小麦K_2型脱水蛋白DHN14响应非生物胁迫的功能分析
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摘要
【目的】研究脱水蛋白DHN14的功能及其在植物响应非生物胁迫中的潜在作用。【方法】从小麦品种"郑引1号"克隆获得脱水蛋白基因DHN14,对其进行生物信息学分析,利用实时定量PCR分析该基因在逆境胁迫下的表达水平,构建脱水蛋白DHN14基因的原核表达载体(重组菌pET28a-DHN14),经IPTG诱导表达后,在非生物胁迫下,研究该蛋白对大肠杆菌及乳酸脱氢酶(LDH)的保护作用。【结果】克隆获得脱水蛋白基因DHN14的CDS为339 bp,编码112个氨基酸,该蛋白含有2个保守的K片段,属于高亲水性无序蛋白,其分子质量为24 ku,等电点(pI)约为6.28;多序列比对分析表明,DHN14蛋白与WCOR726(Triticum aestivum)脱水蛋白的亲缘关系最近;实时定量RT-PCR结果表明,脱水蛋白基因DHN14受干旱、低温和ABA诱导表达;在20 mmol/L金属离子(Co~(2+)、Ni~(2+)、Cu~(2+)、Zn~(2+))胁迫下,表达DHN14重组蛋白的大肠杆菌的活力明显高于对照,表明该蛋白在大肠杆菌中对金属离子胁迫具有保护作用;用1.0 mmol/L过氧化氢进行胁迫处理,重组菌DHN14存活率显著增高,说明脱水蛋白DHN14能够提高大肠杆菌对过氧化氢胁迫的耐受性;在低温和脱水胁迫下,DHN14脱水蛋白对乳酸脱氢酶的活性具有保护作用。【结论】小麦脱水蛋白DHN14能够响应非生物胁迫,提高对低温、干旱、金属离子、过氧化氢的耐受性。
【Objective】 This paper studied the dehydrin function of DHN14 and its potential role in plant responses to abiotic stress.【Method】 The dehydrin DHN14 was isolated from ‘Zhengyin 1' cultivar of Triticum aestivum.Bioinformatics analysis of the dehydrin was conducted,and real-time quantitative PCR was used to demonstrate the expression of gene DHN14 under abiotic stresses.The recombinant plasmid pET28 a-DHN14 was constructed and transformed into the Escherichia coli expression strain for inducible expression of the target protein by IPTG.The protective effect of the protein on E. coli and LDH under abiotic stress was also investigated.【Result】 The dehydrin gene DHN14 was cloned and the full-length of DHN14 CDS was 339 bp encoding 112 amino acids and containing 2 conserved K segments. DHN14 was a highly hydrophilic disorder protein and a 24 ku DHN14 protein induced by IPTG,and the theoretical isoelectric point(pI) value was 6.28.Multiple sequence alignment showed that it had the nearest relationship to a WCOR726 Triticum aestivum.The qRT-PCR assay indicated that the expression of DHN14 gene was induced by low temperature,PEG and ABA.In the E.coli transformants tolerance assays,the relative activity of transgenic E.coli was higher than control in 20 mmol/L(Co~(2+),Ni~(2+),Cu~(2+),and Zn~(2+)) stress,which demonstrated that DHN14 protein could protect the activity of E.coli in metal ion stress.When the hydrogen peroxide concentration was 1.0 mmol/L,the viability of E. coli transformants was significantly increased.DHN14 can improve the tolerance of E. coli to heavy metal and hydrogen peroxide stress.The study on lactate dehydrogenase(LDH) showed that DHN14 dehydrin had protective effect on LDH activity under low temperature and dehydration stress.【Conclusion】 The dehydrin DHN14 can respond to abiotic stress and improve the tolerance of crops in low-temperature,drought,metal ion and hydrogen peroxide stresses.
【Objective】 This paper studied the dehydrin function of DHN14 and its potential role in plant responses to abiotic stress.【Method】 The dehydrin DHN14 was isolated from ‘Zhengyin 1' cultivar of Triticum aestivum.Bioinformatics analysis of the dehydrin was conducted,and real-time quantitative PCR was used to demonstrate the expression of gene DHN14 under abiotic stresses.The recombinant plasmid pET28 a-DHN14 was constructed and transformed into the Escherichia coli expression strain for inducible expression of the target protein by IPTG.The protective effect of the protein on E. coli and LDH under abiotic stress was also investigated.【Result】 The dehydrin gene DHN14 was cloned and the full-length of DHN14 CDS was 339 bp encoding 112 amino acids and containing 2 conserved K segments. DHN14 was a highly hydrophilic disorder protein and a 24 ku DHN14 protein induced by IPTG,and the theoretical isoelectric point(pI) value was 6.28.Multiple sequence alignment showed that it had the nearest relationship to a WCOR726 Triticum aestivum.The qRT-PCR assay indicated that the expression of DHN14 gene was induced by low temperature,PEG and ABA.In the E.coli transformants tolerance assays,the relative activity of transgenic E.coli was higher than control in 20 mmol/L(Co~(2+),Ni~(2+),Cu~(2+),and Zn~(2+)) stress,which demonstrated that DHN14 protein could protect the activity of E.coli in metal ion stress.When the hydrogen peroxide concentration was 1.0 mmol/L,the viability of E. coli transformants was significantly increased.DHN14 can improve the tolerance of E. coli to heavy metal and hydrogen peroxide stress.The study on lactate dehydrogenase(LDH) showed that DHN14 dehydrin had protective effect on LDH activity under low temperature and dehydration stress.【Conclusion】 The dehydrin DHN14 can respond to abiotic stress and improve the tolerance of crops in low-temperature,drought,metal ion and hydrogen peroxide stresses.
引文
[1] Close T J.Dehydrins:emergence of a biochemical role of a family of plant dehydration proteins [J].Physiologia Plantarum,1996,97(4):795-803.
[2] 黄发平,张林生,王路平,等.小麦脱水素基因WZY2的克隆及其序列分析 [J].西北农林科技大学学报(自然科学版),2009,37(2):93-99. Huang F P,Zhang L S,Wang L P,et al.Cloning and sequence analysis of wheat dehydrin gene WZY2 [J].Journal of Northwest A&F University(Nat Sci Ed),2009,37(2):93-99.
[3] Tsvetanov S,Ohno R,Tsuda K,et al.A cold-responsive wheat (Triticum aestivum L.) gene wcor14 identified in a winter-hardy cultivar “Mironovska 808” [J].Genes Genetic Systems,2000,75(1):49-57.
[4] Baker S S,Wilhelm K S,Thomashow M F.The 5′-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold- drought- and ABA-regulated gene expression [J].Plant Molecular Biology,1994,24(5):701-713.
[5] Close T J.Dehydrins:a commonalty in the response of plants to dehydration and low temperature [J].Physiol Plant,1997,100:291-296.
[6] 强治全,梁雅珺,于正阳,等.小麦wzy2-1基因的克隆及功能分析 [J].作物学报,2016,42(8):1253-1258. Qiang Z Q,Liang Y J,Yu Z Y,et al.Cloning and functional analysis of wzy2-1 gene in wheat [J].Crop Science,2016,42(8):1253-1258.
[7] Allagulova C R,Gimalov F R,Shakirova F M,et al.The plant dehydrins: structure and putative functions [J].Biochemistry,2003,68(9):945-951.
[8] 刘浩,杜娅,李核,等.小麦WDHN1基因的克隆、表达及功能分析 [J].农业生物技术学报,2016,24(11):1676-1687. Liu H,Du Y,Li H,et al.Wheat WDHN1 gene cloning, expression and functional analysis [J].Journal of Agricultural Biotechnology,2016,24(11):1676-1687.
[9] Amara I,Odena A,Oliveira E,et al.Insights into maize LEA proteins:from proteomics to functional approaches [J].Plant and Cell Physiology,2012,53(2):312-329.
[10] Brini F,Yamamoto A,Jlaiel L,et al.Pleiotropic effects of the wheat dehydrin DHN-5 on stress responses in Arabidopsis [J].Plant and Cell Physiology,2011,52(4):676-688.
[11] Vaseva I I,Anders I,Yuperlieva-Mateeva,et al.Dehydrin expression as a potential diagnostic tool for cold stress in white clover [J].Plant Physiol Biochem,2014,78:43-48.
[12] 刘兰,张林生,刑媛,等.干旱胁迫下耐旱性小麦在不同生育期脱水素的表达分析 [J].西北植物学报,2011,31(9):1786-1792. Liu L,Zhang L S,Xing Y,et al.Analysis of the expression of dehydrin in drought tolerant wheat at different growth stages under drought stress [J].Acta Botanica Boreali-Occidentalia Sinica,2011,31(9):1786-1792.
[13] 邢鑫,刘洋,李德全.植物脱水素的结构和功能 [J].植物生理学通讯,2010,46(3):268-276. Xing X,Liu Y,Li D Q.The structure and function of plant dehydrin [J].Plant Physiology Communication,2010,46(3):268-276.
[14] Riera M,Figueras M,Lopez C,et al.Protein kinase CK2 modulates developmental functions of the abscisic acid responsive protein Rab17 from maize [J].Proceedings of the National Academy of Sciences of the USA,2004,101(26):9879-9884.
[15] Alsheikh M K,Svensson J T,Randall S K.Phosphorylation regulated ion- binding is a property shared by the acidic subclass dehydrins [J].Plant,Cell and Environment,2005,28(9):1114-1122.
[16] Graether S P,Boddington K F.Disorder and function:a review of the dehydrin protein family [J].Frontiers in Plant Science,2014,5:576.
[17] 孙歆,雷韬,袁澍,等.脱水素研究进展 [J].武汉植物学研究,2005,23(3):299-304. Sun X,Lei T,Yuan S,et al.Progress in the research of dehydrin [J].Wuhan Botany Study,2005,23(3):299-304.
[18] Rinne P L,Kaikuranta P L M,van der Plas L H W,et al.Dehydrins in cold-acclimated apices of birch (Betula pubescens Ehrh.):production,localization and potential role in rescuing enzyme function during dehydration [J].Planta,1999,209(4):377-388.
[19] Hara M,Terashima S,Kuboi T.Characterization and cry-oprotective activity of cold-responsive dehydrin from Citrus unshiu [J].Plant Physiol,2001,158:1333-1339.
[20] Hara M,Fujinaga M,Kuboi T.Radical scavenging activity and oxidative modification of citrus dehydrin [J].Plant Physiol Biochem,2004,42(7):657-662.
[21] 龙火生,杨公社,庞卫军.一种改进的Western杂交方法 [J].畜牧兽医杂志,2003(3):11-13. Long H S,Yang G S,Pang W J.An improved Western hybridization method [J].Journal of Animal Husbandry and Veterinary Medicine,2003(3):11-13.
[22] Nies D H.Microbial heavy-metal resistance [J].Applied Microbiology & Biotechnology,1999,51(6):730-750.
[23] Yang W,Zhang L,Lü H,et al.The K-segments of wheat dehydrin WZY2 are essential for its protective functions under temperature stress [J].Fronteirs in Plant Science,2015,6:406.
[24] Drira M,Saibi W,Amara I,et al.Wheat dehydrin K-segments ensure bacterial stress tolerance,an-tiaggregation and antimicrobial effects [J].Appl Biochem Biotechnol,2015,175(7):3310-3321.
[25] Wang M,Li P,Li C,et al.LEA14,a novel atypical LEA protein, confers abiotic stress resistance in foxtail millet [J].BMC Plant Biology,2014,14:290-306.
[26] Hanin M,Brini F,Ebel C,et al.Plant dehydrins and stress tolerance:versatile proteins for complex mechanisms [J].Plant Signal Behav,2011,6(10):1503-1509.
[27] Vaseva I I,Anders I,Feller U.Identification and expression of different dehydrin subclasses involved in the drought response of Trifolium repens [J].Journal of Plant Physiology,2014,171(3/4):213-224.
[28] Garay-Arroyo A,Colmenoro-Florest J M,Garciarrubio A,et al.Highly hydrophilic proteins in prokaryotes and eucaryotes are common during conditions of water deficit [J].Biological Chemistry,2000,275(8):5668-5674.
[29] Nylander M,Svensson J,Palva E T,et al.Stress-induced accumulation and tissue-specific localization of dehydrins in Arabidopsis thaliana [J].Plant Mol Biol,2001,45(3):263-279.
[30] Davidson W S,Jonas A,Clayton D F,et al.Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes [J].Biological Chemistry,1998,273(16):9443-9449.
[31] Drira M,Saibi W,Brini F,et al.The K-segments of the wheat dehydrin DHN-5 are essential for the protection of lactate dehydrogenase and beta-glucosidase activities in vitro [J].Molecular Biotechnology,2013,54(2):643-650.
[32] 杨文博.温度胁迫下小麦脱水蛋白WZY2的功能及其K片段调控研究 [D].陕西杨凌:西北农林科技大学,2015. Yang W B.Function of wheat dehydrated protein WZY2 and regulation of K fragment under temperature stress [D].Yang-ling,Shaanxi:Northwest Agriculture and Forestry University,2015.
[33] Hara M,Fujinaga M,Kuboi T.Metal binding by citrus dehydrin with histidine-rich domains [J].Journal of Experimental Botany,2005,56(420):2695-2703.
[34] Rahman L N,Bamm V V,Voyer J A M,et al.Zinc induces disorder-to-order transitions in free and membrane-associated Thellungiella salsuginea dehydrins Ts DHN-1 and Ts DHN-2:a solution CD and solid-state ATR-FTIR study [J].Amino Acids,2011,40(5):1485-1502.
[35] Hughes S,Graether S P.Cryoprotective mechanism of a small intrinsically disordered dehydrin protein [J].Protein Science,2011,20(1):42-50.
[36] Zhu W N,Zhang D P,Lu X X,et al.Characterisation of an SKn-type dehydrin promoter from wheat and its responsiveness to various abiotic and biotic stresses [J].Plant Mol Biol Rep,2014,32:664-678.
[37] Kosova K,Vitamvas P,Prasil I T.Wheat and barley dehydrins under cold,drought,and salinity:what can LEA-Ⅱ proteins tell us about plant stress response [J].Front Plant Sci,2014,5:343.
[38] Mouillon J M,Gustafsson P,Harryson P.Structural investigation of disordered stress proteins,comparison of full-length dehydrins with isolated peptides of their conserved segments [J].Plant Physiol,2006,142(2):638-650.
[2] 黄发平,张林生,王路平,等.小麦脱水素基因WZY2的克隆及其序列分析 [J].西北农林科技大学学报(自然科学版),2009,37(2):93-99. Huang F P,Zhang L S,Wang L P,et al.Cloning and sequence analysis of wheat dehydrin gene WZY2 [J].Journal of Northwest A&F University(Nat Sci Ed),2009,37(2):93-99.
[3] Tsvetanov S,Ohno R,Tsuda K,et al.A cold-responsive wheat (Triticum aestivum L.) gene wcor14 identified in a winter-hardy cultivar “Mironovska 808” [J].Genes Genetic Systems,2000,75(1):49-57.
[4] Baker S S,Wilhelm K S,Thomashow M F.The 5′-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold- drought- and ABA-regulated gene expression [J].Plant Molecular Biology,1994,24(5):701-713.
[5] Close T J.Dehydrins:a commonalty in the response of plants to dehydration and low temperature [J].Physiol Plant,1997,100:291-296.
[6] 强治全,梁雅珺,于正阳,等.小麦wzy2-1基因的克隆及功能分析 [J].作物学报,2016,42(8):1253-1258. Qiang Z Q,Liang Y J,Yu Z Y,et al.Cloning and functional analysis of wzy2-1 gene in wheat [J].Crop Science,2016,42(8):1253-1258.
[7] Allagulova C R,Gimalov F R,Shakirova F M,et al.The plant dehydrins: structure and putative functions [J].Biochemistry,2003,68(9):945-951.
[8] 刘浩,杜娅,李核,等.小麦WDHN1基因的克隆、表达及功能分析 [J].农业生物技术学报,2016,24(11):1676-1687. Liu H,Du Y,Li H,et al.Wheat WDHN1 gene cloning, expression and functional analysis [J].Journal of Agricultural Biotechnology,2016,24(11):1676-1687.
[9] Amara I,Odena A,Oliveira E,et al.Insights into maize LEA proteins:from proteomics to functional approaches [J].Plant and Cell Physiology,2012,53(2):312-329.
[10] Brini F,Yamamoto A,Jlaiel L,et al.Pleiotropic effects of the wheat dehydrin DHN-5 on stress responses in Arabidopsis [J].Plant and Cell Physiology,2011,52(4):676-688.
[11] Vaseva I I,Anders I,Yuperlieva-Mateeva,et al.Dehydrin expression as a potential diagnostic tool for cold stress in white clover [J].Plant Physiol Biochem,2014,78:43-48.
[12] 刘兰,张林生,刑媛,等.干旱胁迫下耐旱性小麦在不同生育期脱水素的表达分析 [J].西北植物学报,2011,31(9):1786-1792. Liu L,Zhang L S,Xing Y,et al.Analysis of the expression of dehydrin in drought tolerant wheat at different growth stages under drought stress [J].Acta Botanica Boreali-Occidentalia Sinica,2011,31(9):1786-1792.
[13] 邢鑫,刘洋,李德全.植物脱水素的结构和功能 [J].植物生理学通讯,2010,46(3):268-276. Xing X,Liu Y,Li D Q.The structure and function of plant dehydrin [J].Plant Physiology Communication,2010,46(3):268-276.
[14] Riera M,Figueras M,Lopez C,et al.Protein kinase CK2 modulates developmental functions of the abscisic acid responsive protein Rab17 from maize [J].Proceedings of the National Academy of Sciences of the USA,2004,101(26):9879-9884.
[15] Alsheikh M K,Svensson J T,Randall S K.Phosphorylation regulated ion- binding is a property shared by the acidic subclass dehydrins [J].Plant,Cell and Environment,2005,28(9):1114-1122.
[16] Graether S P,Boddington K F.Disorder and function:a review of the dehydrin protein family [J].Frontiers in Plant Science,2014,5:576.
[17] 孙歆,雷韬,袁澍,等.脱水素研究进展 [J].武汉植物学研究,2005,23(3):299-304. Sun X,Lei T,Yuan S,et al.Progress in the research of dehydrin [J].Wuhan Botany Study,2005,23(3):299-304.
[18] Rinne P L,Kaikuranta P L M,van der Plas L H W,et al.Dehydrins in cold-acclimated apices of birch (Betula pubescens Ehrh.):production,localization and potential role in rescuing enzyme function during dehydration [J].Planta,1999,209(4):377-388.
[19] Hara M,Terashima S,Kuboi T.Characterization and cry-oprotective activity of cold-responsive dehydrin from Citrus unshiu [J].Plant Physiol,2001,158:1333-1339.
[20] Hara M,Fujinaga M,Kuboi T.Radical scavenging activity and oxidative modification of citrus dehydrin [J].Plant Physiol Biochem,2004,42(7):657-662.
[21] 龙火生,杨公社,庞卫军.一种改进的Western杂交方法 [J].畜牧兽医杂志,2003(3):11-13. Long H S,Yang G S,Pang W J.An improved Western hybridization method [J].Journal of Animal Husbandry and Veterinary Medicine,2003(3):11-13.
[22] Nies D H.Microbial heavy-metal resistance [J].Applied Microbiology & Biotechnology,1999,51(6):730-750.
[23] Yang W,Zhang L,Lü H,et al.The K-segments of wheat dehydrin WZY2 are essential for its protective functions under temperature stress [J].Fronteirs in Plant Science,2015,6:406.
[24] Drira M,Saibi W,Amara I,et al.Wheat dehydrin K-segments ensure bacterial stress tolerance,an-tiaggregation and antimicrobial effects [J].Appl Biochem Biotechnol,2015,175(7):3310-3321.
[25] Wang M,Li P,Li C,et al.LEA14,a novel atypical LEA protein, confers abiotic stress resistance in foxtail millet [J].BMC Plant Biology,2014,14:290-306.
[26] Hanin M,Brini F,Ebel C,et al.Plant dehydrins and stress tolerance:versatile proteins for complex mechanisms [J].Plant Signal Behav,2011,6(10):1503-1509.
[27] Vaseva I I,Anders I,Feller U.Identification and expression of different dehydrin subclasses involved in the drought response of Trifolium repens [J].Journal of Plant Physiology,2014,171(3/4):213-224.
[28] Garay-Arroyo A,Colmenoro-Florest J M,Garciarrubio A,et al.Highly hydrophilic proteins in prokaryotes and eucaryotes are common during conditions of water deficit [J].Biological Chemistry,2000,275(8):5668-5674.
[29] Nylander M,Svensson J,Palva E T,et al.Stress-induced accumulation and tissue-specific localization of dehydrins in Arabidopsis thaliana [J].Plant Mol Biol,2001,45(3):263-279.
[30] Davidson W S,Jonas A,Clayton D F,et al.Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes [J].Biological Chemistry,1998,273(16):9443-9449.
[31] Drira M,Saibi W,Brini F,et al.The K-segments of the wheat dehydrin DHN-5 are essential for the protection of lactate dehydrogenase and beta-glucosidase activities in vitro [J].Molecular Biotechnology,2013,54(2):643-650.
[32] 杨文博.温度胁迫下小麦脱水蛋白WZY2的功能及其K片段调控研究 [D].陕西杨凌:西北农林科技大学,2015. Yang W B.Function of wheat dehydrated protein WZY2 and regulation of K fragment under temperature stress [D].Yang-ling,Shaanxi:Northwest Agriculture and Forestry University,2015.
[33] Hara M,Fujinaga M,Kuboi T.Metal binding by citrus dehydrin with histidine-rich domains [J].Journal of Experimental Botany,2005,56(420):2695-2703.
[34] Rahman L N,Bamm V V,Voyer J A M,et al.Zinc induces disorder-to-order transitions in free and membrane-associated Thellungiella salsuginea dehydrins Ts DHN-1 and Ts DHN-2:a solution CD and solid-state ATR-FTIR study [J].Amino Acids,2011,40(5):1485-1502.
[35] Hughes S,Graether S P.Cryoprotective mechanism of a small intrinsically disordered dehydrin protein [J].Protein Science,2011,20(1):42-50.
[36] Zhu W N,Zhang D P,Lu X X,et al.Characterisation of an SKn-type dehydrin promoter from wheat and its responsiveness to various abiotic and biotic stresses [J].Plant Mol Biol Rep,2014,32:664-678.
[37] Kosova K,Vitamvas P,Prasil I T.Wheat and barley dehydrins under cold,drought,and salinity:what can LEA-Ⅱ proteins tell us about plant stress response [J].Front Plant Sci,2014,5:343.
[38] Mouillon J M,Gustafsson P,Harryson P.Structural investigation of disordered stress proteins,comparison of full-length dehydrins with isolated peptides of their conserved segments [J].Plant Physiol,2006,142(2):638-650.