过表达苹果多肽激素基因MdCEP1促进花青苷积累
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摘要
以‘王林’苹果(Malus×domestica‘Orin’)愈伤组织为试材,初步探讨苹果多肽激素Md CEP1(C-TERMINALLY ENCODED PEPTIDE1)在调控花青苷合成方面的作用。分析显示Md CEP1位于苹果第15号染色体,只有1个外显子。蛋白序列比对显示不同物种中CEP结构域非常保守。启动子分析表明,Md CEP1启动子序列包含多个顺式作用元件,包括与分生组织有关的CAT-box元件、赤霉素响应元件(P-box)、光响应元件(MNF1)和与类黄酮合成相关的MYB类蛋白结合位点(MBSI)。通过农杆菌介导的遗传转化获得Md CEP1转基因苹果愈伤组织,进一步分析发现过表达Md CEP1能够明显促进愈伤组织花青苷积累,并且促进花青苷合成相关基因的表达。Md CEP1在拟南芥中异位表达,同样能够促进拟南芥中花青苷的积累,并且促进拟南芥花青苷合成相关基因的表达。研究结果表明,Md CEP1能够正调控苹果花青苷的合成。
The effects of apple peptide hormone MdCEP1(C-TERMINALLY ENCODED PEPTIDE1)on the regulation of anthocyanin synthesis were investigated by using Malus × domestica‘Orin'as the test material,to provide reference for further study on the mechanism of anthocyanin synthesis and characterization of MdCEP1 function. Chromosomal localization analysis revealed that MdCEP1 was located on the chromosome 15 of the apple genome and had only one exon. Protein sequence alignment showed that CEP domains were conserved in different species. Promoter analysis showed that the MdCEP1 promoter sequence contained a number of cis-acting elements,including CAT-box elements associated with meristem,gibberellin response elements(P-boxes),light-responsive elements(MNF1)and the MYB binding site(MBSI)involved in flavonoid biosynthesis. MdCEP1 transgenic apple calli were obtained by Agrobacterium tumefaciens-mediated genetic transformation. Further analysis found that overexpression of MdCEP1 could significantly promote the anthocyanin accumulation,and promote the expression of apple anthocyanin biosynthetic genes. Ectopic expression of MdCEP1 in Arabidopsis thaliana,could also promote the accumulation of anthocyanin,and promote the expression of anthocyanin biosynthetic genes in Arabidopsis thaliana. The results showed that MdCEP1 could positively control the synthesis of anthocyanin.
The effects of apple peptide hormone MdCEP1(C-TERMINALLY ENCODED PEPTIDE1)on the regulation of anthocyanin synthesis were investigated by using Malus × domestica‘Orin'as the test material,to provide reference for further study on the mechanism of anthocyanin synthesis and characterization of MdCEP1 function. Chromosomal localization analysis revealed that MdCEP1 was located on the chromosome 15 of the apple genome and had only one exon. Protein sequence alignment showed that CEP domains were conserved in different species. Promoter analysis showed that the MdCEP1 promoter sequence contained a number of cis-acting elements,including CAT-box elements associated with meristem,gibberellin response elements(P-boxes),light-responsive elements(MNF1)and the MYB binding site(MBSI)involved in flavonoid biosynthesis. MdCEP1 transgenic apple calli were obtained by Agrobacterium tumefaciens-mediated genetic transformation. Further analysis found that overexpression of MdCEP1 could significantly promote the anthocyanin accumulation,and promote the expression of apple anthocyanin biosynthetic genes. Ectopic expression of MdCEP1 in Arabidopsis thaliana,could also promote the accumulation of anthocyanin,and promote the expression of anthocyanin biosynthetic genes in Arabidopsis thaliana. The results showed that MdCEP1 could positively control the synthesis of anthocyanin.
引文
An Jian-ping,Song Lai-qing,Zhao Ling-ling,You Chun-xiang,Wang Xiao-fei,Hao Yu-jin.2017.Effects of overexpression of apple cytokinin response factor gene Md CRF6 on anthocyanins accumulation and salt stress tolerance.Scientia Agricultura Sinica,50(16):3196-3204.(in Chinese)安建平,宋来庆,赵玲玲,由春香,王小非,郝玉金.2017.超表达苹果细胞分裂素响应基因Md CRF6影响花青苷积累和盐胁迫抗性.中国农业科学,50(16):3196-3204.
An X H,Tian Y,Chen K Q,Liu X J,Liu D D,Xie X B,Cheng C G,Cong P H,Hao Y J.2015.Md MYB9 and Md MYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples.Plant&Cell Physiology,56:650.
An Xiu-hong.2013.Studies on the mechanism by which Md TTGl,Md MYB9 and Md MYB11 regulate anthocyanin biosythesis in apple[Ph.D.Dissertation].Tai’an:Shandong Agricultural University.(in Chinese)安秀红.2013.苹果Md TTG1、Md MYB9与Md MYB11基因调控花青苷合成的作用机制研究[博士论文].泰安:山东农业大学.
Blyden E R,Doerner P W,Lamb C L,Dixon R A.1991.Sequence analysis of a chalcone isomerase c DNA of Phaseolus vulgaris L.Plant Molecular Biology,16:167-169.
Chen K Q,Zhao X Y,An X H,Tian Y,Liu D D,You C X,Hao Y J.2017.Md HIR proteins repress anthocyanin accumulation by interacting with the Md JAZ2 protein to inhibit its degradation in apples.Scientific Reports,7:44484.
Farzad M,Griesbach R,Hammond J,Weiss M R,Elmendorf H G.2003.Differential expression of three key anthocyanin biosynthetic genes in a color-changing flower,Viola cornuta cv.Yesterday,Today and Tomorrow.Plant Science,165(6):1333-1342.
Feng Shou-qian,Chen Xue-sen,Zhamg Chun-yu,Liu Xiao-jing,Liu Zun-chun,Wang Hai-bo,Wang Yan-ling,Zhou Chao-hua.2008.A study of the relationship between anthocyanin biosynthesis and related enzymes activity in Pyrus pyrifolia‘Mantianhong’and its bud sports‘Aoguan’.Scientia Agricultura Sinica,41(10):3184-3190.(in Chinese)冯守千,陈学森,张春雨,刘晓静,刘遵春,王海波,王延玲,周朝华.2008.砂梨品种‘满天红’及其芽变品系‘奥冠’花青苷合成与相关酶活性研究.中国农业科学,41(10):3184-3190.
Gould K S,Mckelvie J,Markham K R.2002.Do anthocyanins function as antioxidants in leaves?Imaging of H2O2 in red and green leaves after mechanical injury.Plant Cell&Environment,25:1261-1269.
Hao Hua-ling.2013.The key role of PIF4 in 6-BA induced changes in anthocyanin and chlorophyⅡcontent in Arabidopsis seedlings[M.D.Dissertation].Lanzhou:Lanzhou University.(in Chinese)郝华玲.2013.PIF4在6-BA诱导的拟南芥幼苗花青素及叶绿素含量变化中的重要作用[硕士论文].兰州:兰州大学.
Hu D G,Sun C H,Zhang Q Y,An J P,You C X,Hao Y J.2016.Glucose sensor Md HXK1 phosphorylates and stabilizes Mdb HLH3 to promote anthocyanin biosynthesis in apple.PLo S Genetics,12:e1006273.
Jun J,Fiume E,Roeder A H,Adrienne H,Roeder L M,Vijay K S,Karen S O,Catherine B C,Elliot M M,Lewis J F,Jennifer C F.2010.Comprehensive analysis of CLE polypeptide signaling gene expression and overexpression activity in Arabidopsis.Plant Physiology,154(4):1721-1736.
Katsir L,Davies K A,Bergmann D C,Laux T.2011.Peptide signaling in plant development.Current Biology,21:R356.
Kobayashi S,Ishimaru M,Ding C K,Yakushiji H,Goto N.2001.Comparison of UDP-glucose:flavonoid 3-O-glucosyltransferase(UFGT)gene sequences between white grapes(Vitis vinifera)and their sports with red skin.Plant Science an International Journal of Experimental Plant Biology,160:543.
Li Rui,Li Haohao,An Jianping,You Chunxiang,Wang Xiaofei,Hao Yujin.2017.Apple peptide hormone and its coding gene Md CEP1 regulate root development in Arabidopsis thaliana.Acta Horticulturae Sinica,44(7):1225-1234.(in Chinese)李睿,李浩浩,安建平,由春香,王小非,郝玉金.2017.苹果多肽激素及其编码基因Md CEP1调控拟南芥根系发育.园艺学报,44(7):1225-1234.
Li S.2014.Transcriptional control of flavonoid biosynthesis:fine-tuning of the MYB-b HLH-WD40(MBW)complex.Plant Signaling&Behavior,9:e27522.
Li Y Y,Mao K,Zhao C,Zhao X Y,Zhang H L,Shu H R,Hao Y J.2012.Md COP1 ubiquitin E3 ligases interact with Md MYB1 to regulate light-induced anthocyanin biosynthesis and red fruit coloration in apple.Plant Physiology,160:1011.
Liu Jin,Wei Jing-li,Liu Mei-yan,Song Yang,Feng Shou-qian,Wang Chuan-zeng,Chen Xue-sen.2012.The relationships between the enzyme activity of anthocyanin biosynthesis,ethylene release and anthocyanin accumulation in fruits of precocious apple cultivars.Acta Horticulturae Sinica,39(7):1235-1242.(in Chinese)刘金,魏景立,刘美艳,宋杨,冯守千,王传增,陈学森.2012.早熟苹果花青苷积累与其相关酶活性及乙烯生成之间的关系.园艺学报,39(7):1235-1242.
Peer W A,Brown D E,Tague B W,Muday G K,Taiz L,Murphy A S.2001.Flavonoid accumulation patterns of transparent testa mutants of Arabidopsis.Plant Physiology,126:536.
Rudell D R,Mattheis J P.2008.Synergism exists between ethylene and methyl jasmonate in artificial light-induced pigment enhancement of‘Fuji’apple fruit peel.Postharvest Biology&Technology,47:136-140.
Schenke D,B?ttcher C,Scheel D.2011.Crosstalk between abiotic ultraviolet-B stress and biotic(flg22)stress signalling in Arabidopsis prevents flavonol accumulation in favor of pathogen defence compound production.Plant Cell&Environment,34(11):1849.
Stintzing F C,Carle R.2004.Functional properties of anthocyanins and betalains in plants,food,and in human nutrition.Trends in Food Science&Technology,15:19-38.
Tabata R,Sumida K,Yoshii T,Ohyama K,Shinohara H,Matsubayashi Y.2014.Perception of root-derived peptides by shoot LRR-RKs mediates systemic N-demand signaling.Science,346:343-346.
Takayama S,Sakagami Y.2002.Peptide signalling in plants.Current Opinion in Plant Biology,5:382-387.
Wang J A,Mazza G.2002.Inhibitory effects of anthocyanins and other phenolic compounds on ntric oxide production in LPS/IFN-γ-activated RAW264.7 macrophages.Journal of Agricultural&Food Chemistry,50:850.
Watanabe K,Okumura M,Tominaga F.2013.Red colour distribution,and chlorophyll,carotenoid and anthocyanin pigments of blueberry fruits.Forest Ecology&Management,297:108-119.
Xie X B,Li S,Zhang R F,Zhao J,Chen Y C,Zhao Q,Yao Y X,You C X,Zhang X S,Hao Y J.2012.The b HLH transcription factor Mdb HLH3promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples.Plant Cell&Environment,35:1884-1897.
Wang Yiheng,Xu Haifeng,Wang Nan,Jiang Shenghui,Liu Jingxuan,Wang Deyun,Zuo Weifang,Chen Xuesen.2017.Molecular cloning and expression analysis of an auxin signaling related gene Md ARF3 in red flesh apple.Acta Horticulturae Sinica,44(3):633-643.(in Chinese)王意程,许海峰,王楠,姜生辉,刘静轩,王得云,左卫芳,陈学森.2017.红肉苹果愈伤组织生长素信号相关基因Md ARF3的克隆与表达分析.园艺学报,44(3):633-643.
Zhang Z O,Pang X Q,Xuewu D,Ji Z L,Jiang Y M.2005.Role of peroxidase in anthocyanin degradation in litchi fruit pericarp.Food Chemistry,90:47-52.
An X H,Tian Y,Chen K Q,Liu X J,Liu D D,Xie X B,Cheng C G,Cong P H,Hao Y J.2015.Md MYB9 and Md MYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples.Plant&Cell Physiology,56:650.
An Xiu-hong.2013.Studies on the mechanism by which Md TTGl,Md MYB9 and Md MYB11 regulate anthocyanin biosythesis in apple[Ph.D.Dissertation].Tai’an:Shandong Agricultural University.(in Chinese)安秀红.2013.苹果Md TTG1、Md MYB9与Md MYB11基因调控花青苷合成的作用机制研究[博士论文].泰安:山东农业大学.
Blyden E R,Doerner P W,Lamb C L,Dixon R A.1991.Sequence analysis of a chalcone isomerase c DNA of Phaseolus vulgaris L.Plant Molecular Biology,16:167-169.
Chen K Q,Zhao X Y,An X H,Tian Y,Liu D D,You C X,Hao Y J.2017.Md HIR proteins repress anthocyanin accumulation by interacting with the Md JAZ2 protein to inhibit its degradation in apples.Scientific Reports,7:44484.
Farzad M,Griesbach R,Hammond J,Weiss M R,Elmendorf H G.2003.Differential expression of three key anthocyanin biosynthetic genes in a color-changing flower,Viola cornuta cv.Yesterday,Today and Tomorrow.Plant Science,165(6):1333-1342.
Feng Shou-qian,Chen Xue-sen,Zhamg Chun-yu,Liu Xiao-jing,Liu Zun-chun,Wang Hai-bo,Wang Yan-ling,Zhou Chao-hua.2008.A study of the relationship between anthocyanin biosynthesis and related enzymes activity in Pyrus pyrifolia‘Mantianhong’and its bud sports‘Aoguan’.Scientia Agricultura Sinica,41(10):3184-3190.(in Chinese)冯守千,陈学森,张春雨,刘晓静,刘遵春,王海波,王延玲,周朝华.2008.砂梨品种‘满天红’及其芽变品系‘奥冠’花青苷合成与相关酶活性研究.中国农业科学,41(10):3184-3190.
Gould K S,Mckelvie J,Markham K R.2002.Do anthocyanins function as antioxidants in leaves?Imaging of H2O2 in red and green leaves after mechanical injury.Plant Cell&Environment,25:1261-1269.
Hao Hua-ling.2013.The key role of PIF4 in 6-BA induced changes in anthocyanin and chlorophyⅡcontent in Arabidopsis seedlings[M.D.Dissertation].Lanzhou:Lanzhou University.(in Chinese)郝华玲.2013.PIF4在6-BA诱导的拟南芥幼苗花青素及叶绿素含量变化中的重要作用[硕士论文].兰州:兰州大学.
Hu D G,Sun C H,Zhang Q Y,An J P,You C X,Hao Y J.2016.Glucose sensor Md HXK1 phosphorylates and stabilizes Mdb HLH3 to promote anthocyanin biosynthesis in apple.PLo S Genetics,12:e1006273.
Jun J,Fiume E,Roeder A H,Adrienne H,Roeder L M,Vijay K S,Karen S O,Catherine B C,Elliot M M,Lewis J F,Jennifer C F.2010.Comprehensive analysis of CLE polypeptide signaling gene expression and overexpression activity in Arabidopsis.Plant Physiology,154(4):1721-1736.
Katsir L,Davies K A,Bergmann D C,Laux T.2011.Peptide signaling in plant development.Current Biology,21:R356.
Kobayashi S,Ishimaru M,Ding C K,Yakushiji H,Goto N.2001.Comparison of UDP-glucose:flavonoid 3-O-glucosyltransferase(UFGT)gene sequences between white grapes(Vitis vinifera)and their sports with red skin.Plant Science an International Journal of Experimental Plant Biology,160:543.
Li Rui,Li Haohao,An Jianping,You Chunxiang,Wang Xiaofei,Hao Yujin.2017.Apple peptide hormone and its coding gene Md CEP1 regulate root development in Arabidopsis thaliana.Acta Horticulturae Sinica,44(7):1225-1234.(in Chinese)李睿,李浩浩,安建平,由春香,王小非,郝玉金.2017.苹果多肽激素及其编码基因Md CEP1调控拟南芥根系发育.园艺学报,44(7):1225-1234.
Li S.2014.Transcriptional control of flavonoid biosynthesis:fine-tuning of the MYB-b HLH-WD40(MBW)complex.Plant Signaling&Behavior,9:e27522.
Li Y Y,Mao K,Zhao C,Zhao X Y,Zhang H L,Shu H R,Hao Y J.2012.Md COP1 ubiquitin E3 ligases interact with Md MYB1 to regulate light-induced anthocyanin biosynthesis and red fruit coloration in apple.Plant Physiology,160:1011.
Liu Jin,Wei Jing-li,Liu Mei-yan,Song Yang,Feng Shou-qian,Wang Chuan-zeng,Chen Xue-sen.2012.The relationships between the enzyme activity of anthocyanin biosynthesis,ethylene release and anthocyanin accumulation in fruits of precocious apple cultivars.Acta Horticulturae Sinica,39(7):1235-1242.(in Chinese)刘金,魏景立,刘美艳,宋杨,冯守千,王传增,陈学森.2012.早熟苹果花青苷积累与其相关酶活性及乙烯生成之间的关系.园艺学报,39(7):1235-1242.
Peer W A,Brown D E,Tague B W,Muday G K,Taiz L,Murphy A S.2001.Flavonoid accumulation patterns of transparent testa mutants of Arabidopsis.Plant Physiology,126:536.
Rudell D R,Mattheis J P.2008.Synergism exists between ethylene and methyl jasmonate in artificial light-induced pigment enhancement of‘Fuji’apple fruit peel.Postharvest Biology&Technology,47:136-140.
Schenke D,B?ttcher C,Scheel D.2011.Crosstalk between abiotic ultraviolet-B stress and biotic(flg22)stress signalling in Arabidopsis prevents flavonol accumulation in favor of pathogen defence compound production.Plant Cell&Environment,34(11):1849.
Stintzing F C,Carle R.2004.Functional properties of anthocyanins and betalains in plants,food,and in human nutrition.Trends in Food Science&Technology,15:19-38.
Tabata R,Sumida K,Yoshii T,Ohyama K,Shinohara H,Matsubayashi Y.2014.Perception of root-derived peptides by shoot LRR-RKs mediates systemic N-demand signaling.Science,346:343-346.
Takayama S,Sakagami Y.2002.Peptide signalling in plants.Current Opinion in Plant Biology,5:382-387.
Wang J A,Mazza G.2002.Inhibitory effects of anthocyanins and other phenolic compounds on ntric oxide production in LPS/IFN-γ-activated RAW264.7 macrophages.Journal of Agricultural&Food Chemistry,50:850.
Watanabe K,Okumura M,Tominaga F.2013.Red colour distribution,and chlorophyll,carotenoid and anthocyanin pigments of blueberry fruits.Forest Ecology&Management,297:108-119.
Xie X B,Li S,Zhang R F,Zhao J,Chen Y C,Zhao Q,Yao Y X,You C X,Zhang X S,Hao Y J.2012.The b HLH transcription factor Mdb HLH3promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples.Plant Cell&Environment,35:1884-1897.
Wang Yiheng,Xu Haifeng,Wang Nan,Jiang Shenghui,Liu Jingxuan,Wang Deyun,Zuo Weifang,Chen Xuesen.2017.Molecular cloning and expression analysis of an auxin signaling related gene Md ARF3 in red flesh apple.Acta Horticulturae Sinica,44(3):633-643.(in Chinese)王意程,许海峰,王楠,姜生辉,刘静轩,王得云,左卫芳,陈学森.2017.红肉苹果愈伤组织生长素信号相关基因Md ARF3的克隆与表达分析.园艺学报,44(3):633-643.
Zhang Z O,Pang X Q,Xuewu D,Ji Z L,Jiang Y M.2005.Role of peroxidase in anthocyanin degradation in litchi fruit pericarp.Food Chemistry,90:47-52.