百合花青素苷呈色类型中3种R2R3-MYBs基因的差异表达
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摘要
以亚洲百合系、LA系(麝香百合×亚洲百合)以及野生原种等7个不同呈色类型百合花被片为试验材料,通过RT-PCR扩增检测3种R2R3-MYBs基因Lh MYB12、LhMYB6和LrMYB15在花被片斑点和斑线中的表达,筛选出具有调控作用的LhMYB12和Lh MYB6,并对其进行全长序列的克隆和序列分析。结果表明:LhMYB12在亚洲百合花被片的斑线和溅泼状斑点中表达,LhMYB12和LhMYB6在LA系百合斑线和斑点中表达,在野生原种南川百合(Lilium rosthornii)、湖北百合(L. henryi)和欧洲百合(L. martagon)的花被片不同呈色类型中只有LhMYB12表达,未检测到LrMYB15参与本试验材料的调控。获得的LhMYB12和Lh MYB6全长序列分别为763和797bp,包括ORF区744和705bp,编码247个和234个氨基酸,与已经克隆的百合属LhMYB12、Lh SorMYB12和LhMYB6相似性为78%～88%。表明LhMYB12在百合杂交种和野生原种的呈色类型中均起重要作用,是一个广泛存在的R2R3-MYBs。LhMYB6在LA中与LhMYB12共同发挥作用。本研究揭示了调控百合花被片花青素苷不同呈色类型的R2R3-MYBs在不同品种类群中的差异表达。
Anthocyanin accumulation in different parts of tepal developed various pigmentation patterns,but the expression patterns of R2R3-MYBs regulating various anthocyanin pigmentation patterns in Lilium spp. are not well understood. In this study,expression patterns of LhMYB12,LhMYB6 and LrMYB15 genes regulating various anthocyanin pigmentation patterns were performed in seven different pigmentation patterns of materials[Asiatic hybrids,LA(Lilium longiflorum × Asiatic hybrids)and wild species]. LhMYB12 and LhMYB6,cloned full-length cDNA and analyzed gene sequences,were considered to be regulated gene related to anthocyanin pigmentation patterns. The study showed LhMYB12 was expressed in linear spots at edge of tepals and splatter spots in Asiatic hybrids. But LhMYB12 and LhMYB6 were expressed in linear spots at edge of tepals and splatter spots in LA hybrids. And only LhMYB12 was expressed in anthocyanin pigmentation patterns of wild species(L. rosthornii,L. henryi and L. martagon). However,anthocyanin pigmentation patterns of these parameters were not observed in LrMYB15. The full-length of LhMYB12 and LhMYB6 was 763 and 797 bp containing open reading frame of 744 and 705 bp,which encoded 247 and 234 amino acids respectively. They were homologous sequence with published LhMYB12,LhSorMYB12 and LhMYB6,sharing 78% to 88% similarity with them. The results indicated that LhMYB12 played an important role in the regulation of anthocyanin pigmentation patterns in hybrids and wild species widely. Moreover,LhMYB12 and LhMYB6 regulated anthocyanin in LA hybrids. This study revealed that the expression of R2R3-MYBs was involved in regulating various anthocyanin pigmentation patterns in different species.
Anthocyanin accumulation in different parts of tepal developed various pigmentation patterns,but the expression patterns of R2R3-MYBs regulating various anthocyanin pigmentation patterns in Lilium spp. are not well understood. In this study,expression patterns of LhMYB12,LhMYB6 and LrMYB15 genes regulating various anthocyanin pigmentation patterns were performed in seven different pigmentation patterns of materials[Asiatic hybrids,LA(Lilium longiflorum × Asiatic hybrids)and wild species]. LhMYB12 and LhMYB6,cloned full-length cDNA and analyzed gene sequences,were considered to be regulated gene related to anthocyanin pigmentation patterns. The study showed LhMYB12 was expressed in linear spots at edge of tepals and splatter spots in Asiatic hybrids. But LhMYB12 and LhMYB6 were expressed in linear spots at edge of tepals and splatter spots in LA hybrids. And only LhMYB12 was expressed in anthocyanin pigmentation patterns of wild species(L. rosthornii,L. henryi and L. martagon). However,anthocyanin pigmentation patterns of these parameters were not observed in LrMYB15. The full-length of LhMYB12 and LhMYB6 was 763 and 797 bp containing open reading frame of 744 and 705 bp,which encoded 247 and 234 amino acids respectively. They were homologous sequence with published LhMYB12,LhSorMYB12 and LhMYB6,sharing 78% to 88% similarity with them. The results indicated that LhMYB12 played an important role in the regulation of anthocyanin pigmentation patterns in hybrids and wild species widely. Moreover,LhMYB12 and LhMYB6 regulated anthocyanin in LA hybrids. This study revealed that the expression of R2R3-MYBs was involved in regulating various anthocyanin pigmentation patterns in different species.
引文
Abe H,Nakano M,Nakatsuka A,Nakayama M,Koshioka M,Yamagishi M.2002.Genetic analysis of floral anthocyanin pigmentation traits in Asiatic hybrid lily using molecular linkage maps.Theoretical and Applied Genetics,105(8):1175-1182.
Dai Si-lan,Hong Yan.2016.Molecular breeding for flower colors modification on ornamental plants based on the mechanism of anthocyanins biosynthesis and coloration.Scientia Agricultura Sinica,49(3):529-542.(in Chinese)戴思兰,洪艳.2016.基于花青素苷合成和呈色机理的观赏植物花色改良分子育种.中国农业科学,49(3):529-542.
Davies K M,Albert N W,Schwinn K E.2012.From landing lights to mimicry:the molecular regulation of flower colouration and mechanisms for pigmentation patterning.Functional Plant Biology,39(8):619-638.
Davies K M,Schwinn K E.2003.Transcriptional regulation of secondary metabolism.Functional Plant Biology,30(9):913-925.
Deli J,Molnár P,Matus Z,Tóth G,Steck A,Pfander H.1998.Isolation and characterization of 3,5,6-trihydroxy-carotenoids from petals of Lilium tigrinum.Chromatographia,48(1):27-31.
Ge Cui-lian,Huang Chun-hui,Xu Xiao-biao.2012.Research on anthocyanins biosynthesis in fruit.Acta Horticulturae Sinica,39(9):1655-1664.(in Chinese)葛翠莲,黄春辉,徐小彪.2012.果实花青素生物合成研究进展.园艺学报,39(9):1655-1664.
Goodrich J,Carpenter R,Coen E S.1992.A common gene regulates pigmentation pattern in diverse plant species.Cell,68(5):955-964.
Han Ke-ting,Zhao Li,Tang Xing-jiao,Hu Ke,Dai Si-lan.2012.The relationship between the expression of key genes in anthocyanin biosynthesis and the color of chrysanthemum.Acta Horticulturae Sinica,39(3):516-524.(in Chinese)韩科厅,赵莉,唐杏姣,胡可,戴思兰.2012.菊花花青素苷合成关键基因表达与花色表型的关系.园艺学报,39(3):516-524.
Hichri I,Barrieu F,Bogs J,Kappel C,Delrot S,Lauvergeat V.2011.Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway.Journal of Experimental Botany,62(8):2465-2483.
Kong J M,Chia S,Goh N K,Chia T F,Brouillard R.2003.Analysis and biological activities of anthocyanins.Phytochemistry,64(5):923-933.
Kong Ying,Dou Xiao-ying,Bao Fang,Lang Li-xin,Bai Jin-rong.2015.Advances in flower color mechanism of Lilium.Acta Horticulturae Sinica,42(9):1747-1759.(in Chinese)孔滢,窦晓莹,包放,郎利新,白锦荣.2015.百合花色机理研究进展.园艺学报,42(9):1747-1759.
Lai Y S,Shimoyamada Y,Nakayama M,Yamagishi M.2012.Pigment accumulation and transcription of LhMYB12 and anthocyanin biosynthesis genes during flower development in the Asiatic hybrid lily(Lilium spp.).Plant Science,193:136-147.
Nakatsuka A,Yamagishi M,Nakano M,Tasaki K,Kobayashi N.2009.Light-induced expression of basic helix-loop-helix genes involved in anthocyanin biosynthesis in flowers and leaves of Asiatic hybrid lily.Scientia Horticulturae,121(1):84-91.
N?rb?k R,Kondo T.1999.Anthocyanins from flowers of Lilium(Liliaceae).Phytochemistry,50(7):1181-1184.
Quattrocchio F,Wing J F,Leppen H T,Mol J N,Koes R E.1993.Regulatory genes controlling anthocyanin pigmentation are functionally conserved among plant species and have distinct sets of target genes.The Plant Cell,5(11):1497-1512.
Quattrocchio F,Wing J F,Va K,Mol J N,Koes R.1998.Analysis of bHLH and MYB domain proteins:species-specific regulatory differences are caused by divergent evolution of target anthocyanin genes.The Plant Journal,13(4):475-488.
Spelt C,Quattrocchio F,Mol J N M,Koes R.2000.Anthocyanin1 of petunia encodes a basic helix-loop-helix protein that directly activates transcription of structural anthocyanin genes.The Plant Cell,12(9):1619-1631.
Suzuki K,Suzuki T,Nakatsuka T,Dohra H,Yamagishi M,Matsuyama K,Matsuura H.2016.RNA-seq-based evaluation of bicolor tepal pigmentation in Asiatic hybrid lilies(Lilium spp.).BMC Genomics,17(1):611.
Suzuki K,Tasaki K,Yamagishi M.2015.Two distinct spontaneous mutations involved in white flower development in Lilium speciosum.Molecular Breeding,35(10):193.
Xu L,Xu H,Cao Y,Yang P,Feng Y,Tang Y,Yuan S,Ming J.2017a.Validation of reference genes for quantitative real-time PCR during bicolor tepal development in Asiatic hybrid lilies(Lilium spp.).Frontiers in Plant Science,8:669.
Xu L,Yang P,Feng Y,Xu H,Cao Y,Tang Y,Yuan S,Liu X,Ming J.2017b.Spatiotemporal transcriptome analysis provides insights into bicolor tepal development in Lilium‘Tiny Padhye’.Frontiers in Plant Science,8:398.
Yamagishi M.2011.Oriental hybrid lily Sorbonne homologue of LhMYB12 regulates anthocyanin biosyntheses in flower tepals and tepal spots.Molecular Breeding,28(3):381-389.
Yamagishi M.2013.How genes paint lily flowers:regulation of colouration and pigmentation patterning.Scientia Horticulturae,163:27-36.
Yamagishi M.2016.A novel R2R3-MYB transcription factor regulates light-mediated floral and vegetative anthocyanin pigmentation patterns in Lilium regale.Molecular Breeding,36(1):3.
Yamagishi M.2018.Involvement of a LhMYB18 transcription factor in large anthocyanin spot formation on the flower tepals of the Asiatic hybrid lily(Lilium spp.)cultivar‘Grand Cru’.Molecular Breeding,38(5):60.
Yamagishi M,Akagi K.2013.Morphology and heredity of tepal spots in Asiatic and Oriental hybrid lilies(Lilium spp.).Euphytica,194(3):325-334.
Yamazaki M,Makita Y,Springob K,Saito K.2003.Regulatory mechanisms for anthocyanin biosynthesis in chemotypes of Perilla frutescens var.crispa.Biochemical Engineering Journal,14(3):191-197.
Yamagishi M,Nakatsuka T.2017.LhMYB12,regulating tepal anthocyanin pigmentation in Asiatic hybrid lilies,is derived from Lilium dauricum and L.bulbiferum.The Horticulture Journal,86(4):528-533.
Yamagishi M,Kishimoto S,Nakayama M.2010a.Carotenoid composition and changes in expression of carotenoid biosynthetic genes in tepals of Asiatic hybrid lily.Plant Breeding,129:100-107.
Yamagishi M,Shimoyamada Y,Nakatsuka T,Masuda K.2010b.Two R2R3-MYB genes,homologs of Petunia AN2,regulate anthocyanin biosyntheses in flower tepals,tepal spots and leaves of Asiatic hybrid lily.Plant and Cell Physiology,51:463-474.
Yamagishi M,Toda S,Tasaki K.2014.The novel allele of the LhMYB12 gene is involved in splatter-type spot formation on the flower tepals of Asiatic hybrid lilies(Lilium spp.).New Phytologist,201(3):1009-1020.
Yamagishi M,Yoshida Y,Nakayama M.2012.The transcription factor LhMYB12 determines anthocyanin pigmentation in the tepals of Asiatic hybrid lilies(Lilium spp.)and regulates pigment quantity.Molecular Breeding,30:913-925.
Zhu L,Shan H,Chen S,Jiang J,Gu C,Zhou G,Chen Y,Song A,Chen F.2013.The heterologous expression of the chrysanthemum R2R3-MYBtranscription factor CmMYB1 alters lignin composition and represses flavonoid synthesis in Arabidopsis thaliana.PLoS ONE,8(6):e65680.
Dai Si-lan,Hong Yan.2016.Molecular breeding for flower colors modification on ornamental plants based on the mechanism of anthocyanins biosynthesis and coloration.Scientia Agricultura Sinica,49(3):529-542.(in Chinese)戴思兰,洪艳.2016.基于花青素苷合成和呈色机理的观赏植物花色改良分子育种.中国农业科学,49(3):529-542.
Davies K M,Albert N W,Schwinn K E.2012.From landing lights to mimicry:the molecular regulation of flower colouration and mechanisms for pigmentation patterning.Functional Plant Biology,39(8):619-638.
Davies K M,Schwinn K E.2003.Transcriptional regulation of secondary metabolism.Functional Plant Biology,30(9):913-925.
Deli J,Molnár P,Matus Z,Tóth G,Steck A,Pfander H.1998.Isolation and characterization of 3,5,6-trihydroxy-carotenoids from petals of Lilium tigrinum.Chromatographia,48(1):27-31.
Ge Cui-lian,Huang Chun-hui,Xu Xiao-biao.2012.Research on anthocyanins biosynthesis in fruit.Acta Horticulturae Sinica,39(9):1655-1664.(in Chinese)葛翠莲,黄春辉,徐小彪.2012.果实花青素生物合成研究进展.园艺学报,39(9):1655-1664.
Goodrich J,Carpenter R,Coen E S.1992.A common gene regulates pigmentation pattern in diverse plant species.Cell,68(5):955-964.
Han Ke-ting,Zhao Li,Tang Xing-jiao,Hu Ke,Dai Si-lan.2012.The relationship between the expression of key genes in anthocyanin biosynthesis and the color of chrysanthemum.Acta Horticulturae Sinica,39(3):516-524.(in Chinese)韩科厅,赵莉,唐杏姣,胡可,戴思兰.2012.菊花花青素苷合成关键基因表达与花色表型的关系.园艺学报,39(3):516-524.
Hichri I,Barrieu F,Bogs J,Kappel C,Delrot S,Lauvergeat V.2011.Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway.Journal of Experimental Botany,62(8):2465-2483.
Kong J M,Chia S,Goh N K,Chia T F,Brouillard R.2003.Analysis and biological activities of anthocyanins.Phytochemistry,64(5):923-933.
Kong Ying,Dou Xiao-ying,Bao Fang,Lang Li-xin,Bai Jin-rong.2015.Advances in flower color mechanism of Lilium.Acta Horticulturae Sinica,42(9):1747-1759.(in Chinese)孔滢,窦晓莹,包放,郎利新,白锦荣.2015.百合花色机理研究进展.园艺学报,42(9):1747-1759.
Lai Y S,Shimoyamada Y,Nakayama M,Yamagishi M.2012.Pigment accumulation and transcription of LhMYB12 and anthocyanin biosynthesis genes during flower development in the Asiatic hybrid lily(Lilium spp.).Plant Science,193:136-147.
Nakatsuka A,Yamagishi M,Nakano M,Tasaki K,Kobayashi N.2009.Light-induced expression of basic helix-loop-helix genes involved in anthocyanin biosynthesis in flowers and leaves of Asiatic hybrid lily.Scientia Horticulturae,121(1):84-91.
N?rb?k R,Kondo T.1999.Anthocyanins from flowers of Lilium(Liliaceae).Phytochemistry,50(7):1181-1184.
Quattrocchio F,Wing J F,Leppen H T,Mol J N,Koes R E.1993.Regulatory genes controlling anthocyanin pigmentation are functionally conserved among plant species and have distinct sets of target genes.The Plant Cell,5(11):1497-1512.
Quattrocchio F,Wing J F,Va K,Mol J N,Koes R.1998.Analysis of bHLH and MYB domain proteins:species-specific regulatory differences are caused by divergent evolution of target anthocyanin genes.The Plant Journal,13(4):475-488.
Spelt C,Quattrocchio F,Mol J N M,Koes R.2000.Anthocyanin1 of petunia encodes a basic helix-loop-helix protein that directly activates transcription of structural anthocyanin genes.The Plant Cell,12(9):1619-1631.
Suzuki K,Suzuki T,Nakatsuka T,Dohra H,Yamagishi M,Matsuyama K,Matsuura H.2016.RNA-seq-based evaluation of bicolor tepal pigmentation in Asiatic hybrid lilies(Lilium spp.).BMC Genomics,17(1):611.
Suzuki K,Tasaki K,Yamagishi M.2015.Two distinct spontaneous mutations involved in white flower development in Lilium speciosum.Molecular Breeding,35(10):193.
Xu L,Xu H,Cao Y,Yang P,Feng Y,Tang Y,Yuan S,Ming J.2017a.Validation of reference genes for quantitative real-time PCR during bicolor tepal development in Asiatic hybrid lilies(Lilium spp.).Frontiers in Plant Science,8:669.
Xu L,Yang P,Feng Y,Xu H,Cao Y,Tang Y,Yuan S,Liu X,Ming J.2017b.Spatiotemporal transcriptome analysis provides insights into bicolor tepal development in Lilium‘Tiny Padhye’.Frontiers in Plant Science,8:398.
Yamagishi M.2011.Oriental hybrid lily Sorbonne homologue of LhMYB12 regulates anthocyanin biosyntheses in flower tepals and tepal spots.Molecular Breeding,28(3):381-389.
Yamagishi M.2013.How genes paint lily flowers:regulation of colouration and pigmentation patterning.Scientia Horticulturae,163:27-36.
Yamagishi M.2016.A novel R2R3-MYB transcription factor regulates light-mediated floral and vegetative anthocyanin pigmentation patterns in Lilium regale.Molecular Breeding,36(1):3.
Yamagishi M.2018.Involvement of a LhMYB18 transcription factor in large anthocyanin spot formation on the flower tepals of the Asiatic hybrid lily(Lilium spp.)cultivar‘Grand Cru’.Molecular Breeding,38(5):60.
Yamagishi M,Akagi K.2013.Morphology and heredity of tepal spots in Asiatic and Oriental hybrid lilies(Lilium spp.).Euphytica,194(3):325-334.
Yamazaki M,Makita Y,Springob K,Saito K.2003.Regulatory mechanisms for anthocyanin biosynthesis in chemotypes of Perilla frutescens var.crispa.Biochemical Engineering Journal,14(3):191-197.
Yamagishi M,Nakatsuka T.2017.LhMYB12,regulating tepal anthocyanin pigmentation in Asiatic hybrid lilies,is derived from Lilium dauricum and L.bulbiferum.The Horticulture Journal,86(4):528-533.
Yamagishi M,Kishimoto S,Nakayama M.2010a.Carotenoid composition and changes in expression of carotenoid biosynthetic genes in tepals of Asiatic hybrid lily.Plant Breeding,129:100-107.
Yamagishi M,Shimoyamada Y,Nakatsuka T,Masuda K.2010b.Two R2R3-MYB genes,homologs of Petunia AN2,regulate anthocyanin biosyntheses in flower tepals,tepal spots and leaves of Asiatic hybrid lily.Plant and Cell Physiology,51:463-474.
Yamagishi M,Toda S,Tasaki K.2014.The novel allele of the LhMYB12 gene is involved in splatter-type spot formation on the flower tepals of Asiatic hybrid lilies(Lilium spp.).New Phytologist,201(3):1009-1020.
Yamagishi M,Yoshida Y,Nakayama M.2012.The transcription factor LhMYB12 determines anthocyanin pigmentation in the tepals of Asiatic hybrid lilies(Lilium spp.)and regulates pigment quantity.Molecular Breeding,30:913-925.
Zhu L,Shan H,Chen S,Jiang J,Gu C,Zhou G,Chen Y,Song A,Chen F.2013.The heterologous expression of the chrysanthemum R2R3-MYBtranscription factor CmMYB1 alters lignin composition and represses flavonoid synthesis in Arabidopsis thaliana.PLoS ONE,8(6):e65680.
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