苋菜中甜菜红素代谢相关基因AmMYB2的表达与功能分析
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摘要
为了研究AmMYB2基因在苋菜(Amaranthus tricolor)甜菜红素合成中的作用,对红色苋菜品种‘大红’AmMYB2(登录号为KY814751.1)进行生物信息、基因表达及功能分析。结果表明:AmMYB2的开放阅读框(ORF)全长为678 bp,编码蛋白分子质量为25.96 kD,该蛋白具有R2R3型MYB结构域,属于R2R3型MYB转录因子。核酸序列分析结果显示,AmMYB2与甜菜红素合成相关的甜菜BvMYB1和苋菜AmMYB1的相似度分别达到62.11%和49.93%。亚细胞观察显示,AmMYB2蛋白定位于细胞核。实时荧光定量(qPCR)分析发现,AmMYB2在红色苋菜品种的表达量高于绿色苋菜品种;在‘大红’苋菜中,不同组织以及叶片不同部位中AmMYB2的表达量与甜菜红素含量成正比。构建AmMYB2过表达载体,将其转入烟草和拟南芥中,发现该基因能够在这两种植物中高水平表达,但不会引起颜色变化,揭示AmMYB2可能是苋菜中甜菜红素合成的正调控因子,可能不参与花青素的合成。
In order to explore the role of AmMYB2 in the betalain synthesis in Amaranthus tricolor,the bioinformatics,gene expression and functional analysis of AmMYB2(Genbank accession No. KY814751.1)in the red amaranth cultivar‘Dahong'were conducted. The results showed that the open reading frame(ORF)of the AmMYB2 is 678 bp in length. The coding protein molecular mass fraction is 25.96 kD,which belongs to R2 R3 MYB transcription factor with R2 R3 MYB domain. Subcellular localization assay revealed that the AmMYB2 protein was localized in the nucleus. Nucleotide sequence analysis revealed that the AmMYB2 is relatively close to betalain synthesis-related BvMYB1 and AmMYB1,with an identity of 62.11% and 49.93%,respectively. Quantitative real-time PCR(qRT-PCR)resultsshowed that the expression level of Am MYB2 was higher in red amaranth cultivar than that in green,and was different in different tissues and the different parts of leaves from the‘Dahong'amaranth,consistent with the proportional to the content of betalains. After the over expression vector of AmMYB2 was constructed and transferred to tobacco and Arabidopsis thaliana,the expression level of AmMYB2 was higher in both plants,without color changes. Therefore,the AmMYB2 may be a positive regulatory factor for the synthesis of betalains in amaranth,and maybe not involved in anthocyanin synthesis.
In order to explore the role of AmMYB2 in the betalain synthesis in Amaranthus tricolor,the bioinformatics,gene expression and functional analysis of AmMYB2(Genbank accession No. KY814751.1)in the red amaranth cultivar‘Dahong'were conducted. The results showed that the open reading frame(ORF)of the AmMYB2 is 678 bp in length. The coding protein molecular mass fraction is 25.96 kD,which belongs to R2 R3 MYB transcription factor with R2 R3 MYB domain. Subcellular localization assay revealed that the AmMYB2 protein was localized in the nucleus. Nucleotide sequence analysis revealed that the AmMYB2 is relatively close to betalain synthesis-related BvMYB1 and AmMYB1,with an identity of 62.11% and 49.93%,respectively. Quantitative real-time PCR(qRT-PCR)resultsshowed that the expression level of Am MYB2 was higher in red amaranth cultivar than that in green,and was different in different tissues and the different parts of leaves from the‘Dahong'amaranth,consistent with the proportional to the content of betalains. After the over expression vector of AmMYB2 was constructed and transferred to tobacco and Arabidopsis thaliana,the expression level of AmMYB2 was higher in both plants,without color changes. Therefore,the AmMYB2 may be a positive regulatory factor for the synthesis of betalains in amaranth,and maybe not involved in anthocyanin synthesis.
引文
Aharoni A,De Vos C H,Wein M,Sun Z,Greco R,Kroon A,Mol J N,O'Connell A P.2001.The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco.Plant J,28(3):319-332.
Albert N,Thrimawithana A,Mcghie T.2018.Genetic analysis of the liverwort Marchantia polymorpha reveals that R2R3MYB activation of flavonoid production in response to abiotic stress is an ancient character in land plants.New Phytologist,23(1):554-566.
Ban Y,Honda C,Hatsuyama Y.2007.Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin.Plant&Cell Physiology,48(7):958-970.
Bent A.2006.Arabidopsis thaliana floral dip transformation method.Methods in Molecular Biology,343:87-104.
Brockington S F,Walker R H,Glover B J.2011.Complex pigment evolution in the Caryophyllales.New Phytologist,190(4):854-864.
Chauhan S P,Rathod I S,Suhagia B N.2013.Analysis of betalains from fruits of Opuntia species.Phytochemistry Reviews,12(1):35-45.
Chen N,Yu Z H,Xiao X G.2017.Cytosolic and nuclear co-localization of betalain biosynthetic enzymes in tobacco suggests that betalains are synthesized in the cytoplasm and/or nucleus of betalainic plant cells.Frontiers in Plant Science,8:831.
Deloache W C,Russ Z N,Narcross L.2015.An enzyme-coupled biosensor enables(S)-reticuline production in yeast from glucose.Nature Chemical Biology,11(7):465-471.
Deluc L,Bogs J,Walker A R.2008.The transcription factor VvMYB5b contributes to the regulation of anthocyanin and proanthocyanidin biosynthesis in developing grape berries.Plant Physiology,147(4):2041-2053.
Du X L,Cao X,Yin C R.2016.Comprehensive analysis of R2R3-MYB genes during adventitious root formation in cuttings of Morus alba.Journal of Plant Growth Regulation,36(2):1-10.
Escribano J,Pedre?o M A,García-Carmona F,Mu?oz R.2015.Characterization of the antiradical activity of betalains from Beta vulgaris L.roots.Phytochemical Analysis,9(3):124-127.
Feng S Q,Wang Y L,Yang S.2010.Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor PyMYB10.Planta,232(1):245-255.
Gális I,Simek P,Narisawa T,2010.A novel R2R3 MYB transcription factor NtMYBJS1 is a methyl jasmonate-dependent regulator of phenylpropanoid-conjugate biosynthesis in tobacco.Plant Journal,46(4):573-592.
Gandíaherrero F,Jiménezatiénzar M,Cabanes J.2009.Fluorescence detection of tyrosinase activity on dopamine-betaxanthin purified from Portulaca oleracea(common purslane)flowers.Journal of Agricultural&Food Chemistry,57(6):2523-2528.
Guo Y,Gan S.2011.AtMYB2 regulates whole plant senescence by inhibiting cytokinin-mediated branching at late stages of development in Arabidopsis.Plant Physiology,156(3):1612-1619.
Harris N N.2012.Betalain production is possible in anthocyanin-producing plant species given the presence of DOPA-dioxygenase and L-DOPA.Bmc Plant Biology,12(1):34.
Hatlestad G J,Akhavan N A,Sunnadeniya R M,Elam L,Cargile S,Hembd A,Gonzalez A,McGrath J M,Lloyd A M.2015.The beet Y locus encodes an anthocyanin MYB-like protein that activates the betalain red pigment pathway.Nature Genetics,47(1):92-106.
Hatlestad G J,Sunnadeniya R M,Akhavan N A.2012.The beet R locus encodes a new cytochrome P450 required for red betalain production.Nature Genetics,44(7):816-820.
Huang W,Lv H,Wang Y.2017.Functional characterization of a novel R2R3-MYB transcription factor modulating the flavonoid biosynthetic pathway from Epimedium sagittatum.Frontiers in Plant Science,36(2):813-819.
Jaakola L,Poole M,Jones M O.2010.A SQUAMOSA MADS box gene involved in the regulation of anthocyanin accumulation in bilberry fruits.Plant Physiology,153(4):1619-1629.
Jin H,Martin C.1999.Multifunctionality and diversity within the plant MYB-gene family.Plant Molecular Biology,41(5):577-585.
Liao C,Zheng Y,Guo Y.2017.MYB30 transcription factor regulates oxidative and heat stress responses through ANNEXIN-mediated cytosolic calcium signaling in Arabidopsis.New Phytologist,216(1):163-171.
Meng X,Yin B,Feng H L.2014.Overexpression of R2R3-MYB gene leads to accumulation of anthocyanin and enhanced resistance to chilling and oxidative stress.Biologia Plantarum,58(1):121-130.
Nguyen N,Lee H.2016.MYB-related transcription factors function as regulators of the circadian clock and anthocyanin biosynthesis in Arabidopsis.Plant Signaling&Behavior,11(3):e1139278.
Pérezdíaz J,Pérezdíaz J,Madridespinoza J.2016.New member of the R2R3-MYB transcription factors family in grapevine suppresses the anthocyanin accumulation in the flowers of transgenic tobacco.Plant Molecular Biology,90(1-2):63-76.
Riechmann J L,Heard J,Martin G.2000.Arabidopsis transcription factors:genome-wide comparative analysis among eukaryotes.Science,2000,290(5499):2105-2110.
Sakuta M.2014.Diversity in plant red pigments:anthocyanins and betacyanins.Plant Biotechnology Reports,8(1):37-48.
Seo P,Park C.2010.MYB96-mediated abscisic acid signals induce pathogen resistance response by promoting salicylic acid biosynthesis in Arabidopsis.New Phytologist,186(2):471-483.
Sparkes I A,Runions J,Kearns A.2006.Rapid transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants.Nature Protocols,1(4):2019-2025.
Stintzing F C,Schieber A,Carle R.2002.Betacyanins in fruits from red-purple pitaya,Hylocereus polyrhizus(Weber)Britton&rose.Food Chemistry,77(1):101-106.
Stracke R,Werber M,Weisshaar B.2001.The R2R3-MYB gene family in Arabidopsis thaliana.Current Opinion in Plant Biology,4(5):447-456.
Tian H,Qi T,Li Y.2016.Regulation of the WD-repeat/bHLH/MYB complex by gibberellin and jasmonate.Plant Signal Behavior,11(8):1118-1133.
Tominaga-Wada R,Wada T.2014.Regulation of root hair cell differentiation by R3 MYB transcription factors in tomato and Arabidopsis.Frontiers in Plant Science,5(5):91.
Wang C Q,Chen M,Wang B S.2007.Betacyanin accumulation in the leaves of C3 halophyte Suaeda salsa L.is induced by watering roots with H2O2.Plant Science,172(1):1-7.
Wei Q,Luo Q,Wang R.2017.A Wheat R2R3-type MYB transcription factor TaODORANT1 positively regulates drought and salt stress responses in transgenic tobacco plants.Frontiers in Plant Science,8:1374.
Wen C,Chu F.2017.A R2R3-MYB Gene LfMYB113 is responsible for autumn leaf coloration in formosan sweet gum(Liquidambar formosana Hance).Plant&Cell Physiology,58(3):508-521.
Xie Liyang,Liu Shengcai,Bai Yu.2016.Cloning and expression analysis of betalain-related transcription factor gene AmMYB1 in Amaranthus tricolor L.Acta Botanica Boreali-Occidentalia Sinica,36(6):1080-1090.(in Chinese)谢礼洋,刘生财,白玉.2016.苋菜甜菜红素合成相关基因AmMYB1的克隆及表达分析.西北植物学报,36(6):1080-1090.
Xu K,Huang X,Wu M.2014.A rapid highly efficient and economical method of agrobacterium-mediated in planta transient transformation in living onion epidermis.PLoS ONE,9(1):e83556.
Xu Qing,He Jie,Dong Jianhui,Hou Xiaojin,,Zhang Xian.2018.Genomic survey and expression profiling of the MYB gene family in watermelon.Horticultural Plant Journal,4(1):1-15.
Yang Lifei,Zhu Yuelin,Liu Dandan,Ying Zhengzheng.2015.The invention relates to a method for extracting anthocyanins:China,105199425.2015-12-30.(in Chinese)杨立飞,朱月林,刘丹丹,应铮铮.2015.一种花青素的提取方法:中国,105199425.2015-12-30.
Yasuko K,Katsuyuki M,Akira T.2015.Light and abscisic acid independently regulated FaMYB10,in Fragaria×ananassa fruit.Planta,241(4):953-965.
Zhu N,Cheng S,Liu X.2015.The R2R3-type MYB gene Os MYB91 has a function in coordinating plant growth and salt stress tolerance in rice.Plant Science An International Journal of Experimental Plant Biology,236:146-156.
Zimmermann I M,Heim M A,Weisshaar B.2010.Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like bHLH proteins.Plant Journal,40(1):22-34.
Albert N,Thrimawithana A,Mcghie T.2018.Genetic analysis of the liverwort Marchantia polymorpha reveals that R2R3MYB activation of flavonoid production in response to abiotic stress is an ancient character in land plants.New Phytologist,23(1):554-566.
Ban Y,Honda C,Hatsuyama Y.2007.Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin.Plant&Cell Physiology,48(7):958-970.
Bent A.2006.Arabidopsis thaliana floral dip transformation method.Methods in Molecular Biology,343:87-104.
Brockington S F,Walker R H,Glover B J.2011.Complex pigment evolution in the Caryophyllales.New Phytologist,190(4):854-864.
Chauhan S P,Rathod I S,Suhagia B N.2013.Analysis of betalains from fruits of Opuntia species.Phytochemistry Reviews,12(1):35-45.
Chen N,Yu Z H,Xiao X G.2017.Cytosolic and nuclear co-localization of betalain biosynthetic enzymes in tobacco suggests that betalains are synthesized in the cytoplasm and/or nucleus of betalainic plant cells.Frontiers in Plant Science,8:831.
Deloache W C,Russ Z N,Narcross L.2015.An enzyme-coupled biosensor enables(S)-reticuline production in yeast from glucose.Nature Chemical Biology,11(7):465-471.
Deluc L,Bogs J,Walker A R.2008.The transcription factor VvMYB5b contributes to the regulation of anthocyanin and proanthocyanidin biosynthesis in developing grape berries.Plant Physiology,147(4):2041-2053.
Du X L,Cao X,Yin C R.2016.Comprehensive analysis of R2R3-MYB genes during adventitious root formation in cuttings of Morus alba.Journal of Plant Growth Regulation,36(2):1-10.
Escribano J,Pedre?o M A,García-Carmona F,Mu?oz R.2015.Characterization of the antiradical activity of betalains from Beta vulgaris L.roots.Phytochemical Analysis,9(3):124-127.
Feng S Q,Wang Y L,Yang S.2010.Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor PyMYB10.Planta,232(1):245-255.
Gális I,Simek P,Narisawa T,2010.A novel R2R3 MYB transcription factor NtMYBJS1 is a methyl jasmonate-dependent regulator of phenylpropanoid-conjugate biosynthesis in tobacco.Plant Journal,46(4):573-592.
Gandíaherrero F,Jiménezatiénzar M,Cabanes J.2009.Fluorescence detection of tyrosinase activity on dopamine-betaxanthin purified from Portulaca oleracea(common purslane)flowers.Journal of Agricultural&Food Chemistry,57(6):2523-2528.
Guo Y,Gan S.2011.AtMYB2 regulates whole plant senescence by inhibiting cytokinin-mediated branching at late stages of development in Arabidopsis.Plant Physiology,156(3):1612-1619.
Harris N N.2012.Betalain production is possible in anthocyanin-producing plant species given the presence of DOPA-dioxygenase and L-DOPA.Bmc Plant Biology,12(1):34.
Hatlestad G J,Akhavan N A,Sunnadeniya R M,Elam L,Cargile S,Hembd A,Gonzalez A,McGrath J M,Lloyd A M.2015.The beet Y locus encodes an anthocyanin MYB-like protein that activates the betalain red pigment pathway.Nature Genetics,47(1):92-106.
Hatlestad G J,Sunnadeniya R M,Akhavan N A.2012.The beet R locus encodes a new cytochrome P450 required for red betalain production.Nature Genetics,44(7):816-820.
Huang W,Lv H,Wang Y.2017.Functional characterization of a novel R2R3-MYB transcription factor modulating the flavonoid biosynthetic pathway from Epimedium sagittatum.Frontiers in Plant Science,36(2):813-819.
Jaakola L,Poole M,Jones M O.2010.A SQUAMOSA MADS box gene involved in the regulation of anthocyanin accumulation in bilberry fruits.Plant Physiology,153(4):1619-1629.
Jin H,Martin C.1999.Multifunctionality and diversity within the plant MYB-gene family.Plant Molecular Biology,41(5):577-585.
Liao C,Zheng Y,Guo Y.2017.MYB30 transcription factor regulates oxidative and heat stress responses through ANNEXIN-mediated cytosolic calcium signaling in Arabidopsis.New Phytologist,216(1):163-171.
Meng X,Yin B,Feng H L.2014.Overexpression of R2R3-MYB gene leads to accumulation of anthocyanin and enhanced resistance to chilling and oxidative stress.Biologia Plantarum,58(1):121-130.
Nguyen N,Lee H.2016.MYB-related transcription factors function as regulators of the circadian clock and anthocyanin biosynthesis in Arabidopsis.Plant Signaling&Behavior,11(3):e1139278.
Pérezdíaz J,Pérezdíaz J,Madridespinoza J.2016.New member of the R2R3-MYB transcription factors family in grapevine suppresses the anthocyanin accumulation in the flowers of transgenic tobacco.Plant Molecular Biology,90(1-2):63-76.
Riechmann J L,Heard J,Martin G.2000.Arabidopsis transcription factors:genome-wide comparative analysis among eukaryotes.Science,2000,290(5499):2105-2110.
Sakuta M.2014.Diversity in plant red pigments:anthocyanins and betacyanins.Plant Biotechnology Reports,8(1):37-48.
Seo P,Park C.2010.MYB96-mediated abscisic acid signals induce pathogen resistance response by promoting salicylic acid biosynthesis in Arabidopsis.New Phytologist,186(2):471-483.
Sparkes I A,Runions J,Kearns A.2006.Rapid transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants.Nature Protocols,1(4):2019-2025.
Stintzing F C,Schieber A,Carle R.2002.Betacyanins in fruits from red-purple pitaya,Hylocereus polyrhizus(Weber)Britton&rose.Food Chemistry,77(1):101-106.
Stracke R,Werber M,Weisshaar B.2001.The R2R3-MYB gene family in Arabidopsis thaliana.Current Opinion in Plant Biology,4(5):447-456.
Tian H,Qi T,Li Y.2016.Regulation of the WD-repeat/bHLH/MYB complex by gibberellin and jasmonate.Plant Signal Behavior,11(8):1118-1133.
Tominaga-Wada R,Wada T.2014.Regulation of root hair cell differentiation by R3 MYB transcription factors in tomato and Arabidopsis.Frontiers in Plant Science,5(5):91.
Wang C Q,Chen M,Wang B S.2007.Betacyanin accumulation in the leaves of C3 halophyte Suaeda salsa L.is induced by watering roots with H2O2.Plant Science,172(1):1-7.
Wei Q,Luo Q,Wang R.2017.A Wheat R2R3-type MYB transcription factor TaODORANT1 positively regulates drought and salt stress responses in transgenic tobacco plants.Frontiers in Plant Science,8:1374.
Wen C,Chu F.2017.A R2R3-MYB Gene LfMYB113 is responsible for autumn leaf coloration in formosan sweet gum(Liquidambar formosana Hance).Plant&Cell Physiology,58(3):508-521.
Xie Liyang,Liu Shengcai,Bai Yu.2016.Cloning and expression analysis of betalain-related transcription factor gene AmMYB1 in Amaranthus tricolor L.Acta Botanica Boreali-Occidentalia Sinica,36(6):1080-1090.(in Chinese)谢礼洋,刘生财,白玉.2016.苋菜甜菜红素合成相关基因AmMYB1的克隆及表达分析.西北植物学报,36(6):1080-1090.
Xu K,Huang X,Wu M.2014.A rapid highly efficient and economical method of agrobacterium-mediated in planta transient transformation in living onion epidermis.PLoS ONE,9(1):e83556.
Xu Qing,He Jie,Dong Jianhui,Hou Xiaojin,,Zhang Xian.2018.Genomic survey and expression profiling of the MYB gene family in watermelon.Horticultural Plant Journal,4(1):1-15.
Yang Lifei,Zhu Yuelin,Liu Dandan,Ying Zhengzheng.2015.The invention relates to a method for extracting anthocyanins:China,105199425.2015-12-30.(in Chinese)杨立飞,朱月林,刘丹丹,应铮铮.2015.一种花青素的提取方法:中国,105199425.2015-12-30.
Yasuko K,Katsuyuki M,Akira T.2015.Light and abscisic acid independently regulated FaMYB10,in Fragaria×ananassa fruit.Planta,241(4):953-965.
Zhu N,Cheng S,Liu X.2015.The R2R3-type MYB gene Os MYB91 has a function in coordinating plant growth and salt stress tolerance in rice.Plant Science An International Journal of Experimental Plant Biology,236:146-156.
Zimmermann I M,Heim M A,Weisshaar B.2010.Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like bHLH proteins.Plant Journal,40(1):22-34.