铃铛刺抗逆相关DREB/ERF转录因子基因的克隆与鉴定
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摘要
转录调控是植物应答逆境等一系列基因表达的最主要调控形式,其中DREB和ERF类转录因子主要参与生物和(或)非生物胁迫的响应。本研究以新疆沙生植物铃铛刺为材料,从中分离克隆新的DREB、ERF转录因子基因,以期为利用基因工程改良作物的抗逆性提够优良候选基因,同时为豆科植物耐逆胁迫的分子机制研究和作物抗逆育种提供理论基础。
本研究取得以下成果:
1.据植物中AP2/EREBP类基因DNA结合域保守区设计简并引物,以铃铛刺叶片为材料,RT-PCR扩增出2个AP2/EREBP基因同源片段;据此设计基因特异引物,通过RACE-PCR分离克隆出1个DREB、1个ERF转录因子基因,分别命名为HhDREB2、HhERF2,登陆号为:EU872018和FJ032362。
2.半定量RT-PCR对2个基因的组织特异性表达情况进行了检测,结果在铃铛刺的根、茎、叶中均有表达;胁迫应答检测结果显示,两个基因的表达均受干旱、高盐和低温诱导,HhERF2还受茉莉酸甲酯的诱导。
3.以基因组DNA为模板对HhDREB2、HhERF2进行了PCR扩增,扩增产物经序列分析显示,HhDREB2不含内含子,HhERF2含有1个内含子。
4.酵母单杂交分析结果显示,HhDREB2和HhERF2都具有转录激活功能,β-半乳糖苷酶活性分别为30.37U和49.26U,Whatman滤纸显色均能呈现明显蓝色。
5.构建绿色荧光蛋白融合表达载体,利用基因枪法转化洋葱表皮细胞的瞬时表达结果显示,HhDREB2和HhERF2蛋白均定位于细胞核中。
6.构建植物表达载体pC13rd29A-HhDREB2、pC13rd29A-HhERF2及pC1302-HhERF2,利用Floral-dip法转化野生型拟南芥Columbia-0;通过Hyg抗性、PCR及RT-PCR筛选鉴定阳性苗;耐胁迫检测结果表明,HhDREB2、HhERF2在拟南芥中的表达,增强了转基因拟南芥对干旱、高盐、低温及细菌的胁迫耐受性。
Transcription control is the most vital regulation ways of the genes expressed in plants response to stress. Transcription factors DREB and ERF are involved in the response of organism to biotic and/or abiotic stress. In this study, two novel genes encoding DREB and ERF transcription factors were isolated from salttree cultivars. The purpose of this research was to provide candidate genes to enhance crop tolerance to adverse conditions, meanwhile reveal the mechanism of stress tolerance in leguminous plant and provide a theoretical basis for crop tolerance.
The following results were achieved in this study:
1. According to the conserved regions of the AP2/EREBP DNA binding domains a pair of degenerate primers was designed. Using RT-PCR two fragments were amplified from leaves of salttree cultivars. Two novel genes encoding AP2/EREBP transcription factors, HhDREB2 and HhERF2 were isolated by RACE-PCR. GeneBank numbers are EU872018 and FJ032362, respectively.
2. The expression pattern of HhDREB2 and HhERF2 in different organs was studied using semi-quantitative RT-PCR. It was proved that they expressed in roots, stems, and leaves of salttree, and were greatly induced by drought, high salinity and low temperature. In addition, HhERF2 were greatly induced by methyl jasmonate.
3. HhDREB2 and HhERF2 were amplified using genomic DNA as templates. It was proved that HhDREB2 lacked intron. However, HhERF2 contained one intron.
4. HhDREB2 and HhERF2 proteins both had transcriptional activation confirmed by the yeast one-hybrird method, and theirβ-galactosidase activity were 30.37 U and 49.26 U, respectively. The the yeast transformants of HhDREB2 and HhERF2 showed a clear blue in the colony-lift filters assay.
5. We constructed two green fluorescent protein fusion expression vectors . They were transformed into the epidermal cells of onion by particle bombardmental method. Both HhDREB2 and HhERF2 proteins were localized in cell nucleus.
6. HhDREB2 and HhERF2 were transformed into Arabidopsis thaliana with Agrobacterium tumefaciens C58C1 containing the plant expression vectors pC13rd29A-HhDREB2, pC13rd29A-HhERF2 and pC1302-HhERF2, respectively. The positive Arabidopsis thaliana transformants were selected using MS medium containing hygromycin, PCR and RT-PCR. Results of stress tolerance experiments showed that the expression of HhDREB2 and HhERF2 could improve resistance to drought, high salinity, low temperature and bacteria of transgenic Arabidopsis thaliana obviously.
本研究取得以下成果:
1.据植物中AP2/EREBP类基因DNA结合域保守区设计简并引物,以铃铛刺叶片为材料,RT-PCR扩增出2个AP2/EREBP基因同源片段;据此设计基因特异引物,通过RACE-PCR分离克隆出1个DREB、1个ERF转录因子基因,分别命名为HhDREB2、HhERF2,登陆号为:EU872018和FJ032362。
2.半定量RT-PCR对2个基因的组织特异性表达情况进行了检测,结果在铃铛刺的根、茎、叶中均有表达;胁迫应答检测结果显示,两个基因的表达均受干旱、高盐和低温诱导,HhERF2还受茉莉酸甲酯的诱导。
3.以基因组DNA为模板对HhDREB2、HhERF2进行了PCR扩增,扩增产物经序列分析显示,HhDREB2不含内含子,HhERF2含有1个内含子。
4.酵母单杂交分析结果显示,HhDREB2和HhERF2都具有转录激活功能,β-半乳糖苷酶活性分别为30.37U和49.26U,Whatman滤纸显色均能呈现明显蓝色。
5.构建绿色荧光蛋白融合表达载体,利用基因枪法转化洋葱表皮细胞的瞬时表达结果显示,HhDREB2和HhERF2蛋白均定位于细胞核中。
6.构建植物表达载体pC13rd29A-HhDREB2、pC13rd29A-HhERF2及pC1302-HhERF2,利用Floral-dip法转化野生型拟南芥Columbia-0;通过Hyg抗性、PCR及RT-PCR筛选鉴定阳性苗;耐胁迫检测结果表明,HhDREB2、HhERF2在拟南芥中的表达,增强了转基因拟南芥对干旱、高盐、低温及细菌的胁迫耐受性。
Transcription control is the most vital regulation ways of the genes expressed in plants response to stress. Transcription factors DREB and ERF are involved in the response of organism to biotic and/or abiotic stress. In this study, two novel genes encoding DREB and ERF transcription factors were isolated from salttree cultivars. The purpose of this research was to provide candidate genes to enhance crop tolerance to adverse conditions, meanwhile reveal the mechanism of stress tolerance in leguminous plant and provide a theoretical basis for crop tolerance.
The following results were achieved in this study:
1. According to the conserved regions of the AP2/EREBP DNA binding domains a pair of degenerate primers was designed. Using RT-PCR two fragments were amplified from leaves of salttree cultivars. Two novel genes encoding AP2/EREBP transcription factors, HhDREB2 and HhERF2 were isolated by RACE-PCR. GeneBank numbers are EU872018 and FJ032362, respectively.
2. The expression pattern of HhDREB2 and HhERF2 in different organs was studied using semi-quantitative RT-PCR. It was proved that they expressed in roots, stems, and leaves of salttree, and were greatly induced by drought, high salinity and low temperature. In addition, HhERF2 were greatly induced by methyl jasmonate.
3. HhDREB2 and HhERF2 were amplified using genomic DNA as templates. It was proved that HhDREB2 lacked intron. However, HhERF2 contained one intron.
4. HhDREB2 and HhERF2 proteins both had transcriptional activation confirmed by the yeast one-hybrird method, and theirβ-galactosidase activity were 30.37 U and 49.26 U, respectively. The the yeast transformants of HhDREB2 and HhERF2 showed a clear blue in the colony-lift filters assay.
5. We constructed two green fluorescent protein fusion expression vectors . They were transformed into the epidermal cells of onion by particle bombardmental method. Both HhDREB2 and HhERF2 proteins were localized in cell nucleus.
6. HhDREB2 and HhERF2 were transformed into Arabidopsis thaliana with Agrobacterium tumefaciens C58C1 containing the plant expression vectors pC13rd29A-HhDREB2, pC13rd29A-HhERF2 and pC1302-HhERF2, respectively. The positive Arabidopsis thaliana transformants were selected using MS medium containing hygromycin, PCR and RT-PCR. Results of stress tolerance experiments showed that the expression of HhDREB2 and HhERF2 could improve resistance to drought, high salinity, low temperature and bacteria of transgenic Arabidopsis thaliana obviously.
引文
[1]张木清,陈如凯.作物抗旱分子生理与遗传改良.北京:科学出版社,2006:100-110
[2]杨致荣,王兴春,李西明,等.高等植物转录因子的研究进展[J].遗传,2004,26(3):403-408
[3]Jakoby M.,Weisshaar B.,Droge-laser W.,et al.bZIP transcription factors in Ara-bidopsis[J].Trends Plant Sci,2002,7(3):106-111
[4]陈俊,王宗阳.植物MYB类转录因子的研究进展[J].植物生理学与分子生物学学报,2002,28(2):81-88
[5]Abe H.,Urao T.,Mark D.,et al.AtMYC2(bHLH) and AtMYB2(MYB) function as transcriptional activators in abscisic acid signaling[J].Plant Cell,2003,15(1):63-78
[6]Robatzek S.,Somssich I.E..A new member of the Arabidopsis WRKY transcription factor family,AtWRKY6,is associated with both senescence-and defence-related processes[J].Plant Journal,2001,28:123-133
[7]Kalde M.,Barth M.,Somssich I.E.,et al.Members of the Arabidopsis WRKY group Ⅲtrascription factors are part of different plant defense signaling pathways[J].Mol Plant Microbe Interact,2003,16:295-305
[8]Wu K.L.,Guo Z.J.,Wang H.H.,et al.The WRKY family of transcriptin factors in rice and Arabidopsis and their origins[J].2005,DNA Res,12:9-26
[9]Guo Z.J.,Kan Y.C.,Chen X.J.,et al.Characterization a rice WRKY gene whose expression is induced by pathogen attack and mechanical wounding[J].Acta Bot Sin,2004,46:955-964
[10]Mare C.,Mazzucotelli E.,Crosatti C.,et al.HvWRKY38:a new transcription factor involved in cold- and drought- response in barley[J].Plant Mol Biol,2004,255:399-416
[11]Rizhsky L.,Liang H.,Mittler R.The combined effect of drought stress and heat shock on gene expression in tobacco[J].Plant Physiol,2003,130:1143-1151
[12]仇玉萍,荆邵娟,付坚,等.13个水稻WRKY基因的克隆及其表达谱分析[J].科学通报,2004,49:1860-1869
[13]Rizhsky L.,Davletova S.,Liang H.,et al.The zinc finger protein zet2 is required for cytosolic ascorbate peroxidase expression during oxidative stress in Arabidopsis[J].Biol Chem,2004,279:11736-11743
[14]Wu X.M.,Lim S.H.,Yang W.C.,et al.Characterization expression and phylogenetic study of R2R3-MYB genes in orchid[J].Plant Mol Biol,2003,51(6):959-972
[15] Shen Y.G. An DREBP/AP2 type protein in Triticum aestivum as a DRE binding transcription factor induced by cold, dehydration and ABA stress[J]. Theor Appl Genet, 2003,106: 923-930
[16] Jeong M.P.. Over expression of the tobacco Tsil gene encoding an EREBP/AP2 type transcription factor enhances resistance against pathogen attack and osmotic stress in tabacco[J]. Plant Cell, 2001,13: 1035-1046
[17] Schwechheimer C, Bevan M.. The regulation of transcription factor activity in plants[J]. Trends Plant Sci, 1998, 3: 278-283
[18] Wuitschick J.D., Lindstrim P.R., Meyer A.E., et al. Homing endonulease encoded by germ line limeted genes in Tetrahymena thermophila having APETELA2 DNA binding domains[J]. Eukaryotic Cell, 2004, 3: 685-694
[19] Magnani E., Sjolander K., Hake S., et al. From endonuclease to transcription factors: evolution of the AP2 DNA binding domain in plants[J]. Trends Plant Sci, 2005, 10: 54-56
[20] Mizuno S., Hirasawa Y., Sonoda M.,et al. Isolation and characterization of three DREB/ERF-type transcriptionfactors from melon (Cucumis melo)[J]. Plant Science, 2006,170:1156-1163
[21] Wang Q.Y., Guan Y.C., Wu Y.R., et al. Overexpression of a rice OsDREB1F gene increases salt, drought, and low temperature tolerance in both Arabidopsis and rice[J]. Plant Mol Biol, 2008, 67: 589-602
[22] Schramm F., Larkindale J., Kiehlmann E., et al. A cascade of transcription factor DREB2A and heat stress transcription factor HsfA3 regulates the heat stress response of Arabidopsis[J]. The Plant Journal, 2008, 53: 264-274
[23] Liu L.Q., Zhu K., Yang Y.F., et al. Molecular cloning, expression profiling and trans-activation property studies of a DREB2-like gene from chrysanthemum(Dend-ranthema vestitum)[J]. J Plant Res, 2008, 121: 215-226
[24] Nakano T., Suzuki K., Fujimura T., et al. Genome wide analysis of the ERF gene family in Arabidopsis and rice[J]. Plant Physiol, 2006,140: 411-432
[25] Kasuga M., Liu Q., Miura S., et al. Improving plant drought, salt and freezing tolerance by gene transfer of a single stress inducible transcription factor[J]. Nature Biotech, 1999, 17:287-291
[26] Park J.M., Park C.J., Lee S.B., et al. Over-expression of the tobacco Tsil gene encoding an EREBP/AP2 type transcription factor enhances resistance against pathogen attack and osmotic stress in tobacco[J]. Plant Cell, 2002, 13: 1035-1046
[27] Chakravarthy S., Tuori R.P., Dascenzo M.D., et al. The tomato transcription factor Pti4 regulates defense related genes expression via GCC-box and non-GCC box cis elements[J].Plant Cell,2003,15:3033-3050
[28]Guo Z.J.,Chen X.J.,Wu X.L.,et al.Over-expression of the AP2/EREBP transcription factor OPBP1 enhances disease resistance and salt tolerance stress in tobacco[J].Plant Mol Biol,2004,55:607-618
[29]Wang H.,Huang Z.j.,Huang R.f.,et al.Ectopic over-expression of tomato JERF3 in tobacco activates downstream genes expression and enhances salt tolerance[J].Plant Mol Biol,2004,55:183-192
[30]Xu Z.S.,Xia L.Q.,Chen M.,et al.Isolation and molecular characterization of the Triticum aestivum L.ethylene-responsive factor 1(TaERF1) that increases multiple stress tolerance[J].Plant Mol Biol,2007,58:587-601
[31]ZUO K.J.,Tang K.X.,Chen X.J.,et al.Over-expression GbERF2 transcription factors in tobaco enhances brown spot disease resistance by activating expression of downstream genes[J].Gene,2007,391(12):80-90
[32]Duan H.,Li F..Cloning and characterization of two EREBP transcription factors from cotton(Gossypium hirsutum L.)[J].Biochemistry,2006,71(3):285-293
[33]Cao Y.F.,Song F.M.,Goodman R.M.,et al.Molecular characterization of four rice genes encoding ethylene-responsive transcriptional factors and their expressions in response to biotic and abiotic stress[J].Journal of Plant Physiology,2006,163:1167-1178
[34]Sambrook J..分子克隆实验指南[M].黄培堂译.第三版.北京:科学出版社,2002:1595-1796
[35]曹孜义.实用植物组织培养技术教程[M].修订本.兰州:甘肃科学技术出版社,2002:36-42
[36]杨文杰.大豆MYB转录因子基因的克隆及其表达研究[D].雅安:四川农业大学,2007
[37]赵世杰,李德全.现代植物生理学实验指南[M].北京:科学出版社,1999
[38]王晓飞.大豆转录因子GmDREB的功能分析[D].北京:中国农业科学院,2008
[2]杨致荣,王兴春,李西明,等.高等植物转录因子的研究进展[J].遗传,2004,26(3):403-408
[3]Jakoby M.,Weisshaar B.,Droge-laser W.,et al.bZIP transcription factors in Ara-bidopsis[J].Trends Plant Sci,2002,7(3):106-111
[4]陈俊,王宗阳.植物MYB类转录因子的研究进展[J].植物生理学与分子生物学学报,2002,28(2):81-88
[5]Abe H.,Urao T.,Mark D.,et al.AtMYC2(bHLH) and AtMYB2(MYB) function as transcriptional activators in abscisic acid signaling[J].Plant Cell,2003,15(1):63-78
[6]Robatzek S.,Somssich I.E..A new member of the Arabidopsis WRKY transcription factor family,AtWRKY6,is associated with both senescence-and defence-related processes[J].Plant Journal,2001,28:123-133
[7]Kalde M.,Barth M.,Somssich I.E.,et al.Members of the Arabidopsis WRKY group Ⅲtrascription factors are part of different plant defense signaling pathways[J].Mol Plant Microbe Interact,2003,16:295-305
[8]Wu K.L.,Guo Z.J.,Wang H.H.,et al.The WRKY family of transcriptin factors in rice and Arabidopsis and their origins[J].2005,DNA Res,12:9-26
[9]Guo Z.J.,Kan Y.C.,Chen X.J.,et al.Characterization a rice WRKY gene whose expression is induced by pathogen attack and mechanical wounding[J].Acta Bot Sin,2004,46:955-964
[10]Mare C.,Mazzucotelli E.,Crosatti C.,et al.HvWRKY38:a new transcription factor involved in cold- and drought- response in barley[J].Plant Mol Biol,2004,255:399-416
[11]Rizhsky L.,Liang H.,Mittler R.The combined effect of drought stress and heat shock on gene expression in tobacco[J].Plant Physiol,2003,130:1143-1151
[12]仇玉萍,荆邵娟,付坚,等.13个水稻WRKY基因的克隆及其表达谱分析[J].科学通报,2004,49:1860-1869
[13]Rizhsky L.,Davletova S.,Liang H.,et al.The zinc finger protein zet2 is required for cytosolic ascorbate peroxidase expression during oxidative stress in Arabidopsis[J].Biol Chem,2004,279:11736-11743
[14]Wu X.M.,Lim S.H.,Yang W.C.,et al.Characterization expression and phylogenetic study of R2R3-MYB genes in orchid[J].Plant Mol Biol,2003,51(6):959-972
[15] Shen Y.G. An DREBP/AP2 type protein in Triticum aestivum as a DRE binding transcription factor induced by cold, dehydration and ABA stress[J]. Theor Appl Genet, 2003,106: 923-930
[16] Jeong M.P.. Over expression of the tobacco Tsil gene encoding an EREBP/AP2 type transcription factor enhances resistance against pathogen attack and osmotic stress in tabacco[J]. Plant Cell, 2001,13: 1035-1046
[17] Schwechheimer C, Bevan M.. The regulation of transcription factor activity in plants[J]. Trends Plant Sci, 1998, 3: 278-283
[18] Wuitschick J.D., Lindstrim P.R., Meyer A.E., et al. Homing endonulease encoded by germ line limeted genes in Tetrahymena thermophila having APETELA2 DNA binding domains[J]. Eukaryotic Cell, 2004, 3: 685-694
[19] Magnani E., Sjolander K., Hake S., et al. From endonuclease to transcription factors: evolution of the AP2 DNA binding domain in plants[J]. Trends Plant Sci, 2005, 10: 54-56
[20] Mizuno S., Hirasawa Y., Sonoda M.,et al. Isolation and characterization of three DREB/ERF-type transcriptionfactors from melon (Cucumis melo)[J]. Plant Science, 2006,170:1156-1163
[21] Wang Q.Y., Guan Y.C., Wu Y.R., et al. Overexpression of a rice OsDREB1F gene increases salt, drought, and low temperature tolerance in both Arabidopsis and rice[J]. Plant Mol Biol, 2008, 67: 589-602
[22] Schramm F., Larkindale J., Kiehlmann E., et al. A cascade of transcription factor DREB2A and heat stress transcription factor HsfA3 regulates the heat stress response of Arabidopsis[J]. The Plant Journal, 2008, 53: 264-274
[23] Liu L.Q., Zhu K., Yang Y.F., et al. Molecular cloning, expression profiling and trans-activation property studies of a DREB2-like gene from chrysanthemum(Dend-ranthema vestitum)[J]. J Plant Res, 2008, 121: 215-226
[24] Nakano T., Suzuki K., Fujimura T., et al. Genome wide analysis of the ERF gene family in Arabidopsis and rice[J]. Plant Physiol, 2006,140: 411-432
[25] Kasuga M., Liu Q., Miura S., et al. Improving plant drought, salt and freezing tolerance by gene transfer of a single stress inducible transcription factor[J]. Nature Biotech, 1999, 17:287-291
[26] Park J.M., Park C.J., Lee S.B., et al. Over-expression of the tobacco Tsil gene encoding an EREBP/AP2 type transcription factor enhances resistance against pathogen attack and osmotic stress in tobacco[J]. Plant Cell, 2002, 13: 1035-1046
[27] Chakravarthy S., Tuori R.P., Dascenzo M.D., et al. The tomato transcription factor Pti4 regulates defense related genes expression via GCC-box and non-GCC box cis elements[J].Plant Cell,2003,15:3033-3050
[28]Guo Z.J.,Chen X.J.,Wu X.L.,et al.Over-expression of the AP2/EREBP transcription factor OPBP1 enhances disease resistance and salt tolerance stress in tobacco[J].Plant Mol Biol,2004,55:607-618
[29]Wang H.,Huang Z.j.,Huang R.f.,et al.Ectopic over-expression of tomato JERF3 in tobacco activates downstream genes expression and enhances salt tolerance[J].Plant Mol Biol,2004,55:183-192
[30]Xu Z.S.,Xia L.Q.,Chen M.,et al.Isolation and molecular characterization of the Triticum aestivum L.ethylene-responsive factor 1(TaERF1) that increases multiple stress tolerance[J].Plant Mol Biol,2007,58:587-601
[31]ZUO K.J.,Tang K.X.,Chen X.J.,et al.Over-expression GbERF2 transcription factors in tobaco enhances brown spot disease resistance by activating expression of downstream genes[J].Gene,2007,391(12):80-90
[32]Duan H.,Li F..Cloning and characterization of two EREBP transcription factors from cotton(Gossypium hirsutum L.)[J].Biochemistry,2006,71(3):285-293
[33]Cao Y.F.,Song F.M.,Goodman R.M.,et al.Molecular characterization of four rice genes encoding ethylene-responsive transcriptional factors and their expressions in response to biotic and abiotic stress[J].Journal of Plant Physiology,2006,163:1167-1178
[34]Sambrook J..分子克隆实验指南[M].黄培堂译.第三版.北京:科学出版社,2002:1595-1796
[35]曹孜义.实用植物组织培养技术教程[M].修订本.兰州:甘肃科学技术出版社,2002:36-42
[36]杨文杰.大豆MYB转录因子基因的克隆及其表达研究[D].雅安:四川农业大学,2007
[37]赵世杰,李德全.现代植物生理学实验指南[M].北京:科学出版社,1999
[38]王晓飞.大豆转录因子GmDREB的功能分析[D].北京:中国农业科学院,2008