烟草质膜ATPase4基因的克隆、表达载体构建及表达分析
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摘要
植物细胞质膜ATPase基因表达可以引起细胞向外界环境释放H~+,从而形成细胞膜内外的电化学式梯度,促进各种离子的运输,抵抗外界的各种胁迫。为研究ATPase基因在烟草中的非生物胁迫调控机制及相关生物学功能,本研究采用同源克隆的方法,从普通烟草K326中克隆到了1个ATPase4同源性基因,成功构建ATPase4-pcambia1300过表达载体。利用生物信息学分析ATPase4基因编码蛋白的疏水性、理化性质、蛋白结构预测、信号肽预测、磷酸位点预测及同源性分析,结果显示,该基因与绒毛状烟草(Nicotiana tomentosi-formis)质膜ATPase4具有99%同源性,故命名为质膜ATPase4基因。利用荧光定量PCR(q RT-PCR)测定该基因在烟草组织中的表达量及其在非生物逆境胁迫中的表达模式,结果表明,该基因在根中表达量最高,且能快速响应低钾、高盐、PEG和冷胁迫诱导,说明ATPase4基因在烟草逆境胁迫中发挥着重要的调节作用。
Expression of cytoplasmic membrane ATPase gene in plants can cause cells to release H+to the external environment,thus form the membrane inside and outside electrical chemical formula of the gradient,promote all sorts of ion transport,various stress resistance to the outside world. In order to study the regulatory mechanism of ATPase in tobacco and its related biological functions,this article adopted homologous cloning method to clone a ATPase4 homologous gene from common tobacco K326 and construct ATPase4-pcambia1300 overexpression vector. The bioinformatics software was used to predict the hydrophobic,physical and chemical properties,protein structure,signal peptide,phosphate site and origin of ATPase4. The homology analysis showed that the modified gene had 99% homology with plasma membrane ATPase4 of Nicotiana tomentosiformis,so it was named as plasma membrane ATPase4 gene.Fluorescence quantitative real time PCR( q RT-PCR) was used to determine the relative expression pattern in different tissues under different stresses. The results showed that the length of gene sequence was 2 982 bp,encoding 963 amino acid residues,the gene had the highest expression level in the roots,and it could respond quickly to low potassium,high salt,PEG and cold stress. It was shown that ATPase4 played an important regulatory role in tobacco stress.
Expression of cytoplasmic membrane ATPase gene in plants can cause cells to release H+to the external environment,thus form the membrane inside and outside electrical chemical formula of the gradient,promote all sorts of ion transport,various stress resistance to the outside world. In order to study the regulatory mechanism of ATPase in tobacco and its related biological functions,this article adopted homologous cloning method to clone a ATPase4 homologous gene from common tobacco K326 and construct ATPase4-pcambia1300 overexpression vector. The bioinformatics software was used to predict the hydrophobic,physical and chemical properties,protein structure,signal peptide,phosphate site and origin of ATPase4. The homology analysis showed that the modified gene had 99% homology with plasma membrane ATPase4 of Nicotiana tomentosiformis,so it was named as plasma membrane ATPase4 gene.Fluorescence quantitative real time PCR( q RT-PCR) was used to determine the relative expression pattern in different tissues under different stresses. The results showed that the length of gene sequence was 2 982 bp,encoding 963 amino acid residues,the gene had the highest expression level in the roots,and it could respond quickly to low potassium,high salt,PEG and cold stress. It was shown that ATPase4 played an important regulatory role in tobacco stress.
引文
[1]GAXIOIA R A,PALMGREN M G,SCHUMACHER K.Plant proton pumps[J].FEBS Letters,2007,581(12):2204-2214.
[2]FALHOF J,FUGLSANG A T,PALMGREN M.Plasma membrane H+-ATPase regulation in the center of plant physiology[J].Molecular Plant,2016,9(3):323-337.
[3]BRISKIN D P.The plasma membrane H+-ATPase of higher plant cells:biochemistry and transport function[J].Biochimica et Biophysica Acta(BBA)Bioenergetics,1990,1019(2):95-109.
[4]MUNIZ M N,PAIS S M,TELLEZ M T,et al.Characterization of St PPI1,aproton pump interactor from Solanum tubeosum L.that is up regulated during tuber development and by abiotic stress[J].Planta,2011,233(4):661-674.
[5]MORSOMME P,BOUTRY M.The plant plasma membrane H+-ATPase:structure,function and regulation[J].Biochimica et Biophysica Acta Biomembranes,2000,1465(1/2):1-16.
[6]AYALA F,O'LEARY W,SCHUMAKER K S.Increased vacuolar and plasma membrane H+-ATPase activities in Salicornia bigelovii Torr.in response to Na Cl[J].Journal of Experimental Botany,1996,47(294):25-32.
[7]BINZEL M L.Na Cl-induced accumulation of tonoplast and plasma membrane H+-ATPase message in tomato[J].Physiologia Plantarum,1995,94(4):722-728.
[8]赵昕,谭会娟,丁楠.Na Cl胁迫对盐芥质膜和液泡膜AT-Pase活性的影响[J].西北植物学报,2009,29(4):717-723.ZHAO X,TAN H J,DING N.Changes of PM and TP ATPase activities in Thellungiella seedlings under salt stress[J].Acta Botanica Boreali-Occidentalia Sinica,2009,29(4):717-723.(in Chinese with English abstract)
[9]张敏,黄利斌,季永华,等.Na Cl胁迫对构树质膜H+-AT-Pase活性和表达的影响[J].北京林业大学学报,2011,33(6):21-26.ZHANG M,HUANG L B,JI Y H,et al.Effects of Na Cl stress on activity and expression of plasma membrane H+-AT-Pase in Broussonetia papyrifera[J].Journal of Beijing Forestry University,2011,33(6):21-26.(in Chinese with English abstract)
[10]司丛丛,刘好宝,曲平治.烟草钾离子通道及转基因烟草抗逆性的研究进展[J].中国农学通报,2010,26(2):45-49.SI C C,LIU H B,QU P Z.Research advances in potassium ion channel of tobacco and stress resistance of transgenic tobacco[J].Chinese Agricultural Science Bulletin,2010,26(2):45-49.(in Chinese with English abstract)
[11]HODGES T K,LEONARD R T,BRACKER C E,et al.Purification of an ion-stimulated adenosine triphosphatase from plant roots:association with plasma membranes[J].Proceedings of the National Academy of Sciences of the United States of America,1972,69(11):3307-3311.
[12]SHEN H,HE L F,SASAKI T,et al.Citrate secretion coupled with the modulation of soybean root tip under aluminum stress.Up-regulation of transcription,and threonine-oriented phosphorylation of plasma membrane ATPase[J].Plant Physiology,2005,139(1):287-296.
[13]JUNGMIN O,HYUNSUNG K,HYUNJONG B.Jatropha is vulnerable to cold injury due to impaired activity and expression of plasma membrane H+-ATPase[J].Acta Physiologiae Plantarum,2014,36(1):231-241.
[14]刘健健,刘俊丽,季敏杰,等.番茄质膜ATPase家族基因的鉴定和表达分析[J].浙江农林大学学报,2016,33(5):734-741.LIU J J,LIU J L,JI M J,et al.Identification and expression analysis of tomato plasma membrane H+-ATPase family genes[J].Journal of Zhejiang A&F University,2016,33(5):734-741.(in Chinese with English abstract)
[15]MITO N,WIMMERS L E,BENNETT A B.Sugar regulates m RNA abundance of H+-ATPase gene family members in tomato[J].Plant Physiology,1996,112(3):1229-1236.
[16]武维华.植物生理学[M].北京:科学出版社,2003.
[17]刘建新,胡浩斌,赵国林.Na CI胁迫对骆驼蓬幼苗液泡膜ATPase和ATPase活性的影响[J].中国沙漠,2008,28(2):275-279.LIU J X,HU H B,ZHAO G L.Effects of Na CI stress on the activity of tonoplast ATPase and ATPase in Peganum harmala seedlings[J].Journal of Desert Research,2008,28(2):275-279.(in Chinese with English abstract)
[18]LI X,YANG Y Q,ZHANG M,et al.Effect of PEG priming on plasma membrane H+-ATPase activities and mitochondriu-m function in soybean seeds[J].Seed Science&Technology,2010,38:49-60.
[19]YANG Y Q,WANG X F.Changes of plasma membrane H+-ATPase activities of Glycine max seeds by PEG treatment[J].Forest Studies in China,2005,7(2):7-11.
[20]刘尼歌,王占义,莫丙波,等.质膜ATPase与环境胁迫[J].热带亚热带植物学报,2006,14(3):263-268.LIU N G,WANG Z Y,MO B B,et al.Plasma membrane H+-ATPase and environmental stress[J].Journal of Tropical and Subtropical Botany,2006,14(3):263-268.(in Chinese with English abstract)
[21]BALLESTEROS E,KERKEB B,DONAIRE J P,et al.Effects of salt stress on H+-ATPase activity of plasma membrane-enriched vesicles isolated from sunflower roots[J].Plant Science,1998,134(2):181-190.
[22]PONS R,CORNEIO M J,SANZ A.Differential salinity-induced variations in the activity of H+-pumps and Na+/H+antiporters that are involved in cytoplasm ion homeostasis as a function of genotype and tolerance level in rice cell lines[J].Plant Physiology and Biochemistry,2011,49(12):1399-1409.
[23]JUNG S,HUTSCH B W,SCHUBERT S.Salt stress reduces kernel number of corn by inhibiting plasma membrane H+-ATPase activity[J].Plant Physiology and Biochemistry,2017,113:198-207.
[24]简令成,董合铸,孙龙华.番茄子叶细胞内三磷酸腺苷酶活性的超微结构定位及其在冷害中的变化[J].植物学报,1981,23(4):257-261.JIAN L C,DONG H Z,SUN L H.Ultrastructural localization of adenosine triphosphatase activity in cotyledon cells of tomato and its changes during chilling stress[J].Acta Botanica Sinica,1981,23(4):257-261.(in Chinese with English abstract)
[2]FALHOF J,FUGLSANG A T,PALMGREN M.Plasma membrane H+-ATPase regulation in the center of plant physiology[J].Molecular Plant,2016,9(3):323-337.
[3]BRISKIN D P.The plasma membrane H+-ATPase of higher plant cells:biochemistry and transport function[J].Biochimica et Biophysica Acta(BBA)Bioenergetics,1990,1019(2):95-109.
[4]MUNIZ M N,PAIS S M,TELLEZ M T,et al.Characterization of St PPI1,aproton pump interactor from Solanum tubeosum L.that is up regulated during tuber development and by abiotic stress[J].Planta,2011,233(4):661-674.
[5]MORSOMME P,BOUTRY M.The plant plasma membrane H+-ATPase:structure,function and regulation[J].Biochimica et Biophysica Acta Biomembranes,2000,1465(1/2):1-16.
[6]AYALA F,O'LEARY W,SCHUMAKER K S.Increased vacuolar and plasma membrane H+-ATPase activities in Salicornia bigelovii Torr.in response to Na Cl[J].Journal of Experimental Botany,1996,47(294):25-32.
[7]BINZEL M L.Na Cl-induced accumulation of tonoplast and plasma membrane H+-ATPase message in tomato[J].Physiologia Plantarum,1995,94(4):722-728.
[8]赵昕,谭会娟,丁楠.Na Cl胁迫对盐芥质膜和液泡膜AT-Pase活性的影响[J].西北植物学报,2009,29(4):717-723.ZHAO X,TAN H J,DING N.Changes of PM and TP ATPase activities in Thellungiella seedlings under salt stress[J].Acta Botanica Boreali-Occidentalia Sinica,2009,29(4):717-723.(in Chinese with English abstract)
[9]张敏,黄利斌,季永华,等.Na Cl胁迫对构树质膜H+-AT-Pase活性和表达的影响[J].北京林业大学学报,2011,33(6):21-26.ZHANG M,HUANG L B,JI Y H,et al.Effects of Na Cl stress on activity and expression of plasma membrane H+-AT-Pase in Broussonetia papyrifera[J].Journal of Beijing Forestry University,2011,33(6):21-26.(in Chinese with English abstract)
[10]司丛丛,刘好宝,曲平治.烟草钾离子通道及转基因烟草抗逆性的研究进展[J].中国农学通报,2010,26(2):45-49.SI C C,LIU H B,QU P Z.Research advances in potassium ion channel of tobacco and stress resistance of transgenic tobacco[J].Chinese Agricultural Science Bulletin,2010,26(2):45-49.(in Chinese with English abstract)
[11]HODGES T K,LEONARD R T,BRACKER C E,et al.Purification of an ion-stimulated adenosine triphosphatase from plant roots:association with plasma membranes[J].Proceedings of the National Academy of Sciences of the United States of America,1972,69(11):3307-3311.
[12]SHEN H,HE L F,SASAKI T,et al.Citrate secretion coupled with the modulation of soybean root tip under aluminum stress.Up-regulation of transcription,and threonine-oriented phosphorylation of plasma membrane ATPase[J].Plant Physiology,2005,139(1):287-296.
[13]JUNGMIN O,HYUNSUNG K,HYUNJONG B.Jatropha is vulnerable to cold injury due to impaired activity and expression of plasma membrane H+-ATPase[J].Acta Physiologiae Plantarum,2014,36(1):231-241.
[14]刘健健,刘俊丽,季敏杰,等.番茄质膜ATPase家族基因的鉴定和表达分析[J].浙江农林大学学报,2016,33(5):734-741.LIU J J,LIU J L,JI M J,et al.Identification and expression analysis of tomato plasma membrane H+-ATPase family genes[J].Journal of Zhejiang A&F University,2016,33(5):734-741.(in Chinese with English abstract)
[15]MITO N,WIMMERS L E,BENNETT A B.Sugar regulates m RNA abundance of H+-ATPase gene family members in tomato[J].Plant Physiology,1996,112(3):1229-1236.
[16]武维华.植物生理学[M].北京:科学出版社,2003.
[17]刘建新,胡浩斌,赵国林.Na CI胁迫对骆驼蓬幼苗液泡膜ATPase和ATPase活性的影响[J].中国沙漠,2008,28(2):275-279.LIU J X,HU H B,ZHAO G L.Effects of Na CI stress on the activity of tonoplast ATPase and ATPase in Peganum harmala seedlings[J].Journal of Desert Research,2008,28(2):275-279.(in Chinese with English abstract)
[18]LI X,YANG Y Q,ZHANG M,et al.Effect of PEG priming on plasma membrane H+-ATPase activities and mitochondriu-m function in soybean seeds[J].Seed Science&Technology,2010,38:49-60.
[19]YANG Y Q,WANG X F.Changes of plasma membrane H+-ATPase activities of Glycine max seeds by PEG treatment[J].Forest Studies in China,2005,7(2):7-11.
[20]刘尼歌,王占义,莫丙波,等.质膜ATPase与环境胁迫[J].热带亚热带植物学报,2006,14(3):263-268.LIU N G,WANG Z Y,MO B B,et al.Plasma membrane H+-ATPase and environmental stress[J].Journal of Tropical and Subtropical Botany,2006,14(3):263-268.(in Chinese with English abstract)
[21]BALLESTEROS E,KERKEB B,DONAIRE J P,et al.Effects of salt stress on H+-ATPase activity of plasma membrane-enriched vesicles isolated from sunflower roots[J].Plant Science,1998,134(2):181-190.
[22]PONS R,CORNEIO M J,SANZ A.Differential salinity-induced variations in the activity of H+-pumps and Na+/H+antiporters that are involved in cytoplasm ion homeostasis as a function of genotype and tolerance level in rice cell lines[J].Plant Physiology and Biochemistry,2011,49(12):1399-1409.
[23]JUNG S,HUTSCH B W,SCHUBERT S.Salt stress reduces kernel number of corn by inhibiting plasma membrane H+-ATPase activity[J].Plant Physiology and Biochemistry,2017,113:198-207.
[24]简令成,董合铸,孙龙华.番茄子叶细胞内三磷酸腺苷酶活性的超微结构定位及其在冷害中的变化[J].植物学报,1981,23(4):257-261.JIAN L C,DONG H Z,SUN L H.Ultrastructural localization of adenosine triphosphatase activity in cotyledon cells of tomato and its changes during chilling stress[J].Acta Botanica Sinica,1981,23(4):257-261.(in Chinese with English abstract)