苹果花青苷调控基因MdMYB111的功能鉴定
|
摘要
克隆红肉苹果‘红心16号’果实的MdMYB111,原核诱导并获得其重组蛋白,对MdMYB111进行生物信息学分析,测定其在果肉色泽差异明显的品种‘红心16号’和‘红脆1号’果实中的表达水平,并通过转基因鉴定其功能。结果表明,MdMYB111的开放阅读框为720 bp,编码239个氨基酸,预测其蛋白大小为26.93 kD;进化树分析发现MdMYB111和MdMYB16在同一个进化支上,且其蛋白C端存在EAR抑制序列;‘红心16号’果实成熟期果肉全部呈现红色,并且颜色较深;‘红脆1号’果实成熟期果肉呈现浅红色,红色范围较小。‘红心16号’果实花青苷含量和MdANS、MdUFGT、MdMYB9、MdMYB10、MdMYB11的表达水平均高于‘红脆1号’,但MdMYB16和MdMYB111的表达水平低于‘红脆1号’;在新疆红肉苹果愈伤中过表达MdMYB111,能够抑制MdANS和MdUFGT的表达并降低花青苷的含量。综上所述,MdMYB111可能参与花青苷合成途径并抑制其合成。
The fruit of‘Hongxin 16'apple,a progeny of Malus sieversii f. neidzwetzkyana hybrids,was used as the test material. MdMYB111 was cloned and its recombinant protein was induced and expressed in prokaryotic analysis. The bioinformatic analysis of this gene was conducted,its expression level was determined in the fruits of the varieties of‘Hongxin 16'and‘Hongcui 1'and its function was identified by transgene. The results showed that the open reading frame of MdMYB111 was 720 bp,encoding 239 amino acids,and its predicted protein size was 26.93 kD. Phylogenetic tree analysis showed that MdMYB111 and MdMYB16 were in the same clade and there was an EAR inhibitory sequence at the C-terminus of the protein. The ripened fruit of‘Hongxin 16' is complete red and darker in color. The fruit of‘Hongcui 1'has a light red color and a small red range. On the‘Hongxin 16',both the content of anthocyanin in fruits mature stage and MdANS,MdUFGT,MdMYB9,MdMYB10,MdMYB11 expression levels were higher than that of‘Hongcui 1',but the expression levels of MdMYB16 and MdMYB111 were lower than‘Hongcui 1'. Overexpression of MdMYB111 in red apple callus can inhibit the expression of MdANS and MdUFGT and reduce the content of anthocyanin. In summary,MdMYB111 may be involved in the anthocyanin synthesis pathway and inhibit its synthesis.
The fruit of‘Hongxin 16'apple,a progeny of Malus sieversii f. neidzwetzkyana hybrids,was used as the test material. MdMYB111 was cloned and its recombinant protein was induced and expressed in prokaryotic analysis. The bioinformatic analysis of this gene was conducted,its expression level was determined in the fruits of the varieties of‘Hongxin 16'and‘Hongcui 1'and its function was identified by transgene. The results showed that the open reading frame of MdMYB111 was 720 bp,encoding 239 amino acids,and its predicted protein size was 26.93 kD. Phylogenetic tree analysis showed that MdMYB111 and MdMYB16 were in the same clade and there was an EAR inhibitory sequence at the C-terminus of the protein. The ripened fruit of‘Hongxin 16' is complete red and darker in color. The fruit of‘Hongcui 1'has a light red color and a small red range. On the‘Hongxin 16',both the content of anthocyanin in fruits mature stage and MdANS,MdUFGT,MdMYB9,MdMYB10,MdMYB11 expression levels were higher than that of‘Hongcui 1',but the expression levels of MdMYB16 and MdMYB111 were lower than‘Hongcui 1'. Overexpression of MdMYB111 in red apple callus can inhibit the expression of MdANS and MdUFGT and reduce the content of anthocyanin. In summary,MdMYB111 may be involved in the anthocyanin synthesis pathway and inhibit its 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.The Plant Journal,28(3):319-332.
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.2014.MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples.Plant and Cell Physiology,56(4):650-662.
An X H,Tian Y,Chen K Q,Wang X F,Hao Y J.2012.The apple WD40 protein MdTTG1 interacts with bHLH but not MYB proteins to regulate anthocyanin accumulation.Journal of Plant Physiology,169(7):710-717.
Baudry A,Heim M A,Dubreucq B,Caboche M,Weisshaar B,Lepiniec L.2004.TT2,TT8,and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana.The Plant Journal,39(3):366-380.
Borevitz J O,Xia Y,Blount J,Dixon R A,Lamb C.2000.Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis.The Plant Cell,12(12):2383-2393.
Duan N,Yang B,Sun H,Wang N,Ma Y,Li M.2017.Genome re-sequencing reveals the history of apple and supports a two-stage model for fruit enlargement.Nature Communications,8(1):249.
Espley R.V,Brendolise C,ChagnéD,Kutty-Amma S,Green S,Volz R,Putterill J,Schouten H J,Gardiner S E,Hellens R P,Allan A C.2009.Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples.The Plant Cell,21(1):168-183.
Espley R V,Hellens R P,Putterill J,Stevenson D E,Kutty-Amma S,Allan A C.2007.Red colouration in apple fruit is due to the activity of the MYB transcription factor,MdMYB10.The Plant Journal,49(3):414-427.
Gao J J,Shen X F,Zhang Z,Peng,R H,Xiong A S,Xu J,Zhu B,Zheng J L,Yao Q H.2011.The MYB transcription factor MdMYB6 suppresses anthocyanin biosynthesis in transgenic Arabidopsis.Plant Cell,Tissue and Organ Culture(PCTOC),106(2):235-242.
Grotewold E.2006.The genetics and biochemistry of floral pigments.Annu Rev Plant Biol,57:761-780.
Hiratsu K,Matsui K,Koyama T,Ohme-Takagi M.2003.Dominant repression of target genes by chimeric repressors that include the EAR motif,a repression domain,in Arabidopsis.The Plant Journal,34(5):733-739.
Ji X H,Wang Y T,Zhang R,Wu S J,An M M,Li M,Wang C Z,Chen X L,Zhang Y M,Chen X S.2015.Effect of auxin,cytokinin and nitrogen on anthocyanin biosynthesis in callus cultures of red-fleshed apple(Malus sieversii f.niedzwetzkyana).Plant Cell,Tissue and Organ Culture(PCTOC),120(1):325-337.
Jimenez-Garcia S N,Guevara-Gonzalez R G,Miranda-Lopez R,Feregrino-Perez A A,Torres-Pacheco I,Vazquez-Cruz M A.2013.Functional properties and quality characteristics of bioactive compounds in berries:biochemistry,biotechnology,and genomics.Food Research International,54(1):1195-1207.
Koes R,Verweij W,Quattrocchio F.2005.Flavonoids:a colorful model for the regulation and evolution of biochemical pathways.Trends in Plant Science,10(5):236-242.
Lin-Wang K,Micheletti D,Palmer J,Volz R,Lozano L,Espley R,Hellens R P,ChagnèD,Rowan D D,Troggio M,Iglesias I,Allan A C.2011.High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex.Plant,Cell&Environment,34(7):1176-1190.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method.Methods,25(4):402-408.
Liu Xiaofen,Li Fang,Yin Xueren,Xu Changjie,Chen Kunsong.2013.Recent advances in the transcriptional regulation of anthocyanin biosynthesis.Acta Horticulturae Sinica,40(11):2295-2306.(in Chinese)刘晓芬,李方,殷学仁,徐昌杰,陈昆松.2013.花青苷生物合成转录调控研究进展.园艺学报,40(11):2295-2306.
Matsui K,Umemura Y,Ohme‐Takagi M.2008.AtMYBL2,a protein with a single MYB domain,acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis.The Plant Journal,55(6):954-967.
Martin C,Paz-Ares J.1997.MYB transcription factors in plants.Trends in Genetics,13(2):67-73.
Quattrocchio F,Verweij W,Kroon A,Spelt C,Mol J,Koes R.2006.PH4 of petunia is an R2R3 MYB protein that activates vacuolar acidification through interactions with basic-helix-loop-helix transcription factors of the anthocyanin pathway.The Plant Cell,18(5):1274-1291.
Ramsay N A,Glover B J.2005.MYB-bHLH-WD40 protein complex and the evolution of cellular diversity.Trends in Plant Science,10(2):63-70.
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(1):19-38.
Sun Shasha.2016.The functional characterization of PyMYB10.1 regulating anthocyanin biosynthesis in pear[M.D.Dissertation].Tai’an:Shandong Agricultural University.(in Chinese)孙莎莎.2016.梨花青苷调控基因PyMYB10.1功能鉴定[硕士论文].泰安:山东农业大学.
Wang Z,Meng D,Wang A,Li T,Jiang S,Cong P,Li T.2013.The methylation of the PcMYB10 promoter is associated with green-skinned sport in Max Red Bartlett pear.Plant Physiology,162(2):885-896.
Wang Huicong,Huang Xuming,Hu Gguibing,Huang Huibai.2004.Studies on the relationship between anthocyanin biosynthesis and related enzymes in litchi pericarp.Scientia Agricultura Sinica,37(12):2028-2032.(in Chinese)王惠聪,黄旭明,胡桂兵,黄辉白.2004.荔枝果皮花青苷合成与相关酶的关系研究.中国农业科学,37(12),2028-2032.
Wang Yanling,Zhang Yanmin,Feng shouqian,Tian Changping,Wang Haibo,Liu Zunchun.2010.Cloning,sequence analysis and expression in E.coli of MsMYB10 gene from Malus sieversii f.neidzwetzkyana.Scientia Agricultura Sinica,43(13):2735-2743.(in Chinese)王延玲,张艳敏,冯守千,田长平,王海波,刘遵春.2010.新疆红肉苹果转录因子MsMYB10基因的克隆、序列分析及原核表达.中国农业科学,43(13):2735-2743.
Wang Yicheng,Wang Nan,Xu Haifeng,Zhang Zongying,Jiang Shenghui,Fang Hongcheng,Zhang Jing,Su Mengyu,Xu Lin,Chen Xuesen.2018.Expression of a cold-induced gene MdICE1 in red flesh apple and its protein interaction with MdMYB.Acta Horticulturae Sinica,45(5):817-826.(in Chinese)王意程,王楠,许海峰,张宗营,姜生辉,房鸿成,张静,苏梦雨,许琳,陈学森.2018.红肉苹果冷信号基因MdICE1的表达及其蛋白与MdMYB的互作.园艺学报,45(5):817-826.
Xu H F,Wang N,Liu J X,Qu C Z,Wang Y T,Jiang S H,Chen X S.2017.The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes.Plant Molecular Biology,94(1-2):149-165.
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.
Zhang Bo,Qu Dong,Yang Huijuan,Yang Yazhou,Wang Fan,Zhu Zhenzhen,Zhao Zhengyang.2018.Effects of MdMYB10 gene on phenylalanine metabolism of apple peel.Acta Horticulturae Sinica,45(8):1429-1440.(in Chinese)张波,曲东,杨惠娟,杨亚洲,王帆,朱珍珍,赵政阳.2018.MdMYB10对苹果果皮苯丙氨酸代谢的影响.园艺学报,45(8):1429-1440.
Zhao L,Gao L P,Wang H G,Chen X T,Wang Y S,Yang H,Wei C L,Wan X C,Xia T.2013.The R2R3-MYB,bHLH,WD40,and related transcription factors in flavonoid biosynthesis.Functional&Integrative Genomics,13(1):75-98.
Zhou Lan.2013.Study on the concentration changes and related genes of flavonoids in apple[Ph.D.Dissertation].Beijing:Chinese Academy of Sciences.(in Chinese)周兰.2013.苹果果实发育中类黄酮含量变化及相关基因的研究[博士论文].北京:中国科学院.
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.2014.MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples.Plant and Cell Physiology,56(4):650-662.
An X H,Tian Y,Chen K Q,Wang X F,Hao Y J.2012.The apple WD40 protein MdTTG1 interacts with bHLH but not MYB proteins to regulate anthocyanin accumulation.Journal of Plant Physiology,169(7):710-717.
Baudry A,Heim M A,Dubreucq B,Caboche M,Weisshaar B,Lepiniec L.2004.TT2,TT8,and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana.The Plant Journal,39(3):366-380.
Borevitz J O,Xia Y,Blount J,Dixon R A,Lamb C.2000.Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis.The Plant Cell,12(12):2383-2393.
Duan N,Yang B,Sun H,Wang N,Ma Y,Li M.2017.Genome re-sequencing reveals the history of apple and supports a two-stage model for fruit enlargement.Nature Communications,8(1):249.
Espley R.V,Brendolise C,ChagnéD,Kutty-Amma S,Green S,Volz R,Putterill J,Schouten H J,Gardiner S E,Hellens R P,Allan A C.2009.Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples.The Plant Cell,21(1):168-183.
Espley R V,Hellens R P,Putterill J,Stevenson D E,Kutty-Amma S,Allan A C.2007.Red colouration in apple fruit is due to the activity of the MYB transcription factor,MdMYB10.The Plant Journal,49(3):414-427.
Gao J J,Shen X F,Zhang Z,Peng,R H,Xiong A S,Xu J,Zhu B,Zheng J L,Yao Q H.2011.The MYB transcription factor MdMYB6 suppresses anthocyanin biosynthesis in transgenic Arabidopsis.Plant Cell,Tissue and Organ Culture(PCTOC),106(2):235-242.
Grotewold E.2006.The genetics and biochemistry of floral pigments.Annu Rev Plant Biol,57:761-780.
Hiratsu K,Matsui K,Koyama T,Ohme-Takagi M.2003.Dominant repression of target genes by chimeric repressors that include the EAR motif,a repression domain,in Arabidopsis.The Plant Journal,34(5):733-739.
Ji X H,Wang Y T,Zhang R,Wu S J,An M M,Li M,Wang C Z,Chen X L,Zhang Y M,Chen X S.2015.Effect of auxin,cytokinin and nitrogen on anthocyanin biosynthesis in callus cultures of red-fleshed apple(Malus sieversii f.niedzwetzkyana).Plant Cell,Tissue and Organ Culture(PCTOC),120(1):325-337.
Jimenez-Garcia S N,Guevara-Gonzalez R G,Miranda-Lopez R,Feregrino-Perez A A,Torres-Pacheco I,Vazquez-Cruz M A.2013.Functional properties and quality characteristics of bioactive compounds in berries:biochemistry,biotechnology,and genomics.Food Research International,54(1):1195-1207.
Koes R,Verweij W,Quattrocchio F.2005.Flavonoids:a colorful model for the regulation and evolution of biochemical pathways.Trends in Plant Science,10(5):236-242.
Lin-Wang K,Micheletti D,Palmer J,Volz R,Lozano L,Espley R,Hellens R P,ChagnèD,Rowan D D,Troggio M,Iglesias I,Allan A C.2011.High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex.Plant,Cell&Environment,34(7):1176-1190.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method.Methods,25(4):402-408.
Liu Xiaofen,Li Fang,Yin Xueren,Xu Changjie,Chen Kunsong.2013.Recent advances in the transcriptional regulation of anthocyanin biosynthesis.Acta Horticulturae Sinica,40(11):2295-2306.(in Chinese)刘晓芬,李方,殷学仁,徐昌杰,陈昆松.2013.花青苷生物合成转录调控研究进展.园艺学报,40(11):2295-2306.
Matsui K,Umemura Y,Ohme‐Takagi M.2008.AtMYBL2,a protein with a single MYB domain,acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis.The Plant Journal,55(6):954-967.
Martin C,Paz-Ares J.1997.MYB transcription factors in plants.Trends in Genetics,13(2):67-73.
Quattrocchio F,Verweij W,Kroon A,Spelt C,Mol J,Koes R.2006.PH4 of petunia is an R2R3 MYB protein that activates vacuolar acidification through interactions with basic-helix-loop-helix transcription factors of the anthocyanin pathway.The Plant Cell,18(5):1274-1291.
Ramsay N A,Glover B J.2005.MYB-bHLH-WD40 protein complex and the evolution of cellular diversity.Trends in Plant Science,10(2):63-70.
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(1):19-38.
Sun Shasha.2016.The functional characterization of PyMYB10.1 regulating anthocyanin biosynthesis in pear[M.D.Dissertation].Tai’an:Shandong Agricultural University.(in Chinese)孙莎莎.2016.梨花青苷调控基因PyMYB10.1功能鉴定[硕士论文].泰安:山东农业大学.
Wang Z,Meng D,Wang A,Li T,Jiang S,Cong P,Li T.2013.The methylation of the PcMYB10 promoter is associated with green-skinned sport in Max Red Bartlett pear.Plant Physiology,162(2):885-896.
Wang Huicong,Huang Xuming,Hu Gguibing,Huang Huibai.2004.Studies on the relationship between anthocyanin biosynthesis and related enzymes in litchi pericarp.Scientia Agricultura Sinica,37(12):2028-2032.(in Chinese)王惠聪,黄旭明,胡桂兵,黄辉白.2004.荔枝果皮花青苷合成与相关酶的关系研究.中国农业科学,37(12),2028-2032.
Wang Yanling,Zhang Yanmin,Feng shouqian,Tian Changping,Wang Haibo,Liu Zunchun.2010.Cloning,sequence analysis and expression in E.coli of MsMYB10 gene from Malus sieversii f.neidzwetzkyana.Scientia Agricultura Sinica,43(13):2735-2743.(in Chinese)王延玲,张艳敏,冯守千,田长平,王海波,刘遵春.2010.新疆红肉苹果转录因子MsMYB10基因的克隆、序列分析及原核表达.中国农业科学,43(13):2735-2743.
Wang Yicheng,Wang Nan,Xu Haifeng,Zhang Zongying,Jiang Shenghui,Fang Hongcheng,Zhang Jing,Su Mengyu,Xu Lin,Chen Xuesen.2018.Expression of a cold-induced gene MdICE1 in red flesh apple and its protein interaction with MdMYB.Acta Horticulturae Sinica,45(5):817-826.(in Chinese)王意程,王楠,许海峰,张宗营,姜生辉,房鸿成,张静,苏梦雨,许琳,陈学森.2018.红肉苹果冷信号基因MdICE1的表达及其蛋白与MdMYB的互作.园艺学报,45(5):817-826.
Xu H F,Wang N,Liu J X,Qu C Z,Wang Y T,Jiang S H,Chen X S.2017.The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes.Plant Molecular Biology,94(1-2):149-165.
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.
Zhang Bo,Qu Dong,Yang Huijuan,Yang Yazhou,Wang Fan,Zhu Zhenzhen,Zhao Zhengyang.2018.Effects of MdMYB10 gene on phenylalanine metabolism of apple peel.Acta Horticulturae Sinica,45(8):1429-1440.(in Chinese)张波,曲东,杨惠娟,杨亚洲,王帆,朱珍珍,赵政阳.2018.MdMYB10对苹果果皮苯丙氨酸代谢的影响.园艺学报,45(8):1429-1440.
Zhao L,Gao L P,Wang H G,Chen X T,Wang Y S,Yang H,Wei C L,Wan X C,Xia T.2013.The R2R3-MYB,bHLH,WD40,and related transcription factors in flavonoid biosynthesis.Functional&Integrative Genomics,13(1):75-98.
Zhou Lan.2013.Study on the concentration changes and related genes of flavonoids in apple[Ph.D.Dissertation].Beijing:Chinese Academy of Sciences.(in Chinese)周兰.2013.苹果果实发育中类黄酮含量变化及相关基因的研究[博士论文].北京:中国科学院.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |