FaSPS1基因对草莓果实成熟调控的分子机理
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摘要
该研究以草莓品种‘红颜’(Fragaria×ananassa‘Benihoppe’)为试材,分析了草莓果实发育不同阶段蔗糖磷酸合成酶基因(FaSPS1)的表达量变化,采用PCR方法克隆FaSPS1基因,构建带有报告基因的e-GFP植物表达载体,通过瞬时转基因方法转化草莓果实,采用观察绿色荧光和检测目的基因表达量的方法鉴定转基因植物,并分析FaSPS1基因超表达和反义表达后草莓果实的成熟发育以及与成熟相关的基因表达量变化,探究FaSPS1基因在果实成熟发育中的特殊作用,为深入了解草莓果实发育和成熟调控的分子机理提供思路。结果显示:(1)成功克隆得到FaSPS1基因(GenBank登录号AB267868.1);成功构建带有报告基因e-GFP的FaSPS1基因超表达载体和反义基因表达载体,通过瞬时转基因方法转化并经荧光和目的基因表达量检测的方法鉴定获得转FaSPS1基因草莓植株。(2)与空载对照和非转基因果实相比,FaSPS1基因过表达可促进草莓果实成熟,能够使草莓果实成熟期提前,且果实中蔗糖果糖含量升高;但反义表达后会抑制草莓果实成熟,果实中苹果酸含量升高。(3)基因超表达或者反义表达后,草莓果实成熟相关基因的表达量受到不同程度调控,其中糖代谢基因FaSPS2/3、FaSUT1,果实成软化基因FaEXP1、FaEXP3、FaXYL1以及激素代谢基因FaJAZ1、FaJAZ2、FaJAZ8、FaOPR3、FaPYL1、FaPYL8、FaPYL9、FaNCED1表达量变化最明显。研究推测,FaSPS1基因可能通过影响草莓果实中和成熟相关的糖代谢基因、果实软化基因以及激素代谢基因来调控草莓果实成熟。
This study used strawberry variety ‘Benihoppe' as test material. The FaSPS1 gene expression in different stages of strawberry fruit development was analyzed. The FaSPS1 gene was cloned by PCR and the plant expression vector with reporter e-GFP gene was constructed. The strawberry fruit was transformed by transient transgene method. The aim of this study was to identify the transgenic plants by detecting the expression of FaSPS1 gene and observe green fluorescence. The changes of the expression of FaSPS1 gene during the ripening and development of strawberry fruits were analyzed after the overexpression and antisense expression of FaSPS1 gene. The special role of FaSPS1 gene in the ripening and development of strawberry fruits was explored to provide ideas for further understanding the molecular mechanism of strawberry fruit development and ripening regulation. The results showed that:(1) FaSPS1 gene(GenBank number AB267868.1) was successfully cloned; FaSPS1 gene overexpression vector and antisense gene expression vector with reporter gene e-GFP were successfully constructed, and the transgenic strawberry plants were identified by transient transgene transformation and fluorescence and target gene expression detection.(2) Overexpression of FaSPS1 gene can promote the ripening of strawberry fruit, advance the ripening period of strawberry fruit, and increase the sucrose and fructose contents in the fruit, but antisense expression can inhibit the ripening of strawberry fruit and increase the malic acid content in the fruit.(3) After gene overexpression or antisense expression, the expression of ripening-related genes in strawberry fruits were regulated to varying degrees. Among them, sugar metabolism genes FaSPS2/3, FaSUT1, fruit softening genes FaEXP1, FaEXP3, FaXYL1 and hormone metabolism genes FaJAZ1, FaJAZ2, FaJAZ8, FaOPR3, FaPYL1, FaPYL8, FaPYL9 and FaNCED1 showed the most significant changes. It is speculated that FaSPS1 gene may regulate strawberry fruit ripening by affecting sugar metabolism genes, fruit softening genes and hormone metabolism genes related to fruit ripening.
This study used strawberry variety ‘Benihoppe' as test material. The FaSPS1 gene expression in different stages of strawberry fruit development was analyzed. The FaSPS1 gene was cloned by PCR and the plant expression vector with reporter e-GFP gene was constructed. The strawberry fruit was transformed by transient transgene method. The aim of this study was to identify the transgenic plants by detecting the expression of FaSPS1 gene and observe green fluorescence. The changes of the expression of FaSPS1 gene during the ripening and development of strawberry fruits were analyzed after the overexpression and antisense expression of FaSPS1 gene. The special role of FaSPS1 gene in the ripening and development of strawberry fruits was explored to provide ideas for further understanding the molecular mechanism of strawberry fruit development and ripening regulation. The results showed that:(1) FaSPS1 gene(GenBank number AB267868.1) was successfully cloned; FaSPS1 gene overexpression vector and antisense gene expression vector with reporter gene e-GFP were successfully constructed, and the transgenic strawberry plants were identified by transient transgene transformation and fluorescence and target gene expression detection.(2) Overexpression of FaSPS1 gene can promote the ripening of strawberry fruit, advance the ripening period of strawberry fruit, and increase the sucrose and fructose contents in the fruit, but antisense expression can inhibit the ripening of strawberry fruit and increase the malic acid content in the fruit.(3) After gene overexpression or antisense expression, the expression of ripening-related genes in strawberry fruits were regulated to varying degrees. Among them, sugar metabolism genes FaSPS2/3, FaSUT1, fruit softening genes FaEXP1, FaEXP3, FaXYL1 and hormone metabolism genes FaJAZ1, FaJAZ2, FaJAZ8, FaOPR3, FaPYL1, FaPYL8, FaPYL9 and FaNCED1 showed the most significant changes. It is speculated that FaSPS1 gene may regulate strawberry fruit ripening by affecting sugar metabolism genes, fruit softening genes and hormone metabolism genes related to fruit ripening.
引文
[1] 王晨,房经贵,王涛,等.果树果实中的糖代谢[J].浙江农业学报,2009,21(5):529-534.WANG C,FANG J G,WANG T,et al.The sugar metabolism in fruits[J].Acta Agriculturae Zhejiangensis,2009,21(5):529-534.
[2] 黄东亮,李双喜,廖青,等.植物蔗糖磷酸合成酶研究进展[J].中国生物工程杂志,2012,32(6):109-119.HUANG D L,LI S X,LIAO Q,et al.Advances on sucrose phosphate synthase in plants[J].Chinese Journal of Bioengineering,2012,32(6):109-119.
[3] 刘凌霄,沈法富,卢合全,等.蔗糖代谢中蔗糖磷酸合成酶 (SPS) 的研究进展[J].2005,3(2):275-281.LIU L X,SHEN F F,LU H Q,et al.Research advance on sucrose phosphate synthase in sucrose metabolism[J].Molecular Plant Breeding,2005,3(2):275-281.
[4] 刘洋,林希昊,姚艳丽,等.高等植物蔗糖代谢研究进展[J].中国农学通报,2012,28(6):145-152.LIU Y,LIN X H,YAO Y L,et al.Sucrose metabolism in higher plants[J].Chinese Agricultural Science Bulletin,2012,28(6):145-152.
[5] HASHIDA Y,HIROSE T,OKAMURA M,et al.A reduction of sucrose phosphate synthase (SPS) activity affects sucrose/starch ratio in leaves but does not inhibit normal plant growth in rice [J].Plant Science:an International Journal of Experimental Plant Biology,2016,253(9):40-49.
[6] WORRELL A C,BRUNEAU J M,SUMMERFELT K,et al.Expression of maize sucrosephosphate synthase in tomato alters carbohydrate partitioning[J].The Plant Cell,1991,3(10):1 121-1 130.
[7] HEIM U,WEBER H,WOBUS U.Cloning and characterization of fulllength cDNA encoding sucrose phosphate synthase from faba bean[J].Gene,1996,178(1/2):201-203.
[8] VALDEZ-ALARCóN J J,FERRANDO M,SALERMO G,et al.Characterization of a rice sucrose-phosphate synthase-encoding gene[J].Gene,1996,170(2):217-222.
[9] KOMATSU A,TAKANOKURA Y,OMURA M,et al.Cloning and molecular analysis of cDNAs encoding three sucrose phosphate synthase isoforms from a citrus fruit (Citrus unshiu Marc.)[J].Molecular & General Genetics:MGG,1996,252(3):346-351.
[10] SUGIHARTO B,SAKAKIBARA H,SAUMADI,et al.Differential expression of two genes for sucrose-phosphate synthase in sugarcane:Molecular cloning of the cDNAs and comparative analysis of gene expression[J].Plant & Cell Physiology,1997,38(8):961-965.
[11] 白蓓蓓,耿宏叶,荆永琳,等.芒果MinSPS1 基因的克隆及表达载体的构建[J].分子植物育种,2018-08-30 17:17:06.网络首发地址:http://kns.cnki.net/kcms/detail/46.1068.s.20180829.1617.010.html BAI B B,GENG H Y,JING Y L,et al.Cloning and constructing expression vector of MinSPS1 gene from mango[J].Molecular Plant Breeding,08-30 17:17:06.
[12] WANG D,ZHAO J T,HU B,et al.Identification and expression profile analysis of the sucrose phosphate synthase gene family in Litchi chinensis Sonn [J].Peer J,2018,6(2):e4379.
[13] MCINTYRE C L,GOODE M L,CORDEIRO G,et al.Characterisation of alleles of the sucrose phosphate synthase gene family in sugarcane and their association with sugar-related traits [J].Molecular Breeding,2015,35(3):98-105.
[14] 黄东亮,方锋学,黎涛,等.甘蔗蔗糖磷酸合成酶基因(SofSPSA)的克隆及正反义植物表达载体的构建[J].农业生物技术学报,2011,19(4):624-633.HUANG D L,FANG F X,LI T,et al.Cloning of sucrose phosphate synthase gene (SofSPSA) from sugarcane and construction of its sense and antisense plant expression vector[J].Journal of Agricultural Biotechnology,2011,19(4):624-633.
[15] 苏艳,原牡丹,侯智霞,等.草莓果实发育中糖代谢规律研究[J].江苏农业科学,2011,39(4):147-150 SU Y,YUAN M D,HOU Z X,et al.Study on the regulation of sugar metabolism in strawberry fruit development[J].Jiangsu Agricultural Science,2011,39(4):147-150.
[16] JIA M R,DING N,ZHANG Q,etal.A FERONIA-like receptor kinase regulates strawberry (fragaria × ananassa) fruit ripening and quality formation[J].Frontiers in Plant Science,2017,8:1 099.
[17] 张玲,王延秀,高清华,等.蔗糖转化酶家族基因进化、表达及对草莓果实糖分积累的影响[J].园艺学报,2017,44 (6):1 049-1 060.ZHANG L,WANG Y X,GAO Q H,et al.The phylogeney,expression patterns of alkaline/neutral invertase genes (Fa.A/N-Invs) family and their influence on sugar accumulation in strawberry fruit[J].Acta Horticulturae Sinica,2017,44 (6):1 049-1 060.
[18] 柴叶茂,贾海锋,李春丽,等.草莓果实发育过程中糖代谢相关基因的表达分析[J].园艺学报,2011,38(4):637-643.CHAI Y M,JIA H F,LI C L,et al.Transcriptional analysis of sugar metabolism-related genes during strawberry fruit development[J].Acta Horticulturae Sinica,2011,38(4):637-643.
[19] JIA H F,WANG Y H,SUN M Z,et al.Sucrose functions as a signal involved in the regulation of strawberry fruit development and ripening[J].The New Phytologist,2013,198(2):453-465.
[20] BENíTEZ-BURRACO A,BLANCO-PORTALES R,REDONDO-NEVADO J,et al.Cloning and characterization of two ripening related strawberry (Fragaria×ananassa cv.Chandler) pectate lyase genes[J].Journal of Experimental Botany,2003,54(383):633-645.
[21] SESMERO R,QUESADA M A,MERCADO J A.Antisense inhibition of pectate lyase gene expression in strawberry fruit:characteristic of fruits processed into jam[J].Journal of Food Engineering,2007,79(1):194-199.
[22] YOUSSEF S M,JIMéNEZ-BERM.Fruit yield and quality of strawberry plants transformed with a fruit specific strawberry pectate lyase gene[J].Scientia Horticulturae,2009,119(2):120-125.
[23] FAIT A,HANHINEVA K,BELEGGIA R,et al.Reconfiguration of the achene and receptacle metabolic networks during strawberry fruit development[J].Plant Physiology,2008,148(2):730-750.
[24] 王媛花,贾思振,颜志明,等.SLCBL1基因调控番茄果实成熟发育的分子机理[J].西北植物学报,2016,36(11):2 173-2 181.WANG Y H,JIA S Z,YAN Z M,et al.Molecular mechanism of SlCBL1 gene regulation in tomato fruit development[J].Acta Botanica Boreali-Occidentalia Sinica,2016,36(11):2 173-2 181.
[25] AHARONI A,DE VOS CH,WEIN M,et al.The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco[J].The Plant Journal,2001,28(3):319-332.
[26] CONCHA C M,FIGUEROA N E,POBLETE L A,et al.Methyl jasmonate treatment induces changes in fruit ripening by modifying the expression of several ripening genes in Fragaria chiloensis fruit[J].Plant Physiology and Biochemistry,2013,70(6):433-444.
[27] WEI L Z,MAO W W,JIA M R,et al.FaMYB44.2,a transcriptional repressor,negatively regulates sucrose accumulation in strawberry receptacles through interplay with FaMYB10[J].Journal of Experimental Botany,2018,69(20):4 805-4 820.
[28] CSUKASI F,DONAIRE L,CASA?AL A,et al.Two strawberry miR159 family members display developmental-specific expression patterns in the fruit receptacle and cooperatively regulate Fa-GAMYB[J].The New Phytologist,2012,195(1):47-57.
[29] DU H,ZHANG L,LIU L,et al.Biochemical and molecular characterization of plant MYB transcription factor family[J].Biochemistry Biokhimiia,2009,74(1):1-11.
[30] GüNDüZ K,OZDEMIR E.The effects of genotype and growing conditions on antioxidant capacity,phenolic compounds,organic acid and individual sugars of strawberry[J].Food Chemistry,2014,155(15):298-303.
[31] JIA H F,CHAI Y M,LI C L,et al.Abscisic acid plays an important role in the regulation of strawberry fruit ripening[J].Plant Physiology,2011,157(1):188-199.
[32] REN Y,GUO S G,ZHANG J,et al.A tonoplast sugar transporter underlies a sugar accumulation QTL in watermelon[J].Plant Physiology,2018,176(1):836-850.
[33] ROLLAND F,BAENA-GONZALEZ E,SHEEN J,et al.Sugar sensing and signaling in plants:conserved and novel mechanisms[J].Annual Review of Plant Biology,2006,57:675-709.
[34] GIBSON S I.Control of plant development and gene expression by sugar signaling[J].Current Opinion in Plant Biology,2005,8(1):93-102.
[35] JANG JC,LEON P,ZHOU L,et al.Hexokinase as a sugar sensor in higher plants[J].Plant Cell,1997,9(1):5-19.
[36] LALONDE S,BOLES E,HELLMANN H,et al.The dual function of sugar carriers,transport and sugar sensing[J].The Plant Cell,1999,11(4):707-726.
[37] SMEEKENS S,MA J K,HANSON J,et al.Sugar signals and molecular networks controlling plant growth[J].Current Opinion in Plant Biology,2000,13(3):274-279.
[2] 黄东亮,李双喜,廖青,等.植物蔗糖磷酸合成酶研究进展[J].中国生物工程杂志,2012,32(6):109-119.HUANG D L,LI S X,LIAO Q,et al.Advances on sucrose phosphate synthase in plants[J].Chinese Journal of Bioengineering,2012,32(6):109-119.
[3] 刘凌霄,沈法富,卢合全,等.蔗糖代谢中蔗糖磷酸合成酶 (SPS) 的研究进展[J].2005,3(2):275-281.LIU L X,SHEN F F,LU H Q,et al.Research advance on sucrose phosphate synthase in sucrose metabolism[J].Molecular Plant Breeding,2005,3(2):275-281.
[4] 刘洋,林希昊,姚艳丽,等.高等植物蔗糖代谢研究进展[J].中国农学通报,2012,28(6):145-152.LIU Y,LIN X H,YAO Y L,et al.Sucrose metabolism in higher plants[J].Chinese Agricultural Science Bulletin,2012,28(6):145-152.
[5] HASHIDA Y,HIROSE T,OKAMURA M,et al.A reduction of sucrose phosphate synthase (SPS) activity affects sucrose/starch ratio in leaves but does not inhibit normal plant growth in rice [J].Plant Science:an International Journal of Experimental Plant Biology,2016,253(9):40-49.
[6] WORRELL A C,BRUNEAU J M,SUMMERFELT K,et al.Expression of maize sucrosephosphate synthase in tomato alters carbohydrate partitioning[J].The Plant Cell,1991,3(10):1 121-1 130.
[7] HEIM U,WEBER H,WOBUS U.Cloning and characterization of fulllength cDNA encoding sucrose phosphate synthase from faba bean[J].Gene,1996,178(1/2):201-203.
[8] VALDEZ-ALARCóN J J,FERRANDO M,SALERMO G,et al.Characterization of a rice sucrose-phosphate synthase-encoding gene[J].Gene,1996,170(2):217-222.
[9] KOMATSU A,TAKANOKURA Y,OMURA M,et al.Cloning and molecular analysis of cDNAs encoding three sucrose phosphate synthase isoforms from a citrus fruit (Citrus unshiu Marc.)[J].Molecular & General Genetics:MGG,1996,252(3):346-351.
[10] SUGIHARTO B,SAKAKIBARA H,SAUMADI,et al.Differential expression of two genes for sucrose-phosphate synthase in sugarcane:Molecular cloning of the cDNAs and comparative analysis of gene expression[J].Plant & Cell Physiology,1997,38(8):961-965.
[11] 白蓓蓓,耿宏叶,荆永琳,等.芒果MinSPS1 基因的克隆及表达载体的构建[J].分子植物育种,2018-08-30 17:17:06.网络首发地址:http://kns.cnki.net/kcms/detail/46.1068.s.20180829.1617.010.html BAI B B,GENG H Y,JING Y L,et al.Cloning and constructing expression vector of MinSPS1 gene from mango[J].Molecular Plant Breeding,08-30 17:17:06.
[12] WANG D,ZHAO J T,HU B,et al.Identification and expression profile analysis of the sucrose phosphate synthase gene family in Litchi chinensis Sonn [J].Peer J,2018,6(2):e4379.
[13] MCINTYRE C L,GOODE M L,CORDEIRO G,et al.Characterisation of alleles of the sucrose phosphate synthase gene family in sugarcane and their association with sugar-related traits [J].Molecular Breeding,2015,35(3):98-105.
[14] 黄东亮,方锋学,黎涛,等.甘蔗蔗糖磷酸合成酶基因(SofSPSA)的克隆及正反义植物表达载体的构建[J].农业生物技术学报,2011,19(4):624-633.HUANG D L,FANG F X,LI T,et al.Cloning of sucrose phosphate synthase gene (SofSPSA) from sugarcane and construction of its sense and antisense plant expression vector[J].Journal of Agricultural Biotechnology,2011,19(4):624-633.
[15] 苏艳,原牡丹,侯智霞,等.草莓果实发育中糖代谢规律研究[J].江苏农业科学,2011,39(4):147-150 SU Y,YUAN M D,HOU Z X,et al.Study on the regulation of sugar metabolism in strawberry fruit development[J].Jiangsu Agricultural Science,2011,39(4):147-150.
[16] JIA M R,DING N,ZHANG Q,etal.A FERONIA-like receptor kinase regulates strawberry (fragaria × ananassa) fruit ripening and quality formation[J].Frontiers in Plant Science,2017,8:1 099.
[17] 张玲,王延秀,高清华,等.蔗糖转化酶家族基因进化、表达及对草莓果实糖分积累的影响[J].园艺学报,2017,44 (6):1 049-1 060.ZHANG L,WANG Y X,GAO Q H,et al.The phylogeney,expression patterns of alkaline/neutral invertase genes (Fa.A/N-Invs) family and their influence on sugar accumulation in strawberry fruit[J].Acta Horticulturae Sinica,2017,44 (6):1 049-1 060.
[18] 柴叶茂,贾海锋,李春丽,等.草莓果实发育过程中糖代谢相关基因的表达分析[J].园艺学报,2011,38(4):637-643.CHAI Y M,JIA H F,LI C L,et al.Transcriptional analysis of sugar metabolism-related genes during strawberry fruit development[J].Acta Horticulturae Sinica,2011,38(4):637-643.
[19] JIA H F,WANG Y H,SUN M Z,et al.Sucrose functions as a signal involved in the regulation of strawberry fruit development and ripening[J].The New Phytologist,2013,198(2):453-465.
[20] BENíTEZ-BURRACO A,BLANCO-PORTALES R,REDONDO-NEVADO J,et al.Cloning and characterization of two ripening related strawberry (Fragaria×ananassa cv.Chandler) pectate lyase genes[J].Journal of Experimental Botany,2003,54(383):633-645.
[21] SESMERO R,QUESADA M A,MERCADO J A.Antisense inhibition of pectate lyase gene expression in strawberry fruit:characteristic of fruits processed into jam[J].Journal of Food Engineering,2007,79(1):194-199.
[22] YOUSSEF S M,JIMéNEZ-BERM.Fruit yield and quality of strawberry plants transformed with a fruit specific strawberry pectate lyase gene[J].Scientia Horticulturae,2009,119(2):120-125.
[23] FAIT A,HANHINEVA K,BELEGGIA R,et al.Reconfiguration of the achene and receptacle metabolic networks during strawberry fruit development[J].Plant Physiology,2008,148(2):730-750.
[24] 王媛花,贾思振,颜志明,等.SLCBL1基因调控番茄果实成熟发育的分子机理[J].西北植物学报,2016,36(11):2 173-2 181.WANG Y H,JIA S Z,YAN Z M,et al.Molecular mechanism of SlCBL1 gene regulation in tomato fruit development[J].Acta Botanica Boreali-Occidentalia Sinica,2016,36(11):2 173-2 181.
[25] AHARONI A,DE VOS CH,WEIN M,et al.The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco[J].The Plant Journal,2001,28(3):319-332.
[26] CONCHA C M,FIGUEROA N E,POBLETE L A,et al.Methyl jasmonate treatment induces changes in fruit ripening by modifying the expression of several ripening genes in Fragaria chiloensis fruit[J].Plant Physiology and Biochemistry,2013,70(6):433-444.
[27] WEI L Z,MAO W W,JIA M R,et al.FaMYB44.2,a transcriptional repressor,negatively regulates sucrose accumulation in strawberry receptacles through interplay with FaMYB10[J].Journal of Experimental Botany,2018,69(20):4 805-4 820.
[28] CSUKASI F,DONAIRE L,CASA?AL A,et al.Two strawberry miR159 family members display developmental-specific expression patterns in the fruit receptacle and cooperatively regulate Fa-GAMYB[J].The New Phytologist,2012,195(1):47-57.
[29] DU H,ZHANG L,LIU L,et al.Biochemical and molecular characterization of plant MYB transcription factor family[J].Biochemistry Biokhimiia,2009,74(1):1-11.
[30] GüNDüZ K,OZDEMIR E.The effects of genotype and growing conditions on antioxidant capacity,phenolic compounds,organic acid and individual sugars of strawberry[J].Food Chemistry,2014,155(15):298-303.
[31] JIA H F,CHAI Y M,LI C L,et al.Abscisic acid plays an important role in the regulation of strawberry fruit ripening[J].Plant Physiology,2011,157(1):188-199.
[32] REN Y,GUO S G,ZHANG J,et al.A tonoplast sugar transporter underlies a sugar accumulation QTL in watermelon[J].Plant Physiology,2018,176(1):836-850.
[33] ROLLAND F,BAENA-GONZALEZ E,SHEEN J,et al.Sugar sensing and signaling in plants:conserved and novel mechanisms[J].Annual Review of Plant Biology,2006,57:675-709.
[34] GIBSON S I.Control of plant development and gene expression by sugar signaling[J].Current Opinion in Plant Biology,2005,8(1):93-102.
[35] JANG JC,LEON P,ZHOU L,et al.Hexokinase as a sugar sensor in higher plants[J].Plant Cell,1997,9(1):5-19.
[36] LALONDE S,BOLES E,HELLMANN H,et al.The dual function of sugar carriers,transport and sugar sensing[J].The Plant Cell,1999,11(4):707-726.
[37] SMEEKENS S,MA J K,HANSON J,et al.Sugar signals and molecular networks controlling plant growth[J].Current Opinion in Plant Biology,2000,13(3):274-279.
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