草莓FaMYB10蛋白互作验证及启动子克隆
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摘要
本研究旨在探究光信号如何调控草莓花青素苷合成的分子机制,利用同源克隆法从3个不同颜色的草莓品种(‘红颜’,‘桃熏’,‘白雪公主’)中分离得到FaMYB10基因及其启动子序列,采用酵母双杂交系统验证FaMYB10的转录激活能力及其与FaCOP1的蛋白互作关系,利用红颜草莓FaMYB10基因的启动子序列构建酵母单杂交诱饵载体pHIS2-ProFaMYB10,并验证其在酵母Y187中的自激活现象。结果表明,经测序显示3个不同颜色的草莓品种FaMYB10基因的CDS区域序列完全一致为702 bp,但是在启动子区域存在不同差异。酵母双杂交实验表明Fa MYB10在Y2HGold中存在转录激活能力,FaMYB10同FaCOP1存在蛋白质与蛋白质互作关系。在Y187宿主内,发现当3-AT浓度达到60 mmol/L无法抑制FaMYB10基因启动子带来的自激活现象。本研究结果为理解草莓通过光信号传导调控下游FaMYB10,进而影响花青素苷合成的分子机制提供一定理论参考。
In order to explore the molecular mechanism of light signal affecting anthocyanin synthesis in strawberry, FaMYB10 gene and its promoter sequence were isolated from three strawberry cultivars('Benihoppe','Taoxun', 'Snow Princess') by homologous cloning. Yeast two-hybrid system was used to verify the transcriptional activation ability of FaMYB10 and its protein interaction with FaCOP1. The yeast one-hybrid bait vector pHIS2-ProFaMYB10 was constructed by the promoter sequence of FaMYB10 gene of 'Benihoppe' strawberry, and its self-activation in yeast Y187 was verified. The results showed that the sequence of CDS region of FaMYB10 gene in three strawberry varieties with different colors was 702 bp, but there were differences in promoter region.Yeast two-hybrid experiments indicated that there was a transcriptional activation ability of FaMYB10 in Y2 HGold, and there was a protein-protein interaction between FaCOP1 and FaMYB10. In the host of Y187, it was found that when the concentration of 3-AT reached 60 mmol/L, the self-activation of FaMYB10 gene promoter could not be inhibited. The results of this study could provide some theoretical references for understanding the molecular mechanism of downstream Fa MYB10 regulation by light signal transduction in strawberry, and then affecting the synthesis of anthocyanin glycosides.
In order to explore the molecular mechanism of light signal affecting anthocyanin synthesis in strawberry, FaMYB10 gene and its promoter sequence were isolated from three strawberry cultivars('Benihoppe','Taoxun', 'Snow Princess') by homologous cloning. Yeast two-hybrid system was used to verify the transcriptional activation ability of FaMYB10 and its protein interaction with FaCOP1. The yeast one-hybrid bait vector pHIS2-ProFaMYB10 was constructed by the promoter sequence of FaMYB10 gene of 'Benihoppe' strawberry, and its self-activation in yeast Y187 was verified. The results showed that the sequence of CDS region of FaMYB10 gene in three strawberry varieties with different colors was 702 bp, but there were differences in promoter region.Yeast two-hybrid experiments indicated that there was a transcriptional activation ability of FaMYB10 in Y2 HGold, and there was a protein-protein interaction between FaCOP1 and FaMYB10. In the host of Y187, it was found that when the concentration of 3-AT reached 60 mmol/L, the self-activation of FaMYB10 gene promoter could not be inhibited. The results of this study could provide some theoretical references for understanding the molecular mechanism of downstream Fa MYB10 regulation by light signal transduction in strawberry, and then affecting the synthesis of anthocyanin glycosides.
引文
Bai S.,Sun Y.,Qian M.,Yang F.,Ni J.,and Tao R.,2017,Transcriptome analysis of bagging-treated red chinese sand pear peels reveals light-responsive pathway functions in anthocyanin accumulation,Sci.Rep.,7(1):63
Borevitz J.O.,Xia Y.,Blount J.,Dixon R.A.,and Lamb C.,2000,Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis,Plant Cell,12(12):2383-2394
Chen Q.,Yu H.W.,Wang X.R.,Xie X.L.,Yue X.Y.,and Tang H.R.,2012,An alternative cetyltrimethyl ammonium bromidebased protocol for RNA isolation from blackberry(Rubus L.),Genet.Mol.Res.,11(2):1773-1782
Cominelli E.,Gusmaroli G.,Allegra D.,Galbiati M.,Wade H.K.,and Jenkins G.I.,2008,Expression analysis of anthocyanin regulatory genes in response to different light qualities in Arabidopsis thaliana,J.Plant Physiol.,165(8):886-894
Espley R.V.,Hellens R.P.,Putterill J.,Stevenson D.E.,Kuttyamma S.,and Allan A.C.,2007,Red colouration in apple fruit is due to the activity of the MYB transcription factor,MdMYB10,Plant J.,49(3):414-427
Guan L.,Dai Z.,Wu B.H.,Wu J.,Merlin I.,and Hilbert G.,2016,Anthocyanin biosynthesis is differentially regulated by light in the skin and flesh of white-fleshed and teinturier grape berries,Planta,243(1):23-41
Henrykirk R.A.,Plunkett B.,Hall M.,Mcghie T.,Allan A.C.,and Wargent J.J.,2018,Solar UV light regulates flavonoid metabolism in apple(Malusx domestica),Plant Cell and Environ.,41(3):1-14
Huang X.,Ouyang X.,and Deng X.W.,2014,Beyond repression of photomorphogenesis:role switching of cop/det/fus in light signaling,Curr.Opin.Plant Biol.,21:96-103
Jiao Y.,Lau O.,and Deng X.W.,2007,Light-regulated transcriptional networks in higher plants,Na.Rev.Genet.,8(3):217-230
Jing C.,Ma C.,Zhang J.,Jing S.,Jiang X.,Yang Y.,and Zhao Z.,2016,Effect of debagging time on pigment patterns in the peel and sugar and organic acid contents in the pulp of'golden delicious'and'qinguan'apple fruit at mid and late stages of development,PLoS One,11(10):e0165050
Kadomura-Ishikawa Y.,Miyawaki K.,Takahashi A.,Masuda T.,and Noji S.,2015,Light and abscisic acid independently regulated FaMYB10 in Fragaria×ananassa fruit,Planta.,241(4):953-965
Kondo S.,Tomiyama H.,Rodyoung A.,Okawa K.,Ohara H.,and Sugaya S.,Terahara H.,and Hirai N.,2014,Abscisic acid metabolism and anthocyanin synthesis in grape skin are affected by light emitting diode(led)irradiation at night,J.of Plant Physiol.,171(10):823-829
Lan B.X.,Wang L.,Wu Z.K.,and Jiang B.L.,2015,Rapid miniprep extraction of genomic DNA from micro-endosperm maize with modified CTAB method,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):190-194(蓝碧秀,王凛,吴子恺,姜伯乐,2015,利用改良CTAB法快速小量提取微胚乳玉米基因组DNA,基因组学与应用生物学,34(1):190-194)
Li Y.Y.,Mao K.,Zhao C.,Zhao X.Y.,Zhang H.L.,Shu H.R.,and Hao Y.J.,2012,MdCOP1 ubiquitin E3 ligases interact with MdMYB1 to regulate light-induced anthocyanin biosynthesis and red fruit coloration in apple,Plant Physiol.,160(2):1011-1022
Lin-Wang K.,Bolitho K.,Grafton K.,Kortstee A.,Karunairetnam S.,and Mcghie T.K.,2010,An R2R3 MYB transcrip tion factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae,BMC Plant Biol.,10(1):50
Lin-Wang K.,Mcghie T.K.,Wang M.,Liu Y.,Warren B.,and Storey R.,2014,Engineering the anthocyanin regulatory complex of strawberry(Fragaria vesca),Front.Plant Sci.,5(1):651
Maier A.,Schrader A.,Kokkelink L.,Falke C.,Welter B.,and Iniesto E.,2013,Light and the E3 ubiquitin ligase COP1/SPA control the protein stability of the MYB transcription factors PAP1 and PAP2 involved in anthocyanin accumulation in Arabidopsis,Plant J.,74(4):638-651
Nguyen N.H.,Jeong C.Y.,Kang G.,Yoo S.,Hong S.,and Lee H.,2015,Mybd employed by HY5 increases anthocyanin accumulation via repression of MYBL2 in Arabidopsis,Plant J.,84(6):1192-1205
Shin D.H.,Choi M.G.,Kim K.,Bang G.,Cho M.,and Choi S.B.,2013,Hy5 regulates anthocyanin biosynthesis by inducing the transcriptional activation of the MYB75/PAP1 transcription factor in Arabidopsis,FEBS Lett.,587(10):1543-1547
Tao R.,Bai S.,Ni J.,Yang Q.,Zhao Y.,and Teng Y.,2018,The blue light signal transduction pathway is involved in anthocyanin accumulation in'Red Zaosu'pear,Planta,248(1):37-48
Tohge T.,Nishiyama Y.,Hirai M.Y.,Yano M.,Nakajima J.,and Awazuhara M.,2005,Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor,Plant J.,42(2):218-235
Zhang H.N.,Li W.C.,Wang H.C.,Shi S.Y.,Shu B.,and Liu L.Q.,2016,Transcriptome profiling of light-regulated anthocyanin biosynthesis in Guan he pericarp of litchi,Front.Plant Sci.,7(225):963
Zhang Y.T.,Jiang L.Y.,Li Y.L.,Chen Q.,Ye Y.T.,Zhang Y.,Luo Y.,Sun B.,Wang X.R.,and Tang H.R.,2018,Effect of red and blue light on anthocyanin accumulation and differential gene expression in strawberry(Fragaria×ananassa),Molecule,23(4):1-17
Zhao Y.,Dong W.,Wang K.,Zhang B.,Allan A.C.,and Linwang K.,2017,Differential sensitivity of fruit pigmentation to ultraviolet light between two peach cultivars,Front.Plant Sci.,8:1552
Zoratti L.,Karppinen K.,Luengo A.E.,Haggman H.,and Jaakola L.,2014,Light-controlled flavonoid biosynthesis in fruits,Front.Plant Sci.,5(5):534
Borevitz J.O.,Xia Y.,Blount J.,Dixon R.A.,and Lamb C.,2000,Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis,Plant Cell,12(12):2383-2394
Chen Q.,Yu H.W.,Wang X.R.,Xie X.L.,Yue X.Y.,and Tang H.R.,2012,An alternative cetyltrimethyl ammonium bromidebased protocol for RNA isolation from blackberry(Rubus L.),Genet.Mol.Res.,11(2):1773-1782
Cominelli E.,Gusmaroli G.,Allegra D.,Galbiati M.,Wade H.K.,and Jenkins G.I.,2008,Expression analysis of anthocyanin regulatory genes in response to different light qualities in Arabidopsis thaliana,J.Plant Physiol.,165(8):886-894
Espley R.V.,Hellens R.P.,Putterill J.,Stevenson D.E.,Kuttyamma S.,and Allan A.C.,2007,Red colouration in apple fruit is due to the activity of the MYB transcription factor,MdMYB10,Plant J.,49(3):414-427
Guan L.,Dai Z.,Wu B.H.,Wu J.,Merlin I.,and Hilbert G.,2016,Anthocyanin biosynthesis is differentially regulated by light in the skin and flesh of white-fleshed and teinturier grape berries,Planta,243(1):23-41
Henrykirk R.A.,Plunkett B.,Hall M.,Mcghie T.,Allan A.C.,and Wargent J.J.,2018,Solar UV light regulates flavonoid metabolism in apple(Malusx domestica),Plant Cell and Environ.,41(3):1-14
Huang X.,Ouyang X.,and Deng X.W.,2014,Beyond repression of photomorphogenesis:role switching of cop/det/fus in light signaling,Curr.Opin.Plant Biol.,21:96-103
Jiao Y.,Lau O.,and Deng X.W.,2007,Light-regulated transcriptional networks in higher plants,Na.Rev.Genet.,8(3):217-230
Jing C.,Ma C.,Zhang J.,Jing S.,Jiang X.,Yang Y.,and Zhao Z.,2016,Effect of debagging time on pigment patterns in the peel and sugar and organic acid contents in the pulp of'golden delicious'and'qinguan'apple fruit at mid and late stages of development,PLoS One,11(10):e0165050
Kadomura-Ishikawa Y.,Miyawaki K.,Takahashi A.,Masuda T.,and Noji S.,2015,Light and abscisic acid independently regulated FaMYB10 in Fragaria×ananassa fruit,Planta.,241(4):953-965
Kondo S.,Tomiyama H.,Rodyoung A.,Okawa K.,Ohara H.,and Sugaya S.,Terahara H.,and Hirai N.,2014,Abscisic acid metabolism and anthocyanin synthesis in grape skin are affected by light emitting diode(led)irradiation at night,J.of Plant Physiol.,171(10):823-829
Lan B.X.,Wang L.,Wu Z.K.,and Jiang B.L.,2015,Rapid miniprep extraction of genomic DNA from micro-endosperm maize with modified CTAB method,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):190-194(蓝碧秀,王凛,吴子恺,姜伯乐,2015,利用改良CTAB法快速小量提取微胚乳玉米基因组DNA,基因组学与应用生物学,34(1):190-194)
Li Y.Y.,Mao K.,Zhao C.,Zhao X.Y.,Zhang H.L.,Shu H.R.,and Hao Y.J.,2012,MdCOP1 ubiquitin E3 ligases interact with MdMYB1 to regulate light-induced anthocyanin biosynthesis and red fruit coloration in apple,Plant Physiol.,160(2):1011-1022
Lin-Wang K.,Bolitho K.,Grafton K.,Kortstee A.,Karunairetnam S.,and Mcghie T.K.,2010,An R2R3 MYB transcrip tion factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae,BMC Plant Biol.,10(1):50
Lin-Wang K.,Mcghie T.K.,Wang M.,Liu Y.,Warren B.,and Storey R.,2014,Engineering the anthocyanin regulatory complex of strawberry(Fragaria vesca),Front.Plant Sci.,5(1):651
Maier A.,Schrader A.,Kokkelink L.,Falke C.,Welter B.,and Iniesto E.,2013,Light and the E3 ubiquitin ligase COP1/SPA control the protein stability of the MYB transcription factors PAP1 and PAP2 involved in anthocyanin accumulation in Arabidopsis,Plant J.,74(4):638-651
Nguyen N.H.,Jeong C.Y.,Kang G.,Yoo S.,Hong S.,and Lee H.,2015,Mybd employed by HY5 increases anthocyanin accumulation via repression of MYBL2 in Arabidopsis,Plant J.,84(6):1192-1205
Shin D.H.,Choi M.G.,Kim K.,Bang G.,Cho M.,and Choi S.B.,2013,Hy5 regulates anthocyanin biosynthesis by inducing the transcriptional activation of the MYB75/PAP1 transcription factor in Arabidopsis,FEBS Lett.,587(10):1543-1547
Tao R.,Bai S.,Ni J.,Yang Q.,Zhao Y.,and Teng Y.,2018,The blue light signal transduction pathway is involved in anthocyanin accumulation in'Red Zaosu'pear,Planta,248(1):37-48
Tohge T.,Nishiyama Y.,Hirai M.Y.,Yano M.,Nakajima J.,and Awazuhara M.,2005,Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor,Plant J.,42(2):218-235
Zhang H.N.,Li W.C.,Wang H.C.,Shi S.Y.,Shu B.,and Liu L.Q.,2016,Transcriptome profiling of light-regulated anthocyanin biosynthesis in Guan he pericarp of litchi,Front.Plant Sci.,7(225):963
Zhang Y.T.,Jiang L.Y.,Li Y.L.,Chen Q.,Ye Y.T.,Zhang Y.,Luo Y.,Sun B.,Wang X.R.,and Tang H.R.,2018,Effect of red and blue light on anthocyanin accumulation and differential gene expression in strawberry(Fragaria×ananassa),Molecule,23(4):1-17
Zhao Y.,Dong W.,Wang K.,Zhang B.,Allan A.C.,and Linwang K.,2017,Differential sensitivity of fruit pigmentation to ultraviolet light between two peach cultivars,Front.Plant Sci.,8:1552
Zoratti L.,Karppinen K.,Luengo A.E.,Haggman H.,and Jaakola L.,2014,Light-controlled flavonoid biosynthesis in fruits,Front.Plant Sci.,5(5):534