苹果MpMYB96基因克隆和功能鉴定
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摘要
MpMYB96是拟南芥(Arabidopsis thaliana) AtMYB96的一个同源基因。为了探究MpMYB96在苹果(Malus pumila)中的功能,本文以‘皇家嘎拉’苹果苗的cDNA为模板进行PCR扩增,获得一个约1 000 bp的目的条带,通过测序发现该基因长为1 029 bp,序列与MpMYB96 (MDP0000144744, https://www.rosaceae.org/)的序列一致,该蛋白序列含有两个SANT保守结构域。进化树分析了15个物种MYB96蛋白的亲缘关系,表明MpMYB96在不同物种中的功能是保守的。此外,结果发现苹果MpMYB96与白梨(Pyrus bretschneideri) PbMYB96的亲缘关系最近,说明MpMYB96和PbMYB96可能具有相同的功能。实时荧光定量PCR发现MpMYB96在花中表达量最高,茎中表达量次之,根和叶中表达量均较低,暗示MYB96可能在花中扮演更重要的角色。MpMYB96的表达受脱落酸(ABA)、聚乙二醇(PEG)、水杨酸(SA)处理的诱导,表明MpMYB96的功能可能涉及到了ABA和SA以及PEG模拟的干旱过程。将MpMYB96转化苹果愈伤组织,检测转基因愈伤的表型,发现MpMYB96过表达能够增加植物对ABA和PEG的敏感性,并且能够对轮纹病产生一定的抵抗能力。
MpMYB96 is a homologous gene of Arabidopsis thaliana AtMYB96. In order to explore the function of MpMYB96 in apple(Malus pumila), the cDNA of the apple seedling was used as the template for PCR amplification in this paper, and a target band of about 1 000 bp was obtained. Through sequencing, it was found that the gene was 1 029 bp in length, and is identical to the sequence of MpMYB96(MDP0000144744, https://www.rosaceae.org/). The protein sequence contains two SANT conserved domains. The phylogenetic tree analysis of MYB96 protein in 15 species shows that the function of MYB96 in different species is conservative. In addition, it was found that apple MpMYB96 is most closely related to PbMYB96(Pyrus bretschneideri), suggesting that MpMYB96 and PbMYB96 may have the same function. Real-time fluorescence quantitative PCR showed that the expression of MpMYB96 was highest in flowers, followed by that in stems, and lower in roots and leaves, suggesting that MYB96 may play a more important role in flowers. The expression of MpMYB96 was induced by abscisic acid(ABA), polyethylene glycol(PEG) and salicylic acid(SA) treatments, suggesting that the function of MpMYB96 may involve ABA and SA, as well as the simulated drought process of PEG.MpMYB96 was transformed into apple callus, and the phenotype of the transgenic callus was detected. It was found that the overexpression of MpMYB96 could increase the plant's sensitivity to ABA and PEG, as well as develop a certain resistance to Botryosphaeria dothidea.
MpMYB96 is a homologous gene of Arabidopsis thaliana AtMYB96. In order to explore the function of MpMYB96 in apple(Malus pumila), the cDNA of the apple seedling was used as the template for PCR amplification in this paper, and a target band of about 1 000 bp was obtained. Through sequencing, it was found that the gene was 1 029 bp in length, and is identical to the sequence of MpMYB96(MDP0000144744, https://www.rosaceae.org/). The protein sequence contains two SANT conserved domains. The phylogenetic tree analysis of MYB96 protein in 15 species shows that the function of MYB96 in different species is conservative. In addition, it was found that apple MpMYB96 is most closely related to PbMYB96(Pyrus bretschneideri), suggesting that MpMYB96 and PbMYB96 may have the same function. Real-time fluorescence quantitative PCR showed that the expression of MpMYB96 was highest in flowers, followed by that in stems, and lower in roots and leaves, suggesting that MYB96 may play a more important role in flowers. The expression of MpMYB96 was induced by abscisic acid(ABA), polyethylene glycol(PEG) and salicylic acid(SA) treatments, suggesting that the function of MpMYB96 may involve ABA and SA, as well as the simulated drought process of PEG.MpMYB96 was transformed into apple callus, and the phenotype of the transgenic callus was detected. It was found that the overexpression of MpMYB96 could increase the plant's sensitivity to ABA and PEG, as well as develop a certain resistance to Botryosphaeria dothidea.
引文
An XH,Tian Y,Chen KQ,et al(2014).MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples.Plant Cell Physiol,56(4):650-662
Cao FY,DeFalco TA,Moeder W,et al(2018).Arabidopsis ETHYLENE RESPONSE FACTOR 8(ERF8)has dual functions in ABA signaling and immunity.BMC Plant Biol,18:211
Chen H,Liu L,Wang L,et al(2016).VrDREB2A,a DREB-binding transcription factor from Vigna radiata,increased drought and high-salt tolerance in transgenic Arabidopsis thaliana.J Plant Res,129(2):263-273
Chen XK,Zhang JY,Zhang Z,et al(2012).Overexpressing MhNPR1 in transgenic Fuji apples enhances resistance to apple powdery mildew.Mol Biol Rep,39(8):8083-8089
Dai X,Xu Y,Ma Q,et al(2007).Overexpression of an R1R2R3 MYB gene,OsMYB3R-2,increases tolerance to freezing,drought,and salt stress in transgenic Arabidopsis.Plant Physiol,143(4):1739-1751
Frolov A,Bilova T,Paudel G,et al(2017).Early responses of mature Arabidopsis thaliana plants to reduced water potential in the agar-based polyethylene glycol infusion drought model.J Plant Physiol,208:70-83
Fu L,Ding Z,Han B,et al(2016).Physiological investigation and transcriptome analysis of polyethylene glycol(PEG)-induced dehydration stress in cassava.Int J Mol Sci,17(3):283
Hamayun M,Khan SA,Shinwari ZK,et al(2010).Effect of polyethylene glycol induced drought stress on physio-hormonal attributes of soybean.Pak J Bot,42(2):977-986
He Z,Zhong J,Sun X,et al(2018).The maize ABA receptors ZmPYL8,9,and 12 facilitate plant drought resistance.Front Plant Sci,9:422
Hu DG,Sun CH,Ma QJ,et al(2016).MdMYB1 regulates anthocyanin and malate accumulation by directly facilitating their transport into vacuoles in apples.Plant Physiol,170(3):1315-1330
Huang YJ,Song S,Allan AC,et al(2013).Differential activation of anthocyanin biosynthesis in Arabidopsis and tobacco over-expressing an R2R3 MYB from Chinese bayberry.Plant Cell Tiss Org Cult,113(3):491-499
Jaffar MA,Song A,Faheem M,et al(2016).Involvement of CmWRKY10 in drought tolerance of chrysanthemum through the ABA-signaling pathway.Int J Mol Sci,17(5):693
Kwak JM,Mori IC,Pei ZM,et al(2003).NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis.EMBO J,22(11):2623-2633
Li X,Zhang Y,Yin L,et al(2017).Overexpression of pathogen-induced grapevine TIR-NB-LRR gene VaRGA1enhances disease resistance and drought and salt tolerance in Nicotiana benthamiana.Protoplasma,254(2):957-969
Ning J,Li X,Hicks LM,et al(2010).A Raf-like MAPKKKgene DSM1 mediates drought resistance through reactive oxygen species scavenging in rice.Plant Physiol,152(2):876-890
Pei ZF,Ming DF,Liu D,et al(2010).Silicon improves the tolerance to water-deficit stress induced by polyethylene glycol in wheat(Triticum aestivum L.)seedlings.J Plant Growth Regul,29(1):106-115
Shin D,Moon SJ,Han S,et al(2011).Expression of StMY-B1R-1,a novel potato single MYB-like domain transcription factor,increases drought tolerance.Plant Physiol,155(1):421-432
Wei T,Deng K,Liu D,et al(2016).Ectopic expression of DREB transcription factor,AtDREB1A,confers tolerance to drought in transgenic Salvia miltiorrhiza.Plant Cell Physiol,57(8):1593-1609
Xiong H,Li J,Liu P,et al(2014).Overexpression of OsMYB48-1,a novel MYB-related transcription factor,enhances drought and salinity tolerance in rice.PLoS ONE,9(3):e92913
Zang G,Zou H,Zhang Y,et al(2016).The De-Etiolated 1homolog of Arabidopsis modulates the ABA signaling pathway and ABA biosynthesis in rice.Plant Physiol,171(2):1259-1276
Zhang H,Cui F,Wu Y,et al(2015).The RING finger ubiquitin E3 ligase SDIR1 targets SDIR1-INTERACTINGPROTEIN1 for degradation to modulate the salt stress response and ABA signaling in Arabidopsis.Plant Cell,27(1):214-227
Zhang JY,Qiao YS,Lv D,et al(2012).Malus hupehensis NPR1 induces pathogenesis-related protein gene expression in transgenic tobacco.Plant Biol,14:46-56
Zhao L,Hu Y,Chong K,et al(2010).ARAG1,an ABA-responsive DREB gene,plays a role in seed germination and drought tolerance of rice.Ann Bot,105(3):401-409
Cao FY,DeFalco TA,Moeder W,et al(2018).Arabidopsis ETHYLENE RESPONSE FACTOR 8(ERF8)has dual functions in ABA signaling and immunity.BMC Plant Biol,18:211
Chen H,Liu L,Wang L,et al(2016).VrDREB2A,a DREB-binding transcription factor from Vigna radiata,increased drought and high-salt tolerance in transgenic Arabidopsis thaliana.J Plant Res,129(2):263-273
Chen XK,Zhang JY,Zhang Z,et al(2012).Overexpressing MhNPR1 in transgenic Fuji apples enhances resistance to apple powdery mildew.Mol Biol Rep,39(8):8083-8089
Dai X,Xu Y,Ma Q,et al(2007).Overexpression of an R1R2R3 MYB gene,OsMYB3R-2,increases tolerance to freezing,drought,and salt stress in transgenic Arabidopsis.Plant Physiol,143(4):1739-1751
Frolov A,Bilova T,Paudel G,et al(2017).Early responses of mature Arabidopsis thaliana plants to reduced water potential in the agar-based polyethylene glycol infusion drought model.J Plant Physiol,208:70-83
Fu L,Ding Z,Han B,et al(2016).Physiological investigation and transcriptome analysis of polyethylene glycol(PEG)-induced dehydration stress in cassava.Int J Mol Sci,17(3):283
Hamayun M,Khan SA,Shinwari ZK,et al(2010).Effect of polyethylene glycol induced drought stress on physio-hormonal attributes of soybean.Pak J Bot,42(2):977-986
He Z,Zhong J,Sun X,et al(2018).The maize ABA receptors ZmPYL8,9,and 12 facilitate plant drought resistance.Front Plant Sci,9:422
Hu DG,Sun CH,Ma QJ,et al(2016).MdMYB1 regulates anthocyanin and malate accumulation by directly facilitating their transport into vacuoles in apples.Plant Physiol,170(3):1315-1330
Huang YJ,Song S,Allan AC,et al(2013).Differential activation of anthocyanin biosynthesis in Arabidopsis and tobacco over-expressing an R2R3 MYB from Chinese bayberry.Plant Cell Tiss Org Cult,113(3):491-499
Jaffar MA,Song A,Faheem M,et al(2016).Involvement of CmWRKY10 in drought tolerance of chrysanthemum through the ABA-signaling pathway.Int J Mol Sci,17(5):693
Kwak JM,Mori IC,Pei ZM,et al(2003).NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis.EMBO J,22(11):2623-2633
Li X,Zhang Y,Yin L,et al(2017).Overexpression of pathogen-induced grapevine TIR-NB-LRR gene VaRGA1enhances disease resistance and drought and salt tolerance in Nicotiana benthamiana.Protoplasma,254(2):957-969
Ning J,Li X,Hicks LM,et al(2010).A Raf-like MAPKKKgene DSM1 mediates drought resistance through reactive oxygen species scavenging in rice.Plant Physiol,152(2):876-890
Pei ZF,Ming DF,Liu D,et al(2010).Silicon improves the tolerance to water-deficit stress induced by polyethylene glycol in wheat(Triticum aestivum L.)seedlings.J Plant Growth Regul,29(1):106-115
Shin D,Moon SJ,Han S,et al(2011).Expression of StMY-B1R-1,a novel potato single MYB-like domain transcription factor,increases drought tolerance.Plant Physiol,155(1):421-432
Wei T,Deng K,Liu D,et al(2016).Ectopic expression of DREB transcription factor,AtDREB1A,confers tolerance to drought in transgenic Salvia miltiorrhiza.Plant Cell Physiol,57(8):1593-1609
Xiong H,Li J,Liu P,et al(2014).Overexpression of OsMYB48-1,a novel MYB-related transcription factor,enhances drought and salinity tolerance in rice.PLoS ONE,9(3):e92913
Zang G,Zou H,Zhang Y,et al(2016).The De-Etiolated 1homolog of Arabidopsis modulates the ABA signaling pathway and ABA biosynthesis in rice.Plant Physiol,171(2):1259-1276
Zhang H,Cui F,Wu Y,et al(2015).The RING finger ubiquitin E3 ligase SDIR1 targets SDIR1-INTERACTINGPROTEIN1 for degradation to modulate the salt stress response and ABA signaling in Arabidopsis.Plant Cell,27(1):214-227
Zhang JY,Qiao YS,Lv D,et al(2012).Malus hupehensis NPR1 induces pathogenesis-related protein gene expression in transgenic tobacco.Plant Biol,14:46-56
Zhao L,Hu Y,Chong K,et al(2010).ARAG1,an ABA-responsive DREB gene,plays a role in seed germination and drought tolerance of rice.Ann Bot,105(3):401-409