VIGS诱导PSY基因沉默对枇杷果实类胡萝卜素积累的影响
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摘要
本研究从枇杷(Eriobotrya japonica Lindl.)果肉中克隆得到527 bp的八氢番茄红素合成酶(phytoene synthase,PSY)基因保守片段,通过病毒诱导的基因沉默(virus-induced gene silencing,VIGS)技术干扰枇杷果实中PSY基因的表达,探究PSY基因在枇杷果实类胡萝卜素积累过程中的调控作用。结果表明,干扰枇杷PSY基因表达后,对照果实侵染部位着色为黄色,而沉默果实侵染部位着色为绿色,果肉中均能检测到TRV病毒分子,PSY基因表达量降低55.6%,总类胡萝卜素含量降低46.8%,说明PSY基因对枇杷果实类胡萝卜素积累起着正调控作用。
In this study, a 527 bp phytoene synthase(PSY) gene conservative fragment was cloned from loquat(Eriobotrya japonica) pulp and virus-induced gene silencing(VIGS) was used to interfere the expression of PSY gene to investigate the regulatory function of PSY gene in carotenoid accumulation pathway in loquat fruit. The result showed that after interfering the gene expression of PSY, the infected parts of control fruits were yellow,while those of the silencing fruits were green. TRV molecules could be detected in both of the pulp. The PSY gene expression decreased by 55.6%, and the total carotenoid content decreased by 46.8%, indicating that PSY gene positively regulated the carotenoid accumulation in loquat fruit.
In this study, a 527 bp phytoene synthase(PSY) gene conservative fragment was cloned from loquat(Eriobotrya japonica) pulp and virus-induced gene silencing(VIGS) was used to interfere the expression of PSY gene to investigate the regulatory function of PSY gene in carotenoid accumulation pathway in loquat fruit. The result showed that after interfering the gene expression of PSY, the infected parts of control fruits were yellow,while those of the silencing fruits were green. TRV molecules could be detected in both of the pulp. The PSY gene expression decreased by 55.6%, and the total carotenoid content decreased by 46.8%, indicating that PSY gene positively regulated the carotenoid accumulation in loquat fruit.
引文
Abeysinghe D.C.,Li X.,Sun C.,and Chen K.S.,2008,Pigmen contents and their relationship with hydrophilic and lipophilic antioxidant capacities in seven fruit species,Journal of Fruit Science,25(6):790-796
Bachan S.,and Dineshkumar S.P.,2012,Tobacco rattle virus(trv)-based virus-induced gene silencing,In:Watson J.M.and Wang M.B.(ed.),Antiviral Resistance in Plants,Humana Press,New York City,pp.83-92
Baulcombe D.C.,1999,Fast forward genetics based on virus-induced gene silencing,Current Opinion in Plant Biology,2(2):109-113
Bird C.R.,Ray J.A.,Fletcher J.D.,Boniwell J.M.,Bird A.S.,Teulieres C.,Blain I.,Bramley P.M.,and Schuch W.,1991Using antisense RNA to study gene function:inhibition o carotenoid biosynthesis in Transgenic tomatoes,Nature Biotechnology,9(7):635-639
Bramley P.,Teulieres C.,Blain I.,Bird C.,and Schuch W.,1992,Biochemical characterization of transgenic tomato plants in which carotenoid synthesis has been inhibited through the expression of antisense RNA to p TOM5,Plant J.,2(3):343-349
Brigneti G.,Mratin-Hernández A.M.,Jin H.,Chen J.,Baulcombe D.C.,Baker B.,and Jones J.D.G.,2004,Virus-induced gene silencing in Solanum species,The Plant Journal,39:264-272
Burch-Smith T.M.,Anderson J.C.,Martin G.B.,and Dinesh-Kumar S.P.,2004,Applications and advantages of virus-induced gene silencing for gene function studies in plants,The Plant Journal,39:734-746
Cazzonelli C.I.,and Pogson B.J.,2010,Source to sink:regulation of carotenoid biosynthesis in plants,Trends Plant Sci.,15(5):266-274
Fu D.Q.,Zhu B.Z.,Zhu H.L.,Zhang H.X.,Xie Y.H.,Jiang W.B.,Zhao X.D.,and Luo Y.B.,2006,Enhancement of virus-induced gene silencing in tomato by low temperature and low humidity,Molecules and Cells,21(1):153-160
Fu X.M.,Kong W.B.,Peng G.,Zhou J.Y.,Azam M.,Xu C.J.,Grierson D.,and Chen K.S.,2012,Plastid structure and carotenogenic gene expression in red-and white-fleshed loquat(Eriobotrya japonica)fruits,J.Exp.Bot.,63(1):341-354
Fujisawa M.,and Misawa N.,2010,Enrichment of carotenoids in Flaxseed by introducing a bacterial phytoene synthase gene,In:Arthur G.F.N.(ed.),Plant Secondary Metabolism Engi neering,Humana Press,New York City,pp.201-211
Gao H.J.,Ming J.Q.,Zhang Y.J.,and Xu J.,2015,Regulation of carotenoids biosynthesis in Horticultural crops,Yuanyi Xuebao(Acta Horticulturae Sinica),42(9):1633-1648(高慧君,明家琪,张雅娟,徐娟,2015,园艺植物中类胡萝卜素合成与调控的研究进展,园艺学报,42(9):1633-1648)
Kjemtrup S.,Sampson K.S.,Peele C.G.,Nguyen L.V.,Conkling M.A.,Thompson W.F.,and Robertson D.,1998,Gene silencing from plant DNA carried by a Geminivirus,The Plant Journal,14(1):91-100
Kumagai M.H.,Donson,J.,Della-Cioppa G.,Harvey D.,Hanley K.,and Grill L.K.,1995,Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA,Proc.Natl.Acad.Sci.USA,92(5):1679-1683
Liu J.X.,Chiou C.Y.,Shen C.H.,Chen P.J.,Liu Y.C.,Jian C.D.,Shen X.L.,Shen F.Q.,and Yeh K.W.,2014,RNA interference-based gene silencing of phytoene synthase impairs growth,carotenoids,and plastid phenotype in Oncidium hybrid orchid,Springerplus,3(1):1-13
Liu X.H.,Sun H.Y.,Chen W.W.,Guo Q.G.,Li X.L.,and Liang G.L.,2016,Advances in studies on carotenoids in loquat fruit.Guoshu Xuebao(Journal of Fruit Science),33(7):874-881(刘小洪,孙海艳,陈薇薇,郭启高,李晓林,梁国鲁,2016,枇杷果实类胡萝卜素研究进展,果树学报,33(7):874-881)
Liu Y.L.,Schiff M.,and Dinesh-Kumar S.P.,2002,Virus-induced gene silencing in tomato,The Plant Journal,31(6):777-786
Lu R.,Martin-Hernandez A.M.,Peart J.R.,Malcuit I.,and Baulcombe D.C.,2003,Virus-induced gene silencing in plants,Methods,30(4):296-303
Ratcliff F.,Martin-Hernandez A.M.,and Baulcombe D.C.,2001Tobacco rattle virus as a vector for analysis of gene function by silencing,The Plant Journal,25(2):237-245
Ray J.,Bird C.,Maunders M.,Grierson D.,and Schuch W.1987,Sequence of p TOM5,a ripening related c DNA from tomato,Nucleic Acids Res.,15(24):10587
Ruiz M.T.,Voinnet O.,and Baulcombe D.C.,1998,Initiation and maintenance of virus-induced gene silencing,The Plan Cell,10(6):937-946
Ryu C.M.,Anand A.,Kang L.,and Mysore K.S.,2004,Agrodrench:a novel and efficient agroinoculation method for virus induced gene silencing in plant roots and various Solanaceous species,The Plant Journal,40:322-331
Senthil-Kumar M.,and Mysore K.S.,2011,Virus-induced gene silencing can persist for more than 2 years and also be transmitted to progeny seedlings in Nicotiana benthamiana and tomato,Plant Biotechnology Journal,9(7):797-806
Shewmaker C.K.,Sheehy J.A.,Daley M.,Colburn S.,and Ke DY.,1999,Seed-specific overexpression of phytoene synthase:increase in carotenoids and other metabolic effects Plant Journal,20(4):401-412
Song Z.,Li Z.A.,and Zhou C.Y.,2014,Research Advances of Virus-induced gene silencing(VIGS),Yuanyi Xuebao(Acta Horticulturae Sinica),41(9):1885-1894(宋震,李中安,周常勇,2014,病毒诱导的基因沉默(VIGS)研究进展,园艺学报,41(9):1885-1894)
Wen X.H.,Jiang Y.H.,Zhou K.,and Ren X.L.,2015,Effects of Virus-induced Md HB-1 silencing on apple fruit ripening Xibei Nongye Xuebao(Acta Agriculturae Boreali-occidentalis Sinica),24(11):113-119(温小红,姜永华,周轲,任小林,2015,VIGS诱导Md HB-1沉默对苹果果实成熟的影响,西北农业学报,24(11):113-119)
Xiong Z.M.,Zhou C.H.,and Tao J.,2007,Changes of carotenoid content in Pulp of different Loquat types during fruit coloring,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),40(12):2910-2914(熊作明,周春华,陶俊,2007,不同类型枇杷果实着色期间果肉类胡萝卜素含量的变化,中国农业科学,40(12):2910-2914)
Yang S.Y.,2015,Cloning and expression analysis of anthocyanin biosynthesis related genes in Cerasus humilis,Thesis for M.S.,Shanxi Agricultural University,Supervisor:Zhang J.C.,pp.1-65(杨淑一,2015,欧李果实花色苷生物合成相关结构基因克隆与表达分析,硕士学位论文,山西农业大学,导师:张建成,pp.1-65)
Zhai R.,Wang Z.M.,Zhang S.W.,Meng G.,Song L.Y.,Wang Z.G.,Li P.M.,Ma F.W.,and Xu L.F.,2016,Two MYB transcription factors regulate flavonoid biosynthesis in pear fruit(Pyrus bretschneideri Rehd.),J.Exp.Bot.,67(5):1275-1284
Zhang L.,2012,Analysis of carotenoids accumulation and carotenogenic genes expression during fruit development in Loquat,Thesis for M.S.,South China Agricultural University,Supervisor:Yang X.H.,pp.1-81(张玲,2012,枇杷果实类胡萝卜素积累规律及其生物合成基因的克隆与表达分析,硕士学位论文,华南农业大学,导师:杨向晖,pp.1-81)
Zhang L.,Zhu L.X.,Xu C.,Cui C.M.,Sheng H.Y.,Li R.,and Wang H.Q.,2015,The effect of silencing chalcone synthase on anthocyanin metabolism in Peach,Yuanyi Xuebao(Acta Horticulturae Sinica),42(1):31-37(张蕾,朱立新,徐川,崔春梅,盛宏亚,李瑞,王红清,2015,查尔酮合酶基因对桃果实花色苷代谢的影响,园艺学报,42(1):31-37)
Zhu C.F.,Chen X.,and Wang Y.D.,2004,Carotenoid biosynthesis in plants and application of its relative genes in gene engineering,Zhiwushengli Yu Fenzishengwuxue Xuebao(Journal of Plant Physiology and Molecular Biology),30(6):609-618(朱长甫,陈星,王英典,2004,植物类胡萝卜素生物合成及其相关基因在基因工程中的应用,植物生理与分子生物学学报,30(6):609-618)
Bachan S.,and Dineshkumar S.P.,2012,Tobacco rattle virus(trv)-based virus-induced gene silencing,In:Watson J.M.and Wang M.B.(ed.),Antiviral Resistance in Plants,Humana Press,New York City,pp.83-92
Baulcombe D.C.,1999,Fast forward genetics based on virus-induced gene silencing,Current Opinion in Plant Biology,2(2):109-113
Bird C.R.,Ray J.A.,Fletcher J.D.,Boniwell J.M.,Bird A.S.,Teulieres C.,Blain I.,Bramley P.M.,and Schuch W.,1991Using antisense RNA to study gene function:inhibition o carotenoid biosynthesis in Transgenic tomatoes,Nature Biotechnology,9(7):635-639
Bramley P.,Teulieres C.,Blain I.,Bird C.,and Schuch W.,1992,Biochemical characterization of transgenic tomato plants in which carotenoid synthesis has been inhibited through the expression of antisense RNA to p TOM5,Plant J.,2(3):343-349
Brigneti G.,Mratin-Hernández A.M.,Jin H.,Chen J.,Baulcombe D.C.,Baker B.,and Jones J.D.G.,2004,Virus-induced gene silencing in Solanum species,The Plant Journal,39:264-272
Burch-Smith T.M.,Anderson J.C.,Martin G.B.,and Dinesh-Kumar S.P.,2004,Applications and advantages of virus-induced gene silencing for gene function studies in plants,The Plant Journal,39:734-746
Cazzonelli C.I.,and Pogson B.J.,2010,Source to sink:regulation of carotenoid biosynthesis in plants,Trends Plant Sci.,15(5):266-274
Fu D.Q.,Zhu B.Z.,Zhu H.L.,Zhang H.X.,Xie Y.H.,Jiang W.B.,Zhao X.D.,and Luo Y.B.,2006,Enhancement of virus-induced gene silencing in tomato by low temperature and low humidity,Molecules and Cells,21(1):153-160
Fu X.M.,Kong W.B.,Peng G.,Zhou J.Y.,Azam M.,Xu C.J.,Grierson D.,and Chen K.S.,2012,Plastid structure and carotenogenic gene expression in red-and white-fleshed loquat(Eriobotrya japonica)fruits,J.Exp.Bot.,63(1):341-354
Fujisawa M.,and Misawa N.,2010,Enrichment of carotenoids in Flaxseed by introducing a bacterial phytoene synthase gene,In:Arthur G.F.N.(ed.),Plant Secondary Metabolism Engi neering,Humana Press,New York City,pp.201-211
Gao H.J.,Ming J.Q.,Zhang Y.J.,and Xu J.,2015,Regulation of carotenoids biosynthesis in Horticultural crops,Yuanyi Xuebao(Acta Horticulturae Sinica),42(9):1633-1648(高慧君,明家琪,张雅娟,徐娟,2015,园艺植物中类胡萝卜素合成与调控的研究进展,园艺学报,42(9):1633-1648)
Kjemtrup S.,Sampson K.S.,Peele C.G.,Nguyen L.V.,Conkling M.A.,Thompson W.F.,and Robertson D.,1998,Gene silencing from plant DNA carried by a Geminivirus,The Plant Journal,14(1):91-100
Kumagai M.H.,Donson,J.,Della-Cioppa G.,Harvey D.,Hanley K.,and Grill L.K.,1995,Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA,Proc.Natl.Acad.Sci.USA,92(5):1679-1683
Liu J.X.,Chiou C.Y.,Shen C.H.,Chen P.J.,Liu Y.C.,Jian C.D.,Shen X.L.,Shen F.Q.,and Yeh K.W.,2014,RNA interference-based gene silencing of phytoene synthase impairs growth,carotenoids,and plastid phenotype in Oncidium hybrid orchid,Springerplus,3(1):1-13
Liu X.H.,Sun H.Y.,Chen W.W.,Guo Q.G.,Li X.L.,and Liang G.L.,2016,Advances in studies on carotenoids in loquat fruit.Guoshu Xuebao(Journal of Fruit Science),33(7):874-881(刘小洪,孙海艳,陈薇薇,郭启高,李晓林,梁国鲁,2016,枇杷果实类胡萝卜素研究进展,果树学报,33(7):874-881)
Liu Y.L.,Schiff M.,and Dinesh-Kumar S.P.,2002,Virus-induced gene silencing in tomato,The Plant Journal,31(6):777-786
Lu R.,Martin-Hernandez A.M.,Peart J.R.,Malcuit I.,and Baulcombe D.C.,2003,Virus-induced gene silencing in plants,Methods,30(4):296-303
Ratcliff F.,Martin-Hernandez A.M.,and Baulcombe D.C.,2001Tobacco rattle virus as a vector for analysis of gene function by silencing,The Plant Journal,25(2):237-245
Ray J.,Bird C.,Maunders M.,Grierson D.,and Schuch W.1987,Sequence of p TOM5,a ripening related c DNA from tomato,Nucleic Acids Res.,15(24):10587
Ruiz M.T.,Voinnet O.,and Baulcombe D.C.,1998,Initiation and maintenance of virus-induced gene silencing,The Plan Cell,10(6):937-946
Ryu C.M.,Anand A.,Kang L.,and Mysore K.S.,2004,Agrodrench:a novel and efficient agroinoculation method for virus induced gene silencing in plant roots and various Solanaceous species,The Plant Journal,40:322-331
Senthil-Kumar M.,and Mysore K.S.,2011,Virus-induced gene silencing can persist for more than 2 years and also be transmitted to progeny seedlings in Nicotiana benthamiana and tomato,Plant Biotechnology Journal,9(7):797-806
Shewmaker C.K.,Sheehy J.A.,Daley M.,Colburn S.,and Ke DY.,1999,Seed-specific overexpression of phytoene synthase:increase in carotenoids and other metabolic effects Plant Journal,20(4):401-412
Song Z.,Li Z.A.,and Zhou C.Y.,2014,Research Advances of Virus-induced gene silencing(VIGS),Yuanyi Xuebao(Acta Horticulturae Sinica),41(9):1885-1894(宋震,李中安,周常勇,2014,病毒诱导的基因沉默(VIGS)研究进展,园艺学报,41(9):1885-1894)
Wen X.H.,Jiang Y.H.,Zhou K.,and Ren X.L.,2015,Effects of Virus-induced Md HB-1 silencing on apple fruit ripening Xibei Nongye Xuebao(Acta Agriculturae Boreali-occidentalis Sinica),24(11):113-119(温小红,姜永华,周轲,任小林,2015,VIGS诱导Md HB-1沉默对苹果果实成熟的影响,西北农业学报,24(11):113-119)
Xiong Z.M.,Zhou C.H.,and Tao J.,2007,Changes of carotenoid content in Pulp of different Loquat types during fruit coloring,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),40(12):2910-2914(熊作明,周春华,陶俊,2007,不同类型枇杷果实着色期间果肉类胡萝卜素含量的变化,中国农业科学,40(12):2910-2914)
Yang S.Y.,2015,Cloning and expression analysis of anthocyanin biosynthesis related genes in Cerasus humilis,Thesis for M.S.,Shanxi Agricultural University,Supervisor:Zhang J.C.,pp.1-65(杨淑一,2015,欧李果实花色苷生物合成相关结构基因克隆与表达分析,硕士学位论文,山西农业大学,导师:张建成,pp.1-65)
Zhai R.,Wang Z.M.,Zhang S.W.,Meng G.,Song L.Y.,Wang Z.G.,Li P.M.,Ma F.W.,and Xu L.F.,2016,Two MYB transcription factors regulate flavonoid biosynthesis in pear fruit(Pyrus bretschneideri Rehd.),J.Exp.Bot.,67(5):1275-1284
Zhang L.,2012,Analysis of carotenoids accumulation and carotenogenic genes expression during fruit development in Loquat,Thesis for M.S.,South China Agricultural University,Supervisor:Yang X.H.,pp.1-81(张玲,2012,枇杷果实类胡萝卜素积累规律及其生物合成基因的克隆与表达分析,硕士学位论文,华南农业大学,导师:杨向晖,pp.1-81)
Zhang L.,Zhu L.X.,Xu C.,Cui C.M.,Sheng H.Y.,Li R.,and Wang H.Q.,2015,The effect of silencing chalcone synthase on anthocyanin metabolism in Peach,Yuanyi Xuebao(Acta Horticulturae Sinica),42(1):31-37(张蕾,朱立新,徐川,崔春梅,盛宏亚,李瑞,王红清,2015,查尔酮合酶基因对桃果实花色苷代谢的影响,园艺学报,42(1):31-37)
Zhu C.F.,Chen X.,and Wang Y.D.,2004,Carotenoid biosynthesis in plants and application of its relative genes in gene engineering,Zhiwushengli Yu Fenzishengwuxue Xuebao(Journal of Plant Physiology and Molecular Biology),30(6):609-618(朱长甫,陈星,王英典,2004,植物类胡萝卜素生物合成及其相关基因在基因工程中的应用,植物生理与分子生物学学报,30(6):609-618)