水稻黄素单加氧酶新基因OsFMO1表达特性及其功能初探
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摘要
黄素单加氧酶(flavin-containing monooxygenase, FMO)是一种微粒体酶,目前研究多集中在人类中,植物中研究较少;植物FMOs则与生长素合成、信号传导、极性运输以及病原防御等有关,其中报导过的YU-CCA基因与花发育密切相关。本研究从水稻野生型黎榆B (O. sativa L. ssp. japonica)基因组中克隆了一个未知功能的新基因OsFMO1,生物信息学分析表明,水稻OsFMO1基因位于水稻第9号染色体上,cDNA全长2 100 bp,包含4个外显子,3个内含子,共有519个氨基酸,具有FMO-like家族的保守结构域。本研构建了植物融合表达载体pOsFMO1::GUS和pCaMV35S::OsFMO1:RNAi,用于基因表达和功能分析,使用农杆菌介导法获得了转基因植株。化学组织染色结果表明,OsFMO1在不同时期不同组织中均有表达。在减数分裂期在幼穗和叶茎上表达最为强烈。通过RT-PCR结果显示该目的基因在不同时期在叶片和叶鞘中均有表达,但表达量有明显的不同。结果表明OsFMO1基因可影响植物的生长素合成、信号传导、极性运输以及水稻的生长发育。该研究结果为进一步研究水稻OsFMO1基因在水稻生长发育以及生产量上的研究提供了科学依据。
Flavin-containing monooxygenase(FMO) is a class of microsomal enzyme. At present, most of the research is concentrated in human beings, but few in plants Plant FMOs are associated with auxin synthesis, signal transduction, polar transport, and pathogen defense, among which the YUCCA gene reported is closely related to flower development. In this study, a novel gene OsFMO1 with unknown function was cloned from the genome of LiyuB(O. sativa L. ssp. japonica) in rice. The bioinformatics analysis showed that the rice Os FMO1 gene was located on rice chromosome 9. The full length cDNA was 2 100 bp long with 4 exons and 3 introns and encoding for a protein containing 519 amino acid with a conserved domain of FMO-like family. In this research, the plant fusion expression vectors pOsFMO1::GUS and pCaMV35 S::OsFMO1:RNAi were constructed for gene expression and function analysis. Transgenic plants were generated using Agrobacterium mediated appraoch. GUS staining results showed that OsFMO1 was expressed in different tissues at different developmental stages. The gene was found tobe strongly expressed in developing spikelets and leaf sheaths during meiosis. RT-PCR results showed that the OsFMO1 was expressed in leaves and leaf sheaths, but the expression levels were significantly different.Furthermore, the results also revealed that the OsFMO1 gene could affect auxin synthesis, signal transduction, polar transport, and rice growth and development. This study might provide a scientific basis for further studies on the growth and yield of rice OsFMO1.
Flavin-containing monooxygenase(FMO) is a class of microsomal enzyme. At present, most of the research is concentrated in human beings, but few in plants Plant FMOs are associated with auxin synthesis, signal transduction, polar transport, and pathogen defense, among which the YUCCA gene reported is closely related to flower development. In this study, a novel gene OsFMO1 with unknown function was cloned from the genome of LiyuB(O. sativa L. ssp. japonica) in rice. The bioinformatics analysis showed that the rice Os FMO1 gene was located on rice chromosome 9. The full length cDNA was 2 100 bp long with 4 exons and 3 introns and encoding for a protein containing 519 amino acid with a conserved domain of FMO-like family. In this research, the plant fusion expression vectors pOsFMO1::GUS and pCaMV35 S::OsFMO1:RNAi were constructed for gene expression and function analysis. Transgenic plants were generated using Agrobacterium mediated appraoch. GUS staining results showed that OsFMO1 was expressed in different tissues at different developmental stages. The gene was found tobe strongly expressed in developing spikelets and leaf sheaths during meiosis. RT-PCR results showed that the OsFMO1 was expressed in leaves and leaf sheaths, but the expression levels were significantly different.Furthermore, the results also revealed that the OsFMO1 gene could affect auxin synthesis, signal transduction, polar transport, and rice growth and development. This study might provide a scientific basis for further studies on the growth and yield of rice OsFMO1.
引文
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Chen Q.Q.,Mu J.X.,Zhou H.J.,and Yu S.B.,2007,Genetic effect of Japonica alleles detected in Indica candidate introgression lines,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),40(11):2387-2394(陈庆全,穆俊祥,周红菊,余四斌,2007,利用基础导入系分析粳稻基因的遗传效应,中国农业科学,40(11):2387-2394)
Cheng Y.,Dai X.,and Zhao Y.,2006,Auxin biosynthesis by the YUCCA flavin monooxygenases co ntrols the formation of floral organs and vascular tissues in Arabidopsis,Genes Dev.,20(13):1790-1799
Fujino K.,Matsuda Y.,Ozawa K.,Nishimura T.,Koshiba T.,Fraaije M.W.,and Sekiguchi H.,2008,NARROW LEAF 7 controls leaf shape mediated by auxin in rice,Mol.Genet.Genomics,279(5):499-507
Gallavotti A.,Barazesh S.,Malcomber S.,Hall D.,Jackson D.,Schmidt R.J.,and McSteen P.,2008,Sparse inflorescence1encodes a monocot-specific YUCCA-like gene required for vegetative and reproductive development in maize,Proc.Natl.Acad.Sci.USA,105(39):15196-15201
Jefferson R.A.,1987,Assaying chimeric genes in plants:the GUSgene fusion system,Plant Mol.Biol.Rep.,5(4):387-405
Koch M.,Vorwerk S.,Masur C.,Sharifi-Sirchi G.,Olivieri N.,and Schlaich N.L.,2006,A role for a flavin-containing mono-oxygenase in resistance against microbial pathogens in Arabidopsis,Plant J.,47(4):629-639
Lee D.S.,Chen L.J.,Li C.Y.,Liu Y.,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
Mashiguchi K.,Tanaka K.,Sakai T.,Suqawara S.,Kawaide H.,Natsume M.,Hanada A.,Yaeno T.,Shirasu K.,Yao H.,McSteen P.,Zhao Y.,Hayashi K.,Kamiya Y.,and Kasahara H.,2011,The main auxin biosynthesis pathway in Arabidopsis,Proc.Natl.Acad.Sci.USA,108(45):18512-18517
Stepanova A.N.,Yun J.,Robles L.M.,Novak O.,He W.,Guo H.,Ljung K.,and Alonso J.M.,2011,The Arabidopsis YUCCA1flavin monooxygenase functions in the indole-3-pyruvic acid branch of auxin biosynthesis,Plant Cell,23(11):3961-3973
Tobena-Santamaria R.,Bliek M.,Ljung K.,Sandberq G.,Mol J.N.,Souer E.,and Koes R.,2002,Floozy of petunia is a flavin mono-oxygenase-like protein required for the specification of leaf and flower architecture,Genes Dev.,16(6):753-763
Yamamoto Y.,Kamiya N.,Morinaka Y.,Matsuoka M.,and Sazuka T.,2007,Auxin biosynthesis by the YUCCA genes in rice,Plant Physiol.,143(3):1362-1371
Yi J.,Liu L.,Cao Y.,Li J.,and Mei M.,2013,Cloning characterization and expression of OsFMO(t)in rice encoding a flavin monooxygenase,J.Genet.,92(3):471-480
Zhang S.M.,Liu H.Y.,Cheng J.J.,Feng D.Y.,and Liu Z.,2015,Free-hand section techniques for clear observation of cell structures of millet(Setaria italica)and rice(Oyrza sativa)leaves,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(7):1527-1530(张书敏,刘红云,程金金,冯丹忆,刘征,2015,快速徒手切片法观察谷子和水稻叶片显微结构,基因组学与应用生物学,34(7):1527-1530)
Zhao Y.,Christensen S.K.,Fankhauser C.,Cashman J.R.,Cohen J.D.,Weigel D.,and Chory J.,2001,A role for flavin monooxygenase-like enzymes in auxin biosynthesis,Science,291(5502):306-309
Chen Q.Q.,Mu J.X.,Zhou H.J.,and Yu S.B.,2007,Genetic effect of Japonica alleles detected in Indica candidate introgression lines,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),40(11):2387-2394(陈庆全,穆俊祥,周红菊,余四斌,2007,利用基础导入系分析粳稻基因的遗传效应,中国农业科学,40(11):2387-2394)
Cheng Y.,Dai X.,and Zhao Y.,2006,Auxin biosynthesis by the YUCCA flavin monooxygenases co ntrols the formation of floral organs and vascular tissues in Arabidopsis,Genes Dev.,20(13):1790-1799
Fujino K.,Matsuda Y.,Ozawa K.,Nishimura T.,Koshiba T.,Fraaije M.W.,and Sekiguchi H.,2008,NARROW LEAF 7 controls leaf shape mediated by auxin in rice,Mol.Genet.Genomics,279(5):499-507
Gallavotti A.,Barazesh S.,Malcomber S.,Hall D.,Jackson D.,Schmidt R.J.,and McSteen P.,2008,Sparse inflorescence1encodes a monocot-specific YUCCA-like gene required for vegetative and reproductive development in maize,Proc.Natl.Acad.Sci.USA,105(39):15196-15201
Jefferson R.A.,1987,Assaying chimeric genes in plants:the GUSgene fusion system,Plant Mol.Biol.Rep.,5(4):387-405
Koch M.,Vorwerk S.,Masur C.,Sharifi-Sirchi G.,Olivieri N.,and Schlaich N.L.,2006,A role for a flavin-containing mono-oxygenase in resistance against microbial pathogens in Arabidopsis,Plant J.,47(4):629-639
Lee D.S.,Chen L.J.,Li C.Y.,Liu Y.,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
Mashiguchi K.,Tanaka K.,Sakai T.,Suqawara S.,Kawaide H.,Natsume M.,Hanada A.,Yaeno T.,Shirasu K.,Yao H.,McSteen P.,Zhao Y.,Hayashi K.,Kamiya Y.,and Kasahara H.,2011,The main auxin biosynthesis pathway in Arabidopsis,Proc.Natl.Acad.Sci.USA,108(45):18512-18517
Stepanova A.N.,Yun J.,Robles L.M.,Novak O.,He W.,Guo H.,Ljung K.,and Alonso J.M.,2011,The Arabidopsis YUCCA1flavin monooxygenase functions in the indole-3-pyruvic acid branch of auxin biosynthesis,Plant Cell,23(11):3961-3973
Tobena-Santamaria R.,Bliek M.,Ljung K.,Sandberq G.,Mol J.N.,Souer E.,and Koes R.,2002,Floozy of petunia is a flavin mono-oxygenase-like protein required for the specification of leaf and flower architecture,Genes Dev.,16(6):753-763
Yamamoto Y.,Kamiya N.,Morinaka Y.,Matsuoka M.,and Sazuka T.,2007,Auxin biosynthesis by the YUCCA genes in rice,Plant Physiol.,143(3):1362-1371
Yi J.,Liu L.,Cao Y.,Li J.,and Mei M.,2013,Cloning characterization and expression of OsFMO(t)in rice encoding a flavin monooxygenase,J.Genet.,92(3):471-480
Zhang S.M.,Liu H.Y.,Cheng J.J.,Feng D.Y.,and Liu Z.,2015,Free-hand section techniques for clear observation of cell structures of millet(Setaria italica)and rice(Oyrza sativa)leaves,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(7):1527-1530(张书敏,刘红云,程金金,冯丹忆,刘征,2015,快速徒手切片法观察谷子和水稻叶片显微结构,基因组学与应用生物学,34(7):1527-1530)
Zhao Y.,Christensen S.K.,Fankhauser C.,Cashman J.R.,Cohen J.D.,Weigel D.,and Chory J.,2001,A role for flavin monooxygenase-like enzymes in auxin biosynthesis,Science,291(5502):306-309