沟叶结缕草ZmPSY基因启动子对拟南芥的转化及功能分析
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摘要
利用染色体步移法克隆得到沟叶结缕草(Zoysia matrella)ZmPSY基因的启动子序列。将ZmPSY起始密码子上游的1512bp的启动子序列与GUS基因融合,构建了ZmPSYpro::GUS载体,并利用农杆菌介导的方法转化拟南芥。经潮霉素筛选和PCR鉴定,共获得8个转基因株系。GUS染色结果表明,ZmPSY基因启动子能够驱动GUS基因在拟南芥中表达,且集中在茎叶部位。利用荧光定量技术(qRT-PCR)分析了外施激素和非生物胁迫处理下GUS基因的表达量,结果表明:5μM ABA可诱导GUS表达增强,10μM MeJA和黑暗处理抑制GUS表达,而100μM GA3处理无显著变化。研究成功克隆ZmPSY基因的启动子序列,并证明ZmPSY基因的表达可受ABA、MeJA和黑暗处理的调控,为深入研究ZmPSY基因的转录调控提供了依据。
The promoter of ZmPSY gene was cloned from Zoysia matrella using genome walking method.The 1512 bp upstream sequence in front of the initiation codon of ZmPSY was infused with theβ-glucuronidase(GUS)reporter gene.Then transgenic Arabidopsis was generated using agrobacterium-mediated transformation.After homomycin screening and PCR verification,8 lines expressing ZmPSYpro::GUS were finally selected and used in the following experiments.The results of GUS staining in transgenic plants showed that the ZmPSYpromoter could drive GUSgene expressing in Arabidopsis with the highest abundance in leaves.The qRT-PCR determination indicated that ZmPSY could be induced by exogenous5μM ABA,but suppressed by 10μM MeJA or dark treatment,respectively.However,exogenous 100μM GA3 showed no obvious effect.The study paved the way for further study of the transcriptional regulation mechanism and function of ZmPSY.
The promoter of ZmPSY gene was cloned from Zoysia matrella using genome walking method.The 1512 bp upstream sequence in front of the initiation codon of ZmPSY was infused with theβ-glucuronidase(GUS)reporter gene.Then transgenic Arabidopsis was generated using agrobacterium-mediated transformation.After homomycin screening and PCR verification,8 lines expressing ZmPSYpro::GUS were finally selected and used in the following experiments.The results of GUS staining in transgenic plants showed that the ZmPSYpromoter could drive GUSgene expressing in Arabidopsis with the highest abundance in leaves.The qRT-PCR determination indicated that ZmPSY could be induced by exogenous5μM ABA,but suppressed by 10μM MeJA or dark treatment,respectively.However,exogenous 100μM GA3 showed no obvious effect.The study paved the way for further study of the transcriptional regulation mechanism and function of ZmPSY.
引文
[1]Della Penna D,Pogson B J.Vitamin synthesis in plants:tocopherols and carotenoids[J].Annual Reviews of Plant Biology,2006,57(1):711-738.
[2]Giuliano G,Tavazza R,Diretto G,et al.Metabolic engineering of carotenoid biosynthesis in plants[J].Trends in Biotechnology,2008,26(3):139-145.
[3]Kim O R,Cho M C,Kim B D,et al.A splicing mutation in the gene encoding phytoene synthase causes orange coloration in Habanero pepper fruits[J].Molecules&Cells,2010,30(6):569-574.
[4]Welsch R,Arango J,Br C,et al.Provitamin A accumulation in cassava(Manihot esculenta)roots driven by a single nucleotide polymorphism in a phytoene synthase gene[J].Plant Cell,2010,22(10):3348-3356.
[5]Li F,Vallabhaneni R,Wurtzel ET.PSY3,a new member of the phytoene synthase gene family conserved in the poaceae and regulator of abiotic stress-induced root carotenogenesis[J].Plant Physiology,2008,146(3):1333-1345.
[6]Li F,Vallabhaneni R,Yu J,et al.The maize phytoene synthase gene family:overlapping roles for carotenogenesis in endosperm,photomorphogenesis,and thermal stress tolerance[J].Plant Physiology,2008,147(3):1334-1346.
[7]Welsch R,Medina J,Giuliano G,et al.Structural and functional characterization of the phytoene synthase promoter from Arabidopsis thaliana[J].Planta,2003,216(3):523-534.
[8]Welsch R,Maass D,Voegel T,et al.Transcription factor RAP2.2and its interacting partner SINAT2:stable elements in the carotenogenesis of Arabidopsis leaves[J].Plant Physiology,2007,145(3):1073-1085.
[9]曾文芳.甜橙八氢番茄红素合成酶基因及其启动子的克隆与功能分析[D].武汉:华中农业大学,2013.ZengWenfang.Inditification and functional characterization of promoters and genes of phytoene synthase from sweet orange[D].Wuhan:Huazhong Agricultural University,2013.
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[11]Corona V,Aracri B,Kosturkova G,et al.Regulation of a carotenoid biosynthesis gene promoter during plant development[J].Plant Journal,1996,9(4):505-512.
[12]滕珂,李俊,张兰,等.日本结缕草ZjADH基因的克隆及表达分析[J].中国草地学报,2016,38(3):7-13.Teng Ke,Li Jun,Zhang Lan,et al.Cloning and expression of an alcohol dehydrogenase gene(ZjADH)fromZoysia japonica[J].Chinese Journal of Grassland,2016,38(3):7-13.
[13]任爱琴,易津,高洪文,等.柠条锦鸡儿CkNCED1基因启动子的克隆及表达分析[J].草业学报,2013,22(2):165-170.Ren Aiqin,Yi Jin,Gao Hongwen,et al.Cloning and expression analysis of the promoter of Caragana korshinskii gene[J].Acta Prataculturae Sinica,2013,22(2):165-170.
[14]Clough S J,Bent A F.Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J].Plant Journal for Cell&Molecular Biology,1998,16(6):735-743.
[15]Chen M,Liu H,Kong J,et al.RopGEF7regulates PLETHORA-dependent maintenance of the root stem cell niche in Arabidopsis[J].Plant Cell.2011,23(8):2880-2894.
[16]檀鹏辉,李俊,陈小云,等.草地早熟禾PpGA2ox基因启动子的克隆及功能分析[J].中国草地学报,2017,39(3):8-14.Tan Penghui,LiJun,ChenXiaoyun,et al.Cloning,subcellular localization and expression analysis of PpGA2ox gene in Kentucky bluegrass[J].Chinese Journal of Grassland,2017,39(3):8-14.
[17]张兰,滕珂,尹淑霞.草地早熟禾叶绿素酶1基因PpCHL1的克隆和表达分析[J].中国草地学报,2016,38(4):1-7.Zhang Lan,Teng Ke,Yin Shuxia.Cloning and expression analysis of chlorophyllase1gene PpCHL1fromPoa pratensis L[J].Chinese Journal of Grassland,2016,38(4):1-7.
[18]Giuliano G,Bartley GE,Scolnik PA.Regulation of carotenoid biosynthesis during tomato development[J].Plant Cell,1993,5(4):379-387.
[19]Bouvier F,Hugueney P,D’Harlingue A,et al.Xanthophyll biosynthesis in chromoplasts:isolation and molecular cloning of an enzyme catalyzing the conversion of 5,6-epoxycarotenoid into ketocarotenoid[J].Plant Journal,1994,6(1):45-54.
[20]Ikoma Y,Komatsu A,Kita M,et al.Expression of a phytoene synthase gene and characteristic carotenoid accumulation during citrus fruit development[J].Physiologia Plantarum,2001,111(2):232-238.
[21]Lu S,Li L.Carotenoid metabolism:biosynthesis,regulation,and beyond[J].Journal of Integrative Plant Biology,2008,50(7):778-785.
[22]Bramley PM.Regulation of carotenoid formation during tomato fruit ripening and development[J].Journal of Experimental Botany,2002,53(377):2107-2113.
[23]Toledo-Ortiz G,Huq E,Rodríguez-Concepción M.Direct regulation of phytoene synthase gene expression and carotenoid biosynthesis by phytochrome-interacting factors[J].Proceedings of the National Academy Science of the United States of America,2010,107(25):11626-11631.
[24]Lintig J,Welsch R,Bonk M,et al.Light-dependent regulation of carotenoid biosynthesis occurs at the level of phytoene synthase expression and is mediated by phytochrome in Sinapis alba and Arabidopsis thaliana seedlings[J].Plant Journal,1997,12(3):625-634.
[25]Dellapenna D,Pogson B J.Vitamin synthesis in plants:Tocopherols and carotenoids[J].Annual Review of Plant Biology,2006,57(1):711-738.
[26]Obrero,González-Verdejo C I,Román B,et al.Identification,cloning,and expression analysis of three phytoene synthase genes fromCucurbita pepo[J].Biologia Plantarum,2015,59(2):201-210.
[27]翁倩,周宝利,于洋,等.外源ABA、BR和ETH对番茄果实番茄红素含量的影响[J].沈阳农业大学学报,2007(06):784-787.Weng Qian,Zhou Baoli,Yu Yang,et al.Effects of exogenous ABA,BR and ETH on changes of lycopene's content in fruit of tomato[J].Journal of Shenyang Agricultural University,2007(06):784-787.
[28]Seo M,Koshiba T.Complex regulation of ABA biosynthesis in plants[J].Trends in Plant Science,2002,7(1):41-8.
[29]Wang H,Ou C,Zhuang F,et al.The dual role of phytoene synthase genes in carotenogenesis in carrot roots and leaves[J].Molecular Breeding,2014,34(4):2065-2079.
[30]Rodríguez-VillalónA,Gas E,Rodríguez-Concepción M.Phytoene synthase activity controls the biosynthesis of carotenoids and the supply of their metabolic precursors in darkgrown Arabidopsis seedlings[J].Plant Journal,2009,60(3):424-435.
[2]Giuliano G,Tavazza R,Diretto G,et al.Metabolic engineering of carotenoid biosynthesis in plants[J].Trends in Biotechnology,2008,26(3):139-145.
[3]Kim O R,Cho M C,Kim B D,et al.A splicing mutation in the gene encoding phytoene synthase causes orange coloration in Habanero pepper fruits[J].Molecules&Cells,2010,30(6):569-574.
[4]Welsch R,Arango J,Br C,et al.Provitamin A accumulation in cassava(Manihot esculenta)roots driven by a single nucleotide polymorphism in a phytoene synthase gene[J].Plant Cell,2010,22(10):3348-3356.
[5]Li F,Vallabhaneni R,Wurtzel ET.PSY3,a new member of the phytoene synthase gene family conserved in the poaceae and regulator of abiotic stress-induced root carotenogenesis[J].Plant Physiology,2008,146(3):1333-1345.
[6]Li F,Vallabhaneni R,Yu J,et al.The maize phytoene synthase gene family:overlapping roles for carotenogenesis in endosperm,photomorphogenesis,and thermal stress tolerance[J].Plant Physiology,2008,147(3):1334-1346.
[7]Welsch R,Medina J,Giuliano G,et al.Structural and functional characterization of the phytoene synthase promoter from Arabidopsis thaliana[J].Planta,2003,216(3):523-534.
[8]Welsch R,Maass D,Voegel T,et al.Transcription factor RAP2.2and its interacting partner SINAT2:stable elements in the carotenogenesis of Arabidopsis leaves[J].Plant Physiology,2007,145(3):1073-1085.
[9]曾文芳.甜橙八氢番茄红素合成酶基因及其启动子的克隆与功能分析[D].武汉:华中农业大学,2013.ZengWenfang.Inditification and functional characterization of promoters and genes of phytoene synthase from sweet orange[D].Wuhan:Huazhong Agricultural University,2013.
[10]胡化广,张振铭,季芳芳,等.中国结缕草属植物种质资源水分利用效率变异分析[J].中国草地学报,2015,37(1):45-50.Hu Huaguang,Zhang Zhenming,Ji Fangfang,et al.Variation analysis of water use efficiency in Chinese germplasm of Zoysia Willd[J].Chinese Journal of Grassland,2015,37(1):45-50.
[11]Corona V,Aracri B,Kosturkova G,et al.Regulation of a carotenoid biosynthesis gene promoter during plant development[J].Plant Journal,1996,9(4):505-512.
[12]滕珂,李俊,张兰,等.日本结缕草ZjADH基因的克隆及表达分析[J].中国草地学报,2016,38(3):7-13.Teng Ke,Li Jun,Zhang Lan,et al.Cloning and expression of an alcohol dehydrogenase gene(ZjADH)fromZoysia japonica[J].Chinese Journal of Grassland,2016,38(3):7-13.
[13]任爱琴,易津,高洪文,等.柠条锦鸡儿CkNCED1基因启动子的克隆及表达分析[J].草业学报,2013,22(2):165-170.Ren Aiqin,Yi Jin,Gao Hongwen,et al.Cloning and expression analysis of the promoter of Caragana korshinskii gene[J].Acta Prataculturae Sinica,2013,22(2):165-170.
[14]Clough S J,Bent A F.Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J].Plant Journal for Cell&Molecular Biology,1998,16(6):735-743.
[15]Chen M,Liu H,Kong J,et al.RopGEF7regulates PLETHORA-dependent maintenance of the root stem cell niche in Arabidopsis[J].Plant Cell.2011,23(8):2880-2894.
[16]檀鹏辉,李俊,陈小云,等.草地早熟禾PpGA2ox基因启动子的克隆及功能分析[J].中国草地学报,2017,39(3):8-14.Tan Penghui,LiJun,ChenXiaoyun,et al.Cloning,subcellular localization and expression analysis of PpGA2ox gene in Kentucky bluegrass[J].Chinese Journal of Grassland,2017,39(3):8-14.
[17]张兰,滕珂,尹淑霞.草地早熟禾叶绿素酶1基因PpCHL1的克隆和表达分析[J].中国草地学报,2016,38(4):1-7.Zhang Lan,Teng Ke,Yin Shuxia.Cloning and expression analysis of chlorophyllase1gene PpCHL1fromPoa pratensis L[J].Chinese Journal of Grassland,2016,38(4):1-7.
[18]Giuliano G,Bartley GE,Scolnik PA.Regulation of carotenoid biosynthesis during tomato development[J].Plant Cell,1993,5(4):379-387.
[19]Bouvier F,Hugueney P,D’Harlingue A,et al.Xanthophyll biosynthesis in chromoplasts:isolation and molecular cloning of an enzyme catalyzing the conversion of 5,6-epoxycarotenoid into ketocarotenoid[J].Plant Journal,1994,6(1):45-54.
[20]Ikoma Y,Komatsu A,Kita M,et al.Expression of a phytoene synthase gene and characteristic carotenoid accumulation during citrus fruit development[J].Physiologia Plantarum,2001,111(2):232-238.
[21]Lu S,Li L.Carotenoid metabolism:biosynthesis,regulation,and beyond[J].Journal of Integrative Plant Biology,2008,50(7):778-785.
[22]Bramley PM.Regulation of carotenoid formation during tomato fruit ripening and development[J].Journal of Experimental Botany,2002,53(377):2107-2113.
[23]Toledo-Ortiz G,Huq E,Rodríguez-Concepción M.Direct regulation of phytoene synthase gene expression and carotenoid biosynthesis by phytochrome-interacting factors[J].Proceedings of the National Academy Science of the United States of America,2010,107(25):11626-11631.
[24]Lintig J,Welsch R,Bonk M,et al.Light-dependent regulation of carotenoid biosynthesis occurs at the level of phytoene synthase expression and is mediated by phytochrome in Sinapis alba and Arabidopsis thaliana seedlings[J].Plant Journal,1997,12(3):625-634.
[25]Dellapenna D,Pogson B J.Vitamin synthesis in plants:Tocopherols and carotenoids[J].Annual Review of Plant Biology,2006,57(1):711-738.
[26]Obrero,González-Verdejo C I,Román B,et al.Identification,cloning,and expression analysis of three phytoene synthase genes fromCucurbita pepo[J].Biologia Plantarum,2015,59(2):201-210.
[27]翁倩,周宝利,于洋,等.外源ABA、BR和ETH对番茄果实番茄红素含量的影响[J].沈阳农业大学学报,2007(06):784-787.Weng Qian,Zhou Baoli,Yu Yang,et al.Effects of exogenous ABA,BR and ETH on changes of lycopene's content in fruit of tomato[J].Journal of Shenyang Agricultural University,2007(06):784-787.
[28]Seo M,Koshiba T.Complex regulation of ABA biosynthesis in plants[J].Trends in Plant Science,2002,7(1):41-8.
[29]Wang H,Ou C,Zhuang F,et al.The dual role of phytoene synthase genes in carotenogenesis in carrot roots and leaves[J].Molecular Breeding,2014,34(4):2065-2079.
[30]Rodríguez-VillalónA,Gas E,Rodríguez-Concepción M.Phytoene synthase activity controls the biosynthesis of carotenoids and the supply of their metabolic precursors in darkgrown Arabidopsis seedlings[J].Plant Journal,2009,60(3):424-435.