苦荞FtTCPlike1和FtTCPlike2的克隆及表达分析
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摘要
植物TCP转录因子家族是一类调控植物生长、发育的重要转录因子,主要分为class Ⅰ和class Ⅱ两种类型。在本研究中,我们克隆了苦荞(Fagopyrum tataricum)两个TCP转录因子基因并分别命名为FtTCPlike1和FtTCPlike2。蛋白聚类分析表明FtTCPlike1属于class Ⅱ而FtTCPlike2属于class Ⅰ,并且均含有TCP转录因子典型的保守结构域。亚细胞定位实验也表明FtTCPlike1和FtTCPlike2与报道的其他物种TCP转录因子类似,都定位在细胞核中。本文同时收集了苦荞芽期的子叶和下胚轴、第1~3片长出的真叶以及5个时期由幼嫩到成熟的种子和花共11种样本进行了实时荧光定量PCR,发现FtTCPlike1和FtTCPlike2在苦荞的各个不同时期和不同组织中均有不同的表达,并且在种子的某个特定时期(FtTCPlike1和FtTCPlike2分别在种子发育第3、第4时期和第1、第3时期)有较高的表达量,表明这两个基因可能参与种子发育。从苦荞芽期来看,FtTCPlike1在子叶中表达量较高但在下胚轴表达较低,而FtTCPlike2则相反,这说明FtTCPlike1和FtTCPlike2在苦荞早期形态建成中的调控方式可能有很大区别。
The TCP transcription factor family is largely involved in regulating plant growth and development. Based on the similarity of protein, TCP family can be divided into two classes, class Ⅰ and class Ⅱ. In this study, we isolated two TCP genes from Fagopyrum tataricum, named FtTCPlike1 and FtTCPlike2. Both FtTCPlike1 and FtTCPlike2 contained the reported conserved TCP-domain. Furthermore, phylogenetic tree based on the protein sequences showed that FtTCPlike1 belonged to class Ⅱ, while FtTCPlike2 clustered into class Ⅰ. FtTCPlike1 and FtTCPlike2 were found to be sub-located in the nucleolus similar to the previous reported. Next, we collected cotyledons, hypocotyls, three types of true leaves at different developmental stages, flowers and seeds during five stages of development, isolated the RNA and conducted quantitative real-time PCR to analyze the expression of FtTCPlike1 and FtTCPlike2 in these tissues. The results showed that FtTCPlike1 and FtTCPlike2 were expressed in all the tissues analyzed. In addition, FtTCPlike1 and FtTCPlike2 were expressed highly in specific stages(e.g. FtTCPlike1 in stages 3 and 4; FtTCPlike2 in stages 1 and 3) during seed development, indicating that the two genes might play various roles in seed development. Interestingly, FtTCPlike1 was expressed highly in cotyledons but not in hypocotyls, while the expression of FtTCPlike2 was opposite compared to FtTCPlike1, which indicated the distinct role in early F. tataricum morphogenesis.
The TCP transcription factor family is largely involved in regulating plant growth and development. Based on the similarity of protein, TCP family can be divided into two classes, class Ⅰ and class Ⅱ. In this study, we isolated two TCP genes from Fagopyrum tataricum, named FtTCPlike1 and FtTCPlike2. Both FtTCPlike1 and FtTCPlike2 contained the reported conserved TCP-domain. Furthermore, phylogenetic tree based on the protein sequences showed that FtTCPlike1 belonged to class Ⅱ, while FtTCPlike2 clustered into class Ⅰ. FtTCPlike1 and FtTCPlike2 were found to be sub-located in the nucleolus similar to the previous reported. Next, we collected cotyledons, hypocotyls, three types of true leaves at different developmental stages, flowers and seeds during five stages of development, isolated the RNA and conducted quantitative real-time PCR to analyze the expression of FtTCPlike1 and FtTCPlike2 in these tissues. The results showed that FtTCPlike1 and FtTCPlike2 were expressed in all the tissues analyzed. In addition, FtTCPlike1 and FtTCPlike2 were expressed highly in specific stages(e.g. FtTCPlike1 in stages 3 and 4; FtTCPlike2 in stages 1 and 3) during seed development, indicating that the two genes might play various roles in seed development. Interestingly, FtTCPlike1 was expressed highly in cotyledons but not in hypocotyls, while the expression of FtTCPlike2 was opposite compared to FtTCPlike1, which indicated the distinct role in early F. tataricum morphogenesis.
引文
Aguilar-Martínez JA,Sinha N(2013).Analysis of the role of Arabidopsis class I TCP genes AtTCP7,AtTCP8,AtTCP22 and AtTCP23 in leaf development.Front Plant Sci,4(1):1-13
Bonafaccia G,Gambelli L,Fabjan N,et al(2003).Trace elements in flour and bran from common and tartary buckwheat.Food Chem,83(1):1-5
Braun N,De Saint Germain A,Pillot JP,et al(201 2).The pea TCP trans cription factor PsBRC1 acts downstream of strigolactones to control shoot branching.Plant Physiol,158(1):225-238
Crawford BC,Nath U,Carpenter R,et al(2004).CINCINNATA controls both cell differentiation and growth in petal lobes and leaves of Antirrhinum.Plant Physiol,135(1):244-253
Cubas P,Lauter N,Doebley J,et al(1999).The TCP domain:a motif found in proteins regulating plant growth and development.Plant J,18(2):215-222
Danisman S,van der Wal F,Dhondt S,et al(2012).Arabidopsis class I and class II TCP transcription factors regulate jasmonic acid metabolism and leaf development antagonistically.Plant Physiol,159(4):1511-1523
Doebley J,Stec A,Hubbard L(1997).The evolution of apical dominance in maize.Nature,386:485-488
Efroni I,Blum E,Goldshmidt A,et al(2008).A protracted and dynamic maturation schedule underlies Arabidopsis leaf development.Plant Cell,20:2293-2306
Fabjan N,Rode J,Kosir IJ,et al(2003).Tartary buckwheat(Fagopyrum tataricum Gaertn.)as a source of dietary rutin and quercitrin.J Agric Food Chem,51:6452-6455
Horn S,Pabón-Mora N,Theuβ VS,et al(2015).Analysis of the CYC/TB1 class of TCP transcription factors in basal angiosperms and magnoliids.Plant J,81(4):559-571
Huang D,Li XW,Sun M,et al(2016).Identification and characterization of CYC-like genes in regulation of ray floret development in Chrysanthemum morifolium.Front Plant Sci,7:e1002628
Jin HM,Wei P(2011).Anti-fatigue properties of tartary buckwheat extracts in mice.Int J Mol Sci,12(8):4770-4780
Juntheikki-Palovaara I,Tatiharj u S,Lan T,et al(2012).Functional diversification of duplicated CYC2 cla de genes in regulation of inflorescence development in Gerbera hybrida(Asteraceae).Plant J,79(5):783-796
Koroleva OA,Tomlinson ML,Leader D,et al(2005).Highthroughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFPORFfusions.Plant J,41(1):162-174
Kosugi S,Ohashi Y(19 97).PCF1 and PCF2 specifically bind to cis elements in the rice proliferating cell nuclear antigen gene.Plant Cell,9(9):1607-1619
Kosugi S,Ohashi Y(2002).DNA binding and dimerization specificity and potential targets for the TCP protein family.Plant J,30(3):337-348
Koyama T,Furutani M,Tasaka M,et al(2007).TCPtranscription factors control t he morphol ogy of shoot lateral organs via negative regulation of the expression of boundary-specific genes in Arabidopsis.Plant Cell,19(2):473-484
Lee CC,Shen SR,Lai YJ,et al(2013).Rutin and quercetin,bioactive compounds from tartary buckwheat,prevent liver inflammatory injury.Food Funct,4(5):794-802
Li K,Zhou CJ(2014).Research progress in WRKYtranscription factors in plants.J Plant Physiol,50(9):1329-1335(in Chinese with English abstract)[李岢,周春江(2014).植物WRKY转录因子的研究进展.植物生理学报,50(9):1329-1335]
Liu G,Tan SC,HU XX,et al(2012).A study of the effects of the extract from black buckwheat stem and leaves for regulating blood glucose in hyperglycemia mice.JSouthwest China Norm Univ(Nat Sci Ed),37(2):109-113(in Chinese with English abstract)[刘刚,谭善财,胡细享等(2 012).黑苦荞茎叶提取物对高血糖小鼠降血糖功能研究.西南师范大学学报(自然科学版),37(2):109-113]
Liu LJ,Gao H(2016).Research progress on the family of TCP genes.Biotechnol Bull,32(9):14-22(in Chinese with English abstract)[刘丽娟,高辉(2016).TC P家族基因研究进展.生物技术通报,32(9):14-22]
Lopez JA,Sun Y,Blair PB,et al(2015).TCP three-way handshake:linking developmental processes with plant immunity.Trends Plant Sci,20(4):238-245
Luo D,Carpenter R,Vincent C,et al(1995).Origin of floral asymmetry in Antirrhinum.Nature,383:794-799
Ma XD,Ma JC,Fan D,et al(2016).Genome-wide identification of TCP family transcription factors from Populus euphratica and their involvement in leaf shape regu lation.Sci Rep,6:1-9
Martín-Trillo M,Grandío EG,Serra F,et al(2011).Role of tomato BRANCHED1-like genes in the control of shoot branching.Plant J,67(4):701-714
Nam TG,Lee SM,Park JH,et al(2015).Flavonoid analysis of buckwheat sprouts.Food Chem,170(1):97-101
Navaud O,Dabos P,Carnus E,et al(2007).TCP transcription factors predate the emergence of land plants.J Mol Evol,65(1):23-33
Peng W,Hu CL,Shu ZH,et al(2015).Antitumor activity of tatariside F isolated from roots of Fagopyrum tataricum(L.)Gaertn against H22 hepatocellular carcinoma via upregulation of p53.Phytomedicine,22:730-736
Shi L,Liu C,Liang X,et al(2016).Study on the changes of the contents of rutin and quercetin in buckwheat germination.Food Res Dev,37(15):30-33(in Chinese with English abstract)[石磊,刘超,梁霞等(2016).萌发荞麦中芦丁和槲皮素含量变化的研究.食品研究与开发,37(15):30-33]
Uberti-Manassero NG,Lucero LE,Viola IL,et al(2012).The class I protein AtTCP15 modulates plant development through a pathway that overlaps with the one affected by CIN-like TCP proteins.J Exp Bot,63(2):809-823
Viola IL,Camoirano A,Gonzalez DH(2016).Redoxdependent modulation of anthocyanin biosynthesis by the TCP transcription factor TCP15 during exposure to high light intensity conditions in Arabidopsis.Plant Physiol,170:74-85
Wang ST,Sun XL,Hoshino Y,et al(2014).MicroRNA319positively regulates cold tolerance by targeting OsPCF6and OsTCP21 in rice(Oryza sativa L.).PLoS ONE,9(3):e91357
Zheng CJ,Hu CL,Ma XQ,et al(2012).Cytotoxic phenylpropanoid glycosides from Fagopyrum tataricum(L.)Gaertn.Food Chem,132(1):433-438
Zhu F(2016).Chemical composition and health effects of tartary buckwheat.Food Chem,203:231-245
Bonafaccia G,Gambelli L,Fabjan N,et al(2003).Trace elements in flour and bran from common and tartary buckwheat.Food Chem,83(1):1-5
Braun N,De Saint Germain A,Pillot JP,et al(201 2).The pea TCP trans cription factor PsBRC1 acts downstream of strigolactones to control shoot branching.Plant Physiol,158(1):225-238
Crawford BC,Nath U,Carpenter R,et al(2004).CINCINNATA controls both cell differentiation and growth in petal lobes and leaves of Antirrhinum.Plant Physiol,135(1):244-253
Cubas P,Lauter N,Doebley J,et al(1999).The TCP domain:a motif found in proteins regulating plant growth and development.Plant J,18(2):215-222
Danisman S,van der Wal F,Dhondt S,et al(2012).Arabidopsis class I and class II TCP transcription factors regulate jasmonic acid metabolism and leaf development antagonistically.Plant Physiol,159(4):1511-1523
Doebley J,Stec A,Hubbard L(1997).The evolution of apical dominance in maize.Nature,386:485-488
Efroni I,Blum E,Goldshmidt A,et al(2008).A protracted and dynamic maturation schedule underlies Arabidopsis leaf development.Plant Cell,20:2293-2306
Fabjan N,Rode J,Kosir IJ,et al(2003).Tartary buckwheat(Fagopyrum tataricum Gaertn.)as a source of dietary rutin and quercitrin.J Agric Food Chem,51:6452-6455
Horn S,Pabón-Mora N,Theuβ VS,et al(2015).Analysis of the CYC/TB1 class of TCP transcription factors in basal angiosperms and magnoliids.Plant J,81(4):559-571
Huang D,Li XW,Sun M,et al(2016).Identification and characterization of CYC-like genes in regulation of ray floret development in Chrysanthemum morifolium.Front Plant Sci,7:e1002628
Jin HM,Wei P(2011).Anti-fatigue properties of tartary buckwheat extracts in mice.Int J Mol Sci,12(8):4770-4780
Juntheikki-Palovaara I,Tatiharj u S,Lan T,et al(2012).Functional diversification of duplicated CYC2 cla de genes in regulation of inflorescence development in Gerbera hybrida(Asteraceae).Plant J,79(5):783-796
Koroleva OA,Tomlinson ML,Leader D,et al(2005).Highthroughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFPORFfusions.Plant J,41(1):162-174
Kosugi S,Ohashi Y(19 97).PCF1 and PCF2 specifically bind to cis elements in the rice proliferating cell nuclear antigen gene.Plant Cell,9(9):1607-1619
Kosugi S,Ohashi Y(2002).DNA binding and dimerization specificity and potential targets for the TCP protein family.Plant J,30(3):337-348
Koyama T,Furutani M,Tasaka M,et al(2007).TCPtranscription factors control t he morphol ogy of shoot lateral organs via negative regulation of the expression of boundary-specific genes in Arabidopsis.Plant Cell,19(2):473-484
Lee CC,Shen SR,Lai YJ,et al(2013).Rutin and quercetin,bioactive compounds from tartary buckwheat,prevent liver inflammatory injury.Food Funct,4(5):794-802
Li K,Zhou CJ(2014).Research progress in WRKYtranscription factors in plants.J Plant Physiol,50(9):1329-1335(in Chinese with English abstract)[李岢,周春江(2014).植物WRKY转录因子的研究进展.植物生理学报,50(9):1329-1335]
Liu G,Tan SC,HU XX,et al(2012).A study of the effects of the extract from black buckwheat stem and leaves for regulating blood glucose in hyperglycemia mice.JSouthwest China Norm Univ(Nat Sci Ed),37(2):109-113(in Chinese with English abstract)[刘刚,谭善财,胡细享等(2 012).黑苦荞茎叶提取物对高血糖小鼠降血糖功能研究.西南师范大学学报(自然科学版),37(2):109-113]
Liu LJ,Gao H(2016).Research progress on the family of TCP genes.Biotechnol Bull,32(9):14-22(in Chinese with English abstract)[刘丽娟,高辉(2016).TC P家族基因研究进展.生物技术通报,32(9):14-22]
Lopez JA,Sun Y,Blair PB,et al(2015).TCP three-way handshake:linking developmental processes with plant immunity.Trends Plant Sci,20(4):238-245
Luo D,Carpenter R,Vincent C,et al(1995).Origin of floral asymmetry in Antirrhinum.Nature,383:794-799
Ma XD,Ma JC,Fan D,et al(2016).Genome-wide identification of TCP family transcription factors from Populus euphratica and their involvement in leaf shape regu lation.Sci Rep,6:1-9
Martín-Trillo M,Grandío EG,Serra F,et al(2011).Role of tomato BRANCHED1-like genes in the control of shoot branching.Plant J,67(4):701-714
Nam TG,Lee SM,Park JH,et al(2015).Flavonoid analysis of buckwheat sprouts.Food Chem,170(1):97-101
Navaud O,Dabos P,Carnus E,et al(2007).TCP transcription factors predate the emergence of land plants.J Mol Evol,65(1):23-33
Peng W,Hu CL,Shu ZH,et al(2015).Antitumor activity of tatariside F isolated from roots of Fagopyrum tataricum(L.)Gaertn against H22 hepatocellular carcinoma via upregulation of p53.Phytomedicine,22:730-736
Shi L,Liu C,Liang X,et al(2016).Study on the changes of the contents of rutin and quercetin in buckwheat germination.Food Res Dev,37(15):30-33(in Chinese with English abstract)[石磊,刘超,梁霞等(2016).萌发荞麦中芦丁和槲皮素含量变化的研究.食品研究与开发,37(15):30-33]
Uberti-Manassero NG,Lucero LE,Viola IL,et al(2012).The class I protein AtTCP15 modulates plant development through a pathway that overlaps with the one affected by CIN-like TCP proteins.J Exp Bot,63(2):809-823
Viola IL,Camoirano A,Gonzalez DH(2016).Redoxdependent modulation of anthocyanin biosynthesis by the TCP transcription factor TCP15 during exposure to high light intensity conditions in Arabidopsis.Plant Physiol,170:74-85
Wang ST,Sun XL,Hoshino Y,et al(2014).MicroRNA319positively regulates cold tolerance by targeting OsPCF6and OsTCP21 in rice(Oryza sativa L.).PLoS ONE,9(3):e91357
Zheng CJ,Hu CL,Ma XQ,et al(2012).Cytotoxic phenylpropanoid glycosides from Fagopyrum tataricum(L.)Gaertn.Food Chem,132(1):433-438
Zhu F(2016).Chemical composition and health effects of tartary buckwheat.Food Chem,203:231-245