月季RhMYB96转录激活及表达特性分析
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摘要
月季(Rosa hybrida)种质遗传品性受外界环境条件和基因调控的影响,转录调控在其中发挥重要作用。为了研究月季R2R3-MYB (v-myb avian myeloblastosis viral oncogene homolog)类转录因子的生物学功能,利用转录组测序获得的序列信息,结合c DNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术,从切花月季‘萨蔓莎’中分离得到1个新的MYB类型的转录因子基因,命名为RhMYB96(GenBank No. MF185658)。该基因ORF包含1 065 bp,编码354个氨基酸;蛋白质分子量为39.49 kD,等电点为6.89,分子式为C1693H2655N519O548S14。蛋白序列多重比对发现,RhMYB96在N端具有保守的R2-MYB和R3-MYB结构域,为典型的螺旋-转角-螺旋结构;构建RhMYB96与不同植物R2R3-MYB蛋白C端氨基酸序列的系统发育树,结果表明,Rh MYB96与拟南芥(Arabidopis thaliana) AtMYB96、AtMYB94和AtMYB30聚为一类,属于S1亚组R2R3-MYB类型转录因子。利用qRT-PCR技术分析RhMYB96的表达模式,结果显示,月季RhMYB96在花瓣中的表达量最高;12 h盐胁迫和脱落酸(abscisic acid, ABA)处理可诱导RhMYB96表达,盐胁迫下施加ABA也能诱导RhMYB96表达。构建包含RhMYB96全长等6个不同长度片段的酵母(Yeast)表达载体,利用酵母单杂交技术分析了RhMYB96的转录激活活性,结果显示,RhMYB96是转录激活子,N端没有自激活性,C端具有明显的自激活性。上述结果表明,转录激活子RhMYB96可能通过ABA依赖途径参与盐胁迫应答。本研究为月季RhMYB96转录调控机制研究提供了基础资料,也为月季的分子育种研究提供了理论参考。
External environment and internal gene regulation could influence the germplasm of Rosa hybrida genetic characteristics, and transcription regulation plays vital roles in these biological processes. To determine the biological function of R2R3-MYB(v-myb avian myeloblastosis viral oncogene homolog)transcription factors in rose, a new MYB transcription factor gene named as RhMYB96(GenBank No.MF185658) was cloned from cut rose 'Samantha' based on the expressed sequence tags from transcriptome assembly and rapid amplification of c DNA ends(RACE) methods. The RhMYB96 ORF was 1 065 bp, whichencoded 354 amino acids. Bioinformatics analysis showed that the molecular weight and theoretical isoelectric point of RhMYB96 was 39.49 kD and 6.89, respectively, and the formula was C1693 H2655 N519 O548 S14. Multiply protein alignment revealed that RhMYB96 contained 2 highly conserved domains R2-MYB and R3-MYB in its N terminus regions. The secondary structure of RhMYB96 was typical helix-turn-helix mode. In addition,the C terminal region of RhMYB96 and other plant MYB proteins were used to construct phylogenic tree.RhMYB96 had a close relationship with Arabidopsis AtMYB96, AtMYB94 and AtMYB30, which belonged to the R2 R3-MYB proteins of subgroup 1. qRT-PCR results showed that the expression of RhMYB96 in rose petals was the highest when compared with other organs. In addition, salt stress and abscisic acid(ABA)treatment significantly increased the expression of RhMYB96. Meanwhile, salt stress plus ABA treatment significantly increased the expression of RhMYB96. Moreover, 6 recombinant plasmids in yeast cells, which contained different fragments of RhMYB96 were constructed. The transactivation analysis of RhMYB96 was also examined by yeast one hybrid-assays. The results showed that RhMYB96 was a transcriptional activator,and an activation domain located at the C terminus. The N terminal DNA-binding domain lost transcription activity. Above results showed that transactivator RhMYB96 might participate in salt stress response via ABA dependent pathway. The study provides a foundation for predicting the roles of RhMYB96 in transcription regulatory networks and inferring functions in molecular breeding of rose plant species.
External environment and internal gene regulation could influence the germplasm of Rosa hybrida genetic characteristics, and transcription regulation plays vital roles in these biological processes. To determine the biological function of R2R3-MYB(v-myb avian myeloblastosis viral oncogene homolog)transcription factors in rose, a new MYB transcription factor gene named as RhMYB96(GenBank No.MF185658) was cloned from cut rose 'Samantha' based on the expressed sequence tags from transcriptome assembly and rapid amplification of c DNA ends(RACE) methods. The RhMYB96 ORF was 1 065 bp, whichencoded 354 amino acids. Bioinformatics analysis showed that the molecular weight and theoretical isoelectric point of RhMYB96 was 39.49 kD and 6.89, respectively, and the formula was C1693 H2655 N519 O548 S14. Multiply protein alignment revealed that RhMYB96 contained 2 highly conserved domains R2-MYB and R3-MYB in its N terminus regions. The secondary structure of RhMYB96 was typical helix-turn-helix mode. In addition,the C terminal region of RhMYB96 and other plant MYB proteins were used to construct phylogenic tree.RhMYB96 had a close relationship with Arabidopsis AtMYB96, AtMYB94 and AtMYB30, which belonged to the R2 R3-MYB proteins of subgroup 1. qRT-PCR results showed that the expression of RhMYB96 in rose petals was the highest when compared with other organs. In addition, salt stress and abscisic acid(ABA)treatment significantly increased the expression of RhMYB96. Meanwhile, salt stress plus ABA treatment significantly increased the expression of RhMYB96. Moreover, 6 recombinant plasmids in yeast cells, which contained different fragments of RhMYB96 were constructed. The transactivation analysis of RhMYB96 was also examined by yeast one hybrid-assays. The results showed that RhMYB96 was a transcriptional activator,and an activation domain located at the C terminus. The N terminal DNA-binding domain lost transcription activity. Above results showed that transactivator RhMYB96 might participate in salt stress response via ABA dependent pathway. The study provides a foundation for predicting the roles of RhMYB96 in transcription regulatory networks and inferring functions in molecular breeding of rose plant species.
引文
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牛义岭,姜秀明,许向阳.2016.植物转录因子MYB基因家族的研究进展[J].分子植物育种,(08):2050-2059.(Niu Y L,Jiang X M,Xu X Y.2016.Reaserch advances on transcription factor MYB gene family in plant[J].Molecular Plant Breeding,(08):2050-2059.)
乔孟,于延冲,向凤宁.2009.拟南芥R2R3-MYB类转录因子在环境胁迫中的作用[J].生命科学,21(01):145-150.(Qiao M,Yu Y C,Xiang F N.2009.The roles of the Arabidopsis R2R3-MYB transcription factors in the stress responses[J].Chinese Bulletin Life Science,21(01):145-150.)
荣晶.2014.木薯干旱胁迫相关R2R3-MYB转录因子MeMYB63的基因特性及启动子分析[D].硕士学位论文,海南大学,导师:彭明,pp.4-12.(Rong J.2014Characteristic and promoter analysis of a drought related R2R3-MYB transcription factor MeMYB63 in Cassava(Manihot esculenta Crantz)[D].Thesis for M.S.Hainan University,Supervisor:Peng M,pp.4-12.)
王栋,李利红,陈志玲,等.2001.拟南芥根特异表达转录因子AtMYB305的鉴定及功能研究[J].科学通报,(21)1804-1809.(Wang D,Li L H,Chen Z L,et al.2001Identification and function study of Arabidopsis root specific transcription factor AtMYB305[J].Chinese Science Bulletin,(21):1804-1809.)
吴韵雯,柯蕴倬,冉凤,等.2018.甘蓝型油菜BnMYB193基因的克隆和表达分析[J].农业生物技术学报,26(07)1155-1163.(Wu Y W,Ke Y Z,Ran F,et al.2018.Cloning and expression analysis of BnMYB193 gene in Brassica napus[J].Journal of Agricultural Biotechnology,26(07):1155-1163.)
赵佳,刘荣,杨帆,等.2015.月季花青素苷相关R2R3-MYB蛋白基因的克隆和表达分析[J].中国农业科学,48(07):1392-1404.(Zhao J,Liu R,Yang F,et al.2015Cloning and expression analyses of R2R3-MYB genes related to anthocyanin biosynthesis in rose[J].Scientia Agricultura Sinica,48(07):1392-1404.)
Cominelli E,Galbiati M,Vavasseur A,et al.2005.A guardcell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance[J].Curren Biology,15(13):1196-1200.
Dai F W,Zhang C Q,Jiang X Q,et al.2012.RhNAC2 and RhEXPA4 are involved in the regulation of dehydration tolerance during the expansion of rose petals[J].Plan Physiology,160(4):2064-2082.
Dai X Y,Wang Y Y,Yang A,et al.2012.OsMYB2P-1,a R2R3 MYB transcription factor,is involved in the regulation of phosphate-starvation responses and root architecture in rice[J].Plant Physiology,159(1):169-183.
Dubos C,Stracke R,Grotewold E,et al.2010.MYB transcription factors in Arabidopsis[J].Cell,15(10):1360-1385.
Hobert O.2008.Gene regulation by transcription factors and microRNAs[J].Science,319(5871):1785-1786.
Hu H H,Dai M Q,Yao J L,et al.2006.Over expressing a NAM,ATAF,and CUC(NAC)transcription factor enhances drought resistance and salt tolerance in rice[J]Proceedings of the National Academy of Sciences of the USA,103(35):12987-12992.
Jiang X Q,Li S C,Ding A Q,et al.2018.The novel rose MYB transcription factor RhMYB96 enhances salt tolerance in transgenic Arabidopsis[J].Plant Molecular Biology Reporter,36(3):406-417.
Lee S B,Suh M C.2015.Cuticular wax biosynthesis is upregulated by the MYB94 transcription factor in Arabidopsis[J].Plant Cell Physiology,56(1):48-60.
Li J J,Herskowitz I.1993.Isolation of ORC6,a component of the yeast origin recognition complex by a one-hybrid system[J].Science,262(5141):1870-1873.
Liu J D,Wilson T E,Milbrandt J,et al.1993.Identifying DNA-binding sites and analyzing DNA-binding domains using a yeast selection system[J].Methods,5(2):125-137.
Park J S,Kim J B,Cho K J,et al.2008.Arabidopsis R2R3-MYB transcription factor AtMYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce(Lactuca sativa)[J].Plant Cell Reports,27(6):985-994.
Seo P J,Lee S B,Suh M C,et al.2011.The MYB96 transcription factor regulates cuticular wax biosynthesis under drought conditions in Arabidopsis[J].Plant Cell,23(3):1138-1152.
Sun L,Zhang H,Li D,et al.2013.Functions of rice NACtranscriptional factors,ONAC122 and ONAC131,in defense responses against Magnaporthe grisea[J].Plant Molecular Biology,81(1-2):41-56
Tamura K,Stecher G,Peterson D,et al.2013.MEGA6:Molecular evolutionary genetics analysis version 6.0[J].Molecular Biology and Evolution,30(12):2725-2729.
Wan C Y,Wilkins T A.1994.A modified hot borate method significantly enhances the yield of high-quality RNAfrom cotton(Gossypium hirsutum L.)[J].Analytical Biochemistry,223(1):7-12.
Wei Q H,Zhang F,Sun F S,et al.2017.A wheat MYB transcriptional repressor TaMyb1D regulates phenylpropanoid metabolism and enhances tolerance to drought and oxidative stresses in transgenic tobacco plants[J].Plant Science,265(2017):112-123.
Zhou J N,Li C H,Zhong R Q,et al.2009.MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis[J].Plant Cell,21(01):248-266.
马男,蔡蕾,陆旺金,等.2005.外源乙烯对月季(Rosa hybrida)切花花朵开放的影响与乙烯生物合成相关基因表达的关联[J].中国科学C辑:生命科学,(02):104-114.(Ma N,Cai L,Lu W J,et al.2005.Effects of exogenous ethylene on the flowering of cut flowers of Rosa hybrida and their association with ethylenebiosynthesis related genes[J].Sciencein China(Series C):Life Science,(02):104-114.)
牛义岭,姜秀明,许向阳.2016.植物转录因子MYB基因家族的研究进展[J].分子植物育种,(08):2050-2059.(Niu Y L,Jiang X M,Xu X Y.2016.Reaserch advances on transcription factor MYB gene family in plant[J].Molecular Plant Breeding,(08):2050-2059.)
乔孟,于延冲,向凤宁.2009.拟南芥R2R3-MYB类转录因子在环境胁迫中的作用[J].生命科学,21(01):145-150.(Qiao M,Yu Y C,Xiang F N.2009.The roles of the Arabidopsis R2R3-MYB transcription factors in the stress responses[J].Chinese Bulletin Life Science,21(01):145-150.)
荣晶.2014.木薯干旱胁迫相关R2R3-MYB转录因子MeMYB63的基因特性及启动子分析[D].硕士学位论文,海南大学,导师:彭明,pp.4-12.(Rong J.2014Characteristic and promoter analysis of a drought related R2R3-MYB transcription factor MeMYB63 in Cassava(Manihot esculenta Crantz)[D].Thesis for M.S.Hainan University,Supervisor:Peng M,pp.4-12.)
王栋,李利红,陈志玲,等.2001.拟南芥根特异表达转录因子AtMYB305的鉴定及功能研究[J].科学通报,(21)1804-1809.(Wang D,Li L H,Chen Z L,et al.2001Identification and function study of Arabidopsis root specific transcription factor AtMYB305[J].Chinese Science Bulletin,(21):1804-1809.)
吴韵雯,柯蕴倬,冉凤,等.2018.甘蓝型油菜BnMYB193基因的克隆和表达分析[J].农业生物技术学报,26(07)1155-1163.(Wu Y W,Ke Y Z,Ran F,et al.2018.Cloning and expression analysis of BnMYB193 gene in Brassica napus[J].Journal of Agricultural Biotechnology,26(07):1155-1163.)
赵佳,刘荣,杨帆,等.2015.月季花青素苷相关R2R3-MYB蛋白基因的克隆和表达分析[J].中国农业科学,48(07):1392-1404.(Zhao J,Liu R,Yang F,et al.2015Cloning and expression analyses of R2R3-MYB genes related to anthocyanin biosynthesis in rose[J].Scientia Agricultura Sinica,48(07):1392-1404.)
Cominelli E,Galbiati M,Vavasseur A,et al.2005.A guardcell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance[J].Curren Biology,15(13):1196-1200.
Dai F W,Zhang C Q,Jiang X Q,et al.2012.RhNAC2 and RhEXPA4 are involved in the regulation of dehydration tolerance during the expansion of rose petals[J].Plan Physiology,160(4):2064-2082.
Dai X Y,Wang Y Y,Yang A,et al.2012.OsMYB2P-1,a R2R3 MYB transcription factor,is involved in the regulation of phosphate-starvation responses and root architecture in rice[J].Plant Physiology,159(1):169-183.
Dubos C,Stracke R,Grotewold E,et al.2010.MYB transcription factors in Arabidopsis[J].Cell,15(10):1360-1385.
Hobert O.2008.Gene regulation by transcription factors and microRNAs[J].Science,319(5871):1785-1786.
Hu H H,Dai M Q,Yao J L,et al.2006.Over expressing a NAM,ATAF,and CUC(NAC)transcription factor enhances drought resistance and salt tolerance in rice[J]Proceedings of the National Academy of Sciences of the USA,103(35):12987-12992.
Jiang X Q,Li S C,Ding A Q,et al.2018.The novel rose MYB transcription factor RhMYB96 enhances salt tolerance in transgenic Arabidopsis[J].Plant Molecular Biology Reporter,36(3):406-417.
Lee S B,Suh M C.2015.Cuticular wax biosynthesis is upregulated by the MYB94 transcription factor in Arabidopsis[J].Plant Cell Physiology,56(1):48-60.
Li J J,Herskowitz I.1993.Isolation of ORC6,a component of the yeast origin recognition complex by a one-hybrid system[J].Science,262(5141):1870-1873.
Liu J D,Wilson T E,Milbrandt J,et al.1993.Identifying DNA-binding sites and analyzing DNA-binding domains using a yeast selection system[J].Methods,5(2):125-137.
Park J S,Kim J B,Cho K J,et al.2008.Arabidopsis R2R3-MYB transcription factor AtMYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce(Lactuca sativa)[J].Plant Cell Reports,27(6):985-994.
Seo P J,Lee S B,Suh M C,et al.2011.The MYB96 transcription factor regulates cuticular wax biosynthesis under drought conditions in Arabidopsis[J].Plant Cell,23(3):1138-1152.
Sun L,Zhang H,Li D,et al.2013.Functions of rice NACtranscriptional factors,ONAC122 and ONAC131,in defense responses against Magnaporthe grisea[J].Plant Molecular Biology,81(1-2):41-56
Tamura K,Stecher G,Peterson D,et al.2013.MEGA6:Molecular evolutionary genetics analysis version 6.0[J].Molecular Biology and Evolution,30(12):2725-2729.
Wan C Y,Wilkins T A.1994.A modified hot borate method significantly enhances the yield of high-quality RNAfrom cotton(Gossypium hirsutum L.)[J].Analytical Biochemistry,223(1):7-12.
Wei Q H,Zhang F,Sun F S,et al.2017.A wheat MYB transcriptional repressor TaMyb1D regulates phenylpropanoid metabolism and enhances tolerance to drought and oxidative stresses in transgenic tobacco plants[J].Plant Science,265(2017):112-123.
Zhou J N,Li C H,Zhong R Q,et al.2009.MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis[J].Plant Cell,21(01):248-266.