小麦VP基因的克隆、生物信息学分析及功能初探
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摘要
为研究小麦液泡膜H~+转运无机焦磷酸酶基因VP在植物中的作用,根据大麦、水稻等植物VP基因序列设计简并引物,用RACE法扩增小麦VP基因全长,对VP蛋白进行生物信息学分析,构建VP基因植物表达载体,转化拟南芥,对阳性转基因纯合体株系进行筛选和耐盐性鉴定。结果表明,小麦VP基因编码区序列全长2 286 bp,共编码761个氨基酸,等电点为5.17。VP蛋白为无信号肽的疏水性蛋白,主要含α螺旋。转VP基因拟南芥在NaCl胁迫下的萌发率、绿苗率均高于野生型拟南芥,成苗生长状态优于野生型拟南芥,说明小麦VP基因提高了转基因拟南芥的耐盐性。
In order to study the role of wheat vacuolar H~+-pyrophosphatase gene VP in the plant,degenerate primers were designed according to the VP gene sequences of barley,rice and so on,and used to amplify the VP gene in wheat by RACE method.VP protein was analyzed by bioinformatics,and plant expression vector was constructed,then transfered into Arabidopsis thaliana.The positive transgenic homozygous lines overexpressing VP were screened,and their salt tolerance was identified.The results showed that full-length coding sequence of wheat VP gene was 2 286 bp,encoded 761 amino acids,and the pI of VP protein was 5.17.VP protein is a hydrophobic protein without signal peptide,and mainly contains α helix.The germination rate and green seedlings rate of transgenic Arabidopsis thaliana overexpressing VP were higher compared with wild Arabidopsis thaliana,and transgenic Arabidopsis thaliana overexpressing VP grew better compared with wild Arabidopsis thaliana under NaCl stress.These results showed that wheat VP gene increased tolerance to salt in transgenic Arabidopsis thaliana overexpressing VP.
In order to study the role of wheat vacuolar H~+-pyrophosphatase gene VP in the plant,degenerate primers were designed according to the VP gene sequences of barley,rice and so on,and used to amplify the VP gene in wheat by RACE method.VP protein was analyzed by bioinformatics,and plant expression vector was constructed,then transfered into Arabidopsis thaliana.The positive transgenic homozygous lines overexpressing VP were screened,and their salt tolerance was identified.The results showed that full-length coding sequence of wheat VP gene was 2 286 bp,encoded 761 amino acids,and the pI of VP protein was 5.17.VP protein is a hydrophobic protein without signal peptide,and mainly contains α helix.The germination rate and green seedlings rate of transgenic Arabidopsis thaliana overexpressing VP were higher compared with wild Arabidopsis thaliana,and transgenic Arabidopsis thaliana overexpressing VP grew better compared with wild Arabidopsis thaliana under NaCl stress.These results showed that wheat VP gene increased tolerance to salt in transgenic Arabidopsis thaliana overexpressing VP.
引文
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[16] 包爱科.拟南芥液泡膜H+-焦磷酸酶基因AVP1改良紫花苜蓿(Medicago sativa L.)抗逆性的研究[D].兰州:兰州大学,2009.
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[26] GUO S,YIN H,ZHANG X,et al.Molecular cloning and characterization of a vacuolar H+-pyrophosphatase gene,SsVP,from the halophyte Suaeda aralocaspica and its overexpression increases salt and drought tolerance of Arabidopsis[J].Plant Mol Biol,2006,60(1):41-50.
[27] GAO F,GAO Q,DUAN X G,et al.Cloning of an H+-PPase gene from Thellungiella halophila and its heterologous expression to improve tobacco salt tolerance[J].J Exp Bot,2006,57(12):3259-3270.
[28] 王建武,沈裕虎,王沛,等.截形苜蓿液泡膜H+-PPase基因克隆与序列分析[J].西北植物学报,2009,29(3):435-442.
[29] 董建辉,陈明,徐兆师,等.披碱草(Elymus dahuricus)焦磷酸化酶基因EdHP1的克隆及功能鉴定[J].麦类作物学报,2008,28(3):364-371.
[30] 朱春利,张登峰,刘颖慧,等.玉米液泡膜焦磷酸酶基因ZmVPP1的克隆及逆境下的表达分析[J].植物遗传资源学报,2011,12(1):107-112.
[31] LI Y F.cDNA cloning of a vacuolar H+-pyrophosphatase and its expression in Hordeum brevisubulatum (Trin.)link in response to salt stress[J].Agr Sci China,2005,4(4):247-251.
[32] BRINI F,GAXIOLA R A,Berkowitz G A,et al.Cloning and characterization of a wheat vacuolar cation/proton antiporter and pyrophosphatase proton pump[J].Plant Physiol Biochem,2005,43(4):347-354.
[33] MENG X,XU Z,SONG R.Molecular cloning and characterization of a vacuolar H+-pyrophosphatase from Dunaliella viridis[J].Mol Biol Rep,2011,38(5):3375-3382.
[2] FEKI K,QUINTERO F,PARDO J,et al.Regulation of durum wheat Na+/H+ exchanger TdSOS1 by phosphorylation[J].Plant Mol Biol,2011,76(6):545-556.
[3] FEKI K,FARHAT-KHEMAKHEM A,KAMOUN Y,et al.Responses of transgenic Arabidopsis plants and recombinant yeast cells expressing a novel durum wheat manganese superoxide dismutase DWMSOD to various abiotic stresses[J].J Plant Physiol,2016,198:56-68.
[4] JAMES R A,DAVENPORT R J,MUNNS R.Physiological characterization of two genes for Na+ exclusion in durum wheat,Nax1 and Nax2[J].Plant Physiol,2006,142(4):1537-1547.
[5] TOUNSI S,BEN AMAR S,MASMOUDI K,et al.Characterisation of two HKT1;4 transporters from Triticum monococcum to elucidate the determinants of the wheat salt tolerance Nax1 QTL[J].Plant Cell Physiol,2016,57(10):2047-2057.
[6] QIN F,SHINOZAKI K,YAMAGUCHI-SHINOZAKI K.Achievements and challenges in understanding plant abiotic stress responses and tolerance[J].Plant and Cell Physio-logy,2011,52:1569-1582.
[7] MAESHIMA M.Vacuolar H+-pyrophosphatase[J].Biochem Biophys Acta,2000,1465:37-51 .
[8] 包爱科,张金林,郭正刚,等.液泡膜H+-PPase与植物耐盐性[J].植物生理学通讯,2006,42(4):777-783.
[9] MAESHIMA M.Molecular cloning of cDNA for vacuolar membrane proton-translocating inorganic pyrophosphatase in Hordeum vulgare[J].Biochem Biophys Res Commun,1993,190:1110-1114.
[10] SONNEWALD U.Molecular cloning,characterization and expression analysis of isoforms encoding tonoplast-bound proton-translocating inorganic pyrophosphatase in tobacco[J].Plant Mol Biol,1995,29:833-840.
[11] KASAMO K.Isolation and characterization of cDNAs encoding vacuolar H+-pyrophos-phates isoforms from rice [J].Plant Mol Biol,1996,31:1029-1038.
[12] LI B,WEI A,SONG C,et al.Heterologous expression of the TsVP gene improves the drought resistance of maize[J].Plant Biotechnology Journal,2008,6(2):146-159.
[13] BRINI F,HANIN M,MEZGHANI I,et al.Overexpression of wheat Na+/H+ antiporter TNHX1 and H+-pyrophosphatase TVP1 improve salt-and drought-stress tolerance in Arabidopsis thaliana plants[J].Journal of Expe-rimental Botany,2007,58(2):301-308.
[14] LI Z,BALDWIN C M,HU Q,et al.Heterologous expression of Arabidopsis H+-PPase enhances salt tolerance in transgenic creeping bentgrass[J].Plant Cell Environ,2010,33(2):272-289.
[15] 刘岩,计东风,朱燕,等.超表达桑树MaNHX1和拟南芥AVP1基因增强拟南芥耐盐性的研究[J].蚕业科学,2016,42(3):386-392.
[16] 包爱科.拟南芥液泡膜H+-焦磷酸酶基因AVP1改良紫花苜蓿(Medicago sativa L.)抗逆性的研究[D].兰州:兰州大学,2009.
[17] YAO M H,ZENG Y L,LIU L,et al.Overexpression of the halophyte Kalidium foliatum H+-pyrophosphatase gene confers salt and drought tolerance in Arabidopsis thaliana[J].Molecular Biology Reports,2012,39(8):7989-7996.
[18] 李杉杉,郭静雅,郭强,等.过量表达马蔺VP基因增强烟草抗旱耐盐性研究[J].草学,2017(2):21-28.
[19] LIU L,FAN X D,WANG F W,et al.Coexpression of ScNHX1 and ScVP in transgenic hybrids improves salt and saline-alkali tolerance in alfalfa (Medicago sativa L.) [J].Journal of Plant Growth Regulation,2013,32(1):1-8.
[20] ANIANEVULU E,REDDY P S,SUNITA M S,et al.Salt tolerance and activity of antioxidative enzymes of transgenic finger millet overexpressing a vacuolar H+-pyrophosphatase gene(SbVPPase) from Sorghum bicolor[J].J Plant Phy-siol,2014,171(10):789-798.
[21] MENG L,LI S,GUO J,et al.Molecular cloning and functional characterisation of an H+-pyrophosphatase from Iris lactea[J].Sci Rep,2017,7(1):17779-17780.
[22] CHEN Y,LI L,ZONG J,et al.Heterologous expression of the halophyte Zoysia matrella H+-pyrophosphatase gene improved salt tolerance in Arabidopsis thaliana[J].Plant Physiol Biochem,2015,91(1):49-55.
[23] YANG Y,TANG R J,LI B,et al.Overexpression of a Populus trichocarpa H+-pyrophosphatase gene PtVP1.1 confers salt tolerance on transgenic poplar[J].Tree Physiol,2015,35(6):663-677.
[24] GAXIOLA R A,LI J,UNDURRAGA S,et al.Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump[J].Proc Natl Acad Sci,2001,98(20):11444-11449.
[25] PARK S,LI J,PITTMAN J K,et al.Up-regulation of a H+-pyrophosphatase (H+-PPase) as a strategy to engineer drought-resistant crop plants[J].Proc Natl Acad Sci,2005,102(52):18830-18835.
[26] GUO S,YIN H,ZHANG X,et al.Molecular cloning and characterization of a vacuolar H+-pyrophosphatase gene,SsVP,from the halophyte Suaeda aralocaspica and its overexpression increases salt and drought tolerance of Arabidopsis[J].Plant Mol Biol,2006,60(1):41-50.
[27] GAO F,GAO Q,DUAN X G,et al.Cloning of an H+-PPase gene from Thellungiella halophila and its heterologous expression to improve tobacco salt tolerance[J].J Exp Bot,2006,57(12):3259-3270.
[28] 王建武,沈裕虎,王沛,等.截形苜蓿液泡膜H+-PPase基因克隆与序列分析[J].西北植物学报,2009,29(3):435-442.
[29] 董建辉,陈明,徐兆师,等.披碱草(Elymus dahuricus)焦磷酸化酶基因EdHP1的克隆及功能鉴定[J].麦类作物学报,2008,28(3):364-371.
[30] 朱春利,张登峰,刘颖慧,等.玉米液泡膜焦磷酸酶基因ZmVPP1的克隆及逆境下的表达分析[J].植物遗传资源学报,2011,12(1):107-112.
[31] LI Y F.cDNA cloning of a vacuolar H+-pyrophosphatase and its expression in Hordeum brevisubulatum (Trin.)link in response to salt stress[J].Agr Sci China,2005,4(4):247-251.
[32] BRINI F,GAXIOLA R A,Berkowitz G A,et al.Cloning and characterization of a wheat vacuolar cation/proton antiporter and pyrophosphatase proton pump[J].Plant Physiol Biochem,2005,43(4):347-354.
[33] MENG X,XU Z,SONG R.Molecular cloning and characterization of a vacuolar H+-pyrophosphatase from Dunaliella viridis[J].Mol Biol Rep,2011,38(5):3375-3382.