木薯SWEET1基因的分子克隆、亚细胞定位与功能分析
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摘要
SWEET基因家族为新发现的一类糖转运蛋白家族,具有转运蔗糖及单糖的功能,在真核生物中广泛存在,其特点为具有1~2个MtN3_slv结构域及7个跨膜结构域,对植物生长发育、生理代谢及植物-病原菌互作等方面具有重要的调控作用。为了探索热带作物木薯中SWEET基因的功能,本研究采用RT-PCR技术,从木薯Ku50成熟叶片中克隆得到MeSWEET1基因的编码序列,并对其进行了生物信息学预测分析和功能初步研究。本研究结果表明:MeSWEET1蛋白含有植物SWEET家族两个保守的MtN3_Slv功能域,还具有7个跨膜结构域。系统进化树分析显示,MeSWEET1与拟南芥AtSWEET1、水稻OsSWEET1a和OsSWEET1b亲缘关系最近,属于SWEET蛋白家族进化树的Clade I分枝class II小家族。亚细胞定位结果表明,MeSWEET1蛋白定位于细胞膜上。组织特异性表达分析揭示,MeSWEET1在木薯成熟叶片中相对表达量最高,而在新叶和果实中相对表达量最低。本研究的结果将为进一步研究木薯MeSWEET1基因在糖转运及与病原互作等方面的功能奠定了初步的理论基础。
SWEET gene famil y is a novel identified sugar transporter family.SWEET protein can selectively transport sucrose or monosaccharide and it is widely present in higher eukaryotes.SWEET protein harbors one or two Mt N3_slv motif and contains seven transmembrane domains, which play a crucial role in plant development,physiological processes and plant-microbe interaction. In order to elucidate the function ofSWEET gene in tropical plant Cassava, a cDNA of full-length MeSWEET1 gene was cloned by RT-PCR technique and its primarily function was explored in the present study. Our results showed: MeSWEET1 protein contains two conserved Mt N3-slv motifs and seven transmembrane domains, which are the characteristic feathers of plantSWEET proteins. Phylogenetic analysis showed that MeSWEET1 belongs to Clade I branch, Class II sub-family inSWEET evolutionary tree and was relatively clo se d to AtSWEET1, OsSWEET1 a and OsSWEET1 b in evolution. Tissue specific expression pattern showed that MeSWEET1 gene was differentially expressed in tested five organs of Cassava. The expression level of MeSWEE1 was relatively most abundant in mature leaf and was relatively lowestin young leaf and fruit. Our results provided a solid foundation for further investigate of the biological function of MeSWEET1 involved in sugar translocation and Cassava-microbe interaction fields.
SWEET gene famil y is a novel identified sugar transporter family.SWEET protein can selectively transport sucrose or monosaccharide and it is widely present in higher eukaryotes.SWEET protein harbors one or two Mt N3_slv motif and contains seven transmembrane domains, which play a crucial role in plant development,physiological processes and plant-microbe interaction. In order to elucidate the function ofSWEET gene in tropical plant Cassava, a cDNA of full-length MeSWEET1 gene was cloned by RT-PCR technique and its primarily function was explored in the present study. Our results showed: MeSWEET1 protein contains two conserved Mt N3-slv motifs and seven transmembrane domains, which are the characteristic feathers of plantSWEET proteins. Phylogenetic analysis showed that MeSWEET1 belongs to Clade I branch, Class II sub-family inSWEET evolutionary tree and was relatively clo se d to AtSWEET1, OsSWEET1 a and OsSWEET1 b in evolution. Tissue specific expression pattern showed that MeSWEET1 gene was differentially expressed in tested five organs of Cassava. The expression level of MeSWEE1 was relatively most abundant in mature leaf and was relatively lowestin young leaf and fruit. Our results provided a solid foundation for further investigate of the biological function of MeSWEET1 involved in sugar translocation and Cassava-microbe interaction fields.
引文
Ainsworth E.A.,and Bush D.R.,2011,Carbohydrate export from the leaf:a highly regulated process and target to enhanc photosynthesis and productivity,Plant Physiol.,155(1)64-69
Artero R.D.,Terol-Alcayde J.,Paricio N.,Ring J.,Bargues M.Torres A.,and Perez-Alonso M.,1998,Saliva,a new Drosophila gene expressed in the embryonic salivary gland with homologues in plants and vertebrates,Mechanisms o Development,75(1-2):159-162
Ayre B.G.,2011,Membrane-transport systems for sucrose in re lation to whole-plant carbon partitioning,Molecular Plant,4(3):377-394
Braun D.M.,2012,SWEET!The pathway is complete,Science335(6065):173-174
Braun D.M.,Wang L.,and Ruan Y.L.,2014,Understanding and manipulating sucrose phloem loading,unloading,meta bolism,and signalling to enhance crop yield and food secu rity,J.Exp.Bot.,65(7):1713-1735
Chardon F.,Bedu M.,Calenge F.,Klemens P.A.,Spinner L.Clement G.,Chietera G.,Leran S.,Ferrand M.,Lacombe B.Loudet O.,Dinant S.,Bellini C.,Neuhaus H.E.,Daniel Vedele F.,and Krapp A.,2013,Leaf fructose content is con trolled by the vacuolar transporter SWEET17 in Arabidopsis Current Biology,23(8):697-702
Chen L.Q.,2014,SWEET sugar transporters for phloem transpor and pathogen nutrition,New Phytol.,201(4):1150-1155
Chen L.Q.,Hou B.H.,Lalonde S.,Takanaga H.,Hartung M.L.Qu X.Q.,Guo W.J.,Kim J.G.,Underwood W.,Chaudhur B.,Chermak D.,Antony G.,White F.F.,Somerville S.C.Mudgett M.B.,and Frommer W.B.,2010,Sugar transporter for intercellular exchange and nutrition of pathogens,Na ture,468(7323):527-532
Chen L.Q.,Lin I.W.,Qu X.Q.,Sosso D.,Mc Farlane H.E.,Lon dono A.,Samuels A.L.,and Frommer W.B.,2015,A cas cade of sequentially expressed sucrose transporters in th seed coat and endosperm provides nutrition for the Ara bidopsis embryo,The Plant Cell,27(3):607-619
Chen L.Q.,Qu X.Q.,Hou B.H.,Sosso D.,Osorio S.,Fernie A.R.and Frommer W.B.,2012,Sucrose efflux mediated by SWEET proteins as a key step for phloem transport,Sci ence,335(6065):207-211
Chong J.,Piron M.C.,Meyer S.,Merdinoglu D.,Bertsch C.,and Mestre P.,2014,The SWEET family of sugar transporters in grapevine:Vv SWEET4 is involved in the interaction with Botrytis cinerea,J.Exp.Bot.,65(22):6589-6601
Feng C.Y.,Han J.X.,Han X.X.,and Jiang J.,2015,Genome wide identification,phylogeny,and expression analysis o the SWEET gene family in tomato,Gene,573(2):261-272
Ferrari S.,Galletti R.,Denoux C.,De Lorenzo G.,Ausubel F.M.and Dewdney J.,2007,Resistance to Botrytis cinerea in duced in Arabidopsis by elicitors is independent of salicyli acid,ethylene,or jasmonate signaling but requires PHYTOALEXIN DEFICIENT3,Plant Physiol.,144(1):367-379
Frank Baker R.,Leach K.A.,and Braun D.M.,2012,SWEET a sugar:new sucrose effluxers in plants,Molecular Plant,5(4)766-768
Gamas P.,Niebel F.C.,Lescure N.,and Cullimore J.V.,1996Use of a subtractive hybridization approach to identify new Medicago truncatula genes induced during root nodule de velopment,Mol.Plant Microbe Inter.,9(4):233-242
Guan Y.F.,Huang X.Y.,Zhu J.,Gao J.F.,Zhang H.X.,and Yang Z.N.,2008,RUPTURED POLLEN GRAIN1,a member o the Mt N3/saliva gene family,is crucial for exine pattern for mation and cell integrity of microspores in Arabidopsis Plant Physiol.,147(2):852-863
Guo W.J.,Nagy R.,Chen H.Y.,Pfrunder S.,Yu Y.C.,Santelia D.Frommer W.B.,and Martinoia E.,2014,SWEET17,a facili tative transporter,mediates fructose transport across th tonoplast of Arabidopsis roots and leaves,Plant Physiol.164(2):777-789
Han J.X.,and Jiang J.,2015,Genome-Wide analysis of SWEETgene family in Arabidopsis thaliana,Oryza sativa and Lycopersicum esculentum,Fenzi Zhiwu Yuzhong(Molecu lar Plant Breeding),13(3):581-588(韩佳轩,姜晶,2015,拟南芥、水稻和番茄SWEET/Mt N3/saliva基因家族的分析分子植物育种,13(3):581-588)
Kehr J.,2009,Long-distance transport of macromolecule through the phloem,F1000 Biology Reports,1:31
Klemens P.A.,Patzke K.,Deitmer J.,Spinner L.,Le Hir R.,Belli ni C.,Bedu M.,Chardon F.,Krapp A.,and Neuhaus H.E.2013,Overexpression of the vacuolar sugar carrie At SWEET16 modifies germination,growth,and stress toler ance in Arabidopsis,Plant Physiol.,163(3):1338-1352
Kuhn C.,and Grof C.P.,2010,Sucrose transporters of highe plants,Current Opinion in Plant Biology,13(3):288-298
Lin I.W.,Sosso D.,Chen L.Q.,Gase K.,Kim S.G.,Kessler D.Klinkenberg P.M.,Gorder M.K.,Hou B.H.,Qu X.Q.,Carte C.J.,Baldwin I.T.,and Frommer W.B.,2014,Nectar secre tion requires sucrose phosphate synthases and the suga transporter SWEET9,Nature,508(7497):546-549
Mizuno H.,Kasuga S.,and Kawahigashi H.,2016,The sorghum SWEET gene family:stem sucrose accumulation as revealed through transcriptome profiling,Biotechnology for Biofuels9:127
Patil G.,Valliyodan B.,Deshmukh R.,Prince S.,Nicander B.Zhao M.,Sonah H.,Song L.,Lin L.,Chaudhary J.,Liu Y.Joshi T.,Xu D.,and Nguyen H.T.,2015,Soybean(Glycin max)SWEET gene family:insights through comparative ge nomics,transcriptome profiling and whole genome re-se quence analysis,BMC Genomics,16:520
Rolland F.,Baena-Gonzalez E.,and Sheen J.,2006,Sugar sens ing and signaling in plants:conserved and novel mecha nisms,Annual Review of Plant Biology,57:675-709
Rolland F.,Moore B.,and Sheen J.,2002,Sugar sensing and sig naling in plants,The Plant Cell,14(S):185-205
Ruan Y.L.,2014,Sucrose metabolism:gateway to diverse carbon use and sugar signaling,Annual Review of Plant Biology65:33-67
Slewinski T.L.,2011,Diverse functional roles of monosaccharid transporters and their homologs in vascular plants:a physio logical perspective,Molecular Plant,4(4):641-662
Sun W.J.,and Zuo K.J.,2016,The finding of SWEET transporte family and the research advance on its structure and func tion,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(4):878-885(孙文杰,左开井,2016,SWEET转运蛋白家族的发现、结构及功能研究进展,分子植物育种,14(4)878-885)
Turgeon R.,and Wolf S.,2009,Phloem transport:cellular path ways and molecular trafficking,Annual Review of Plant Bi ology,60:207-221
Yuan M.,and Wang S.P.,2013,Rice Mt N3/saliva/SWEET family genes and their homologs in cellular organisms,Molecula Plant,6(3):665-674
Yuan M.,Zhao J.,Huang R.,Li X.,Xiao J.,and Wang S.P.2014,Rice Mt N3/saliva/SWEET gene family:evolution,ex pression profiling,and sugar transport,Journal of Integrativ Plant Biology,56(6):559-570
Zhou Y.,Liu L.,Huang W.,Yuan M.,Zhou F.,Li X.,and Lin YJ.,2014,Overexpression of Os SWEET5 in rice cause growth retardation and precocious senescence,PLo S One,9(4):e94210
Artero R.D.,Terol-Alcayde J.,Paricio N.,Ring J.,Bargues M.Torres A.,and Perez-Alonso M.,1998,Saliva,a new Drosophila gene expressed in the embryonic salivary gland with homologues in plants and vertebrates,Mechanisms o Development,75(1-2):159-162
Ayre B.G.,2011,Membrane-transport systems for sucrose in re lation to whole-plant carbon partitioning,Molecular Plant,4(3):377-394
Braun D.M.,2012,SWEET!The pathway is complete,Science335(6065):173-174
Braun D.M.,Wang L.,and Ruan Y.L.,2014,Understanding and manipulating sucrose phloem loading,unloading,meta bolism,and signalling to enhance crop yield and food secu rity,J.Exp.Bot.,65(7):1713-1735
Chardon F.,Bedu M.,Calenge F.,Klemens P.A.,Spinner L.Clement G.,Chietera G.,Leran S.,Ferrand M.,Lacombe B.Loudet O.,Dinant S.,Bellini C.,Neuhaus H.E.,Daniel Vedele F.,and Krapp A.,2013,Leaf fructose content is con trolled by the vacuolar transporter SWEET17 in Arabidopsis Current Biology,23(8):697-702
Chen L.Q.,2014,SWEET sugar transporters for phloem transpor and pathogen nutrition,New Phytol.,201(4):1150-1155
Chen L.Q.,Hou B.H.,Lalonde S.,Takanaga H.,Hartung M.L.Qu X.Q.,Guo W.J.,Kim J.G.,Underwood W.,Chaudhur B.,Chermak D.,Antony G.,White F.F.,Somerville S.C.Mudgett M.B.,and Frommer W.B.,2010,Sugar transporter for intercellular exchange and nutrition of pathogens,Na ture,468(7323):527-532
Chen L.Q.,Lin I.W.,Qu X.Q.,Sosso D.,Mc Farlane H.E.,Lon dono A.,Samuels A.L.,and Frommer W.B.,2015,A cas cade of sequentially expressed sucrose transporters in th seed coat and endosperm provides nutrition for the Ara bidopsis embryo,The Plant Cell,27(3):607-619
Chen L.Q.,Qu X.Q.,Hou B.H.,Sosso D.,Osorio S.,Fernie A.R.and Frommer W.B.,2012,Sucrose efflux mediated by SWEET proteins as a key step for phloem transport,Sci ence,335(6065):207-211
Chong J.,Piron M.C.,Meyer S.,Merdinoglu D.,Bertsch C.,and Mestre P.,2014,The SWEET family of sugar transporters in grapevine:Vv SWEET4 is involved in the interaction with Botrytis cinerea,J.Exp.Bot.,65(22):6589-6601
Feng C.Y.,Han J.X.,Han X.X.,and Jiang J.,2015,Genome wide identification,phylogeny,and expression analysis o the SWEET gene family in tomato,Gene,573(2):261-272
Ferrari S.,Galletti R.,Denoux C.,De Lorenzo G.,Ausubel F.M.and Dewdney J.,2007,Resistance to Botrytis cinerea in duced in Arabidopsis by elicitors is independent of salicyli acid,ethylene,or jasmonate signaling but requires PHYTOALEXIN DEFICIENT3,Plant Physiol.,144(1):367-379
Frank Baker R.,Leach K.A.,and Braun D.M.,2012,SWEET a sugar:new sucrose effluxers in plants,Molecular Plant,5(4)766-768
Gamas P.,Niebel F.C.,Lescure N.,and Cullimore J.V.,1996Use of a subtractive hybridization approach to identify new Medicago truncatula genes induced during root nodule de velopment,Mol.Plant Microbe Inter.,9(4):233-242
Guan Y.F.,Huang X.Y.,Zhu J.,Gao J.F.,Zhang H.X.,and Yang Z.N.,2008,RUPTURED POLLEN GRAIN1,a member o the Mt N3/saliva gene family,is crucial for exine pattern for mation and cell integrity of microspores in Arabidopsis Plant Physiol.,147(2):852-863
Guo W.J.,Nagy R.,Chen H.Y.,Pfrunder S.,Yu Y.C.,Santelia D.Frommer W.B.,and Martinoia E.,2014,SWEET17,a facili tative transporter,mediates fructose transport across th tonoplast of Arabidopsis roots and leaves,Plant Physiol.164(2):777-789
Han J.X.,and Jiang J.,2015,Genome-Wide analysis of SWEETgene family in Arabidopsis thaliana,Oryza sativa and Lycopersicum esculentum,Fenzi Zhiwu Yuzhong(Molecu lar Plant Breeding),13(3):581-588(韩佳轩,姜晶,2015,拟南芥、水稻和番茄SWEET/Mt N3/saliva基因家族的分析分子植物育种,13(3):581-588)
Kehr J.,2009,Long-distance transport of macromolecule through the phloem,F1000 Biology Reports,1:31
Klemens P.A.,Patzke K.,Deitmer J.,Spinner L.,Le Hir R.,Belli ni C.,Bedu M.,Chardon F.,Krapp A.,and Neuhaus H.E.2013,Overexpression of the vacuolar sugar carrie At SWEET16 modifies germination,growth,and stress toler ance in Arabidopsis,Plant Physiol.,163(3):1338-1352
Kuhn C.,and Grof C.P.,2010,Sucrose transporters of highe plants,Current Opinion in Plant Biology,13(3):288-298
Lin I.W.,Sosso D.,Chen L.Q.,Gase K.,Kim S.G.,Kessler D.Klinkenberg P.M.,Gorder M.K.,Hou B.H.,Qu X.Q.,Carte C.J.,Baldwin I.T.,and Frommer W.B.,2014,Nectar secre tion requires sucrose phosphate synthases and the suga transporter SWEET9,Nature,508(7497):546-549
Mizuno H.,Kasuga S.,and Kawahigashi H.,2016,The sorghum SWEET gene family:stem sucrose accumulation as revealed through transcriptome profiling,Biotechnology for Biofuels9:127
Patil G.,Valliyodan B.,Deshmukh R.,Prince S.,Nicander B.Zhao M.,Sonah H.,Song L.,Lin L.,Chaudhary J.,Liu Y.Joshi T.,Xu D.,and Nguyen H.T.,2015,Soybean(Glycin max)SWEET gene family:insights through comparative ge nomics,transcriptome profiling and whole genome re-se quence analysis,BMC Genomics,16:520
Rolland F.,Baena-Gonzalez E.,and Sheen J.,2006,Sugar sens ing and signaling in plants:conserved and novel mecha nisms,Annual Review of Plant Biology,57:675-709
Rolland F.,Moore B.,and Sheen J.,2002,Sugar sensing and sig naling in plants,The Plant Cell,14(S):185-205
Ruan Y.L.,2014,Sucrose metabolism:gateway to diverse carbon use and sugar signaling,Annual Review of Plant Biology65:33-67
Slewinski T.L.,2011,Diverse functional roles of monosaccharid transporters and their homologs in vascular plants:a physio logical perspective,Molecular Plant,4(4):641-662
Sun W.J.,and Zuo K.J.,2016,The finding of SWEET transporte family and the research advance on its structure and func tion,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(4):878-885(孙文杰,左开井,2016,SWEET转运蛋白家族的发现、结构及功能研究进展,分子植物育种,14(4)878-885)
Turgeon R.,and Wolf S.,2009,Phloem transport:cellular path ways and molecular trafficking,Annual Review of Plant Bi ology,60:207-221
Yuan M.,and Wang S.P.,2013,Rice Mt N3/saliva/SWEET family genes and their homologs in cellular organisms,Molecula Plant,6(3):665-674
Yuan M.,Zhao J.,Huang R.,Li X.,Xiao J.,and Wang S.P.2014,Rice Mt N3/saliva/SWEET gene family:evolution,ex pression profiling,and sugar transport,Journal of Integrativ Plant Biology,56(6):559-570
Zhou Y.,Liu L.,Huang W.,Yuan M.,Zhou F.,Li X.,and Lin YJ.,2014,Overexpression of Os SWEET5 in rice cause growth retardation and precocious senescence,PLo S One,9(4):e94210