甜橙成花关键基因CsFT的克隆分析与表达载体构建
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摘要
FT被认为是汇集各个开花途径的关键因子,其超量表达可促进植物提早开花。本研究通过RT-PCR方法,以纽荷尔脐橙(Citrus sinensis (L.) Osbeck)叶片为材料,成功地克隆到了2个FT基因的cDNA序列,分别为534 bp和537 bp,各自编码177个和178个氨基酸,并对其进行了生物信息学相关分析。荧光定量分析了2个CsFT基因在脐橙不同器官的表达情况,结果表明二者在果皮、果肉、茎、叶、花中均有表达,但CsFT1基因在果肉中的表达量最高,CsFT2基因在叶中的表达量最高;这2个基因在不同花器官中均有表达,CsFT1基因在花瓣中表达最高,CsFT2基因雌蕊中表达最高,并都在开花后第7天的花器官中达到最大,随后下降。将2个基因连接到植物表达载体pCAMBIA1301上,得到了过量表达载体pCAMBIA1301-35S-CsFT1和pCAMBIA1301-35S-CsFT2,并成功转化农杆菌,为下一步转化柑橘获得转基因植株提供了技术参考。
The FT is c onsidered to be a key factor in the convergence of various flowering pathways, and its over-expression can promote early flowering of plants. In this study, the leaves of Citrus sinensis(L.) Osbeck were used as materials, and the cDNA sequence of two FT genes were cloned through RT-PCR technology. They were534 bp and 537 bp, respectively, encoding 177 and 178 amino acids, and the bioinformatics correlation analysis of them was carried out. The expression of two CsFT genes in different organs of Citrus sinensis was analyzed using real-time PCR, of which the results showed that the two genes were expressed in peel, pulp, stem, leaf and flower.However, the expression of CsFT1 gene was the highest in the pulp, while that of CsFT2 gene was in the leaves.Both of them were expressed in different floral organs, and Cs FT1 gene expressed the highest in petals, and the expression of CsFT2 gene was the highest in pistil. The expression of these two genes both reached the highest in the flower organ in the seventh day after flowering and then decreased. The over-expression vector pCAMBIA1301-35S-CsFT1 and pCAMBIA1301-35S-CsFT2 were obtained by connecting these two genes to the plant expression vector pCAMBIA1301, and Agrobacterium was successfully transformed, which could provide scientific reference for the further transformation of citrus to obtain transgenic plants.
The FT is c onsidered to be a key factor in the convergence of various flowering pathways, and its over-expression can promote early flowering of plants. In this study, the leaves of Citrus sinensis(L.) Osbeck were used as materials, and the cDNA sequence of two FT genes were cloned through RT-PCR technology. They were534 bp and 537 bp, respectively, encoding 177 and 178 amino acids, and the bioinformatics correlation analysis of them was carried out. The expression of two CsFT genes in different organs of Citrus sinensis was analyzed using real-time PCR, of which the results showed that the two genes were expressed in peel, pulp, stem, leaf and flower.However, the expression of CsFT1 gene was the highest in the pulp, while that of CsFT2 gene was in the leaves.Both of them were expressed in different floral organs, and Cs FT1 gene expressed the highest in petals, and the expression of CsFT2 gene was the highest in pistil. The expression of these two genes both reached the highest in the flower organ in the seventh day after flowering and then decreased. The over-expression vector pCAMBIA1301-35S-CsFT1 and pCAMBIA1301-35S-CsFT2 were obtained by connecting these two genes to the plant expression vector pCAMBIA1301, and Agrobacterium was successfully transformed, which could provide scientific reference for the further transformation of citrus to obtain transgenic plants.
引文
Aki T.,Shigyo M.,Nakano R.,Yoneyama T.,and Yanagisawa S.,2008,Nano scale proteomics revealed the presence of regulatory proteins including three FT-like proteins in phloem and xylem saps from rice,Plant Cell Physiol.,49(5):767-790
Chen X.,Li S.Y.,Wu L.C.,Wang C.L.,Chen Y.H.,2006,Molecular mechanisms of photoperiod actions on plant flowering,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),26(7):1490-1499(陈晓,李思远,吴连成,王翠玲,陈彦惠,2006,光周期影响植物花时的分子机制,西北植物学报,26(7):1490-1499)
Endo T.,Shimada T.,Fujii H.,Kobayashi Y.,Araki T.,and Omura M.,2005,Ectopic expression of an FT homolog from Citrus confers an early flowering phenotype on trifoliate orange(Poncirus trifoliata L.Raf.),Trans.Res.,14(5):703-712
Endo T.,Shimada T.,Fujii H.,Nishikawa F.,Sugiyama A.,Nakano M.,Shimizu T.,Kobayashi Y.,Araki T.,Pea L.,and Omura M.,2009,Development of a Ci FT co-expression system for function analysis of gene in citrus flowers and fruit,J.Japan.Soc.Hort.Sci.,78(1):74-83
Guo W.F.,Liu D.C.,Yang L.,Zhuang X.,Zhang J.J.,Wang S.S.,and Liu Y.,2015,Cloning and expression analysis of new stress-resistant NAC83 gene from Citrus,Yuanyi Xuebao(Acta Horticulturae Sinica),42(3):445-454(郭文芳,刘德春,杨莉,庄霞,张涓涓,王书胜,刘勇,2015,柑橘抗逆基因NAC83的克隆与表达分析,园艺学报,34(3):445-454)
Hemming M.N.,Peacock W.J.,Dennis E.S.,and Trevaskis B.,2008,Low-temperature and daylength cues are integrated to regulate FLOWERING LOCUS T in barley,Plant Physiol.,147(1):355-366
Imamura T.,Nakatsuka T.,Higuchi A.,Nishihara M.,and Takahashi H.T.,2011,The gentian orthologs of the FT/TFL1 gene family control floral initiation in Gentiana,Plant Cell Physiol.,52(6):1031-1041
Kikuchi R.,Kawahigashi H.,Ando T.,Tonooka T.,and Handa H.,2009,Molecular and functional characterization of PEBPgenes in barley reveal the diversification of their roles in flowering,Plant Physiol.,149(3):1341-1353
Kong F.,Liu B.,Xia Z.,Sato S.,Kim B.M.,Watanabe S.,Yamada T.,Tabata S.,Kanazawa A.,Harada K.,and Abe J.,2010,Two coordinately regulated homologs of FLOWER-ING LOCUS T are involved in the control of photoperiodic flowering in soybean,Plant Physiol.,154(3):1220-1231
Kotoda N.,Hayashi H.,Suzuki M.,Igarashi M.,Hatsuyama Y.,Kidou S.,Igasaki T.,Nishiguchi M.,Yano K.,Shimizu T.,Takahashi S.,Iwanami H.,Moriya S.,and Abe K.,2010,Molecular characterization of FLOWERING LOCUS T-like genes of apple(Malus domestica Borkh.),Plant Cell Physiol.,51(4):561-575
Laurie R.E.,Diwadkar P.,Jaudal M.,Zhang L.,Hecht V.,Wen J.,Tadege M.,Mysore K.S.,Putterill J.,Weller J.L.,and Macknight R.C.,2011,The medicago FLOWERING LOCUS Thomology,MtFTa1,is a key regulator of flowering time,Plant Physiol.,156(4):2207-2224
Li X.F.,Jia L.Y.,Xu J.,Deng X.J.,Wang Y.,Zhang W.,Zhang X.P.,Fang Q.,Zhang D.M.,Sun Y.,and Xu L.,2013,FT-like NFT1 gene may play a role in flower transition induced by heat accumulation in Narcissus tazetta var.chinensis,Plant Cell Physiol.,54(2):270-281
Marchlerbauer A.,Bo Y.,Han L.,He J.,Lanczycki C.J.,Lu S.,Chitsaz F.,Derbyshire M.K.,Geer R.C.,Gonzales N.R.,Gwadz M.,Hurwitz D.I.,Lu F.,Marchler G.H.,Song J.S.,Thanki N.,Wang Z.,Yamashita R.A.,Zhang D.,Zheng C.,Geer L.Y.,and Bryant S.H.,2017,Cdd/sparcle:functional classification of proteins via subfamily domain architectures,Nucleic Acids Research,45(Database issue):D200-D203
Meng X.,Muszynski M.G.,and Danilevskaya O.N.,2011,The FT-like ZCN8 gene functions as a floral activator and is involved in photoperiod sensitivity in maize,Plant Cell,23(3):942-960
Nakano Y.,Higuchi Y.,Sumitomo K.,and Hisamatsu T.,2013,Flowering retardation by high temperature in chrysanthemums:involvement of FLOWERING LOCUS T-like 3 gene repression,J.Exp.Bot.,64(4):909-920
Nishikawa F.,Endo T.,Shimada T.,Fujii H.,Shimizu T.,Omura M.,and Ikoma Y.,2007,Increased Ci FT abundance in the stem correlates with floral induction by low temperature in satsuma mandarin(Citrus unshiu Marc.),J.Exp.Bot.,58(14):3915-3927
Nishikawa F.,2013,Regulation of floral induction in citrus,J.Japan.Soc.Hort.Sci.,82(4):283-292
Pillitteri L.,Lovatt C.,and Walling L.,2004,Isolation and characterization of a TERMINAL FLOWER homolog and its correla tion with juvenility in citrus,Plant Physiol.,135(3):1540-1551
Shao J.R.,Song C.B.,Bian K.,Chen W.,and Yang Z.F.,2016,Expression responses of SUMO E3 ligase(SIZ1)to low temperature stress and exogenous melatonin in postharvest peach fruit,Yuanyi Xuebao(Acta Horticulturae Sinica),43(7):1257-1266(邵佳蓉,宋春波,卞坤,陈伟,杨振峰,2016,桃果实PpSIZ1基因对低温和外源褪黑素处理的响应,园艺学报,43(7):1257-1266)
Song G.,Walworth A.,Zhao D.,Jiang N.,and Hancock J.F.,2013,The vaccinium corymbosum FLOWERING LOCUST-like gene(VcFT):a flowering activator reverses photoperiodic and chilling requirements in blueberry,Plant Cell Report,32(11):1759-1769
Tamaki S.,Matsuo S.,Wong H.L.,Yokoi S.,and Shimamoto K.,2007,Hd3a protein is a mobile flowering signal in rice,Science,316(5827):1033-1036
Wu L.,Liu D.F.,Wu J.J.,Zhang R.Z.,Qin Z.R.,Liu D.M.,Li A.L.,Fu D.L.,Zhai W.X.,and Mao L.,2013,Regulation of FLOWERING LOCUS T by a microRNA in Brachypodium distachyon,Plant Cell,25(11):4363-4377
Xue D.L.,Zeng S.H.,and Wang Y.,2015,Molecular cloning and expression analysis of Flowering locus T in lycium ruthenicum Murr.and L.barbarum L.,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(3):565-570(薛定磊,曾少华,王瑛,2015,黑果枸杞和宁夏枸杞FT(Flowering Locus T)基因的克隆及表达分析,基因组学与应用生物学,34(3):565-570)
Yang L.,Liu D.C.,and Liu Y.,2017,Advances in the study of citrus orthologue FT gene,Jiangxi Nongye Daxue Xuebao(Acta Agriculturae Universitatis Jiangxiensis),39(6):1067-1074(杨莉,刘德春,刘勇,2017,柑橘FT同源基因的研究进展,江西农业大学学报,39(6):1067-1074)
Yasushi K.,and Weigel D.,2007,Move on up,it's time for changemobile signals controlling photoperiod-dependent flowering,Genes Dev.,21(19):2371-2384
Zhang Y.,He X.H.,Hu Y.,Dan S.N.,Xu C.,and Lin W.,2016,Clone and expression analysis of the key flowering gene FcFT1 in kumquat,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(3):562-569(张奕,何新华,胡颖,旦帅男,徐趁,林蔚,2016,金柑开花关键基因FcFT1的克隆及表达分析,分子植物育种,14(3):562-569)
Zhou C.M.,and Wang J.W.,2013,Regulation of flowering time by microRNAs,J.Genetic Genomics,40(5):211-215
Chen X.,Li S.Y.,Wu L.C.,Wang C.L.,Chen Y.H.,2006,Molecular mechanisms of photoperiod actions on plant flowering,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),26(7):1490-1499(陈晓,李思远,吴连成,王翠玲,陈彦惠,2006,光周期影响植物花时的分子机制,西北植物学报,26(7):1490-1499)
Endo T.,Shimada T.,Fujii H.,Kobayashi Y.,Araki T.,and Omura M.,2005,Ectopic expression of an FT homolog from Citrus confers an early flowering phenotype on trifoliate orange(Poncirus trifoliata L.Raf.),Trans.Res.,14(5):703-712
Endo T.,Shimada T.,Fujii H.,Nishikawa F.,Sugiyama A.,Nakano M.,Shimizu T.,Kobayashi Y.,Araki T.,Pea L.,and Omura M.,2009,Development of a Ci FT co-expression system for function analysis of gene in citrus flowers and fruit,J.Japan.Soc.Hort.Sci.,78(1):74-83
Guo W.F.,Liu D.C.,Yang L.,Zhuang X.,Zhang J.J.,Wang S.S.,and Liu Y.,2015,Cloning and expression analysis of new stress-resistant NAC83 gene from Citrus,Yuanyi Xuebao(Acta Horticulturae Sinica),42(3):445-454(郭文芳,刘德春,杨莉,庄霞,张涓涓,王书胜,刘勇,2015,柑橘抗逆基因NAC83的克隆与表达分析,园艺学报,34(3):445-454)
Hemming M.N.,Peacock W.J.,Dennis E.S.,and Trevaskis B.,2008,Low-temperature and daylength cues are integrated to regulate FLOWERING LOCUS T in barley,Plant Physiol.,147(1):355-366
Imamura T.,Nakatsuka T.,Higuchi A.,Nishihara M.,and Takahashi H.T.,2011,The gentian orthologs of the FT/TFL1 gene family control floral initiation in Gentiana,Plant Cell Physiol.,52(6):1031-1041
Kikuchi R.,Kawahigashi H.,Ando T.,Tonooka T.,and Handa H.,2009,Molecular and functional characterization of PEBPgenes in barley reveal the diversification of their roles in flowering,Plant Physiol.,149(3):1341-1353
Kong F.,Liu B.,Xia Z.,Sato S.,Kim B.M.,Watanabe S.,Yamada T.,Tabata S.,Kanazawa A.,Harada K.,and Abe J.,2010,Two coordinately regulated homologs of FLOWER-ING LOCUS T are involved in the control of photoperiodic flowering in soybean,Plant Physiol.,154(3):1220-1231
Kotoda N.,Hayashi H.,Suzuki M.,Igarashi M.,Hatsuyama Y.,Kidou S.,Igasaki T.,Nishiguchi M.,Yano K.,Shimizu T.,Takahashi S.,Iwanami H.,Moriya S.,and Abe K.,2010,Molecular characterization of FLOWERING LOCUS T-like genes of apple(Malus domestica Borkh.),Plant Cell Physiol.,51(4):561-575
Laurie R.E.,Diwadkar P.,Jaudal M.,Zhang L.,Hecht V.,Wen J.,Tadege M.,Mysore K.S.,Putterill J.,Weller J.L.,and Macknight R.C.,2011,The medicago FLOWERING LOCUS Thomology,MtFTa1,is a key regulator of flowering time,Plant Physiol.,156(4):2207-2224
Li X.F.,Jia L.Y.,Xu J.,Deng X.J.,Wang Y.,Zhang W.,Zhang X.P.,Fang Q.,Zhang D.M.,Sun Y.,and Xu L.,2013,FT-like NFT1 gene may play a role in flower transition induced by heat accumulation in Narcissus tazetta var.chinensis,Plant Cell Physiol.,54(2):270-281
Marchlerbauer A.,Bo Y.,Han L.,He J.,Lanczycki C.J.,Lu S.,Chitsaz F.,Derbyshire M.K.,Geer R.C.,Gonzales N.R.,Gwadz M.,Hurwitz D.I.,Lu F.,Marchler G.H.,Song J.S.,Thanki N.,Wang Z.,Yamashita R.A.,Zhang D.,Zheng C.,Geer L.Y.,and Bryant S.H.,2017,Cdd/sparcle:functional classification of proteins via subfamily domain architectures,Nucleic Acids Research,45(Database issue):D200-D203
Meng X.,Muszynski M.G.,and Danilevskaya O.N.,2011,The FT-like ZCN8 gene functions as a floral activator and is involved in photoperiod sensitivity in maize,Plant Cell,23(3):942-960
Nakano Y.,Higuchi Y.,Sumitomo K.,and Hisamatsu T.,2013,Flowering retardation by high temperature in chrysanthemums:involvement of FLOWERING LOCUS T-like 3 gene repression,J.Exp.Bot.,64(4):909-920
Nishikawa F.,Endo T.,Shimada T.,Fujii H.,Shimizu T.,Omura M.,and Ikoma Y.,2007,Increased Ci FT abundance in the stem correlates with floral induction by low temperature in satsuma mandarin(Citrus unshiu Marc.),J.Exp.Bot.,58(14):3915-3927
Nishikawa F.,2013,Regulation of floral induction in citrus,J.Japan.Soc.Hort.Sci.,82(4):283-292
Pillitteri L.,Lovatt C.,and Walling L.,2004,Isolation and characterization of a TERMINAL FLOWER homolog and its correla tion with juvenility in citrus,Plant Physiol.,135(3):1540-1551
Shao J.R.,Song C.B.,Bian K.,Chen W.,and Yang Z.F.,2016,Expression responses of SUMO E3 ligase(SIZ1)to low temperature stress and exogenous melatonin in postharvest peach fruit,Yuanyi Xuebao(Acta Horticulturae Sinica),43(7):1257-1266(邵佳蓉,宋春波,卞坤,陈伟,杨振峰,2016,桃果实PpSIZ1基因对低温和外源褪黑素处理的响应,园艺学报,43(7):1257-1266)
Song G.,Walworth A.,Zhao D.,Jiang N.,and Hancock J.F.,2013,The vaccinium corymbosum FLOWERING LOCUST-like gene(VcFT):a flowering activator reverses photoperiodic and chilling requirements in blueberry,Plant Cell Report,32(11):1759-1769
Tamaki S.,Matsuo S.,Wong H.L.,Yokoi S.,and Shimamoto K.,2007,Hd3a protein is a mobile flowering signal in rice,Science,316(5827):1033-1036
Wu L.,Liu D.F.,Wu J.J.,Zhang R.Z.,Qin Z.R.,Liu D.M.,Li A.L.,Fu D.L.,Zhai W.X.,and Mao L.,2013,Regulation of FLOWERING LOCUS T by a microRNA in Brachypodium distachyon,Plant Cell,25(11):4363-4377
Xue D.L.,Zeng S.H.,and Wang Y.,2015,Molecular cloning and expression analysis of Flowering locus T in lycium ruthenicum Murr.and L.barbarum L.,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(3):565-570(薛定磊,曾少华,王瑛,2015,黑果枸杞和宁夏枸杞FT(Flowering Locus T)基因的克隆及表达分析,基因组学与应用生物学,34(3):565-570)
Yang L.,Liu D.C.,and Liu Y.,2017,Advances in the study of citrus orthologue FT gene,Jiangxi Nongye Daxue Xuebao(Acta Agriculturae Universitatis Jiangxiensis),39(6):1067-1074(杨莉,刘德春,刘勇,2017,柑橘FT同源基因的研究进展,江西农业大学学报,39(6):1067-1074)
Yasushi K.,and Weigel D.,2007,Move on up,it's time for changemobile signals controlling photoperiod-dependent flowering,Genes Dev.,21(19):2371-2384
Zhang Y.,He X.H.,Hu Y.,Dan S.N.,Xu C.,and Lin W.,2016,Clone and expression analysis of the key flowering gene FcFT1 in kumquat,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(3):562-569(张奕,何新华,胡颖,旦帅男,徐趁,林蔚,2016,金柑开花关键基因FcFT1的克隆及表达分析,分子植物育种,14(3):562-569)
Zhou C.M.,and Wang J.W.,2013,Regulation of flowering time by microRNAs,J.Genetic Genomics,40(5):211-215
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