independent 1) is a histidine kinase protein involved in the two-component system, which can activate two-component signaling via the downstream histidine phospho-transfer proteins, playing the essential roles in central cell fate determination and development regulation in embryo sacs. However, studies on CKI1 upstream transcription regulators are still limited. In the present study, promoter activities with varying fragments were investigated, and CKI1 upstream transcription regulators were screened and identified by the yeast-one hybrid technique.Results indicated F5/R2 fragments located in the intron region showed promoter activities in embryo sacs, which is consistent with CKI1 full-length promoters. Then three tandem repeats of F5/R2 fragments were used to construct the bait expression vector, and Arabidopsis pistils were collected for cDNA library construction. Totally, 226 positive clones were screened by the yeast-one hybrid technique, 66 readable sequences were retrieved after removing sequences with low quality and redundant repeats, among which eight proteins could act as DNA-binding proteins. These results provided some important clues to study the molecular function of CKI1 in the transcription regulation network.
引文
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[24]Xie XZ,Wu NH.Isolation of protease inhibitor II and functional analysis of its intron in tomato.Chin Sci Bull,2001,46(11):934-938.谢先芝,吴乃虎.番茄蛋白酶抑制剂Ⅱ基因的分离及其内含子功能.科学通报,2001,46(11):934-938.
[25]Wang Y,Joh K,Masuko S,Yatsuki H,Soejima H,Nabetani A,Beechey CV,Okinami S,Mukai T.The mouse Murr1 gene is imprinted in the adult brain,presumably due to transcriptional interference by the antisenseoriented U2af1-rs1 gene.Mol Cell Biol,2004,24(1):270-279.
[26]Okada T,Endo M,Singh MB,Bhalla PL.Analysis of the histone H3 gene family in Arabidopsis and identification of the male-gamete-specific variant AtMGH3.Plant J,2005,44(4):557-568.
[27]Wituszyńska W,Szechyńska-hebda M,Sobczak M,Rusaczonek A,Koz?owska-makulska A,WitońD,Karpiński S.LESION SIMULATING DISEASE 1 and ENHANCED DISEASE SUSCEPTIBILITY 1 differentially regulate UV-C-induced photooxidative stress signalling and programmed cell death in Arabidopsis thaliana.Plant Cell Environ,2015,38(2):315-330.
[28]Ou B,Yin KQ,Liu SN,Yang Y,Gu T,Wing Hui JM,Zhang L,Miao J,Kondou Y,Matsui M,Gu HY,Qu LJ.Ahigh-throughput screening system for Arabidopsis transcription factors and its application to Med25-dependent transcriptional regulation.Mol Plant,2011,4(3):546-555.
[2]Kakimoto T.CKI1,a histidine kinase homolog implicated in cytokinin signal transduction.Science,1996,274(5289):982-985.
[3]Hwang I,Sheen J.Two-component circuitry in Arabidopsis cytokinin signal transduction.Nature,2001,413(6854):383-389.
[4]Rabiger DS,Drews GN.MYB64 and MYB119 are required for cellularization and differentiation during female gametogenesis in Arabidopsis thaliana.PLoSGenet,2013,9(9):e1003783.
[5]Yuan L,Liu ZN,Song XY,Johnson C,Yu XL,Sundaresan V.The CKI1 histidine kinase specifies the female gametic precursor of the endosperm.Dev Cell,2016,37(1):34-46.
[6]Deng Y,Dong H,Mu J,Ren B,Zheng B,Ji Z,Yang WC,Liang Y,Zuo J.Arabidopsis histidine kinase CKI1 acts upstream of histidine phosphotransfer proteins to regulate female gametophyte development and vegetative growth.The Plant Cell,2010,22(4):1232-1248.
[7]Hejátko J.PernisováM,Eneva T,Palme K,BrzobohatyB.The putative sensor histidine kinase CKI1 is involved in female gametophyte development in Arabidopsis.Mol Genet Genomics,2003,269(4):443-453.
[8]Hejátko J,Ryu H,Kim GT,DobesováR,Choi S,Choi SM,Soucek P,Horák J,PekárováB,Palme K,Brzobohaty B,Hwang I.The histidine kinases CYTOKININ-INDEPENDENT1 and ARABIDOPSIS HISTIDINEKINASE2 and 3 regulate vascular tissue development in Arabidopsis shoots.The Plant Cell,2009,21(7):2008-2021.
[9]Pischke MS,Jones LG,Otsuga D,Fernandez DE,Drews GN,Sussman MR.An Arabidopsis histidine kinase is essential for megagametogenesis.Proc Natl Acad Sci USA,2002,99(24):15800-15805.
[10]Ito Y,Kurata N.Identification and characterization of cytokinin-signalling gene families in rice.Gene,2006,382,57-65.
[11]Liu ZN,Yuan L,Song XY,Yu XL,Sundaresan V.AHP2,AHP3,and AHP5 act downstream of CKI1 in Arabidopsis female gametophyte development.J Exp Bot,2017,68(13):3365-3373.
[12]Liu ZN,Yuan L,Sundaresan V,Yu X.Arabidopsis CKI1mediated two-component signaling in the specification of female gametophyte.Plant Signal Behav,2018,13(10):e1469360.
[13]Dobisova T,Hrdinova V,Cuesta C,Michlickova S,Urbankova I,Hejatkova R,Zadnikova P,Pernisova M,Benkova E,Hejatko J.Light controls cytokinin signaling via transcriptional regulation of constitutively active sensor histidine kinase CKI1.Plant Physiol,2017,174(1):387-404.
[14]Li JJ,Herskowitz I.Isolation of ORC6,a component of the yeast origin recognition complex by a one-hybrid system.Science,1993,262(5141):1870-1874.
[15]Pruneda-Paz JL,Breton G,Nagel DH,Kang SE,Bonaldi K,Doherty CJ,Ravelo S,Galli M,Ecker,JR,Kay SA.Agenome-scale resource for the functional characterization of Arabidopsis transcription factors.Cell Rep,2014,8(2):622-632.
[16]Castrillo G,Turck F,Leveugle M,Lecharny A,Carbonero P,Coupland G,Paz-Ares J,O?ate-Sánchez L.Speeding cis-trans regulation discovery by phylogenomic analyses coupled with screenings of an arrayed library of Arabidopsis transcription factors.PLoS One,2011,6(6):e21524.
[17]Clough SJ,Bent AF.Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J,1998,16(6):735-743.
[18]Zhang X,Henriques R,Lin SS,Niu QW,Chua NH.Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method.Nat Protoc,2006,1(2):641-646.
[19]Smyth DR,Bowman JL,Meyerowitz EM.Early flower development in Arabidopsis.Plant Cell,1990,2(8):755-767.
[20]Schmid MW,Schmidt A,Klostermeier UC,Barann M,Rosenstiel P,Grossniklaus U.A powerful method for transcriptional profiling of specific cell types in eukaryotes:laser-assisted microdissection and RNASequencing.PLoS One,2012:7(1):e29685.
[21]Nolte-’t Hoen EN,Buermans HP,Waasdorp M,Stoorvogel W,Wauben MH,’t Hoen PA.Deep sequencing of RNAfrom immune cell-derived vesicles uncovers the selective incorporation of small non-coding RNA biotypes with potential regulatory functions.Nucleic Acids Res,2012,40(18):9272-9285.
[22]Chang KC.Critical regulatory domains in intron 2 of a porcine sarcomeric myosin heavy chain gene.J Muscle Res Cell M,2000,21(5):451-461.
[23]Salgueiro S,Pignocchi C,Parry MA.Intron-mediated gusA expression in tritordeum and wheat resulting from particle bombardment.Plant Mol Biol,2000,42(4):615-622.
[24]Xie XZ,Wu NH.Isolation of protease inhibitor II and functional analysis of its intron in tomato.Chin Sci Bull,2001,46(11):934-938.谢先芝,吴乃虎.番茄蛋白酶抑制剂Ⅱ基因的分离及其内含子功能.科学通报,2001,46(11):934-938.
[25]Wang Y,Joh K,Masuko S,Yatsuki H,Soejima H,Nabetani A,Beechey CV,Okinami S,Mukai T.The mouse Murr1 gene is imprinted in the adult brain,presumably due to transcriptional interference by the antisenseoriented U2af1-rs1 gene.Mol Cell Biol,2004,24(1):270-279.
[26]Okada T,Endo M,Singh MB,Bhalla PL.Analysis of the histone H3 gene family in Arabidopsis and identification of the male-gamete-specific variant AtMGH3.Plant J,2005,44(4):557-568.
[27]Wituszyńska W,Szechyńska-hebda M,Sobczak M,Rusaczonek A,Koz?owska-makulska A,WitońD,Karpiński S.LESION SIMULATING DISEASE 1 and ENHANCED DISEASE SUSCEPTIBILITY 1 differentially regulate UV-C-induced photooxidative stress signalling and programmed cell death in Arabidopsis thaliana.Plant Cell Environ,2015,38(2):315-330.
[28]Ou B,Yin KQ,Liu SN,Yang Y,Gu T,Wing Hui JM,Zhang L,Miao J,Kondou Y,Matsui M,Gu HY,Qu LJ.Ahigh-throughput screening system for Arabidopsis transcription factors and its application to Med25-dependent transcriptional regulation.Mol Plant,2011,4(3):546-555.
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