水稻LysM结构域包含基因OsEMSA1的克隆及过表达株系构建
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摘要
LysM结构域包含蛋白(lysM domain containing protein)为植物中公认的病原菌信号受体蛋白。本研究在水稻雌性不育基因FST遗传调控网中筛选获得一个关键靶基因,暂命名为OsEMSA1,该基因编码包含一个LysM结构域的未知功能蛋白质。蛋白质序列分析表明,OsEMSA1蛋白N端包含一个信号肽序列,具备跨膜结构,LysM结构域位于蛋白C端,为胞外结构。启动子顺式作用元件分析表明,光响应元件、激素应答元件、生长调节元件在OsEMSA1启动子区有很高的分布。电子表达谱分析表明,OsEMSA1基因在野生型水稻日本晴多组织中均有不同程度的表达,而根中和开花前的胚囊中表达量相对较高,可能参与调控水稻根和雌配子发育,同时逆境胁迫、激素信号以及病菌侵害也能不同程度的诱导OsEMSA1基因的表达。基因共表达分析显示,OsEMSA1基因与激素信号传导响应、逆境胁迫应答以及抵御真菌病害的基因存在互作。本研究成功构建了由OsEMSA1基因自身启动子驱动的过表达转基因水稻株系,为进一步分析OsEMSA1基因功能奠定了实验基础,并为发掘LysM结构域包含蛋白的潜在功能提供了一定的理论依据。
LysM domain-c ontaining protein is generally recognized as a pathogen signal receptor protein in plants.In this study,a key target gene was screened from the genetic regulatory network of female sterile gene FST in rice,which was temporarily named as OsEMSA1.The gene encoded a function-unknown protein that contained a LysM domain.Protein sequence analysis showed that the N-terminus of OsEMSA1 protein contained a signal peptide sequence with a transmembrane structure.The LysM domain was located at the C-terminus of OsEMSA1 protein,which was an extracellular structure.The cis-acting element analysis for promoter indicated that the lightresponsive elements,hormone responsive elements and growth regulatory elements were highly distributed in the OsEMSA1 promoter region.The electronic expression profile analysis suggested that OsEMSA1 was expressed with various degrees in different tissues of Nipponbare,and the expression levels of OsEMSA1 in root as well as in pre-flowering embryosac were higher than those in others,which implied that OsEMSA1 might be involved in the regulation of root and female gamete development.Moreover,adversity stress,hormone signaling and pathogen infections could induce the expression of OsEMSA1 in different degrees.Gene co-expression analysis revealed that OsEMSA1 interacted with some other genes which were involved in hormone signaling,abiotic stress response and pathogen defense.In this study,an overexpression transgenic rice strain driven by OsEMSA1 promoter was successfully constructed,which established an experimental basis for further research of analysis of OsEMSA1 function and provided a theoretical basis for exploring the potential function of LysM domain-containing protein in rice.
LysM domain-c ontaining protein is generally recognized as a pathogen signal receptor protein in plants.In this study,a key target gene was screened from the genetic regulatory network of female sterile gene FST in rice,which was temporarily named as OsEMSA1.The gene encoded a function-unknown protein that contained a LysM domain.Protein sequence analysis showed that the N-terminus of OsEMSA1 protein contained a signal peptide sequence with a transmembrane structure.The LysM domain was located at the C-terminus of OsEMSA1 protein,which was an extracellular structure.The cis-acting element analysis for promoter indicated that the lightresponsive elements,hormone responsive elements and growth regulatory elements were highly distributed in the OsEMSA1 promoter region.The electronic expression profile analysis suggested that OsEMSA1 was expressed with various degrees in different tissues of Nipponbare,and the expression levels of OsEMSA1 in root as well as in pre-flowering embryosac were higher than those in others,which implied that OsEMSA1 might be involved in the regulation of root and female gamete development.Moreover,adversity stress,hormone signaling and pathogen infections could induce the expression of OsEMSA1 in different degrees.Gene co-expression analysis revealed that OsEMSA1 interacted with some other genes which were involved in hormone signaling,abiotic stress response and pathogen defense.In this study,an overexpression transgenic rice strain driven by OsEMSA1 promoter was successfully constructed,which established an experimental basis for further research of analysis of OsEMSA1 function and provided a theoretical basis for exploring the potential function of LysM domain-containing protein in rice.
引文
Agrawal G.K.,Jwa N.S.,Shibato J.,Han O.,Iwahashi H.,and Rakwal R.,2003,Diverse environmental cues transiently regulate OsOPR1 of the“octadecanoid pathway”revealing its importance in rice defense/stress and development,Biochem.Biophys.Res.Commun.,310(4):1073-1082
Fan J.,2015,Cloning and sequence analysis of the 5'upstream regulatory region of seed-specific OsEm gene in rice,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(4):797-805(范晶,2015,水稻种子特异表达OsEm基因5'上游调控区的克隆与序列分析,基因组学与应用生物学,34(4):797-805)
Fujita M.,Horiuchi Y.,Ueda Y.,Mizuta Y.,Kubo T.,Yano K.,Yamaki S.,Tsuda K.,Nagata T.,Niihama M.,Kato H.,Kikuchi S.,Hamada K.,Mochizuki T.,Ishimizu T.,Iwai H.,Tsutsumi N.,and Kurata N.,2010,Rice expression atlas in reproductive development,Plant Cell Physiol.,51(12):2060-2081
Garvey K.J.,Saedi M.S.,and Ito J.,1986,Nucleotide sequence of Bacillus phage phi 29 genes 14 and 15:homology of gene15 with other phage lysozymes,Nucleic Acids Res.,14(24):10001-10008
Gust A.A.,Willmann R.,Desaki Y.,Grabherr H.M.,and Nürnberger T.,2012,Plant LysMproteins:modulesmediating symbiosis and immunity,Trends Plant Sci.,17(8):495-502
Hiei Y.,Ohta S.,Komari T.,and Kumashiro T.,1994,Effcient transformation of rice(Oryza sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA,Plant J.,6(2):271-282
Hirose N.,Makita N.,Kojima M.,Kamada-Nobusada T.,and Sakakibara H.,2007,Overexpression of a type-A response regulator alters rice morphology and cytokinin metabolism,Plant Cell Physiol.,48(3):523-539
Kaku H.,Nishizawa Y.,Ishii-minami N.,Akimoto-Tomiyama C.,Dohmae N.,Takio K.,Minami E.,and Shibuya N.,2006,Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor,Proc.Natl.Acad.Sci.USA,103(29):11086-11091
Kang H.G.,and Klessig D.F.,2005,Salicylic acid-inducible Arabidopsis CK2-like activity phosphorylates TGA2,Plant Mol.Biol.,57(4):541-557
Laroche F.J.,Tulotta C.,Lamers G.E.,Meijer A.H.,Yang P.,Verbeek F.J.,Blaise M.,Stougaard J.,and Spaink H.P.,2013,The embryonic expression patterns of zebrafish genes encodingLysM-domains,Gene Expression Patterns,13(7):212-224
Lasanthi-Kudahettige R.,Magneschi L.,Loreti E.,Gonzali S.,Licausi F.,Novi G.,Beretta O.,Vitulli F.,Alpi A.,and Perata P.,2007,Transcript profiling of the anoxic rice coleoptile,Plant Physiol.,144(1):218-231
Lee D.K.,Kim H.I.,Jang G.,Chung P.J.,Jeong J.S.,Kim Y.S.,Bang S.W.,Jung H.,Choi Y.D.,and Kim J.K.,2015,The NF-YA transcription factor OsNF-YA7 confers drought s tress tolerance of rice in an abscisic acid independent manner,Plant Sci.,241:199-210
Lee D.S.,Chen L.J.,Li C.Y.,Liu Y.S.,Tan X.L.,Lu B.R.,Li J.,Gan S.X.,Kang S.G.,Suh H.S.,and Zhu Y.,2013,The Bsister MADS gene FST determines ovule patterning and development of the zygotic embryo and endosperm,PLoSOne,8(3):e58748
Li D.X.,Li J.,Gan S.X.,Tan X.L.,and Chen L.J.,2010,A rapid sampling device for leaf PCR,China Patent,CN201512535U(李东宣,李娟,甘树仙,谭学林,陈丽娟,2010,一种叶片PCR快速取样装置,中国专利,CN201512535U)
Li J.,Liang D.,Li M.,and Ma F.,2013,Light and abiotic stresses regulate the expression of GDP-L-galactose phosphorylase and levels of ascorbic acid in two kiwifruit genotypes via light-responsive and stress-inducible cis-elements in their promoters,Planta,238(3):535-547
Liu B.,Li J.F.,Ao Y.,Qu J.,Li Z.,Su J.,Zhang Y.,Liu J.,Feng D.,Qi K.,He Y.,Wang J.,and Wang H.B.,2012,Lysin motif-containing proteins LYP4 and LYP6 play dual roles in peptidoglycan and chitin perception in rice innate immunity,Plant Cell,24(8):3406-3419
Miya A.,Albert P.,Shinya T.,Desaki Y.,Ichimura K.,Shirasu K.,Narusaka Y.,Kawakami N.,Kaku H.,and Shibuya N.,2007,CERK1,a LysM receptor kinase,is essential for chitin elicitor signaling in Arabidopsis,Proc.Natl.Acad.Sci.USA,104(49):19613-19618
Miyata K.,Kozaki T.,Kouzai Y.,Ozawa K.,Ishii K.,Asamizu E.,Okabe Y.,Umehara Y.,Miyamoto A.,Kobae Y.,Akiyama K.,Kaku H.,Nishizawa Y.,Shibuya N.,and Nakagawa T.,2014,The bifunctional plant receptor,OsCERK1,regulates both chitin-triggered immunity and arbuscular mycorrhizal symbiosis in rice,Plant Cell Physiol.,55(11):1864-1872
Mutwil M.,Obro J.,Willats W.G.,and Persson S.,2008,GeneCAT--novel webtools that combine BLAST and co-expression analyses,Nucleic Acids Res.,36:W320-W326
Norton G.J.,Lou-Hing D.E.,Meharg A.A.,and Price A.H.,2008,Rice-arsenate interactions in hydroponics:whole genome transcriptional analysis,J.Exp.Bot.,59(8):2267-2276
Pei H.,Ma N.,Tian J.,Luo J.,Chen J.,Li J.,Zheng Y.,Chen X.,Fei Z.,and Gao J.,2013,An NAC transcription factor controls ethylene-regulated cell expansion in flower petals,Plant Physiol.,163(2):775-791
Ribot C.,Hirsch J.,Balzergue S.,Tharreau D.,Nottéghem J.L.,Lebrun M.H.,and Morel J.B.,2008,Susceptibility of rice to the blast fungus,Magnaporthe grisea,J.Plant Physiol.,165(1):114-124
Roy S.,Choudhury S.R.,Singh S.K.,and Das K.P.,2012,Functional analysis of light-regulated promoter region of AtPolλgene,Planta,235(2):411-432
Shi X.Z.,Feng X.W.,Sun J.J.,Yang M.C.,Lan J.F.,Zhao X.F.,and Wang J.X.,2016,Involvement of a LysM and putative peptidoglycan-binding domain-containing protein in the antibacterial immune response of kuruma shrimp Marsupenaeus japonicus,Fish Shellfish Immunol.,54:489-498
Shimizu T.,Nakano T.,Takamizawa D.,Desaki Y.,Ishii-Minami N.,Nishizawa Y.,Minami E.,Okada K.,Yamane H.,Kaku H.,and Shibuya N.,2010,Two LysM receptor molecules,CEBiP and OsCERK1,cooperatively regulate chitin elicitor signaling in rice,Plant J.,64(2):204-214
Tanaka K.,Nguyen C.T.,Liang Y.,Cao Y.,and Stacey G.,2013,Role of LysM receptors in chitin-triggered plant innate immunity,Plant Signal.Behav.,8(1):e22598
Wan J.,Zhang X.C.,Neece D.,Ramonell K.M.,Clough S.,Kim S.Y.,StaceyM.G.,and Stacey G.,2008,A LysM receptor-like kinase plays a critical role in chitin signaling and fungal resistance in Arabidopsis,Plant Cell,20(2):471-481
Wang J.,Li H.,Han Z.,Zhang H.,Wang T.,Lin G.,Chang J.,Yang W.,and Chai J.,2015,Allosteric receptor activation by the plant peptide hormone phytosulfokine,Nature,525(7568):265-268
Wang Y.,Liu K.,Liao H.,Zhuang C.,Ma H.,and Yan X.,2008,The plant WNK gene family and regulation of flowering time in Arabidopsis,Plant Biol.,10(5):548-562
Yazaki J.,Shimatani Z.,Hashimoto A.,Nagata Y.,Fujii F.,Kojima K.,Suzuki K.,Taya T.,Tonouchi M.,Nelson C.,Nakagawa A.,Otomo Y.,Murakami K.,Matsubara K.,Kawai J.,Carninci P.,Hayashizaki Y.,and Kikuchi S.,2004,Transcriptional profiling of genes responsive to abscisic acid and gibberellin in rice:phenotyping and comparative analysis between rice and Arabidopsis,Physiol Genomics.,17(2):87-100
Yuan J.,Chen D.,Ren Y.,Zhang X.,and Zhao J.,2008,Characteristic and expression analysis of a metallothionein gene,OsMT2b,down-regulated by cytokinin suggests functions in root development and seed embryo germination of rice,Plant Physiol.,146(4):1637-1650
Fan J.,2015,Cloning and sequence analysis of the 5'upstream regulatory region of seed-specific OsEm gene in rice,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(4):797-805(范晶,2015,水稻种子特异表达OsEm基因5'上游调控区的克隆与序列分析,基因组学与应用生物学,34(4):797-805)
Fujita M.,Horiuchi Y.,Ueda Y.,Mizuta Y.,Kubo T.,Yano K.,Yamaki S.,Tsuda K.,Nagata T.,Niihama M.,Kato H.,Kikuchi S.,Hamada K.,Mochizuki T.,Ishimizu T.,Iwai H.,Tsutsumi N.,and Kurata N.,2010,Rice expression atlas in reproductive development,Plant Cell Physiol.,51(12):2060-2081
Garvey K.J.,Saedi M.S.,and Ito J.,1986,Nucleotide sequence of Bacillus phage phi 29 genes 14 and 15:homology of gene15 with other phage lysozymes,Nucleic Acids Res.,14(24):10001-10008
Gust A.A.,Willmann R.,Desaki Y.,Grabherr H.M.,and Nürnberger T.,2012,Plant LysMproteins:modulesmediating symbiosis and immunity,Trends Plant Sci.,17(8):495-502
Hiei Y.,Ohta S.,Komari T.,and Kumashiro T.,1994,Effcient transformation of rice(Oryza sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA,Plant J.,6(2):271-282
Hirose N.,Makita N.,Kojima M.,Kamada-Nobusada T.,and Sakakibara H.,2007,Overexpression of a type-A response regulator alters rice morphology and cytokinin metabolism,Plant Cell Physiol.,48(3):523-539
Kaku H.,Nishizawa Y.,Ishii-minami N.,Akimoto-Tomiyama C.,Dohmae N.,Takio K.,Minami E.,and Shibuya N.,2006,Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor,Proc.Natl.Acad.Sci.USA,103(29):11086-11091
Kang H.G.,and Klessig D.F.,2005,Salicylic acid-inducible Arabidopsis CK2-like activity phosphorylates TGA2,Plant Mol.Biol.,57(4):541-557
Laroche F.J.,Tulotta C.,Lamers G.E.,Meijer A.H.,Yang P.,Verbeek F.J.,Blaise M.,Stougaard J.,and Spaink H.P.,2013,The embryonic expression patterns of zebrafish genes encodingLysM-domains,Gene Expression Patterns,13(7):212-224
Lasanthi-Kudahettige R.,Magneschi L.,Loreti E.,Gonzali S.,Licausi F.,Novi G.,Beretta O.,Vitulli F.,Alpi A.,and Perata P.,2007,Transcript profiling of the anoxic rice coleoptile,Plant Physiol.,144(1):218-231
Lee D.K.,Kim H.I.,Jang G.,Chung P.J.,Jeong J.S.,Kim Y.S.,Bang S.W.,Jung H.,Choi Y.D.,and Kim J.K.,2015,The NF-YA transcription factor OsNF-YA7 confers drought s tress tolerance of rice in an abscisic acid independent manner,Plant Sci.,241:199-210
Lee D.S.,Chen L.J.,Li C.Y.,Liu Y.S.,Tan X.L.,Lu B.R.,Li J.,Gan S.X.,Kang S.G.,Suh H.S.,and Zhu Y.,2013,The Bsister MADS gene FST determines ovule patterning and development of the zygotic embryo and endosperm,PLoSOne,8(3):e58748
Li D.X.,Li J.,Gan S.X.,Tan X.L.,and Chen L.J.,2010,A rapid sampling device for leaf PCR,China Patent,CN201512535U(李东宣,李娟,甘树仙,谭学林,陈丽娟,2010,一种叶片PCR快速取样装置,中国专利,CN201512535U)
Li J.,Liang D.,Li M.,and Ma F.,2013,Light and abiotic stresses regulate the expression of GDP-L-galactose phosphorylase and levels of ascorbic acid in two kiwifruit genotypes via light-responsive and stress-inducible cis-elements in their promoters,Planta,238(3):535-547
Liu B.,Li J.F.,Ao Y.,Qu J.,Li Z.,Su J.,Zhang Y.,Liu J.,Feng D.,Qi K.,He Y.,Wang J.,and Wang H.B.,2012,Lysin motif-containing proteins LYP4 and LYP6 play dual roles in peptidoglycan and chitin perception in rice innate immunity,Plant Cell,24(8):3406-3419
Miya A.,Albert P.,Shinya T.,Desaki Y.,Ichimura K.,Shirasu K.,Narusaka Y.,Kawakami N.,Kaku H.,and Shibuya N.,2007,CERK1,a LysM receptor kinase,is essential for chitin elicitor signaling in Arabidopsis,Proc.Natl.Acad.Sci.USA,104(49):19613-19618
Miyata K.,Kozaki T.,Kouzai Y.,Ozawa K.,Ishii K.,Asamizu E.,Okabe Y.,Umehara Y.,Miyamoto A.,Kobae Y.,Akiyama K.,Kaku H.,Nishizawa Y.,Shibuya N.,and Nakagawa T.,2014,The bifunctional plant receptor,OsCERK1,regulates both chitin-triggered immunity and arbuscular mycorrhizal symbiosis in rice,Plant Cell Physiol.,55(11):1864-1872
Mutwil M.,Obro J.,Willats W.G.,and Persson S.,2008,GeneCAT--novel webtools that combine BLAST and co-expression analyses,Nucleic Acids Res.,36:W320-W326
Norton G.J.,Lou-Hing D.E.,Meharg A.A.,and Price A.H.,2008,Rice-arsenate interactions in hydroponics:whole genome transcriptional analysis,J.Exp.Bot.,59(8):2267-2276
Pei H.,Ma N.,Tian J.,Luo J.,Chen J.,Li J.,Zheng Y.,Chen X.,Fei Z.,and Gao J.,2013,An NAC transcription factor controls ethylene-regulated cell expansion in flower petals,Plant Physiol.,163(2):775-791
Ribot C.,Hirsch J.,Balzergue S.,Tharreau D.,Nottéghem J.L.,Lebrun M.H.,and Morel J.B.,2008,Susceptibility of rice to the blast fungus,Magnaporthe grisea,J.Plant Physiol.,165(1):114-124
Roy S.,Choudhury S.R.,Singh S.K.,and Das K.P.,2012,Functional analysis of light-regulated promoter region of AtPolλgene,Planta,235(2):411-432
Shi X.Z.,Feng X.W.,Sun J.J.,Yang M.C.,Lan J.F.,Zhao X.F.,and Wang J.X.,2016,Involvement of a LysM and putative peptidoglycan-binding domain-containing protein in the antibacterial immune response of kuruma shrimp Marsupenaeus japonicus,Fish Shellfish Immunol.,54:489-498
Shimizu T.,Nakano T.,Takamizawa D.,Desaki Y.,Ishii-Minami N.,Nishizawa Y.,Minami E.,Okada K.,Yamane H.,Kaku H.,and Shibuya N.,2010,Two LysM receptor molecules,CEBiP and OsCERK1,cooperatively regulate chitin elicitor signaling in rice,Plant J.,64(2):204-214
Tanaka K.,Nguyen C.T.,Liang Y.,Cao Y.,and Stacey G.,2013,Role of LysM receptors in chitin-triggered plant innate immunity,Plant Signal.Behav.,8(1):e22598
Wan J.,Zhang X.C.,Neece D.,Ramonell K.M.,Clough S.,Kim S.Y.,StaceyM.G.,and Stacey G.,2008,A LysM receptor-like kinase plays a critical role in chitin signaling and fungal resistance in Arabidopsis,Plant Cell,20(2):471-481
Wang J.,Li H.,Han Z.,Zhang H.,Wang T.,Lin G.,Chang J.,Yang W.,and Chai J.,2015,Allosteric receptor activation by the plant peptide hormone phytosulfokine,Nature,525(7568):265-268
Wang Y.,Liu K.,Liao H.,Zhuang C.,Ma H.,and Yan X.,2008,The plant WNK gene family and regulation of flowering time in Arabidopsis,Plant Biol.,10(5):548-562
Yazaki J.,Shimatani Z.,Hashimoto A.,Nagata Y.,Fujii F.,Kojima K.,Suzuki K.,Taya T.,Tonouchi M.,Nelson C.,Nakagawa A.,Otomo Y.,Murakami K.,Matsubara K.,Kawai J.,Carninci P.,Hayashizaki Y.,and Kikuchi S.,2004,Transcriptional profiling of genes responsive to abscisic acid and gibberellin in rice:phenotyping and comparative analysis between rice and Arabidopsis,Physiol Genomics.,17(2):87-100
Yuan J.,Chen D.,Ren Y.,Zhang X.,and Zhao J.,2008,Characteristic and expression analysis of a metallothionein gene,OsMT2b,down-regulated by cytokinin suggests functions in root development and seed embryo germination of rice,Plant Physiol.,146(4):1637-1650
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