白菜型冬油菜响应干旱胁迫差异蛋白质组学和HSC70-1基因克隆及表达分析
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摘要
为探索白菜型冬油菜抗旱机理,采用双向电泳结合液相色谱-质谱技术分析抗旱冬油菜品系DR-5差异蛋白质组变化,克隆其差异蛋白热激同源蛋白70(heat shock cognate protein 70)基因HSC70-1,研究干旱胁迫对白菜型冬油菜HSC70-1表达的影响。质谱鉴定出23个差异蛋白质,分别参与刺激响应(5)、糖/能量代谢(6)、脂代谢(2)、信号转导(1)、氨基酸/蛋白质代谢(2)、核酸代谢(1)、细胞骨架(1)、光合作用(2)、伴侣蛋白(3)。同源克隆得到1 911bp的HSC70-1基因完整开放阅读框,编码637个氨基酸残基。所编码的蛋白HSC70是稳定的疏水性蛋白质,含有HSP蛋白家族特有的核酸结合功能区及底物结合功能区,有5个活性口袋,氨基酸序列与白菜型油菜(Brassica rapa)HSC70氨基酸序列相似度达98%。实时荧光定量和半定量分析HSC70-1基因干旱胁迫下表达,发现HSC70-1在叶片中上调表达。本研究结果为冬油菜抗旱育种提供了理论基础。
In this study, we analyzed the differential proteome of winter rapeseed(Brassica rapa) by two-dimensional gel electrophoresis(2 D-DIGE) technology combined with the liquid chromatography-mass spectrometry(LC-MS) technology to detect the changes of differential proteome in DR-5(drought-resistant Winter Rapeseed line) under drought stress, and cloned the heat shock cognate protein 70 gene(HSC70-1) and analyzed the expression of this gene under drought stress. In order to provide a theoretical base for drought resistance breeding of winter rapeseed, the effect of moderate drought stress on winter rapeseed was studied. We obtained the coding region sequence of HSC70-1 in winter rapeseed by homologous cloning, and analysed the physicochemical properties, secondary structure, tertiary structure, signal peptide, transmembrane structure, conserved region, active pocket and phylogenetic conservatism of this gene encoding protein HSC70 by bioinformatics analysis methods. The real-time fluorescence quantitative and semi-quantitative analysis found the changes of HSC70-1 gene expression levels under drought stress. There were 23 differentially expressed proteins successfully identified by Mass spectrometry, which were involved in stimulation response(5), glucose/energy metabolism(6), lipid metabolism(2), signal transduction(1), amino acid/protein metabolism(2), nucleic acid metabolism(1), cytoskeleton(1), photosynthesis(2) and chaperone protein(3). We obtained the 1 911 bp complete open reading frame of HSC70-1 gene via cloning,which encoded 637 amino acid sequences. HSC70 is a stable hydrophobic protein,and it contains the unique nucleic acid and substrate binding functional area of HSP protein family,it have five active pockets. The amino acid sequence was highly similar to HSC70 in Brassica rape,and the similarity was 98%,which is highly conserved. The expression of HSC70-1 in winter rapeseed leaves was up-regulated under drought stress,which improved the tolerance of drought stress in winter rapeseed.
In this study, we analyzed the differential proteome of winter rapeseed(Brassica rapa) by two-dimensional gel electrophoresis(2 D-DIGE) technology combined with the liquid chromatography-mass spectrometry(LC-MS) technology to detect the changes of differential proteome in DR-5(drought-resistant Winter Rapeseed line) under drought stress, and cloned the heat shock cognate protein 70 gene(HSC70-1) and analyzed the expression of this gene under drought stress. In order to provide a theoretical base for drought resistance breeding of winter rapeseed, the effect of moderate drought stress on winter rapeseed was studied. We obtained the coding region sequence of HSC70-1 in winter rapeseed by homologous cloning, and analysed the physicochemical properties, secondary structure, tertiary structure, signal peptide, transmembrane structure, conserved region, active pocket and phylogenetic conservatism of this gene encoding protein HSC70 by bioinformatics analysis methods. The real-time fluorescence quantitative and semi-quantitative analysis found the changes of HSC70-1 gene expression levels under drought stress. There were 23 differentially expressed proteins successfully identified by Mass spectrometry, which were involved in stimulation response(5), glucose/energy metabolism(6), lipid metabolism(2), signal transduction(1), amino acid/protein metabolism(2), nucleic acid metabolism(1), cytoskeleton(1), photosynthesis(2) and chaperone protein(3). We obtained the 1 911 bp complete open reading frame of HSC70-1 gene via cloning,which encoded 637 amino acid sequences. HSC70 is a stable hydrophobic protein,and it contains the unique nucleic acid and substrate binding functional area of HSP protein family,it have five active pockets. The amino acid sequence was highly similar to HSC70 in Brassica rape,and the similarity was 98%,which is highly conserved. The expression of HSC70-1 in winter rapeseed leaves was up-regulated under drought stress,which improved the tolerance of drought stress in winter rapeseed.
引文
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[18] 梁建萍,贾小云,刘亚令,等.干旱胁迫对蒙古黄芪生长及根部次生代谢物含量的影响[J].生态学报,2016,36(14):4 415-4 422.
[19] 米超,赵艳宁,刘自刚,等.白菜型冬油菜RuBisCo蛋白亚基基因rbcL和rbcS的克隆及其在干旱胁迫下的表达[J].作物学报,2018,44(12):1 882-1 890.
[20] 刘晓东,李月,王若仲,等.过表达GH3-5提高拟南芥抗旱的分子机制[J].南京农业大学学报,2016,39(4):557-562.
[21] 侯典云,杨萌萌,李炯,等.小麦返白系HSP70基因cDNA克隆及序列分析[J].基因组学与应用生物学,2018,37(9):3 874-3 880.
[22] 马盼盼.干旱胁迫下白芥差异表达蛋白的研究[D].郑州:郑州大学,2012:45-48.
[23] 李国龙,吴海霞,孙亚卿,等.甜菜叶片应答干旱胁迫的差异蛋白质组学分析[J].作物杂志,2015(5):63-68.
[24] Liu C Q,Zhang X K,Zhang K,et al.Comparative analysis of the Brassica napus root and leaf transcript profiling in response to drought stress[J].Int J Mol Sci,2015,16(8):18 752-18 777.
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[26] 刘晓东,王若仲,焦彬彬,等.拟南芥IAA酰胺合成酶GH3-6负调控干旱和盐胁迫的反应[J].植物学报,2016,51(5):586-593.
[27] Galvis M L E,Marttila S,H?kansson G,et al.Heat stress response in pea involves interaction of mitochondrial nucleoside diphosphate Kinase with a Novel 86-Kilodalton Protein[J].Plant Physiol,2001,126(1):69-77.
[28] Haejeong M,Boyoung L,Giltsu C,et al.NDP kinase 2 interacts with two oxidative stress-activated MAPKs to regulate cellular redox state and enhances multiple stress tolerance in transgenic plants[J].PNAS,2003,100(1):58-63.
[29] Guo X J,Feng J N.Comparisons of expression levels of heat shock proteins (hsp70 and hsp90) from anaphothrips obscurus (Thysanoptera:Thripidae) in polymorphic adults exposed to different heat shock treatments[J].J Insect Sci,2018,18(3):1-10.
[30] Marriott A S,Vasieva O,Fang Y,et al.NUDT2 disruption elevates diadenosine tetraphosphate (Ap4A) and down-regulates immune response and cancer promotion genes[J].PLoS One,2016,11(5):e0154674.
[31] 闫龙凤,杨青川,韩建国,等.植物半胱氨酸蛋白酶研究进展[J].草业学报,2005,14(5):11-19.
[2] 王向辉.西北地区环境变迁与农业可持续发展研究[D].杨凌:西北农林科技大学,2012:25-30.
[3] 孔德晶,王月,孙万仓,等.北方白菜型冬油菜F2主要生理生化特性的变异与抗寒性相关分析[J].草业学报,2014,23(4):79-86.
[4] 梁潘霞,黄杏,李杨瑞.甘蔗小分子量热激蛋白(sHSP)基因克隆及水分胁迫下的表达分析[J].生物技术通报,2016,32(10):163-169.
[5] 李雅博,李婷,韩莹琰,等.叶用莴苣热激蛋白基因LsHsp70-2711的克隆及高温胁迫下的功能分析[J].中国农业科学,2017,50(8):1 486-1 494.
[6] 裴丽丽,徐兆师,尹丽娟,等.植物热激蛋白90的分子作用机理及其利用研究进展[J].植物遗传资源学报,2013,14(1):109-114.
[7] 安艳秋,蔺瑞明,芦冯晶,等.小麦热激蛋白基因TaHSP70克隆及其在植物防卫和抗逆反应中的表达分析[J].分子植物育种,2011,9(4):402-409.
[8] 王明强,张道远.植物热激蛋白70基因家族及其生物学功能研究进展[J].基因组学与应用生物学,2015,34(2):421-428.
[9] 樊芳菲,杨暹,康云艳,等.植物DnaJ蛋白的研究进展[J].分子植物育种,2018,16(6):2 028-2 034.
[10] 王利彬,董爽爽,王灿国,等.黑麦热激蛋白ScHsp90-1基因的克隆及表达分析[J].山东农业科学,2018,50(3):1-5.
[11] Wang N B,Zhao J,He X Y,et al.Comparative proteomic analysis of drought tolerance in the two contrasting Tibetan wild genotypes and cultivated genotype[J].BMC Genomics,2015,16(1):1-19.
[12] Xie H,Yang D H,Yao H,et al.iTRAQ-based quantitative proteomic analysis reveals proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum) in response to drought stress[J].Biochem Bioph Res,2016,469(3):768-775.
[13] Liu H,Muhammad A R F S,Liu X L,et al.Physiological and comparative proteomic analysis reveals different drought responses in roots and leaves of drought-tolerant wild wheat (Triticum boeoticum)[J].PLoS One,2015,10(4):e0121852.
[14] Ankur R B,Gopal J,Bharti K,et al.Global insights into high temperature and drought stress regulated genes by RNA-Seq in economically important oilseed crop Brassica juncea[J].BMC Plant Biol,2015,15(1):9.
[15] Lu K,Zhang L,Qu C M,et al.Identification of drought stress-responsive genes in leaves of Brassica napus by RNA sequencing[J].Sci Agric Sin,2015,48:630-645.
[16] Yong H Y,Zou Z W,Eng-Piew Kok,et al.Comparative transcriptome analysis of leaves and roots in response to sudden increase in salinity in Brassica napus by RNA-seq[J].Biomed Res Int,2014:467395.
[17] 崔大练,马玉心,石戈,等.紫穗槐幼苗叶片对不同干旱梯度胁迫的生理生态响应[J].水土保持研究,2010,17(2):178-181.
[18] 梁建萍,贾小云,刘亚令,等.干旱胁迫对蒙古黄芪生长及根部次生代谢物含量的影响[J].生态学报,2016,36(14):4 415-4 422.
[19] 米超,赵艳宁,刘自刚,等.白菜型冬油菜RuBisCo蛋白亚基基因rbcL和rbcS的克隆及其在干旱胁迫下的表达[J].作物学报,2018,44(12):1 882-1 890.
[20] 刘晓东,李月,王若仲,等.过表达GH3-5提高拟南芥抗旱的分子机制[J].南京农业大学学报,2016,39(4):557-562.
[21] 侯典云,杨萌萌,李炯,等.小麦返白系HSP70基因cDNA克隆及序列分析[J].基因组学与应用生物学,2018,37(9):3 874-3 880.
[22] 马盼盼.干旱胁迫下白芥差异表达蛋白的研究[D].郑州:郑州大学,2012:45-48.
[23] 李国龙,吴海霞,孙亚卿,等.甜菜叶片应答干旱胁迫的差异蛋白质组学分析[J].作物杂志,2015(5):63-68.
[24] Liu C Q,Zhang X K,Zhang K,et al.Comparative analysis of the Brassica napus root and leaf transcript profiling in response to drought stress[J].Int J Mol Sci,2015,16(8):18 752-18 777.
[25] 顾颖慧.龙须菜热激蛋白70(HSP70)基因克隆及热激下的表达模式分析[D].青岛:中国海洋大学,2011:56-59.
[26] 刘晓东,王若仲,焦彬彬,等.拟南芥IAA酰胺合成酶GH3-6负调控干旱和盐胁迫的反应[J].植物学报,2016,51(5):586-593.
[27] Galvis M L E,Marttila S,H?kansson G,et al.Heat stress response in pea involves interaction of mitochondrial nucleoside diphosphate Kinase with a Novel 86-Kilodalton Protein[J].Plant Physiol,2001,126(1):69-77.
[28] Haejeong M,Boyoung L,Giltsu C,et al.NDP kinase 2 interacts with two oxidative stress-activated MAPKs to regulate cellular redox state and enhances multiple stress tolerance in transgenic plants[J].PNAS,2003,100(1):58-63.
[29] Guo X J,Feng J N.Comparisons of expression levels of heat shock proteins (hsp70 and hsp90) from anaphothrips obscurus (Thysanoptera:Thripidae) in polymorphic adults exposed to different heat shock treatments[J].J Insect Sci,2018,18(3):1-10.
[30] Marriott A S,Vasieva O,Fang Y,et al.NUDT2 disruption elevates diadenosine tetraphosphate (Ap4A) and down-regulates immune response and cancer promotion genes[J].PLoS One,2016,11(5):e0154674.
[31] 闫龙凤,杨青川,韩建国,等.植物半胱氨酸蛋白酶研究进展[J].草业学报,2005,14(5):11-19.