霸王、沙冬青、小叶锦鸡儿CBF/DREB1转录因子cDNA全长的克隆及生物信息学分析
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摘要
霸王(Zygophyllum xanthoxylon)、沙冬青(Ammopiptanthus mongolicus)、小叶锦鸡儿(Caragana microphylia Lam)是我国西北荒漠区典型的资源植物,不但具有重要的生态和经济价值,而且抗逆境能力强,是非常好的研究抗逆的实验材料。
CBF/DREB1(C-repeat binding factor/dehydration resistance element binding protein 1)基因是一类抗逆相关转录因子,它们的表达可以激活下游一系列逆境应答基因的表达,从整体水平提高植株的抗逆性。
本文利用RT-PCR和RACE技术首次克隆了霸王、沙冬青、小叶锦鸡儿CBF/DREB1基因cDNA全序列,并对其推导的氨基酸序列进行了相关生物信息学分析,确认其具有AP2结构域,且AP2域的两端拥有CBF家族特有的PKK/RPAGRxKFxETRHP和DSAWR短多肽序列,属于CBF家族。
通过对霸王、沙冬青、小叶锦鸡儿CBF/DREB1与其它植物来源的蛋白全序列比较发现,与霸王CBF/DREB1亲缘关系最近的是白桦(Betula platyphylla)的BpCBF,二者蛋白序列同源性为60.1%。沙冬青、小叶锦鸡儿因为都属于豆科,它们的CBF/DREB1与野生大豆(Glycine soja)CBF/DREB1的GsCBF1亲缘关系最近,蛋白序列同源性分别为49.6%和66.8%。
Zygophyllum xanthoxylon , Ammopiptanthus mongolicus and Caragana microphylia Lam are typical stress-resisting plants in the arid area of Northwest China. They are not only playing an important role in local ecosystem and economy, but also valuable material for stess resistance research.
CBF/DREB1(C-repeat binding factor/dehydration resistance element binding protein 1)is a family of transcription factors related to stress resistance, whose expression can trigger the activation of downstream genes involving in stress response.
In this paper, full-length cDNAs of the CBFs- ZxCBF from Zygophyllum xanthoxylon, AmCBF from Ammopiptanthus mongolicus and CmCBF from Caragana microphylia Lam were cloned by RT-PCR and RACE. The deduced proteins have the typilcal AP2/EREBP domain, and show significant sequence similarity with those known CBFs, especially in the conserved AP2/EREBP DNA-binding domain. Further bioinformatics analysis showes these ZxCBF , AmCBF and CmCBF belong to the CBF family of AP2-DNA-binding protein superfamily. It has an AP2 binding region with two special sects of short polypeptides of PKK/RPAGRxKFxETRHP and DSAWR.
Amino acid homology analysis showed the sequence of amino acids of ZxCBF , AmCBF and CmCBF share 69.1%, 60.1%, and 70.6% identity with proteins BpCBF(Betula platyphylla), GsCBF(Glycine soja), GsCBF, respectively.
CBF/DREB1(C-repeat binding factor/dehydration resistance element binding protein 1)基因是一类抗逆相关转录因子,它们的表达可以激活下游一系列逆境应答基因的表达,从整体水平提高植株的抗逆性。
本文利用RT-PCR和RACE技术首次克隆了霸王、沙冬青、小叶锦鸡儿CBF/DREB1基因cDNA全序列,并对其推导的氨基酸序列进行了相关生物信息学分析,确认其具有AP2结构域,且AP2域的两端拥有CBF家族特有的PKK/RPAGRxKFxETRHP和DSAWR短多肽序列,属于CBF家族。
通过对霸王、沙冬青、小叶锦鸡儿CBF/DREB1与其它植物来源的蛋白全序列比较发现,与霸王CBF/DREB1亲缘关系最近的是白桦(Betula platyphylla)的BpCBF,二者蛋白序列同源性为60.1%。沙冬青、小叶锦鸡儿因为都属于豆科,它们的CBF/DREB1与野生大豆(Glycine soja)CBF/DREB1的GsCBF1亲缘关系最近,蛋白序列同源性分别为49.6%和66.8%。
Zygophyllum xanthoxylon , Ammopiptanthus mongolicus and Caragana microphylia Lam are typical stress-resisting plants in the arid area of Northwest China. They are not only playing an important role in local ecosystem and economy, but also valuable material for stess resistance research.
CBF/DREB1(C-repeat binding factor/dehydration resistance element binding protein 1)is a family of transcription factors related to stress resistance, whose expression can trigger the activation of downstream genes involving in stress response.
In this paper, full-length cDNAs of the CBFs- ZxCBF from Zygophyllum xanthoxylon, AmCBF from Ammopiptanthus mongolicus and CmCBF from Caragana microphylia Lam were cloned by RT-PCR and RACE. The deduced proteins have the typilcal AP2/EREBP domain, and show significant sequence similarity with those known CBFs, especially in the conserved AP2/EREBP DNA-binding domain. Further bioinformatics analysis showes these ZxCBF , AmCBF and CmCBF belong to the CBF family of AP2-DNA-binding protein superfamily. It has an AP2 binding region with two special sects of short polypeptides of PKK/RPAGRxKFxETRHP and DSAWR.
Amino acid homology analysis showed the sequence of amino acids of ZxCBF , AmCBF and CmCBF share 69.1%, 60.1%, and 70.6% identity with proteins BpCBF(Betula platyphylla), GsCBF(Glycine soja), GsCBF, respectively.
引文
1 Pradeep K A , Parinita A ,et al.(2006). Role of DREB transcription factors in abiotic and biotic stress tolerance in plants[J]. Plant Cell Rep, 25:1263-1274.
2刘强,赵南明等. DREB转录因子在提高植物耐逆性中的作用[J].科学通报,2000,45:11-16).
3 Kanaya E,Nakajima N,et al. (1999) Characterization of the transcriptional activator CBF1 from Arabidopsis thaliana. Evidence for cold denaturation in regions outside Of the DNA binding domain[J]. J Biol Chem 274: 16068-16076.
4 Stockinger E J,GiImour S J,et al. (1997) Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE,a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[J] .Proc Natl Acad Sci U S A 94:1035-1040
5 Haake V,Cook D,et al. (2002) Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis[J]. Plant Physiol 130: 639-648
6 Zhou N,Wu G,et al. (1998) Molecular cloning of a cDNA encoding dehydration responsive element (DRE) binding protein in Brassica napus[J]. NCBI GenBank accession number AF084185
7 Jaglo K R,Kleff S,et al. (2001) Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plants species[J]. Plant Physiol 127:910-917.
8 Liu Q,Kasuga M,et al.. Two transcription factors,DREB1 and DREB,with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought and low-temperature- responsive gene expression,respectively, in Arabidopsis[J]. Plant Cell, 1998, 10 (8) :1391-1406
9 Medina J,Bargues M, et al. The Arabidopsis CBF gene family is composed of three genes eneoding AP2 domain containing proteins whose expression is regulated by low temperature but not by ABA or dehydration[J]. Plant Physiol, 1999, 119:463-470
10 Gao M J, Allard G Byass L et al.. Regulation and characterization of four CBF transcription factors from Brassica napus[J]. Plant Mol Biol, 2002, 49:459-471
11 Riechmann J L, Meyerowitz E M. The AP2/EREBP family of Plant transcription factors[J]. Binchem, 1998 13 (9) :633-646
12 Sakuma Y, Maruyama K, et al. Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in drought–responsive gene expression[J]. Plant Cell 2006,18: 1292-1309.
13 Dubouzet J G, Sakuma Y, et al. OsDREB genes in rice,Oryza sativa L.,encode transcription activators that function in drought-,high-salt- and cold-responsive gene expression[J]. Plant J, 2003,33:751-763
14 Xue G P. The DNA-binding activity of an AP2 transcription activator HvCBF2 involved in regulation of low–temperature responsive genes in barley is modulated by temperature [J].Plant J,2003,33: 373-383
15 Feng Q, Yoh S,et al. Cloning and Functional Analysis of a Novel DREB1/CBF Transeription Factor Involved in cold- Responsive Gene Expression in Zea mays L[J]. Plant Cell physiol. 2004 45 (8): 1042-1052
16 Gilmour S J,Zarka D G, et al. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step incold- induced COR gene expression[J]. Plant J, 1998, 16:433-442
17 Gilmour S J,Sebolt A M,et al. Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation[J]. Plant Physiol, 2000, 124:1854-1865
18 Kasuga M,Liu Q,et al. Improving plant drought,salt,and freezing tolerance by gene transfer of a single stress- inducible transcription factor[J]. Nat Biotech, 1999, 17:287-291
19 Kirsten R,Jaglo-Ottosen,et al. Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance[J]. Seienee, 1997, 280:104-106
20 Chinnusamy V, Ohta M et al. KICEI: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis[J]. Genes Dev, 2003, 17:1043-1054
21 Ishitani M, Xiong L, et al. HOSI, a genetic locus involved in cold-responsive gene expression in Arabdopsis[J]. Plant Cell, 1998, 10:1151-1161
22 Lee H,Xiong L, et al.The Arabidopsis HOS1 gene negatively regulates cold signal transduction and encodes a RING finger protein that displays cold-regulated nucleo-cytoplasmic partitioning[J]. Genes Dev, 2001, 15:912-924
23 MeKown R,Kuroki G, et al. Cold responses of Arabidopsis mutants impaired in freezing tolerance[J]. J Exp Bot, 1996, 47:1919-1925
24 Thomashow M F. So whatˊs new in the field of Plant cold acclimation? Lots! [J] Plant Physiol, 2001, 125:89-9
25 Thomashow M F. Role of cold- responsive genes in plant freezing toleranee[J]. PlantPhysiol, 1998, 118:1-7
26 Wanner L A, Junttila O. Cold- induced freezing tolerance in Arabidopsis[J]. Plant Physiol, 1999, 120:391-400
27吴彩霞,周志宇等.强旱生植物霸王和红砂地上部营养物质含量及其季节动态[J].草业科学, 2004, 21(3:)30-34.
28董学军.九种沙生灌木水分关系参数的实验测定及生态意义[J].植物学报,1998,40(7):657-664.
29黄振英.新疆10种沙生植物旱生结构的解剖学研究[J].西北植物学报, 1995, 15(6):56-61.
30黄振英. 30种新疆沙生植物的结构及其对沙漠环境的适应[J].植物生态学报, 1997,21(6):521-530.
31李景平,杨鑫光等.阿拉善荒漠区3种旱生植物体内主要渗透调节物质的含量和分配特征[J].草业科, 2005, 22(19):35-35.
32李正理,李荣敖.对甘肃九种旱生植物同化枝解剖结构的观察研究.植物学报,1981, 23 (3) :181-183
33刘娥娥,汪沛宏等.植物的干旱诱导蛋白[J].植物生理学通讯, 2001, 37(2):155-160
34卢存福,简令成等.高山植物的抗寒抗冻特性[J].植物学通报, 1998,15(3):17-22.
35刘美芹,卢存福等.珍稀濒危植物沙冬青生物学特性及抗逆性研究进展[J].应用与环境生物学报, 2004,10(3):384-388.
36刘家琼,丘明新.我国荒漠特有的常绿植物—沙冬青的生态生理及解剖学特征[J].植物学报,1982,24(6):568-573.
37张利平,王新平等.沙漠地区沙冬青的气体交换特性研究[J].西北植物学报,1998,18(2):207-213.
38张涛.沙冬青生理结构特性的研究[J].林业科学,1988,24(4):508-509.
39费云标,孙龙华等.沙冬青高活性抗冻蛋白的发现[J].植物学报, 1994, (36):649-650.
40费云标,魏令波等.沙冬青抗冻蛋白的分离、纯化及其理化特性分析[J].科学通报,2000,45(20):2185-2189.
41蒋志荣.沙区常绿灌木沙冬青的防风固沙改土效能研究[J].甘肃农业大学学报,1994,29(1):
83-86.
42蒋志荣.沙冬青抗旱机理的探讨[J].中国沙漠, 2000, 20(1): 71-74.
43牛西午著.柠条的栽培与利用[M].太原:山西科学教育出版社, 1988.
44牛西午著.柠条研究论文集[M].太原:山西科学技术出版社, 2003:163-164
45牛西午.柠条研究[M],北京:科学出版社, 2003
46牛西午,杨慧珍等.小叶锦鸡儿的组织培养和快速繁殖[J].西北植物学报, 2004, 24 (08):502-1505
47徐世健,安黎哲等.两种沙生植物抗旱指标的比较研究[J].西北植物学, 2000,20(2):224-228
48李文亭,苗艳等.锦鸡儿研究概况[J].特产研究, 1995(04):43-44, 46
49伊莉佳.柠条锦鸡儿CBF/DREB1转录因子cDNA全长的克隆及生物信息学分析[D].内蒙古农业大学硕士学位论文,2008.
50潘瑞炽.植物组织培养[M].广东高等教育出版社,2000.
51郭荣起.利用RNAi技术对拟南芥液泡H+-ATPase c亚基功能的初步研究[D].内蒙古农业大学硕士学位论文,2008.
52王福慧.利用烟草和拟南芥建立BSFE生物反应器[D].内蒙古农业大学硕士学位论文,2008.
2刘强,赵南明等. DREB转录因子在提高植物耐逆性中的作用[J].科学通报,2000,45:11-16).
3 Kanaya E,Nakajima N,et al. (1999) Characterization of the transcriptional activator CBF1 from Arabidopsis thaliana. Evidence for cold denaturation in regions outside Of the DNA binding domain[J]. J Biol Chem 274: 16068-16076.
4 Stockinger E J,GiImour S J,et al. (1997) Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE,a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[J] .Proc Natl Acad Sci U S A 94:1035-1040
5 Haake V,Cook D,et al. (2002) Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis[J]. Plant Physiol 130: 639-648
6 Zhou N,Wu G,et al. (1998) Molecular cloning of a cDNA encoding dehydration responsive element (DRE) binding protein in Brassica napus[J]. NCBI GenBank accession number AF084185
7 Jaglo K R,Kleff S,et al. (2001) Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plants species[J]. Plant Physiol 127:910-917.
8 Liu Q,Kasuga M,et al.. Two transcription factors,DREB1 and DREB,with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought and low-temperature- responsive gene expression,respectively, in Arabidopsis[J]. Plant Cell, 1998, 10 (8) :1391-1406
9 Medina J,Bargues M, et al. The Arabidopsis CBF gene family is composed of three genes eneoding AP2 domain containing proteins whose expression is regulated by low temperature but not by ABA or dehydration[J]. Plant Physiol, 1999, 119:463-470
10 Gao M J, Allard G Byass L et al.. Regulation and characterization of four CBF transcription factors from Brassica napus[J]. Plant Mol Biol, 2002, 49:459-471
11 Riechmann J L, Meyerowitz E M. The AP2/EREBP family of Plant transcription factors[J]. Binchem, 1998 13 (9) :633-646
12 Sakuma Y, Maruyama K, et al. Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in drought–responsive gene expression[J]. Plant Cell 2006,18: 1292-1309.
13 Dubouzet J G, Sakuma Y, et al. OsDREB genes in rice,Oryza sativa L.,encode transcription activators that function in drought-,high-salt- and cold-responsive gene expression[J]. Plant J, 2003,33:751-763
14 Xue G P. The DNA-binding activity of an AP2 transcription activator HvCBF2 involved in regulation of low–temperature responsive genes in barley is modulated by temperature [J].Plant J,2003,33: 373-383
15 Feng Q, Yoh S,et al. Cloning and Functional Analysis of a Novel DREB1/CBF Transeription Factor Involved in cold- Responsive Gene Expression in Zea mays L[J]. Plant Cell physiol. 2004 45 (8): 1042-1052
16 Gilmour S J,Zarka D G, et al. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step incold- induced COR gene expression[J]. Plant J, 1998, 16:433-442
17 Gilmour S J,Sebolt A M,et al. Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation[J]. Plant Physiol, 2000, 124:1854-1865
18 Kasuga M,Liu Q,et al. Improving plant drought,salt,and freezing tolerance by gene transfer of a single stress- inducible transcription factor[J]. Nat Biotech, 1999, 17:287-291
19 Kirsten R,Jaglo-Ottosen,et al. Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance[J]. Seienee, 1997, 280:104-106
20 Chinnusamy V, Ohta M et al. KICEI: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis[J]. Genes Dev, 2003, 17:1043-1054
21 Ishitani M, Xiong L, et al. HOSI, a genetic locus involved in cold-responsive gene expression in Arabdopsis[J]. Plant Cell, 1998, 10:1151-1161
22 Lee H,Xiong L, et al.The Arabidopsis HOS1 gene negatively regulates cold signal transduction and encodes a RING finger protein that displays cold-regulated nucleo-cytoplasmic partitioning[J]. Genes Dev, 2001, 15:912-924
23 MeKown R,Kuroki G, et al. Cold responses of Arabidopsis mutants impaired in freezing tolerance[J]. J Exp Bot, 1996, 47:1919-1925
24 Thomashow M F. So whatˊs new in the field of Plant cold acclimation? Lots! [J] Plant Physiol, 2001, 125:89-9
25 Thomashow M F. Role of cold- responsive genes in plant freezing toleranee[J]. PlantPhysiol, 1998, 118:1-7
26 Wanner L A, Junttila O. Cold- induced freezing tolerance in Arabidopsis[J]. Plant Physiol, 1999, 120:391-400
27吴彩霞,周志宇等.强旱生植物霸王和红砂地上部营养物质含量及其季节动态[J].草业科学, 2004, 21(3:)30-34.
28董学军.九种沙生灌木水分关系参数的实验测定及生态意义[J].植物学报,1998,40(7):657-664.
29黄振英.新疆10种沙生植物旱生结构的解剖学研究[J].西北植物学报, 1995, 15(6):56-61.
30黄振英. 30种新疆沙生植物的结构及其对沙漠环境的适应[J].植物生态学报, 1997,21(6):521-530.
31李景平,杨鑫光等.阿拉善荒漠区3种旱生植物体内主要渗透调节物质的含量和分配特征[J].草业科, 2005, 22(19):35-35.
32李正理,李荣敖.对甘肃九种旱生植物同化枝解剖结构的观察研究.植物学报,1981, 23 (3) :181-183
33刘娥娥,汪沛宏等.植物的干旱诱导蛋白[J].植物生理学通讯, 2001, 37(2):155-160
34卢存福,简令成等.高山植物的抗寒抗冻特性[J].植物学通报, 1998,15(3):17-22.
35刘美芹,卢存福等.珍稀濒危植物沙冬青生物学特性及抗逆性研究进展[J].应用与环境生物学报, 2004,10(3):384-388.
36刘家琼,丘明新.我国荒漠特有的常绿植物—沙冬青的生态生理及解剖学特征[J].植物学报,1982,24(6):568-573.
37张利平,王新平等.沙漠地区沙冬青的气体交换特性研究[J].西北植物学报,1998,18(2):207-213.
38张涛.沙冬青生理结构特性的研究[J].林业科学,1988,24(4):508-509.
39费云标,孙龙华等.沙冬青高活性抗冻蛋白的发现[J].植物学报, 1994, (36):649-650.
40费云标,魏令波等.沙冬青抗冻蛋白的分离、纯化及其理化特性分析[J].科学通报,2000,45(20):2185-2189.
41蒋志荣.沙区常绿灌木沙冬青的防风固沙改土效能研究[J].甘肃农业大学学报,1994,29(1):
83-86.
42蒋志荣.沙冬青抗旱机理的探讨[J].中国沙漠, 2000, 20(1): 71-74.
43牛西午著.柠条的栽培与利用[M].太原:山西科学教育出版社, 1988.
44牛西午著.柠条研究论文集[M].太原:山西科学技术出版社, 2003:163-164
45牛西午.柠条研究[M],北京:科学出版社, 2003
46牛西午,杨慧珍等.小叶锦鸡儿的组织培养和快速繁殖[J].西北植物学报, 2004, 24 (08):502-1505
47徐世健,安黎哲等.两种沙生植物抗旱指标的比较研究[J].西北植物学, 2000,20(2):224-228
48李文亭,苗艳等.锦鸡儿研究概况[J].特产研究, 1995(04):43-44, 46
49伊莉佳.柠条锦鸡儿CBF/DREB1转录因子cDNA全长的克隆及生物信息学分析[D].内蒙古农业大学硕士学位论文,2008.
50潘瑞炽.植物组织培养[M].广东高等教育出版社,2000.
51郭荣起.利用RNAi技术对拟南芥液泡H+-ATPase c亚基功能的初步研究[D].内蒙古农业大学硕士学位论文,2008.
52王福慧.利用烟草和拟南芥建立BSFE生物反应器[D].内蒙古农业大学硕士学位论文,2008.