拟南芥卵形家族蛋白AtOFP1与KNAT5互作及功能初步分析
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摘要
拟南芥卵形家族蛋白(Arabidopsis thaliana ovate family protein, AtOFP)是一种新型的植物特有的转录因子,AtOFP1为其中重要一员。TALE类同源结构域蛋白(3 amino-acid loop extension homeodomain protein)在植物发育过程中具有核心作用,其中KNAT5 (homeobox protein knotted-1-like 5)在植物侧根原基表达,可能参与侧根发生,对植株生长发育有重要影响。本实验针对AtOFP1与KNAT5是否具有相互作用,及其相互作用有何功能进行了研究。首先应用酵母双杂交技术和双分子荧光互补技术(bimolecular fluorescence complementation, BiFC)分别证实二者具有相互作用;同时,筛选knat5纯合突变体,获得其纯合突变体拟南芥植株,并利用qRT-PCR方法确定OFP1基因和KNAT5基因在拟南芥根和叶中协同表达,影响侧根数目和根长,在植物生长发育中具有重要作用。本研究为进一步探究AtOFP1与KNAT5互作机制及其对植物生长发育的调控机理奠定了基础。
Arabidopsis thaliana Ovate Family Proteins(AtOFPs) is a novel plant-specific transcription factor.As one of important member of the OFPs, AtOFP1 is the earliest and most studied transcriptional repressor. It has been proved that AtOFP1 can directly regulate the transcriptional activity of AtGA20 ox, a key gene of GA synthase, and inhibit cell elongation. 3-Amino-acid loop extension homeodomain protein(TALE) plays a central role in plant development. It has a potential interaction with AtOFP1, and existing studies have shown that 9 OFPs and TALE proteins have a close functional association with their homeodomains. The literature showed that the ovate family proteins have potential interactions with TALE transcription factor, and identified 13 transcription factors that interact with AtOFP1 by the way of a screening library which include KNAT5. KNAT5 is expressed in the lateral root primordium and may participate in the occurrence of lateral roots, which has an important influence on plant growth and development. In order to further verify the interaction between AtOFP1 and KNAT5 and the effects of the interactions on Arabidopsis thaliana, we first verified the interaction between AtOFP1 and AtKNAT5 by yeast two-hybrid, then their biocompatibility was further verified by bimolecular fluorescence complementation(BiFC). In addition, the knat5 homozygousmutant was screened, and the gene expression of OFP1 and KNAT5 in Col-0, knat5 mutant, ofp1 mutant and HAOFP1 overexpression plant was analyzed by q RT-PCR to compare the expression pattern. Similar expression patterns in the roots and leaves of the plants were found. The existed synergistic expression in roots and leaves of Arabidopsis thaliana could affect the number of lateral roots and root length, which play an important role in plant growth and development. The present study lays the foundation for further exploring the mechanism of AtOFP1-KNAT5 interaction and its regulation function in plant growth and development.
Arabidopsis thaliana Ovate Family Proteins(AtOFPs) is a novel plant-specific transcription factor.As one of important member of the OFPs, AtOFP1 is the earliest and most studied transcriptional repressor. It has been proved that AtOFP1 can directly regulate the transcriptional activity of AtGA20 ox, a key gene of GA synthase, and inhibit cell elongation. 3-Amino-acid loop extension homeodomain protein(TALE) plays a central role in plant development. It has a potential interaction with AtOFP1, and existing studies have shown that 9 OFPs and TALE proteins have a close functional association with their homeodomains. The literature showed that the ovate family proteins have potential interactions with TALE transcription factor, and identified 13 transcription factors that interact with AtOFP1 by the way of a screening library which include KNAT5. KNAT5 is expressed in the lateral root primordium and may participate in the occurrence of lateral roots, which has an important influence on plant growth and development. In order to further verify the interaction between AtOFP1 and KNAT5 and the effects of the interactions on Arabidopsis thaliana, we first verified the interaction between AtOFP1 and AtKNAT5 by yeast two-hybrid, then their biocompatibility was further verified by bimolecular fluorescence complementation(BiFC). In addition, the knat5 homozygousmutant was screened, and the gene expression of OFP1 and KNAT5 in Col-0, knat5 mutant, ofp1 mutant and HAOFP1 overexpression plant was analyzed by q RT-PCR to compare the expression pattern. Similar expression patterns in the roots and leaves of the plants were found. The existed synergistic expression in roots and leaves of Arabidopsis thaliana could affect the number of lateral roots and root length, which play an important role in plant growth and development. The present study lays the foundation for further exploring the mechanism of AtOFP1-KNAT5 interaction and its regulation function in plant growth and development.
引文
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李帅,蒋西子,梁伟芳,等.2017.利用酵母双杂交系统筛选与草莓镶脉病毒P6蛋白互作的森林草莓寄主因子[J].中国农业科学,50(18):3519-3528.(Li S,Jiang X Z,Liang W F,et al.2017.Yeast two-hybrid system screening for forest strawberry host factor interacting with strawberry vein virus P6 protein[J].Chinese Agricultural Sciences,50(18):3519-3528.)
刘杨杨.2017.拟南芥Armadillo蛋白ZAK IXIK与F-box蛋白SAP相互作用参与花器官发育[D].博士学位论文中国农业大学,导师:叶德,pp.50-65.(Liu Y Y.2017Arabidopsis Armadillo protein ZAK IXIK interacts with F-box protein SAP in flower organ development[D]Thesis for Ph.D.,China Agricultural University,Supervisor:YE D,pp.50-65.)
沈珂,吴群英,蒋晓山.2015.双分子荧光互补技术在蛋白质相互作用研究中的应用[J].中国医学创新,12(12)132-135.(Shen K,Wu Q Y,Jiang X S.2015.Application of bimolecular fluorescence complementary technology in protein interaction research[J].Chinese Medical Innovation,12(12):132-135.)
叶曙光,宰文珊,熊自力,等.2017.番茄KNOX基因家族鉴定及茄科作物KNOX基因进化关系分析[J].核农学报,31(7):1263-1271.(Ye S G,Zai W S,Xiong Z Let al.2017.Identification of KNOX gene family in tomato and evolution of KNOX gene in Solanaceae[J]Journal of Nuclear Agricultural Sciences,31(7):1263-1271.)
訾亮,洪灏,翟金玲,等.2013.拟南芥NiNJA基因酵母双杂诱饵载体构建及互作蛋白的筛选[J].热带生物学报,4(1):31-35.(Zi L,Hong H,Zhai J L et al.2013.Construction of Arabidopsis thaliana NiNJA yeast two-hybrid bait vector and screening of interaction proteins[J]Journal of Tropical Biology,4(1):31-35.)
Gong S Y,Huang G Q,Sun X,et al.2014.Cotton KNL1,encoding a class II KNOX transcription factor,is involved in regulation of fibre development[J].Journal of Experimental Botany,65(15):4133-4147.
Hackbusch J,Richter K,Müller J,et al.2005.A central role of Arabidopsis thaliana ovate family proteins in networking and subcellular localization of 3-aa loop extension homeodomain proteins[J].Proceedings of the Nationa Academy of Sciences of the United States of America102(13):4908-4912.
Li E Y,Wang S C,Liu Y Y,et al.2011.Ovate family protein 4(OFP4)interaction with KNAT7 regulates secondary cell wall formation in Arabidopsis thaliana[J].Plant Journal,67(2):328-341.
Li Z,Li B,Shen W H,et al.2012.TCP transcription factors interact with AS2 in the repression of class-ⅠKNOXgene in Arabidopsis thaliana[J].Plant Journal,71(1):99-107
Li Z,Li B,Liu J,et al.2016.Transcription factors AS1 and AS2 interact with LHP1 to repress KNOX genes in Arabidopsis[J].Journal of Integrative Plant Biology,58(12):959-970.
Liu Y,Douglas C J.2015.A role for Ovate family protein1(OFP1)and OFP4 in a BLH6-KNAT7 multi-protein complex regulating secondary cell wall formation in Arabidopsis thaliana[J].Plant Signaling and Behavior,10(7):e1033126
Pagnussat G C,Yu H J,Sundaresan V.2007.Cell-fate switch of synergid to egg cell in Arabidopsis eostre mutant embryo sacs arises from misexpression of the BEL1-like homeodomain gene BLH1[J].Plant Cell,19(11):3578-3592.
Rastsomssich M I,Broholm S,Jenkins H,et al.2015.Alternate wiring of a KNOXI genetic network underlies differences in leaf development of A.thaliana and C.hirsuta.Genes and Development,29(22):2391-2404.
Truernit E,Siemering K R,Hodge S,et al.2006.A map of KNAT gene expression in the Arabidopsis root[J].Plant Molecular Biology,60(1):1-20.
Wang S C,Chang Y,Ellis B E.2016.Overview of ovate family proteins,a novel class of plant-specific growth regulators[J].Frontiers in Plant Science,7:e0123870.
Wang S C,Chang Y,Guo J J,et al.2011.Arabidopsis ovate family proteins,a novel transcriptional repressor family,control multiple aspects of plant growth and development[J].Plos One,6(8):e23896.
Wang S C,Chang Y,Guo,J J,et al.2007.Arabidopsis ovate family protein 1 is a transcriptional repressor that suppresses cell elongation[J].The Plant Journal,50(5):858-872.
Wang Y K,Chang W C,Liu P F,et al.2010.Ovate family protein 1 as a plant Ku70 interacting protein involved in DNA double-strand break repair[J].Plant Molecular Biology,74(4-5):453-466.
Zhang L G,Zhang X F,Ju H X,et al.2016.Ovate family protein1 interaction with BLH3 regulates transition timing from vegetative to reproductive phase in Arabidopsis[J].Biochemical and Biophysical Research Communications,470(3):492-497.
崔喜艳,赵吉元,胡广宇,等.2015.玉米AGPasebt2与GPN1蛋白互作的双分子荧光互补技术研究[J].西北农林科技大学学报(自然科学版),43(6):99-104.(Cui X Y,Zhao J Y,Hu G Y et al.2015.Study on two-molecule fluorescence complementation technique for interaction between maize AGPasebt2 and GPN1 protein[J].Journal of Northwest Sci-Tech University of Agriculture and Forestry,43(6):99-104.)
李帅,蒋西子,梁伟芳,等.2017.利用酵母双杂交系统筛选与草莓镶脉病毒P6蛋白互作的森林草莓寄主因子[J].中国农业科学,50(18):3519-3528.(Li S,Jiang X Z,Liang W F,et al.2017.Yeast two-hybrid system screening for forest strawberry host factor interacting with strawberry vein virus P6 protein[J].Chinese Agricultural Sciences,50(18):3519-3528.)
刘杨杨.2017.拟南芥Armadillo蛋白ZAK IXIK与F-box蛋白SAP相互作用参与花器官发育[D].博士学位论文中国农业大学,导师:叶德,pp.50-65.(Liu Y Y.2017Arabidopsis Armadillo protein ZAK IXIK interacts with F-box protein SAP in flower organ development[D]Thesis for Ph.D.,China Agricultural University,Supervisor:YE D,pp.50-65.)
沈珂,吴群英,蒋晓山.2015.双分子荧光互补技术在蛋白质相互作用研究中的应用[J].中国医学创新,12(12)132-135.(Shen K,Wu Q Y,Jiang X S.2015.Application of bimolecular fluorescence complementary technology in protein interaction research[J].Chinese Medical Innovation,12(12):132-135.)
叶曙光,宰文珊,熊自力,等.2017.番茄KNOX基因家族鉴定及茄科作物KNOX基因进化关系分析[J].核农学报,31(7):1263-1271.(Ye S G,Zai W S,Xiong Z Let al.2017.Identification of KNOX gene family in tomato and evolution of KNOX gene in Solanaceae[J]Journal of Nuclear Agricultural Sciences,31(7):1263-1271.)
訾亮,洪灏,翟金玲,等.2013.拟南芥NiNJA基因酵母双杂诱饵载体构建及互作蛋白的筛选[J].热带生物学报,4(1):31-35.(Zi L,Hong H,Zhai J L et al.2013.Construction of Arabidopsis thaliana NiNJA yeast two-hybrid bait vector and screening of interaction proteins[J]Journal of Tropical Biology,4(1):31-35.)
Gong S Y,Huang G Q,Sun X,et al.2014.Cotton KNL1,encoding a class II KNOX transcription factor,is involved in regulation of fibre development[J].Journal of Experimental Botany,65(15):4133-4147.
Hackbusch J,Richter K,Müller J,et al.2005.A central role of Arabidopsis thaliana ovate family proteins in networking and subcellular localization of 3-aa loop extension homeodomain proteins[J].Proceedings of the Nationa Academy of Sciences of the United States of America102(13):4908-4912.
Li E Y,Wang S C,Liu Y Y,et al.2011.Ovate family protein 4(OFP4)interaction with KNAT7 regulates secondary cell wall formation in Arabidopsis thaliana[J].Plant Journal,67(2):328-341.
Li Z,Li B,Shen W H,et al.2012.TCP transcription factors interact with AS2 in the repression of class-ⅠKNOXgene in Arabidopsis thaliana[J].Plant Journal,71(1):99-107
Li Z,Li B,Liu J,et al.2016.Transcription factors AS1 and AS2 interact with LHP1 to repress KNOX genes in Arabidopsis[J].Journal of Integrative Plant Biology,58(12):959-970.
Liu Y,Douglas C J.2015.A role for Ovate family protein1(OFP1)and OFP4 in a BLH6-KNAT7 multi-protein complex regulating secondary cell wall formation in Arabidopsis thaliana[J].Plant Signaling and Behavior,10(7):e1033126
Pagnussat G C,Yu H J,Sundaresan V.2007.Cell-fate switch of synergid to egg cell in Arabidopsis eostre mutant embryo sacs arises from misexpression of the BEL1-like homeodomain gene BLH1[J].Plant Cell,19(11):3578-3592.
Rastsomssich M I,Broholm S,Jenkins H,et al.2015.Alternate wiring of a KNOXI genetic network underlies differences in leaf development of A.thaliana and C.hirsuta.Genes and Development,29(22):2391-2404.
Truernit E,Siemering K R,Hodge S,et al.2006.A map of KNAT gene expression in the Arabidopsis root[J].Plant Molecular Biology,60(1):1-20.
Wang S C,Chang Y,Ellis B E.2016.Overview of ovate family proteins,a novel class of plant-specific growth regulators[J].Frontiers in Plant Science,7:e0123870.
Wang S C,Chang Y,Guo J J,et al.2011.Arabidopsis ovate family proteins,a novel transcriptional repressor family,control multiple aspects of plant growth and development[J].Plos One,6(8):e23896.
Wang S C,Chang Y,Guo,J J,et al.2007.Arabidopsis ovate family protein 1 is a transcriptional repressor that suppresses cell elongation[J].The Plant Journal,50(5):858-872.
Wang Y K,Chang W C,Liu P F,et al.2010.Ovate family protein 1 as a plant Ku70 interacting protein involved in DNA double-strand break repair[J].Plant Molecular Biology,74(4-5):453-466.
Zhang L G,Zhang X F,Ju H X,et al.2016.Ovate family protein1 interaction with BLH3 regulates transition timing from vegetative to reproductive phase in Arabidopsis[J].Biochemical and Biophysical Research Communications,470(3):492-497.