核桃V-ATPase c亚基基因(JrVHAc4)的克隆和抗旱功能分析
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摘要
【目的】V-ATPase是植物逆境响应的重要酶,有必要了解V-ATPase相关亚基参与核桃响应逆境胁迫的功能机制。【方法】从‘香玲’核桃转录组中克隆获得1条V-ATPase c亚基基因(命名为JrVHAc4),通过分析启动子预测其逆境响应功能。对JrVHAc4基因进行干旱胁迫下的表达分析,同时构建JrVHAc4酵母表达载体转入酵母表达系统研究其抗旱功能。【结果】JrVHAc4基因开放读码框(ORF)全长495 bp,拟推导的蛋白分子量为16 527.61 u,包含164个氨基酸,理论等电点为8.62;其上游1 047 bp启动子包含MYC、DOF、MYB等与干旱响应相关的顺式作用元件。干旱胁迫下,JrVHAc4基因在核桃叶和根中均被显著诱导,并存在差异。对JrVHAc4转基因酵母进行不同浓度甘露醇胁迫,与对照酵母相比,发现JrVHAc4基因的表达能显著提高转基因酵母的生长活性。【结论】JrVHAc4基因能响应干旱胁迫,并能提高酵母的抗旱能力,JrVHAc4可作为核桃抗逆重要候选基因。
【Objective】 V-ATPase is an important enzyme in plant stress response. This study is understand the functional mechanism of V-ATPase subunits in response to stress in walnuts. 【Method】 A V-ATPase c subunit gene(named JrVHAc4) was cloned from the ‘Xiangling' walnut transcriptome. The stress response function was predicted by analyzing its promoter and expression under drought stress. Meanwhile, JrVHAc4 was inserted into a yeast expression vector pYES2 and transformed into yeast INVSC1 to study its drought resistance function. 【Result】 The open reading frame(ORF) of JrVHAc4 was 495 bp in length, the deduced protein was 16 527.61 u and contained 164 amino acids, whose theoretical isoelectric point(pI) was 8.62. An upstream 1 047 bp promoter segment was identified and found to contain abundant cis-elements, among which some are related to drought response, such as MYC, DOF and MYB. JrVHAc4 was significantly induced in leaves and roots by drought stress, and the induction in leaves and roots were different. Compared with control yeast, we found that expression of JrVHAc4 increased the growth activity of transgenic yeast significantly under different concentrations of mannitol stress. 【Conclusion】 JrVHAc4 can respond to drought stress and improve the drought resistance of transgenic yeasts, JrVHAc4 can be used as an important candidate gene for walnut resistance.
【Objective】 V-ATPase is an important enzyme in plant stress response. This study is understand the functional mechanism of V-ATPase subunits in response to stress in walnuts. 【Method】 A V-ATPase c subunit gene(named JrVHAc4) was cloned from the ‘Xiangling' walnut transcriptome. The stress response function was predicted by analyzing its promoter and expression under drought stress. Meanwhile, JrVHAc4 was inserted into a yeast expression vector pYES2 and transformed into yeast INVSC1 to study its drought resistance function. 【Result】 The open reading frame(ORF) of JrVHAc4 was 495 bp in length, the deduced protein was 16 527.61 u and contained 164 amino acids, whose theoretical isoelectric point(pI) was 8.62. An upstream 1 047 bp promoter segment was identified and found to contain abundant cis-elements, among which some are related to drought response, such as MYC, DOF and MYB. JrVHAc4 was significantly induced in leaves and roots by drought stress, and the induction in leaves and roots were different. Compared with control yeast, we found that expression of JrVHAc4 increased the growth activity of transgenic yeast significantly under different concentrations of mannitol stress. 【Conclusion】 JrVHAc4 can respond to drought stress and improve the drought resistance of transgenic yeasts, JrVHAc4 can be used as an important candidate gene for walnut resistance.
引文
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[11] 肖忠意,谭琨岭,胡明瑜,等.过量表达棉花液泡 H+-ATPase c 亚基基因促进酵母细胞伸长和提高盐胁迫耐受性[J].遗传,2008,30(4):495-500.DOI:10.3321/j.issn:0253-9772.2008.04.017.XIAO Z Y,TAN K L,HU M Y,et al.Overexpression of cotton vacuole H+-ATPase c subunit gene promotes yeast cell elongation and increases salt stress tolerance[J].Genetic,2008,30(4):495-500.
[12] BAISAKH N,RAMANARAO M V,RAJASEKARAN K,et al.Enhanced salt stress tolerance of rice plants expressing a vacuolar H+-ATPase subunit c1 (SaVHAc1) gene from the halophyte grass Spartina alterniflora Loisel[J].Plant Biotechnology Journal,2012,10(4):453-464.DOI:10.1111/j.1467-7652.2012.00678.x.
[13] 高向倩,李忆林,贾彩霞,等.核桃抗逆基因JrGSTU23的克隆及表达分析[J].浙江农林大学学报,2018,35(4):589-595.DOI:10.11833/j.issn.2095-0756.2018.04.002.GAO X Q,LI Y L,JIA C X,et al.Identification and expression analysis of the stress resistance gene JrGSTU23 from Juglans regia[J].Journal of Zhejiang A&F University,2018,35(4):589-595.
[14] 陈淑雯,郝茜珣,贾彩霞,等.核桃WD40转录因子JrATG18a基因的克隆及逆境响应[J].植物遗传资源学报,2018,19(5):979-986.DOI:10.13430/j.cnki.jpgr.20180121002.CHEN S W,HAO X X,JIA C X,et al.Identification and stress response analysis of a WD40 transcription factor JrATG18a gene from Juglans regia[J].Journal of Plant Genetic Resources,2018,19(5):979-986.
[15] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method[J].Methods,2001,25(4):402-408.
[16] 粟莉圆,李孝哲,陈淑雯,等.核桃JrGRAS2基因响应热胁迫的表达及功能分析[J].植物研究,2018,38(1):125-131.DOI:10.7525/j.issn.1673-5102.2018.01.015.SU L Y,LI X Z,CHEN S W,et al.Expression and function analysis of walnut JrGRAS2 gene under heat stress[J].Bulletin of Botanical Research,2018,38(1):125-131.
[17] GAO C Q,JIANG B,WANG Y C,et al.Overexpression of a heat shock protein (ThHSP18.3) from Tamarix hispida confers stress tolerance to yeast[J].Molecular Biology Reports,2012,39(4):4889-4897.DOI:10.1007/s11033-011-1284-2.
[18] ZANG D,WANG L,ZHANG Y,et al.ThDof1.4 and ThZFP1 constitute a transcriptional regulatory cascade involved in salt or osmotic stress in Tamarix hispida[J].Plant Molecular Biology,2017,94(4/5):495-507.DOI:10.1007/s11103-017-0620-x.
[19] YIN X,CUI Y,WANG M,et al.Overexpression of a novel MYB-related transcription factor,OsMYBR1,confers improved drought tolerance and decreased ABA sensitivity in rice[J].Biochemical and Biophysical Research Communications,2017,490(4):1355-1361.DOI:10.1016/j.bbrc.2017.07.029.
[20] HUANG C,ZHOU J,JIE Y,et al.A ramie bZIP transcription factor BnbZIP2 is involved in drought,salt,and heavy metal stress response[J].DNA and Cell Biology,2016,35(12):776-786.DOI:10.1089/dna.2016.3251.
[21] GAO C Q,WANG Y C,JIANG B,et al.A novel vacuolar membrane H+-ATPase c subunit gene (ThVHAc1) from Tamarix hispida confers tolerance to several abiotic stresses in Saccharomyces cerevisiae[J].Molecular Biology Reports,2011,38(2):957-963.DOI:10.1007/s11033-010-0189-9.
[22] 张博,王莎莎,王君珂,等.小桐子JcLEA4 基因克隆及表达和功能分析[J].基因组学与应用生物学,2018,37(8):3473-3481.DOI:10.13417/j.gab.037.003473.ZHANG B,WANG S S,WANG J K,et al.Cloning,expression and functional analysis of Jatropha JcLEA4 gene[J].Genomics and Applied Biology,2018,37(8):3473-3481.
[23] 粟莉圆,李大培,常远,等.核桃JrGSTTau1基因的克隆及转入酵母的抗逆功能分析[J].西北林学院学报,2016,31(3):98-102.DOI:10.3969/j.issn.1001-7461.2016.03.16.SU L Y,LI D P,CHANG Y,et al.Cloning of walnut JrGSTau1 gene and analysis of its anti-reverse function into yeast[J].Journal of Northwest Forestry University,2016,31(3):98-102.
[24] 杨桂燕,于丽丽,赵玉琳,等.刚毛柽柳TheIF1A基因转入酵母的抗逆能力分析[J].南京林业大学学报(自然科学版),2014,38(5):62-66.DOI:10.3969/j.issn.1000-2006.2014.05.013.YANG G Y,YU L L,ZHAO Y L,et al.Stress tolerance analysis of a Tamarix hispida TheIF1A in Saccharomyces cerevisiae[J].Journal of Nanjing Forestry University (Natural Sciences Edition),2014,38(5):62-66.
[25] 周爱民,卜媛媛,张欣欣,等.过量表达星星草(Puccinellia tenuiflora)液泡型 H+-ATP 酶c亚基基因提高转基因酵母的耐盐性[J].分子植物育种,2015,13(2):409-414.DOI:10.13271/j.mpb.013.000409.ZHOU A M,BU Y Y,ZHANG X X,et al.Overexpression of Puccinellia tenuiflora vacuolar H+-ATPase c subunit gene enhances salt tolerance of transgenic yeast[J].Molecular Plant Breeding,2015,13(2):409-414.
[2] KABALA K,JANICKA R-RUSSAK M.Differential regulation of vacuolar H+-ATPase and H+-PPase in Cucumis sativus roots by zinc and nickel[J].Plant Science,2011,180(3):531-539.DOI:10.1016/j.plantsci.2010.11.013.
[3] YOICHIRO F,ALI F.V-ATPase dys function under excess zinc inhibits Arabidopsis cell expansion[J].Plant Signaling & Behavior,2011,6(9):1253-1255.DOI:10.4161/psb.6.9.16529.
[4] YANG G Y,WANG C,WANG Y C,et al.Overexpression of ThVHAc1 and its potential upstream regulator,ThWRKY7,improved plant tolerance of cadmium stress[J].Scientific Reports,2016,6(18752):1-17.DOI:10.1038/srep18752.
[5] XU Z G,GE Y,ZHANG W,et al.The walnut JrVHAG1 gene is involved in cadmium stress response through ABA-signal pathway and MYB transcription regulation[J].BMC Plant Biology,2018,18(19):2-13.DOI:10.1186/s12870-018-1231-7.
[6] XU Z G,ZHAO Y L,GE Y,et al.Characterization of a vacuolar H+-ATPase G subunit gene from Juglans regia (JrVHAG1) involved in mannitol-induced osmotic stress tolerance[J].Plant Cell Reports,2017,36(3):407-418.DOI:10.1007/s00299-016-2090-z.
[7] HANITZSCH M,SCHNITZER D,SEIDEL T,et al.Transcript level regulation of the vacuolar H(+)-ATPase subunit isoforms VHA-a,VHA-E and VHA-G in Arabidopsis thaliana[J].Molecular Membrane Biology,2007,24(5/6):507-518.DOI:10.1080/09687680701447393.
[8] ZHOU A,BU Y,TAKANO T,et al.Conserved V-ATPase c subunit plays a role in plant growth by influencing V-ATPase-dependent endosomal trafficking[J].Plant Biotechnology Journal,2015,28(10):12381.DOI:10.1111/pbi.12381.
[9] GAXIOLA R A,PALMGREN M G,SCHUMACHER K.Plant proton pumps[J].Febs Letters,2007,581(12):2204-2214.DOI:10.1016/j.febslet.2007.03.050.
[10] 徐萍,刘瑞香,曾卫军,等.拟南芥VHA-c基因在非生物胁迫响应中的作用[J].华北农学报,2006,21(1):19-22.DOI:10.3321/j.issn:1000-7091.2006.01.005.XU P,LIU R X,ZENG W J,et al.The role of VHA-c gene in Arabidopsis thaliana in abiotic stress response[J].North China Agricultural Journal,2006,21(1):19-22.
[11] 肖忠意,谭琨岭,胡明瑜,等.过量表达棉花液泡 H+-ATPase c 亚基基因促进酵母细胞伸长和提高盐胁迫耐受性[J].遗传,2008,30(4):495-500.DOI:10.3321/j.issn:0253-9772.2008.04.017.XIAO Z Y,TAN K L,HU M Y,et al.Overexpression of cotton vacuole H+-ATPase c subunit gene promotes yeast cell elongation and increases salt stress tolerance[J].Genetic,2008,30(4):495-500.
[12] BAISAKH N,RAMANARAO M V,RAJASEKARAN K,et al.Enhanced salt stress tolerance of rice plants expressing a vacuolar H+-ATPase subunit c1 (SaVHAc1) gene from the halophyte grass Spartina alterniflora Loisel[J].Plant Biotechnology Journal,2012,10(4):453-464.DOI:10.1111/j.1467-7652.2012.00678.x.
[13] 高向倩,李忆林,贾彩霞,等.核桃抗逆基因JrGSTU23的克隆及表达分析[J].浙江农林大学学报,2018,35(4):589-595.DOI:10.11833/j.issn.2095-0756.2018.04.002.GAO X Q,LI Y L,JIA C X,et al.Identification and expression analysis of the stress resistance gene JrGSTU23 from Juglans regia[J].Journal of Zhejiang A&F University,2018,35(4):589-595.
[14] 陈淑雯,郝茜珣,贾彩霞,等.核桃WD40转录因子JrATG18a基因的克隆及逆境响应[J].植物遗传资源学报,2018,19(5):979-986.DOI:10.13430/j.cnki.jpgr.20180121002.CHEN S W,HAO X X,JIA C X,et al.Identification and stress response analysis of a WD40 transcription factor JrATG18a gene from Juglans regia[J].Journal of Plant Genetic Resources,2018,19(5):979-986.
[15] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method[J].Methods,2001,25(4):402-408.
[16] 粟莉圆,李孝哲,陈淑雯,等.核桃JrGRAS2基因响应热胁迫的表达及功能分析[J].植物研究,2018,38(1):125-131.DOI:10.7525/j.issn.1673-5102.2018.01.015.SU L Y,LI X Z,CHEN S W,et al.Expression and function analysis of walnut JrGRAS2 gene under heat stress[J].Bulletin of Botanical Research,2018,38(1):125-131.
[17] GAO C Q,JIANG B,WANG Y C,et al.Overexpression of a heat shock protein (ThHSP18.3) from Tamarix hispida confers stress tolerance to yeast[J].Molecular Biology Reports,2012,39(4):4889-4897.DOI:10.1007/s11033-011-1284-2.
[18] ZANG D,WANG L,ZHANG Y,et al.ThDof1.4 and ThZFP1 constitute a transcriptional regulatory cascade involved in salt or osmotic stress in Tamarix hispida[J].Plant Molecular Biology,2017,94(4/5):495-507.DOI:10.1007/s11103-017-0620-x.
[19] YIN X,CUI Y,WANG M,et al.Overexpression of a novel MYB-related transcription factor,OsMYBR1,confers improved drought tolerance and decreased ABA sensitivity in rice[J].Biochemical and Biophysical Research Communications,2017,490(4):1355-1361.DOI:10.1016/j.bbrc.2017.07.029.
[20] HUANG C,ZHOU J,JIE Y,et al.A ramie bZIP transcription factor BnbZIP2 is involved in drought,salt,and heavy metal stress response[J].DNA and Cell Biology,2016,35(12):776-786.DOI:10.1089/dna.2016.3251.
[21] GAO C Q,WANG Y C,JIANG B,et al.A novel vacuolar membrane H+-ATPase c subunit gene (ThVHAc1) from Tamarix hispida confers tolerance to several abiotic stresses in Saccharomyces cerevisiae[J].Molecular Biology Reports,2011,38(2):957-963.DOI:10.1007/s11033-010-0189-9.
[22] 张博,王莎莎,王君珂,等.小桐子JcLEA4 基因克隆及表达和功能分析[J].基因组学与应用生物学,2018,37(8):3473-3481.DOI:10.13417/j.gab.037.003473.ZHANG B,WANG S S,WANG J K,et al.Cloning,expression and functional analysis of Jatropha JcLEA4 gene[J].Genomics and Applied Biology,2018,37(8):3473-3481.
[23] 粟莉圆,李大培,常远,等.核桃JrGSTTau1基因的克隆及转入酵母的抗逆功能分析[J].西北林学院学报,2016,31(3):98-102.DOI:10.3969/j.issn.1001-7461.2016.03.16.SU L Y,LI D P,CHANG Y,et al.Cloning of walnut JrGSTau1 gene and analysis of its anti-reverse function into yeast[J].Journal of Northwest Forestry University,2016,31(3):98-102.
[24] 杨桂燕,于丽丽,赵玉琳,等.刚毛柽柳TheIF1A基因转入酵母的抗逆能力分析[J].南京林业大学学报(自然科学版),2014,38(5):62-66.DOI:10.3969/j.issn.1000-2006.2014.05.013.YANG G Y,YU L L,ZHAO Y L,et al.Stress tolerance analysis of a Tamarix hispida TheIF1A in Saccharomyces cerevisiae[J].Journal of Nanjing Forestry University (Natural Sciences Edition),2014,38(5):62-66.
[25] 周爱民,卜媛媛,张欣欣,等.过量表达星星草(Puccinellia tenuiflora)液泡型 H+-ATP 酶c亚基基因提高转基因酵母的耐盐性[J].分子植物育种,2015,13(2):409-414.DOI:10.13271/j.mpb.013.000409.ZHOU A M,BU Y Y,ZHANG X X,et al.Overexpression of Puccinellia tenuiflora vacuolar H+-ATPase c subunit gene enhances salt tolerance of transgenic yeast[J].Molecular Plant Breeding,2015,13(2):409-414.