小麦TaDREB1的功能标记作图和表达特性分析
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摘要
DREB (dehydration responsive element binding)是植物应答干旱、高盐、高温和低温等非生物逆境的一类重要的转录因子,该基因在不同作物间存在抗逆复杂性和局限性。本研究以普通小麦(Triticum aestivum)及其野生近缘种的二倍体和四倍体为材料,克隆TaDREB1基因(GenBank No. DQ195070.1),发现在六倍体小麦中有3种TaDREB1的DNA序列,分别对应于A、B和D基因组,命名为TaDREB1-a/b/d。根据TaDREB1-b在不同材料间的多态性位点开发功能标记,利用加倍单倍体(doubled haploid, DH)和重组自交系(recombinant inbred lines, RIL)群体将TaDREB1-b定位在染色体3B上,与两侧标记P2449-185和WMC231、Xfbb117和Xgwm566的距离为19.2和16.0 cM、3.4和6.4 cM;同时以小麦品种旱选10号为材料,分别设计TaDREB1-a/b/d的特异引物,通过qRT-PCR分析其在4种胁迫下的表达模式发现,TaDREB1-a/b/d在小麦根、叶中的表达时间和峰值均存在差异。其中,在PEG、NaCl和ABA胁迫处理下,叶片中的TaDREB1-a/b表现为上调表达,TaDREB1-d则为下调表达;低温胁迫下,叶片中的TaDREB1-a/b/d均为上调表达。在4种处理模式下,小麦幼根中的TaDREB1-a/b/d都为上调表达。本研究开发的TaDREB1-b功能标记为分子标记辅助选择育种提供了支持,同时从基因组水平上初步阐释了TaDREB1-a/b/d对非生物胁迫的应答模式,为深入探讨小麦抗逆分子机制提供了基础资料。
DREB(dehydration responsive element binding) in plant plays important roles in resisting drought, high salinity and low temperature, etc. However, the further study on its family showed that the resistance of this gene to different crops was complicated and limited. TaDREB1 was cloned by hexaploid wheat(Triticum aestivum) and its diploid and tetraploid wild relative species as materials(GenBank No.DQ195070.1). It was found that there were 3 DNA sequences in the hexaploid wheat, corresponding to the 3genomes A, B and D, and were named as TaDREB1-a/b/d, respectively. Develop functional markers according to polymorphic sites of TaDREB1-b between different materials, TaDREB1-b was mapped using a doubled haploid(DH) and recombinant inbred lines(RIL) population. TaDREB1-b was mapped in the intervals between markers P2449-185 and WMC231 on chromosome 3 B, with 19.2 and 16.0 cM from the flanking markers using DH population, and mapped in the intervals between markers Xfbb117 and Xgwm566 on chromosome 3 B, with 3.4 and 6.4 cM from the flanking markers using RIL population; At the same time, the specific primers of TaDREB1-a/b/d were designed by using wheat variety Hanxuan 10 as the material, and the expression pattern of TaDREB1-a/b/d under 4 stresses was analyzed by qRT-PCR. The expression peak and degree of TaDREB1-a/b/d were different in leaves and roots. TaDREB1-a/b expression were up-regulated in leaves under PEG, NaCl and ABA condition, while the expression of TaDREB1-d were down-regulated.TaDREB1-a/b/d was up-regulated under low temperature in leaves. In roots, the expression of TaDREB1-a/b/d were up-regulated in all condition. TaDREB1-a/b/d were all involved in responding to PEG, NaCl, ABA and4 ℃ stresses, but different in expression patterns. The functional markers developed in this study provides the tools for molecular marker-assisted selective breeding. Meanwhile, the relationship between TaDREB1-a/b/d and abiotic stress response is preliminarily explained at the genome level, laying a foundation for in-depth exploration of the molecular mechanism of wheat stress resistance.
DREB(dehydration responsive element binding) in plant plays important roles in resisting drought, high salinity and low temperature, etc. However, the further study on its family showed that the resistance of this gene to different crops was complicated and limited. TaDREB1 was cloned by hexaploid wheat(Triticum aestivum) and its diploid and tetraploid wild relative species as materials(GenBank No.DQ195070.1). It was found that there were 3 DNA sequences in the hexaploid wheat, corresponding to the 3genomes A, B and D, and were named as TaDREB1-a/b/d, respectively. Develop functional markers according to polymorphic sites of TaDREB1-b between different materials, TaDREB1-b was mapped using a doubled haploid(DH) and recombinant inbred lines(RIL) population. TaDREB1-b was mapped in the intervals between markers P2449-185 and WMC231 on chromosome 3 B, with 19.2 and 16.0 cM from the flanking markers using DH population, and mapped in the intervals between markers Xfbb117 and Xgwm566 on chromosome 3 B, with 3.4 and 6.4 cM from the flanking markers using RIL population; At the same time, the specific primers of TaDREB1-a/b/d were designed by using wheat variety Hanxuan 10 as the material, and the expression pattern of TaDREB1-a/b/d under 4 stresses was analyzed by qRT-PCR. The expression peak and degree of TaDREB1-a/b/d were different in leaves and roots. TaDREB1-a/b expression were up-regulated in leaves under PEG, NaCl and ABA condition, while the expression of TaDREB1-d were down-regulated.TaDREB1-a/b/d was up-regulated under low temperature in leaves. In roots, the expression of TaDREB1-a/b/d were up-regulated in all condition. TaDREB1-a/b/d were all involved in responding to PEG, NaCl, ABA and4 ℃ stresses, but different in expression patterns. The functional markers developed in this study provides the tools for molecular marker-assisted selective breeding. Meanwhile, the relationship between TaDREB1-a/b/d and abiotic stress response is preliminarily explained at the genome level, laying a foundation for in-depth exploration of the molecular mechanism of wheat stress resistance.
引文
冯军,郑彩霞.2011.DREB转录因子在植物非生物胁迫中的作用及应用研究[J].植物生理学报,47(5):437-442.(Fen J,Zheng C X.2011.Research and application prospect of DREB transcription factor in plant abiotic stress resistance[J].Plant Physiology Journal,47(5):437-442.)
洪波,仝征,马男,等.2006.AtDREB1A基因在菊花中的异源表达提高了植株对干旱和盐渍胁迫的耐性[J].中国科学C辑(生命科学),36(3):223-231.(Hong B,Tong Z,Ma N,et al.2006.Heterologous expression of AtDREB1A gene in chrysanthemum increases plant tolerance to drought and salt stress[J].Science in China Series C(Life Sciences),36(3):223-231.)
李付振,邱新棉,刘传亮.2010.棉花中一个新的类DREB转录因子GhDREB2B的克隆、序列特征及表达分析[J].浙江农业学报,22(5):564-569.(Li F Z,Qiu X M,Liu C L.2010.Molecular cloning,characterization and expression of a novel DREB transcript factor GhDREB2B in cotton[J].Acta Agriculturae Zhejiang Gensis,22(5):564-569.)
李健,王雅晴,刘洋,等.2017.CBF转录因子在植物抗逆和生长发育中的重要功能[J].植物生理学报,53(12):2045-2056.(Li J,Wang Y Q,Liu Y,et al.2017.Important functions of CBF transcription factor in plant stress resistance and growth[J].Plant Physiology Journal,53(12):2045-2056.)
李小双,梁玉青,高贝,等.2017.植物抗逆相关转录因子基因DREB的研究展望[J].分子植物育种,15(7):2612-2622.(Li X S,Liang Y Q,Gao B,et al.2017.Research advance of the plant stress-related DREB transcriptional factor genes[J].Molecular Plant Breeding,15(7):2612-2622.)
刘晓颖,陈丽媛,张竞秋,等.2013.白菜脱水应答转录因子BpDREB1基因的克隆及功能研究[J].作物学报,39(2):230-237.(Liu X Y,Chen L Y,Zhang J Q,et al.2013.Isolation and functional analysis of a new DREB transcription factor(BpDREB1)from Brassica pekinensis[J].Acta Agronomica Sinica,39(2):230-237.)
倪志勇,徐兆师,刘丽,等.2008.小麦转录因子TaDREB6基因的克隆及鉴[J].麦类作物学报,28(3):357-363.(Ni Z Y,Xu Z S,Liu L,et al.2008.Isolation and characterization of a transcription factor TaDREB6 gene from Triticum aestivum L.[J].Journal o f Triticeae Crops,28(3):357-363.)
普正菲.2016.小麦抗逆相关转录因子基因DREB4A、W17转化株系筛选及其抗旱性鉴定[D].硕士学位论文,西北农林科技大学,导师:闵东红,pp.1-2.(Pu Z F.2016.Screening and identification of drought resistance with transcription factor genes DREB 4A and W17 transgenic lines in wheat[D].Thesis for M.S.,Northwest A&F University,Supervisor:Min D H,pp.1-2.)
谢登雷,崔江慧,常金华.2013.高粱中SbDREB2基因的克隆与表达分析[J].作物学报,39(8):1352-1359.(Xie D LCui J H,Chang J H.2013.Cloning and expression analysis of sbDREB2 gene from Sorghum bicolor[J].Acta Agronomica Sinica,39(8):1352-1359.)
Challam C,Ghosh T,Rai M,et al.2015.Allele mining across DREB1A and DREB1B in diverse rice genotypes sugges a highly conserved pathway inducible by low temperature[J].Journal of Genetics,2(94):231-238.
Charfeddine M,Bouaziz D,Charfeddine S,et al.2015.Overexpression of dehydration-responsive element-binding 1protein(DREB1)in transgenic Solanum tuberosum enhances tolerance to biotic stress[J].Plant Biotechnology Reports,9:79-88.
Chen J Q,Meng X P,Zhang Y,et al.2008.Over-expression of OsDREB genes lead to enhanced drought tolerance in rice[J].Biotechnology Letters,30(12):2191-2198.
Chen M,Xu Z S,Xia L Q,et al.2009.Cold-induced modulation and functional analyses of the DRE-binding transcription factor gene,GmDREB3,in soybean(Glycine max L.)[J].Journal of Experimental Botany,60(1):121-135.
Dary J,Somers,Peter I,et al.2004.A high-density microsatellite consensus map for bread wheat(Triticum aestivum L.)[J].Theoretical and Applied Genetics,109:1105-1114.
Hao Z F,Chang X P,Guo X J,et al.2003.QTL mapping for drought tolerance at stages of germination and seedling in wheat(Triticum aestivum L.)using a DH population[J].Agricultural Sciences in China(English),(2):943-949.
Kim Y H,Yang K S,Ryu S H,et al.2008.Molecular characterization of a cDNA encoding DRE-binding transcription factor from dehydration-treated fibrous roots of swee potato[J].Plant Physiology and Biochemistry,46(2)196-204.
Li X P,Tian A G,Luo G Z,et al.2005.Soybean DRE-binding transcription factors that are responsive to abiotic stresses[J].Theoretical and Applied Genetics,110:1355-1362.
Liang M X,Chen D,Lin M M,et al.2014.Isolation and characterization of two DREB1 genes encoding dehydrationresponsive element binding proteins in chicory(Cichoriumintybus)[J].Plant Growth Regulation,73:45-55.
Liu N,Zhong N Q,Wang G L,et al.2007.Cloning and functional characterization of PpDBF1 gene encoding a DRE-binding transcription factor from physcomitrella patens[J].Planta,226:827-838.
Liu Q,Kasuga M,Sakuma Y,et al.1998.Two transcription factors,DREB1 and DREB2,with an EREBP/AP2 DNAbinding domain separate two cellular signal transduction pathways in drought and low temperature responsive gene expression,respectively,in Arabidopsis[J].Plant Cell,10:1391-1406.
Marion S R,Victor K,Katja W,et al.1998.A microsatellite map of wheat[J].Genetics,149:2007-2023.
Mondini L,Nachit M M,Pagnotta M A.2015.Allelic variants in durum wheat(Triticum turgidum L.var.durum)DREB genes conferring tolerance to abiotic stresses[J].Molecular Genetics and Genomics,290:531-544.
Qin F,Sakuma Y,Li J,et al.2004.Cloning and functional analysis of a novel DREB1/CBF transcription factor involved in cold-responsive gene expression in Zea mays L.[J].Plant Cell Physiol,45(8):1042-1052.
Rehman S,Mahmood T.2015.Functional role of DREB and ERF transcription factors:Regulating stress-responsive network in plants[J].Acta Physiol Plant,37:178.
Shen Y G,Zhang W K,He S J,et al.2003.An EREBP/AP2-type protein in Triticum aestivum was a DRE-binding transcription factor induced by cold,dehydration and ABA stress[J].Theoretical and Applied Genetics,106:923-930.
Su J Y,Zheng Q,Li H W,et al.2009.Detection of QTLs for phosphorus use efficiency in relation to agronomic performance of wheat grown under phosphorus sufficient and limited conditions[J].Plant Science,176:824-836.
Wang J W,Yang F P,Chen X Q.2006.Induced expression of DREB transcriptional factor and study on its physiologycal effects of drought tolerance in transgenic wheat[J].Acta Genetica Sinica,33(5):468-476.
Wei B,Jing R L,Wang C S,et al.2009.DREB1 genes in wheat(Triticum aestivum L.):Development of functional markers and gene mapping based on SNPs[J].Molecular Breeding,23:13-22.
Wu X S,Chang X P,Jing R L.2012.Genetic insight into yieldassociated traits of wheat grown in multiple tainfed environments[J].PLoS One,7:e31249.
洪波,仝征,马男,等.2006.AtDREB1A基因在菊花中的异源表达提高了植株对干旱和盐渍胁迫的耐性[J].中国科学C辑(生命科学),36(3):223-231.(Hong B,Tong Z,Ma N,et al.2006.Heterologous expression of AtDREB1A gene in chrysanthemum increases plant tolerance to drought and salt stress[J].Science in China Series C(Life Sciences),36(3):223-231.)
李付振,邱新棉,刘传亮.2010.棉花中一个新的类DREB转录因子GhDREB2B的克隆、序列特征及表达分析[J].浙江农业学报,22(5):564-569.(Li F Z,Qiu X M,Liu C L.2010.Molecular cloning,characterization and expression of a novel DREB transcript factor GhDREB2B in cotton[J].Acta Agriculturae Zhejiang Gensis,22(5):564-569.)
李健,王雅晴,刘洋,等.2017.CBF转录因子在植物抗逆和生长发育中的重要功能[J].植物生理学报,53(12):2045-2056.(Li J,Wang Y Q,Liu Y,et al.2017.Important functions of CBF transcription factor in plant stress resistance and growth[J].Plant Physiology Journal,53(12):2045-2056.)
李小双,梁玉青,高贝,等.2017.植物抗逆相关转录因子基因DREB的研究展望[J].分子植物育种,15(7):2612-2622.(Li X S,Liang Y Q,Gao B,et al.2017.Research advance of the plant stress-related DREB transcriptional factor genes[J].Molecular Plant Breeding,15(7):2612-2622.)
刘晓颖,陈丽媛,张竞秋,等.2013.白菜脱水应答转录因子BpDREB1基因的克隆及功能研究[J].作物学报,39(2):230-237.(Liu X Y,Chen L Y,Zhang J Q,et al.2013.Isolation and functional analysis of a new DREB transcription factor(BpDREB1)from Brassica pekinensis[J].Acta Agronomica Sinica,39(2):230-237.)
倪志勇,徐兆师,刘丽,等.2008.小麦转录因子TaDREB6基因的克隆及鉴[J].麦类作物学报,28(3):357-363.(Ni Z Y,Xu Z S,Liu L,et al.2008.Isolation and characterization of a transcription factor TaDREB6 gene from Triticum aestivum L.[J].Journal o f Triticeae Crops,28(3):357-363.)
普正菲.2016.小麦抗逆相关转录因子基因DREB4A、W17转化株系筛选及其抗旱性鉴定[D].硕士学位论文,西北农林科技大学,导师:闵东红,pp.1-2.(Pu Z F.2016.Screening and identification of drought resistance with transcription factor genes DREB 4A and W17 transgenic lines in wheat[D].Thesis for M.S.,Northwest A&F University,Supervisor:Min D H,pp.1-2.)
谢登雷,崔江慧,常金华.2013.高粱中SbDREB2基因的克隆与表达分析[J].作物学报,39(8):1352-1359.(Xie D LCui J H,Chang J H.2013.Cloning and expression analysis of sbDREB2 gene from Sorghum bicolor[J].Acta Agronomica Sinica,39(8):1352-1359.)
Challam C,Ghosh T,Rai M,et al.2015.Allele mining across DREB1A and DREB1B in diverse rice genotypes sugges a highly conserved pathway inducible by low temperature[J].Journal of Genetics,2(94):231-238.
Charfeddine M,Bouaziz D,Charfeddine S,et al.2015.Overexpression of dehydration-responsive element-binding 1protein(DREB1)in transgenic Solanum tuberosum enhances tolerance to biotic stress[J].Plant Biotechnology Reports,9:79-88.
Chen J Q,Meng X P,Zhang Y,et al.2008.Over-expression of OsDREB genes lead to enhanced drought tolerance in rice[J].Biotechnology Letters,30(12):2191-2198.
Chen M,Xu Z S,Xia L Q,et al.2009.Cold-induced modulation and functional analyses of the DRE-binding transcription factor gene,GmDREB3,in soybean(Glycine max L.)[J].Journal of Experimental Botany,60(1):121-135.
Dary J,Somers,Peter I,et al.2004.A high-density microsatellite consensus map for bread wheat(Triticum aestivum L.)[J].Theoretical and Applied Genetics,109:1105-1114.
Hao Z F,Chang X P,Guo X J,et al.2003.QTL mapping for drought tolerance at stages of germination and seedling in wheat(Triticum aestivum L.)using a DH population[J].Agricultural Sciences in China(English),(2):943-949.
Kim Y H,Yang K S,Ryu S H,et al.2008.Molecular characterization of a cDNA encoding DRE-binding transcription factor from dehydration-treated fibrous roots of swee potato[J].Plant Physiology and Biochemistry,46(2)196-204.
Li X P,Tian A G,Luo G Z,et al.2005.Soybean DRE-binding transcription factors that are responsive to abiotic stresses[J].Theoretical and Applied Genetics,110:1355-1362.
Liang M X,Chen D,Lin M M,et al.2014.Isolation and characterization of two DREB1 genes encoding dehydrationresponsive element binding proteins in chicory(Cichoriumintybus)[J].Plant Growth Regulation,73:45-55.
Liu N,Zhong N Q,Wang G L,et al.2007.Cloning and functional characterization of PpDBF1 gene encoding a DRE-binding transcription factor from physcomitrella patens[J].Planta,226:827-838.
Liu Q,Kasuga M,Sakuma Y,et al.1998.Two transcription factors,DREB1 and DREB2,with an EREBP/AP2 DNAbinding domain separate two cellular signal transduction pathways in drought and low temperature responsive gene expression,respectively,in Arabidopsis[J].Plant Cell,10:1391-1406.
Marion S R,Victor K,Katja W,et al.1998.A microsatellite map of wheat[J].Genetics,149:2007-2023.
Mondini L,Nachit M M,Pagnotta M A.2015.Allelic variants in durum wheat(Triticum turgidum L.var.durum)DREB genes conferring tolerance to abiotic stresses[J].Molecular Genetics and Genomics,290:531-544.
Qin F,Sakuma Y,Li J,et al.2004.Cloning and functional analysis of a novel DREB1/CBF transcription factor involved in cold-responsive gene expression in Zea mays L.[J].Plant Cell Physiol,45(8):1042-1052.
Rehman S,Mahmood T.2015.Functional role of DREB and ERF transcription factors:Regulating stress-responsive network in plants[J].Acta Physiol Plant,37:178.
Shen Y G,Zhang W K,He S J,et al.2003.An EREBP/AP2-type protein in Triticum aestivum was a DRE-binding transcription factor induced by cold,dehydration and ABA stress[J].Theoretical and Applied Genetics,106:923-930.
Su J Y,Zheng Q,Li H W,et al.2009.Detection of QTLs for phosphorus use efficiency in relation to agronomic performance of wheat grown under phosphorus sufficient and limited conditions[J].Plant Science,176:824-836.
Wang J W,Yang F P,Chen X Q.2006.Induced expression of DREB transcriptional factor and study on its physiologycal effects of drought tolerance in transgenic wheat[J].Acta Genetica Sinica,33(5):468-476.
Wei B,Jing R L,Wang C S,et al.2009.DREB1 genes in wheat(Triticum aestivum L.):Development of functional markers and gene mapping based on SNPs[J].Molecular Breeding,23:13-22.
Wu X S,Chang X P,Jing R L.2012.Genetic insight into yieldassociated traits of wheat grown in multiple tainfed environments[J].PLoS One,7:e31249.