两个烟草YUCCA6基因的克隆及分析
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摘要
为了研究烟草生长素合成关键基因NtYUCCA6,本研究通过同源克隆从烟草品种云烟87中克隆了2个YUCCA6基因:NtYUCCA6-1和NtYUCCA6-2。分析了NtYUCCA6-1和NtYUCCA6-2基因编码的蛋白质基本理化性质、二级结构等。保守基元分析表明烟草NtYUCCA6-1和NtYUCCA6-2蛋白含有FAD结合位点和还原型辅酶Ⅱ结合位点,可能烟草的两个YUCCA6基因都具有生物学功能。进化分析表明烟草YUCCA6蛋白与番茄YUCCA6蛋白亲缘关系最近。另外,本研究对克隆基因在烟草根、茎、叶及腋芽中的表达模式进行了q PCR检测,结果显示:两个基因主要都在根中表达,但NtYUCCA6-2在所检测的组织中都有表达,而NtYUCCA6-1在茎中基本不表达。试验结果将有助于研究烟草YUCCA6基因在烟草生长发育中的具体功能。
In order to understand the function of YUCCA6 in tobacco, two YUCCA6 s, designated as NtYUCCA6-1 and NtYUCCA6-2, were cloned from cultured tobacco variety: Yunyan 87 in this study by BLAST against the NCBI database using the nucleotide sequence of At YUCCA6. The basic protein analyses including the physical and chemical features, secondary molecular structure were performed. The conservative motif analysis revealed that both proteins have FAD binding sites and NADPH binding sites, implying that both genes function in tobacco. The evolutionary analysis showed that YUCCA6 s from tobacco were most closely related to that from tomato. In addition, both genes are expressed most abundantly in root but displayed different tissue expression patterns in other tissues. NtYUCCA6-2 was expressed in all tissues tested whereas NtYUCCA6-1 expression couldn't be detected in the stem. Results from this study might contribute the further functional characterization of both genes in tobacco.
In order to understand the function of YUCCA6 in tobacco, two YUCCA6 s, designated as NtYUCCA6-1 and NtYUCCA6-2, were cloned from cultured tobacco variety: Yunyan 87 in this study by BLAST against the NCBI database using the nucleotide sequence of At YUCCA6. The basic protein analyses including the physical and chemical features, secondary molecular structure were performed. The conservative motif analysis revealed that both proteins have FAD binding sites and NADPH binding sites, implying that both genes function in tobacco. The evolutionary analysis showed that YUCCA6 s from tobacco were most closely related to that from tomato. In addition, both genes are expressed most abundantly in root but displayed different tissue expression patterns in other tissues. NtYUCCA6-2 was expressed in all tissues tested whereas NtYUCCA6-1 expression couldn't be detected in the stem. Results from this study might contribute the further functional characterization of both genes in tobacco.
引文
Cheng Y.,Dai X.,and Zhao Y.,2006,Auxin biosynthesis by th YUCCA flavin monooxygenases controls the formation o floral organs and vascular tissues in Arabidopsis,Gene.Dev.20(13):1790-1799
D ai X.,Mashiguchi K.,Chen Q.,Kasahara H.,Kamiya Y.,Ojha SDu Bois J.,Ballou D.,and Zhao Y.,2013,The biochemica mechanism of auxin biosynthesis by an Arabidopsis YUCCAflavin-containing monooxygenase,J.Biol.Chem.,288(31448-1457
Ding Z.,and Friml J.,2010,Auxin regulates distal stem cell differ entiation in Arabidopsis roots,Proc.Natl.Acad.Sci.USA27(6)12046-12051
Expósito-Rodríguez M.,Borges A.A.,Borges-Pérez A.,and Pérez J.A.,2011,Gene structure and spatiotemporal ex pression profile of tomato genes encoding YUCCA-lik flavin monooxygenases:the To FZY gene family,Plan Physiol.Biochem.,49(7):782-791
Gallavotti A.,Barazesh S.,Malcomber S.,Hall D.,Jackson D.Schmidt R.J.,and Mc Steen P.,2008,Sparse inflorescence 1encodes a monocot-specific YUCCA-like gene required fo vegetative and reproductive development in maize,Proc Natl.Acad.Sci.USA,105(39):15196-15201
Kim J.,Baek D.,Park H.C.,Chun H.J.,Oh D.H.,Lee M.K.,Ch J.Y.,Kim W.Y.,Kim M.C.,and Chung W.S.,2013,Overex pression of Arabidopsis YUCCA6 in potato results in high-auxin developmental phenotypes and enhanced resistance to water deficit,Molecular Plant,6(2):337-349
Liu H.,Xie W.F.,Zhang L.,Valpuesta V.,Ye Z.W.,Gao Q.H.and Duan K.,2014,Auxin biosynthesis by the YUCCA6flavin monooxygenase gene in woodland strawberry,J.Integr Plant Biol.,56(4):350-363
Liu H.,Ying Y.Y.,Zhang L.,Gao Q.H.,Li J.,Zhang Z.,Fang JG.,and Duan K.,2012,Isolation and characterization of two YUCCA flavin monooxygenase genes from cultivated strawberry(Fragaria×ananassa Duch.),Plant Cell Rep.,31(8):1425-1435
Mashiguchia K.,Tanaka K.,Sakaic T.,Sugawaraa S.,Kawaideb HNatsumeb M.,Hanadaa A.,Yaenoa T.,Shirasua K.,Yao HMc Steen P.,Zhao Y.,Hayashi K.,Kamiya Y.,and Kasahara H2011,The main auxin biosynthesis pathway in Arabidopsis Proc.Natl.Acad.Sci.USA,108(54):18512-18517
Rozov S.M.,Zagorskaya A.A.,Shcherbakov D.N.,Belavin P.A.Deineko E.V.,and Shumnyi V.K.,2012,Identification o the NtF ZY gene familyin tobacco(Nicotiana tabacum)involved in the tryptophan-dependent auxin biosynthesis path way Doklady Biochemistry and Biophysics,444(1):140-143
Tobe觡a-Santamaria R.,Bliek M.,Ljung K.,Sandberg G.,Mol JN.,and Souer E.,2002,FLOOZY of petunia is a flavin mono-oxygenase-like protein required for the specification of leaf and flower architecture,Gene.Dev.,16(6):753-763
Wang W.,Xu B.,Wang H.,Li J.,Huang H.,and Xu L.,2011YUCCA genes are expressed in response to leaf adaxialabaxial juxtaposition and are required for leaf margin development,Plant Physiol.,157(4):1805-1819
Won C.,Shen X.,Mashiguchi K.,Zheng Z.,Dai X.,Cheng Y.Kasahara H.,Kamiya Y.,Chory J.,and Zhao Y.,2011Conversion of tryptophan to indole-3-acetic acid by Tryptophan aminotransferases of Arabidopsis and YUCCAs in Arabidopsis,Proc.Natl.Acad.Sci.USA,108(45):18518-18523
Woo Y.M.,Park H.J.,Su'udi M.,Yang J.I.,Park J.J.,Back K.Park Y.M.,and An G.,2007,Constitutively wilted 1,a member of the rice YUCCA gene family,is required fo maintaining water homeostasis and an appropriate root to shoot ratio,Plant Mol.Biol.,65(1)125-136
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
Ye X.,Kang B.G.,Osburn L.D.,Li Y.,and Max C.Z.M.,2009Identification of the flavin-dependent monooxygenase-en coding YUCCA gene family in Populus trichocarpa and thei expression in vegetative tissues and in response to hormon and environmental stresses,Plant Cell Tiss.Org.Cult.,97(3):271-283
You X.Q.,Sang X.C.,Zhao F.M.,Ling Y.H.,Li Y.F.,Yang Z.L.Li N.,and He G.H.,2010,Genetic analysis and gene mapping of a narrow leaf mutant(nal7(t))in rice(Oryza sativa L.),Fenz Zhiwu Yuzhong(Molecular Plant Breeding),8(2):240-244(游小庆,桑贤春,赵芳明,凌英华,李云峰,杨正林,李楠何光华,2010,水稻窄叶突变体nal7(t)的遗传分析与基因定位,分子植物育种,8(2):240-244)
Zhao Y.,2010,Auxin biosynthesis and its role in plant development,Annu.Rev.Plant Biol.,61:49-64
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,Science291(5502):306-309
D ai X.,Mashiguchi K.,Chen Q.,Kasahara H.,Kamiya Y.,Ojha SDu Bois J.,Ballou D.,and Zhao Y.,2013,The biochemica mechanism of auxin biosynthesis by an Arabidopsis YUCCAflavin-containing monooxygenase,J.Biol.Chem.,288(31448-1457
Ding Z.,and Friml J.,2010,Auxin regulates distal stem cell differ entiation in Arabidopsis roots,Proc.Natl.Acad.Sci.USA27(6)12046-12051
Expósito-Rodríguez M.,Borges A.A.,Borges-Pérez A.,and Pérez J.A.,2011,Gene structure and spatiotemporal ex pression profile of tomato genes encoding YUCCA-lik flavin monooxygenases:the To FZY gene family,Plan Physiol.Biochem.,49(7):782-791
Gallavotti A.,Barazesh S.,Malcomber S.,Hall D.,Jackson D.Schmidt R.J.,and Mc Steen P.,2008,Sparse inflorescence 1encodes a monocot-specific YUCCA-like gene required fo vegetative and reproductive development in maize,Proc Natl.Acad.Sci.USA,105(39):15196-15201
Kim J.,Baek D.,Park H.C.,Chun H.J.,Oh D.H.,Lee M.K.,Ch J.Y.,Kim W.Y.,Kim M.C.,and Chung W.S.,2013,Overex pression of Arabidopsis YUCCA6 in potato results in high-auxin developmental phenotypes and enhanced resistance to water deficit,Molecular Plant,6(2):337-349
Liu H.,Xie W.F.,Zhang L.,Valpuesta V.,Ye Z.W.,Gao Q.H.and Duan K.,2014,Auxin biosynthesis by the YUCCA6flavin monooxygenase gene in woodland strawberry,J.Integr Plant Biol.,56(4):350-363
Liu H.,Ying Y.Y.,Zhang L.,Gao Q.H.,Li J.,Zhang Z.,Fang JG.,and Duan K.,2012,Isolation and characterization of two YUCCA flavin monooxygenase genes from cultivated strawberry(Fragaria×ananassa Duch.),Plant Cell Rep.,31(8):1425-1435
Mashiguchia K.,Tanaka K.,Sakaic T.,Sugawaraa S.,Kawaideb HNatsumeb M.,Hanadaa A.,Yaenoa T.,Shirasua K.,Yao HMc Steen P.,Zhao Y.,Hayashi K.,Kamiya Y.,and Kasahara H2011,The main auxin biosynthesis pathway in Arabidopsis Proc.Natl.Acad.Sci.USA,108(54):18512-18517
Rozov S.M.,Zagorskaya A.A.,Shcherbakov D.N.,Belavin P.A.Deineko E.V.,and Shumnyi V.K.,2012,Identification o the NtF ZY gene familyin tobacco(Nicotiana tabacum)involved in the tryptophan-dependent auxin biosynthesis path way Doklady Biochemistry and Biophysics,444(1):140-143
Tobe觡a-Santamaria R.,Bliek M.,Ljung K.,Sandberg G.,Mol JN.,and Souer E.,2002,FLOOZY of petunia is a flavin mono-oxygenase-like protein required for the specification of leaf and flower architecture,Gene.Dev.,16(6):753-763
Wang W.,Xu B.,Wang H.,Li J.,Huang H.,and Xu L.,2011YUCCA genes are expressed in response to leaf adaxialabaxial juxtaposition and are required for leaf margin development,Plant Physiol.,157(4):1805-1819
Won C.,Shen X.,Mashiguchi K.,Zheng Z.,Dai X.,Cheng Y.Kasahara H.,Kamiya Y.,Chory J.,and Zhao Y.,2011Conversion of tryptophan to indole-3-acetic acid by Tryptophan aminotransferases of Arabidopsis and YUCCAs in Arabidopsis,Proc.Natl.Acad.Sci.USA,108(45):18518-18523
Woo Y.M.,Park H.J.,Su'udi M.,Yang J.I.,Park J.J.,Back K.Park Y.M.,and An G.,2007,Constitutively wilted 1,a member of the rice YUCCA gene family,is required fo maintaining water homeostasis and an appropriate root to shoot ratio,Plant Mol.Biol.,65(1)125-136
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
Ye X.,Kang B.G.,Osburn L.D.,Li Y.,and Max C.Z.M.,2009Identification of the flavin-dependent monooxygenase-en coding YUCCA gene family in Populus trichocarpa and thei expression in vegetative tissues and in response to hormon and environmental stresses,Plant Cell Tiss.Org.Cult.,97(3):271-283
You X.Q.,Sang X.C.,Zhao F.M.,Ling Y.H.,Li Y.F.,Yang Z.L.Li N.,and He G.H.,2010,Genetic analysis and gene mapping of a narrow leaf mutant(nal7(t))in rice(Oryza sativa L.),Fenz Zhiwu Yuzhong(Molecular Plant Breeding),8(2):240-244(游小庆,桑贤春,赵芳明,凌英华,李云峰,杨正林,李楠何光华,2010,水稻窄叶突变体nal7(t)的遗传分析与基因定位,分子植物育种,8(2):240-244)
Zhao Y.,2010,Auxin biosynthesis and its role in plant development,Annu.Rev.Plant Biol.,61:49-64
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,Science291(5502):306-309