播娘蒿转录因子DsCBF1突变体的构建及其蛋白质的EMSA试验分析
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摘要
CBF(C-repeat binding factor)是调控植物冷驯化相关基因表达的调控转录激活因子,可与下游冷应答基因启动子的核心序列CBT/DRE元件(CCGAC)特异性地结合,启动耐寒基因的表达,提高植物的抗寒能力.为揭示播娘蒿(Descurainia sophia)的抗寒机制,研究播娘蒿转录因子Ds CBF1蛋白AP2区域编码氨基酸对启动下游抗寒基因表达的作用.首先分析Ds CBF1蛋白AP2区域编码氨基酸的疏水性,设计重叠延伸PCR引物,定点突变Ds CBF1基因;然后构建p ET32a-Ds CBF1突变体原核表达载体,通过冻融法转化入宿主大肠杆菌BL21(DE3)中,进行原核表达,并通过镍离子亲和层析纯化Ds CBF1突变体蛋白;最后利用化学发光法EMSA试验(Electrophoretic mobility shift assay)方法分析Ds CBF1蛋白与含有CRT/DRE元件的DNA探针之间的相互作用.EMSA试验分析显示,播娘蒿Ds CBF1的AP2区域的57位苯丙氨酸F(TTT)和68位缬氨酸V(GTT)分别突变为精氨酸R(CGT)和谷氨酸E(GAA),导致播娘蒿顺式调控因子Ds CBF1蛋白与下游冷响应基因的CRT/DRE元件结合效率出现明显下降趋势,说明播娘蒿Ds CBF1蛋白AP2区域编码氨基酸位点的突变影响了播娘蒿的抗寒能力.本研究表明播娘蒿Ds CBF1的AP2区域的57位苯丙氨酸F(TTT)和68位缬氨酸V(GTT)位点对于播娘蒿的冷驯化具有十分重要的作用.
CBF(C-repeat binding factor) is a transcriptional activator related to the regulation of plant cold acclimation gene expression, and AP2 domain of CBF protein can specifically bind with CBT/DRE element of cold-responsive gene promoter, which switches on the expression of anti-cold gene and improves plant cold hardiness. To reveal the anti-cold mechanism of Descurainia sophia and the mechanism of how AP2 domain of CBF protein activates the expression of its downstream genes, we analyzed the hydrophobic amino acids of AP2 domain of CBF protein, designed PCR primer to mutant Ds CBF1 gene, constructed mutant p ET32a-Ds CBF1 prokaryotic expression vector, transformed it into E. coli BL21(DE3) by freeze-thaw method, expressed and purified Ds CBF1 mutant proteins by nickel ion affinity chromatography. After that we analyzed using EMSA the interaction between Ds CBF1 mutant protein and DNA probe. The EMSA results showed that the mutation of the 57 th and the 68 th amino acid sites in the AP2 region of D. sophia from F(TTT) and V(GTT) to R(CGT) and E(GAA) respectively, leading to an obvious decline of binding Ds CBF1 with its downstream genes, thereby affecting the freeze resistance of D. sophia. The reults suggested that the 57 th amino acid site F and the 68 th amino acid site V play an important role in the cold acclimation of D. sophia.
CBF(C-repeat binding factor) is a transcriptional activator related to the regulation of plant cold acclimation gene expression, and AP2 domain of CBF protein can specifically bind with CBT/DRE element of cold-responsive gene promoter, which switches on the expression of anti-cold gene and improves plant cold hardiness. To reveal the anti-cold mechanism of Descurainia sophia and the mechanism of how AP2 domain of CBF protein activates the expression of its downstream genes, we analyzed the hydrophobic amino acids of AP2 domain of CBF protein, designed PCR primer to mutant Ds CBF1 gene, constructed mutant p ET32a-Ds CBF1 prokaryotic expression vector, transformed it into E. coli BL21(DE3) by freeze-thaw method, expressed and purified Ds CBF1 mutant proteins by nickel ion affinity chromatography. After that we analyzed using EMSA the interaction between Ds CBF1 mutant protein and DNA probe. The EMSA results showed that the mutation of the 57 th and the 68 th amino acid sites in the AP2 region of D. sophia from F(TTT) and V(GTT) to R(CGT) and E(GAA) respectively, leading to an obvious decline of binding Ds CBF1 with its downstream genes, thereby affecting the freeze resistance of D. sophia. The reults suggested that the 57 th amino acid site F and the 68 th amino acid site V play an important role in the cold acclimation of D. sophia.
引文
1 Zhu J,Dong CH,Zhu J K.Inter play bet ween cold-responsive gene regulation,meta bolism and RNA processing during plant cold acclimation[J].Curr Opin Plant Biol,2007,10(3):290-295
2 Stockinger EJ,Gilmour SJ,Thomashow MF.Arabidopsis thaliana CBF1encodes 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 defi cit[J].Proc Nat Acad Sci,1997,94(3):1035-1040
3 Jaglo-Ottosen K R,Gilmour S J,Zarka D G,Schabenberger O,Thomashow MF.Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance[J].Science,1998,280(5360):104-106
4 Liu Q,Kasuga M,Sakuma Y,Abe H,Miura S,Yamaguchi-Shinozaki K,Shinozaki K.Two transcription factors,DREB1 and DREB2,with an ER EBP/A P2 DNA binding domain separate t wo cellular sig nal transduction pathways in drought-and low-temperature-responsive gene expression,respectively,in Arabidopsis[J].Plant Cell,1998,10(8):1391-1406
5 Jaglo KR,Kleff S,Amundsen KL,Zhang X,Haake V,Zhang JZ,Deits T,Thomashow MF.Components of the Arabidopsis C-repeat/dehydrationresponsive element binding factor cold-response pathway are conserved inbrassica napus and other plant species[J].Plant Physiol,2001,127(3):910-917
6 王乐,李晶,曹瑜,张玲,巨艳,乔代蓉,曹毅.播娘蒿转录因子Ds CBF与CRT/DRE元件的相互作用[J].应用与环境生物学报,2010,16(5):609-612[Wang L,Li J,Cao Y,Zhang L,Ju Y,Qiao DR,Cao Y.Interaction of Ds CBF of Descurainia sophia with CRT/DRE element[J].Chin J Appl Environ Biol,2010,16(5):609-612]
7 Neuteboom ST,Murre C.Interaction between the tobacco DNA-binding activity CBF and the cyt-1 promoter element of the Agrobacterium tumefaciens T-DNA gene T-CYT correlates with cyt-1 directed gene expression in multiple tobacco tissue types[J].Plant J,1993,4(3):525-534
8 Gupta K,Dey A,Gupta B.Plant polyamines in abiotic stress responses[J].Acta Physiol Plant,2013,35(7):2015-2036
9 雒丽娜,王盛,王玉炯.利用重叠延伸PCR技术进行定点突变研究[J].安徽农业科学与技术,2012,13(4):719-722[Luo LN,Wang S,Wang YJ.Site-directed mutagenesis based on overlap extension PCR[J].J Anhui Agric Sci,2012,13(4):719-722]
10 叶程,邵坤彦,王亚南,覃桂,代风娇,何冬兰.重叠延伸PCR法定点突变微生物产谷氨酰胺转氨酶基因[J].中国生物制品学杂志,2013,26(5):670-674[Ye C,Shao KY,Wang YN,Tan G,He DL.Overlap extension PCR method for the production of glutamine aminotransferase gene in fi xed point mutant[J].China J Biol Prod,2013,26(5):670-674]
11 刘凯,胡春华,魏岳荣,易干军,邵秀红.拟南芥CBF1基因植物表达载体构建及其对野生蕉的遗传转化[J].湖南农业大学学报(自然科学版),2011,37(3):248-252[Liu K,Hu CH,Wei Y R,Yi GJ,Shao XH.The plant Arabidopsis CBF1 gene expression vector construction and genetic transformation of wild banana[J].J Hunan Agric Univ(Nat Sci Ed),2011,37(3):248-252]
12 Zhang CP,Shu LZ,Zhang QJ,Lu CG,Cai XN.Cloning of CBF3 gene from Arabidopsis thaliana and construction of plant expression vector[J].Agric Biotechnol,2011,12(5):670-673].
13 王晓燕,杜改亮,韩瑶瑶,刘梦欣,马燕勤,冯欢,徐子勤.拟南芥Hy PR P蛋白A Z I1在大肠杆菌中的表达及其抗真菌活性分析[J].植物生理学报,2015,51(1):63-72[Wang XY,Du GL,Han YY,Liu MX,Ma Yanqin,Feng H,Xu ZQ.Expression of AZI1 protein Hy PRP in Escherichia coli and analysis of its antifungal activity[J].J Plant Physiol,2015,51(1):63-72]
14 陈勇,应汉杰.亲和层析研究进展[J].离子交换与吸附,2001,17(3):276-280[Chen Y,Ying HJ.Affinity chromatography.Res Progr Ion Exchange Adsorption,2001,17(3):276-280]
15 Hellman LM,Fried MG.Electro phoretic Mobility Shift Assay(EMSA)for detecting protein-nucleic acid interactions[J].NIH Public Access,2007,2(8):1849-1861
16 Sakuma Y,Liu Q,Dubouzet JG,Abe H,Shinozaki K.YamaguchiShinozaki K.DNA-binding specif icity of the ER F/AP2 domain of Arabidopsis DREBs,transcription factors involved in dehydrationand cold-inducible gene expression[J].Biochem Biophys Res Commun,2002,290(3):998-1009
17 Ohme-Takagi M,Shinshi H.Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[J].Plant Cell,1995,7(2):173-182
18 Weigel D.The APETA LA2 domain is related to a novel type of DNA binding domain[J].Plant Cell,1995,7(4):388
19 Kanaya E,Nakajima N,Morikawa K,Okada K,Shimura Y.Characterization of the transcriptional activator CBF1 from Arabidopsis thaliana evidence for cold denaturation in regions outs ide of the DNA binding domain[J].J Biol Chem,1999,274(23):16068-16076
2 Stockinger EJ,Gilmour SJ,Thomashow MF.Arabidopsis thaliana CBF1encodes 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 defi cit[J].Proc Nat Acad Sci,1997,94(3):1035-1040
3 Jaglo-Ottosen K R,Gilmour S J,Zarka D G,Schabenberger O,Thomashow MF.Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance[J].Science,1998,280(5360):104-106
4 Liu Q,Kasuga M,Sakuma Y,Abe H,Miura S,Yamaguchi-Shinozaki K,Shinozaki K.Two transcription factors,DREB1 and DREB2,with an ER EBP/A P2 DNA binding domain separate t wo cellular sig nal transduction pathways in drought-and low-temperature-responsive gene expression,respectively,in Arabidopsis[J].Plant Cell,1998,10(8):1391-1406
5 Jaglo KR,Kleff S,Amundsen KL,Zhang X,Haake V,Zhang JZ,Deits T,Thomashow MF.Components of the Arabidopsis C-repeat/dehydrationresponsive element binding factor cold-response pathway are conserved inbrassica napus and other plant species[J].Plant Physiol,2001,127(3):910-917
6 王乐,李晶,曹瑜,张玲,巨艳,乔代蓉,曹毅.播娘蒿转录因子Ds CBF与CRT/DRE元件的相互作用[J].应用与环境生物学报,2010,16(5):609-612[Wang L,Li J,Cao Y,Zhang L,Ju Y,Qiao DR,Cao Y.Interaction of Ds CBF of Descurainia sophia with CRT/DRE element[J].Chin J Appl Environ Biol,2010,16(5):609-612]
7 Neuteboom ST,Murre C.Interaction between the tobacco DNA-binding activity CBF and the cyt-1 promoter element of the Agrobacterium tumefaciens T-DNA gene T-CYT correlates with cyt-1 directed gene expression in multiple tobacco tissue types[J].Plant J,1993,4(3):525-534
8 Gupta K,Dey A,Gupta B.Plant polyamines in abiotic stress responses[J].Acta Physiol Plant,2013,35(7):2015-2036
9 雒丽娜,王盛,王玉炯.利用重叠延伸PCR技术进行定点突变研究[J].安徽农业科学与技术,2012,13(4):719-722[Luo LN,Wang S,Wang YJ.Site-directed mutagenesis based on overlap extension PCR[J].J Anhui Agric Sci,2012,13(4):719-722]
10 叶程,邵坤彦,王亚南,覃桂,代风娇,何冬兰.重叠延伸PCR法定点突变微生物产谷氨酰胺转氨酶基因[J].中国生物制品学杂志,2013,26(5):670-674[Ye C,Shao KY,Wang YN,Tan G,He DL.Overlap extension PCR method for the production of glutamine aminotransferase gene in fi xed point mutant[J].China J Biol Prod,2013,26(5):670-674]
11 刘凯,胡春华,魏岳荣,易干军,邵秀红.拟南芥CBF1基因植物表达载体构建及其对野生蕉的遗传转化[J].湖南农业大学学报(自然科学版),2011,37(3):248-252[Liu K,Hu CH,Wei Y R,Yi GJ,Shao XH.The plant Arabidopsis CBF1 gene expression vector construction and genetic transformation of wild banana[J].J Hunan Agric Univ(Nat Sci Ed),2011,37(3):248-252]
12 Zhang CP,Shu LZ,Zhang QJ,Lu CG,Cai XN.Cloning of CBF3 gene from Arabidopsis thaliana and construction of plant expression vector[J].Agric Biotechnol,2011,12(5):670-673].
13 王晓燕,杜改亮,韩瑶瑶,刘梦欣,马燕勤,冯欢,徐子勤.拟南芥Hy PR P蛋白A Z I1在大肠杆菌中的表达及其抗真菌活性分析[J].植物生理学报,2015,51(1):63-72[Wang XY,Du GL,Han YY,Liu MX,Ma Yanqin,Feng H,Xu ZQ.Expression of AZI1 protein Hy PRP in Escherichia coli and analysis of its antifungal activity[J].J Plant Physiol,2015,51(1):63-72]
14 陈勇,应汉杰.亲和层析研究进展[J].离子交换与吸附,2001,17(3):276-280[Chen Y,Ying HJ.Affinity chromatography.Res Progr Ion Exchange Adsorption,2001,17(3):276-280]
15 Hellman LM,Fried MG.Electro phoretic Mobility Shift Assay(EMSA)for detecting protein-nucleic acid interactions[J].NIH Public Access,2007,2(8):1849-1861
16 Sakuma Y,Liu Q,Dubouzet JG,Abe H,Shinozaki K.YamaguchiShinozaki K.DNA-binding specif icity of the ER F/AP2 domain of Arabidopsis DREBs,transcription factors involved in dehydrationand cold-inducible gene expression[J].Biochem Biophys Res Commun,2002,290(3):998-1009
17 Ohme-Takagi M,Shinshi H.Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[J].Plant Cell,1995,7(2):173-182
18 Weigel D.The APETA LA2 domain is related to a novel type of DNA binding domain[J].Plant Cell,1995,7(4):388
19 Kanaya E,Nakajima N,Morikawa K,Okada K,Shimura Y.Characterization of the transcriptional activator CBF1 from Arabidopsis thaliana evidence for cold denaturation in regions outs ide of the DNA binding domain[J].J Biol Chem,1999,274(23):16068-16076
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