陆地棉HRD转录因子基因原核表达与亚细胞定位分析
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摘要
【目的】AP2/ERF家族基因在植物生长和逆境胁迫中起重要作用,GhHRD转录因子基因是该家族的一员。本试验首次克隆了GhHRD基因,为研究其功能提供理论依据。【方法】选用相对耐旱的新陆中36作为试验材料,通过反转录聚合酶链式反应的方法获得GhHRD基因,并通过生物信息学、原核表达、亚细胞定位和酵母杂交分析预测其结构和功能。【结果】GhHRD基因开放阅读框为555 bp,编码184个氨基酸,相对分子质量为19.539×10~3 Da,等电点为5,具有典型的AP2/ERF保守结构域。原核表达分析发现,pET-28a(+)-GhHRD可以被诱导表达,且在37℃、IPTG浓度1 mmol·L~(-1)条件下高效表达。与GFP融合的重组蛋白GhHRD-GFP定位在细胞核,且具有明显的转录自激活特征。【结论】推测GhHRD可能参与了棉花逆境胁迫应答反应,为进一步探索该转录因子的DNA结合位点和转录调控网络奠定了基础。
[Objective] The members of AP2/ERF gene family play an important role in plant growth and stress, and the GhHRD gene is a member of this family. The purpose of our search was to study the function of GhHRD in upland cotton. [Method] The GhHRD gene was cloned by reverse transcription polymerase chain reaction technology and its structure and function were predicted by bioinformatics methods, prokaryotic expression, subcellular localization and yeast hybridization analysis. [Result]The Open read frame of GhHRD gene was 555 bp, encoding 184 amino acids, with relative molecular mass 19.539 k Da,isoelectric point 5, and a typical AP2/ERF conservative domain. The prokaryotic expression analysis showed that expression of pET-28(+)-GhHRD could be efficiently induced under the condition of 37 ℃ and 1 mmol·L~(-1) IPTG. The recombinant protein GhHRD-GFP is sublocated in the nucleus and has obvious transcriptional self-activation characteristics. [Conclusion] It is speculated that GhHRD may be involved in response to stress responses in cotton. The results laid a foundation for further exploration of DNA binding sites and transcriptional regulatory networks.
[Objective] The members of AP2/ERF gene family play an important role in plant growth and stress, and the GhHRD gene is a member of this family. The purpose of our search was to study the function of GhHRD in upland cotton. [Method] The GhHRD gene was cloned by reverse transcription polymerase chain reaction technology and its structure and function were predicted by bioinformatics methods, prokaryotic expression, subcellular localization and yeast hybridization analysis. [Result]The Open read frame of GhHRD gene was 555 bp, encoding 184 amino acids, with relative molecular mass 19.539 k Da,isoelectric point 5, and a typical AP2/ERF conservative domain. The prokaryotic expression analysis showed that expression of pET-28(+)-GhHRD could be efficiently induced under the condition of 37 ℃ and 1 mmol·L~(-1) IPTG. The recombinant protein GhHRD-GFP is sublocated in the nucleus and has obvious transcriptional self-activation characteristics. [Conclusion] It is speculated that GhHRD may be involved in response to stress responses in cotton. The results laid a foundation for further exploration of DNA binding sites and transcriptional regulatory networks.
引文
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[13]陈琴,李星星,童婷,等.干旱胁迫对转CpHRD基因烟草的影响[J].植物生理学报,2015,51(5):715-720.Chen Qin,Li Xingxing,Tong Ting,et al.Influence of drought stress on transgenic CpHRD Gene in tobacco[J].Plant Physiology Journal,2015,51(5):715-720.
[14]吴文超,曲延英,高文伟,等.不同棉花品种对盐、旱胁迫的光合响应及抗逆性评价[J].新疆农业科学,2016,53(9):1569-1579.Wu Wenchao,Qu Yanying,Gao Wenwei,et al.Photosynthetic response and stress resistance of different cotton varieties to salt and drought stress[J].Xinjiang Agricultural Science,2016,53(9):1569-1579.
[15]Sakuma Y,Liu Q,Dubouzet J G,et al.DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs,transcription factors involved in dehydration-and cold-inducible gene expression[J].Biochemical and Biophysical Research Communications,2002,290(3):998-1009.
[16]Phukan U J,Jeena G S,Tripathi V,et al.Regulation of Apetala2/Ethylene response factors in plants[J].Frontiers in Plant Science,2017,8:150.
[17]Liu Q,Kasuga M,Sakuma Y,et al.Two transcription factors,DREB1 and DREB2,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].The Plant Cell,1998,10(8):1391-1406.
[18]Liu X Q,Liu C Y,Guo Q,et al.Mulberry transcription factor MnDREB4A confers tolerance to multiple abiotic stresses in transgenic tobacco[J/OL].PLoS ONE,2015,10(12):e0145619(2015-12-22)[2018-06-07].https://doi.org/10.1371/journal.pone.0145619
[19]Huang B,Liu J Y.Cloning and functional analysis of the novel gene GhDBP3 encoding a DRE-binding transcription factor from Gossypium hirsutum[J].Biochimica et Biophysica Acta,2006,1759(6):263-269.
[20]Huang B O,Jin L G,Liu J Y.Molecular cloning and functional characterization of a DREB1/CBF-like gene(GhDREB1L)from cotton[J].Science in China Series C:Life Sciences,2007,50(1):7-14.
[21]Huang B,Jin L,Liu J Y.Identification and characterization of the novel gene GhDBP2 encoding a DRE-binding protein from cotton(Gossypium hirsutum)[J].Journal of Plant Physiology,2008,165(2):214-223.
[22]Liu C,Zhang T.Expansion and stress responses of the AP2/EREBP superfamily in cotton[J].BMC Genomics,2017,18(1):118.
[2]Seki M,Umezawa T,Urano K,et al.Regulatory metabolic networks in drought stress responses[J].Current Opinion in Plant Biology,2007,10(3):296-302.
[3]Singh D,Laxmi A.Transcriptional regulation of drought response:A tortuous network of transcriptional factors[J].Frontiers in Plant Science,2015,6:895.
[4]Joshi R,Wani S H,Singh B,et al.Transcription factors and plants response to drought stress:Current understanding and future directions[J].Frontiers in Plant Science,2016,7:1029.
[5]Yan H,Jia H,Chen X,et al.The cotton WRKY transcription factor GhWRKY17 functions in drought and salt stress in transgenic Nicotiana benthamiana through ABA signaling and the modulation of reactive oxygen species production[J].Plant and Cell Physiology,2014,55(12):2060-2076.
[6]Mizoi J,Shinozaki K,Yamaguchi-Shinozaki K.AP2/ERF family transcription factors in plant abiotic stress responses[J].Biochimica et Biophysica Acta,2012,1819(2):86-96.
[7]Jisha V,Dampanaboina L,Vadassery J,et al.Overexpression of an AP2/ERF type transcription factor Os EREBP1 confers biotic and abiotic stress tolerance in rice[J/OL].PLoS ONE,2015,10(6):e0127831(2015-06-02)[2018-06-07].https://doi.org/10.1371/journal.pone.0127831
[8]Jofuku K D,Den Boer B G,Van Montagu M,et al.Control of Arabidopsis flower and seed development by the homeotic gene APETALA2[J].The Plant Cell,1994,6(9):1211-1225.
[9]Ohme-Takagi M,Shinshi H.Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[J].The Plant Cell,1995,7(2):173-182.
[10]Shaheen T,Mahmood-ur-Rahman M F A,Zaib P,et al.Identification,characterization,homology modeling and protein-protein interactions of cotton(Gossypium arboreum L.)DREB gene[J].International Journal of Agriculture and Biology,2018,20(5):1055-1061.
[11]Karaba A,Dixit S,Greco R,et al.Improvement of water use efficiency in rice by expression of HARDY,an Arabidopsis drought and salt tolerance gene[J].Proceedings of the National Academy of Sciences,2007,104(39):15270-15275.
[12]Abogadallah G M,Nada R M,Malinowski R,et al.Overexpression of HARDY,an AP2/ERF gene from Arabidopsis,improves drought and salt tolerance by reducing transpiration and sodium uptake in transgenic Trifolium alexandrinum L.[J].Planta,2011,233(6):1265-1276.
[13]陈琴,李星星,童婷,等.干旱胁迫对转CpHRD基因烟草的影响[J].植物生理学报,2015,51(5):715-720.Chen Qin,Li Xingxing,Tong Ting,et al.Influence of drought stress on transgenic CpHRD Gene in tobacco[J].Plant Physiology Journal,2015,51(5):715-720.
[14]吴文超,曲延英,高文伟,等.不同棉花品种对盐、旱胁迫的光合响应及抗逆性评价[J].新疆农业科学,2016,53(9):1569-1579.Wu Wenchao,Qu Yanying,Gao Wenwei,et al.Photosynthetic response and stress resistance of different cotton varieties to salt and drought stress[J].Xinjiang Agricultural Science,2016,53(9):1569-1579.
[15]Sakuma Y,Liu Q,Dubouzet J G,et al.DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs,transcription factors involved in dehydration-and cold-inducible gene expression[J].Biochemical and Biophysical Research Communications,2002,290(3):998-1009.
[16]Phukan U J,Jeena G S,Tripathi V,et al.Regulation of Apetala2/Ethylene response factors in plants[J].Frontiers in Plant Science,2017,8:150.
[17]Liu Q,Kasuga M,Sakuma Y,et al.Two transcription factors,DREB1 and DREB2,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].The Plant Cell,1998,10(8):1391-1406.
[18]Liu X Q,Liu C Y,Guo Q,et al.Mulberry transcription factor MnDREB4A confers tolerance to multiple abiotic stresses in transgenic tobacco[J/OL].PLoS ONE,2015,10(12):e0145619(2015-12-22)[2018-06-07].https://doi.org/10.1371/journal.pone.0145619
[19]Huang B,Liu J Y.Cloning and functional analysis of the novel gene GhDBP3 encoding a DRE-binding transcription factor from Gossypium hirsutum[J].Biochimica et Biophysica Acta,2006,1759(6):263-269.
[20]Huang B O,Jin L G,Liu J Y.Molecular cloning and functional characterization of a DREB1/CBF-like gene(GhDREB1L)from cotton[J].Science in China Series C:Life Sciences,2007,50(1):7-14.
[21]Huang B,Jin L,Liu J Y.Identification and characterization of the novel gene GhDBP2 encoding a DRE-binding protein from cotton(Gossypium hirsutum)[J].Journal of Plant Physiology,2008,165(2):214-223.
[22]Liu C,Zhang T.Expansion and stress responses of the AP2/EREBP superfamily in cotton[J].BMC Genomics,2017,18(1):118.