厚藤胚胎发生晚期丰富蛋白基因IpLEA的克隆与功能分析
|
摘要
LEA(Late embryogenesis abundant)基因是生物体中广泛存在的一类广泛响应逆境胁迫的功能基因,主要参与调控植物对缺水等逆境胁迫的耐受。为了探索厚藤LEA基因的抗逆分子机制,本研究通过前期对厚藤(Ipomoea pes-caprae L.)c DNA文库的筛选,获得了一个编码厚藤LEA基因的全长c DNA,命名为IpLEA(Gen Bank登录号:MF680612)。通过在酵母中表达,表明IpLEA能够提高转基因酵母的耐盐性及抗氧化能力。以厚藤成年植株及幼苗为材料进行实时荧光定量RT-PCR分析的结果表明:IpLEA基因在厚藤各组织中广泛表达;在厚藤幼苗中的表达受到高盐、甘露醇胁迫和ABA处理的诱导。推测IpLEA基因参与厚藤应答高盐、干旱等逆境胁迫,并响应ABA信号的传递。本研究为今后厚藤IpLEA基因的进一步功能发掘和厚藤响应极端逆境的机理研究提供了理论依据。
LEA(Late embryogenesis abundant) gene is a functional gene widely responding to adversity stress which generally exists in living organisms, and is mainly involved in regulating the tolerance of plants to stresses such as water shortage. Through the screening of cDNA library of Ipomoea pes-caprae in early stage, the study obtained a full length c DNA encoding LEA protein of I. pes-caprae, and named as IpLEA(Gen Bank accession number: MF680612) with the purpose to reveal the stress resistance mechanism of LEA gene in I. pes-caprae. The expression of IpLEA in yeast indicated that IpLEA could improve the salt tolerance and antioxidant ability of transgenic yeast. Using adult plants and seedlings of I. pes-caprae as materials to carry out real-time fluorescence quantitative RT-PCR analysis, the result showed that IpLEA gene was widely expressed in various tissues in I.pes-caprae, and the expression in seedling was induced under high salt, mannitol stress, and ABA treatment. It was speculated that IpLEA gene might play important roles in responding to adversity stresses such as high salt and drought, and ABA signal transmission. This study might provide a theoretical basis for further functional excavation of IpLEA genes and the mechanisms of responding to extreme adversity in I. pes-caprae.
LEA(Late embryogenesis abundant) gene is a functional gene widely responding to adversity stress which generally exists in living organisms, and is mainly involved in regulating the tolerance of plants to stresses such as water shortage. Through the screening of cDNA library of Ipomoea pes-caprae in early stage, the study obtained a full length c DNA encoding LEA protein of I. pes-caprae, and named as IpLEA(Gen Bank accession number: MF680612) with the purpose to reveal the stress resistance mechanism of LEA gene in I. pes-caprae. The expression of IpLEA in yeast indicated that IpLEA could improve the salt tolerance and antioxidant ability of transgenic yeast. Using adult plants and seedlings of I. pes-caprae as materials to carry out real-time fluorescence quantitative RT-PCR analysis, the result showed that IpLEA gene was widely expressed in various tissues in I.pes-caprae, and the expression in seedling was induced under high salt, mannitol stress, and ABA treatment. It was speculated that IpLEA gene might play important roles in responding to adversity stresses such as high salt and drought, and ABA signal transmission. This study might provide a theoretical basis for further functional excavation of IpLEA genes and the mechanisms of responding to extreme adversity in I. pes-caprae.
引文
Bao F.,Du D.L.,An Y.,Yang W.R.,Wang J.,Cheng T.R.,and Zhang Q.X.,2017,Overexpression of Prunus mume dehydrin genes in tobacco enhances tolerance to cold and drought,Front.Plant Sci.,8:151
Chen Y.,Liu Y.S.,and Zeng J.G.,2014,Progresses on plant genome sequencing profile,Shengming Kexue Yanjiu(Life Science Research),18(1):66-74(陈勇,柳亦松,曾建国,2014,植物基因组测序的研究进展,生命科学研究,18(1):66-74)
Dure L.,Greenway S.C.,and Galau G.A.,1981,Developmental biochemistry of cotton seed embryogenesis and germination:changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein synthesis,Biochemistry,20(14):4162-4168
Guo Y.,Zhang H.,Jian S.G.,Xia K.F.,and Zhang M.,2017,Construction of a c DNA library of Ipomoea pes-caprae L.and screening of cadmium tolerant genes,Zhiwu Kexue Xuebao(Plant Science Journal),35(3):372-379(郭艳,张会,简曙光,夏快飞,张美,2017,厚藤c DNA文库的构建和重金属镉耐受相关基因的筛选,植物科学学报,35(3):372-379)
Hand S.C.,Menze M.A.,Toner M.,Boswell L.,and Moore D.,2011,LEA proteins during water stress:not just for plants anymore,Annu.Rev.Physiol.,73:115-134
Hincha D.K.,and Thalhammer A.,2012,LEA proteins:IDPs with versatile functions in cellular dehydration tolerance,Biochem.Soc.T.,40(5):1000-1003
Hughes D.W.,and Galau G.A.,1991,Developmental and environmental induction of Lea and Lea A mR NAs and the postabscission program during embryo culture,Plant Cell,3(6):605-618
Miryeganeh M.,Takayama K.,Tateishi Y.,and Kajita T.,2014,Long-distance dispersal by sea-drifted seeds has maintained the global distribution of Ipomoea pes-caprae subsp.brasiliensis(Convolvulaceae),PLo S One,9(4):e91836
Ouyang P.Y.,Liu N.,Zhang W.W.,Wang J.,and Jian S.G.,2011,Biological and eco-physiological characteristics of a beach plant Ipomoea pescaprae,Hunan Keji Daxue Xuebao(Journal of Hunan University of Science and Technology),26(4):117-121(欧阳蒲月,刘楠,张伟伟,王俊,简曙光,2011,海滩植物厚藤(Ipomoea pescaprae)的生物学及生理生态特性,湖南科技大学学报,26(4):117-121)
Quintero F.J.,Blatt M.R.,and Pardo J.M.,2000,Functional conservation between yeast and plant endosomal Na+/H+antiporters,FEBS Lett.,471(2-3):224-228
Sharma A.,Kumar D.,Kumar S.,Rampuria S.,Reddy A.R.,and Kirti P.B.,2016,Ectopic expression of an atypical hydrophobic group 5 LEA protein from wild peanut,Arachis diogoi confers abiotic stress tolerance in tobacco,PLo S One,11 (3):e0150609
Singh S.,Cornilescu C.C.,Tyler R.C.,Cornilescu G.,Tonelli M.,Lee M.S.,and Markley J.L.,2005,Solution structure of a late embryogenesis abundant protein(LEA14)from Arabidopsis thaliana,a cellular stress-related protein,Protein Sci.,14(10):2601-2609
Sun X.L.,Erik H.A.,Rikkerink,William T.J.,and Vladimir N.U.,2013,Multifarious roles of intrinsic disorder in proteins illustrate its broad impact on plant biology,Plant Cell,25(1):38-55
Tunnacliffe A.,Hincha D.K.,Leprince O.,and Macherel D.,2010,LEA proteins:versatility of form and function,In:Lubzens E.,Cerda J.,and Clark M.(eds.),Dormancy and resistance in harsh environments,Springer,Berlin Heidelberg,Germany,pp.91-108
Van den Dries N.,Facchinelli F.,Giarola V.,Phillips J.R.,and Bartels D.,2011,Comparative analysis of LEA-like 11-24gene expression and regulation in related plant species within the linderniaceae that differ in desiccation tolerance,NewPhytol.,190(1):75-88
Wang H.Q.,Wu Y.C.,Yang X.B.,Guo X.R.,and Cao X.Y.,2017,Sm LEA2,a gene for late embryogenesis abundant protein isolated from Salvia miltiorrhiza,confers tolerance to drought and salt stress in Escherichia coli and S.miltiorrhiza,Protoplasma,254(2):685-696
Wise M.J.,and Tunnacliffe A.,2004,POPP the question:what do LEA proteins do?Trends Plant Sci.,9(1):13-17
Yang J.,Fan W.J.,Wang H.X.,Zhao S.S.,Wu Y.L.,and Zhang P.,2017,Advances in genome sequencing of Ipomoea plants,Zhiwu Shengli Xuebao(Plant Physiology Journal),53(5):768-771(杨俊,范维娟,王红霞,赵姗姗,吴银亮,张鹏,2017,番薯属植物基因组测序最新进展,植物生理学报,53(5):768-771)
Zhang Y.,Li Y.,Lai J.,Zhang H.,Liu Y.,Liang L.,and Xie Q.,2012,Ectopic expression of a LEA protein gene Ts LEA1from Thellungiella salsuginea confers salt-tolerance in yeast and Arabidopsis,Mol.Biol.Rep.,39(4):4627-4633
Zhou Y.,Yin X.C.,Duan R.J.,Hao G.P.,Guo J.,and Jiang X.Y.,2015,Sp AHA1 and Sp SOS1 coordinate in transgenic yeast to improve salt tolerance,PLo S One,10(9):e0137447
Chen Y.,Liu Y.S.,and Zeng J.G.,2014,Progresses on plant genome sequencing profile,Shengming Kexue Yanjiu(Life Science Research),18(1):66-74(陈勇,柳亦松,曾建国,2014,植物基因组测序的研究进展,生命科学研究,18(1):66-74)
Dure L.,Greenway S.C.,and Galau G.A.,1981,Developmental biochemistry of cotton seed embryogenesis and germination:changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein synthesis,Biochemistry,20(14):4162-4168
Guo Y.,Zhang H.,Jian S.G.,Xia K.F.,and Zhang M.,2017,Construction of a c DNA library of Ipomoea pes-caprae L.and screening of cadmium tolerant genes,Zhiwu Kexue Xuebao(Plant Science Journal),35(3):372-379(郭艳,张会,简曙光,夏快飞,张美,2017,厚藤c DNA文库的构建和重金属镉耐受相关基因的筛选,植物科学学报,35(3):372-379)
Hand S.C.,Menze M.A.,Toner M.,Boswell L.,and Moore D.,2011,LEA proteins during water stress:not just for plants anymore,Annu.Rev.Physiol.,73:115-134
Hincha D.K.,and Thalhammer A.,2012,LEA proteins:IDPs with versatile functions in cellular dehydration tolerance,Biochem.Soc.T.,40(5):1000-1003
Hughes D.W.,and Galau G.A.,1991,Developmental and environmental induction of Lea and Lea A mR NAs and the postabscission program during embryo culture,Plant Cell,3(6):605-618
Miryeganeh M.,Takayama K.,Tateishi Y.,and Kajita T.,2014,Long-distance dispersal by sea-drifted seeds has maintained the global distribution of Ipomoea pes-caprae subsp.brasiliensis(Convolvulaceae),PLo S One,9(4):e91836
Ouyang P.Y.,Liu N.,Zhang W.W.,Wang J.,and Jian S.G.,2011,Biological and eco-physiological characteristics of a beach plant Ipomoea pescaprae,Hunan Keji Daxue Xuebao(Journal of Hunan University of Science and Technology),26(4):117-121(欧阳蒲月,刘楠,张伟伟,王俊,简曙光,2011,海滩植物厚藤(Ipomoea pescaprae)的生物学及生理生态特性,湖南科技大学学报,26(4):117-121)
Quintero F.J.,Blatt M.R.,and Pardo J.M.,2000,Functional conservation between yeast and plant endosomal Na+/H+antiporters,FEBS Lett.,471(2-3):224-228
Sharma A.,Kumar D.,Kumar S.,Rampuria S.,Reddy A.R.,and Kirti P.B.,2016,Ectopic expression of an atypical hydrophobic group 5 LEA protein from wild peanut,Arachis diogoi confers abiotic stress tolerance in tobacco,PLo S One,11 (3):e0150609
Singh S.,Cornilescu C.C.,Tyler R.C.,Cornilescu G.,Tonelli M.,Lee M.S.,and Markley J.L.,2005,Solution structure of a late embryogenesis abundant protein(LEA14)from Arabidopsis thaliana,a cellular stress-related protein,Protein Sci.,14(10):2601-2609
Sun X.L.,Erik H.A.,Rikkerink,William T.J.,and Vladimir N.U.,2013,Multifarious roles of intrinsic disorder in proteins illustrate its broad impact on plant biology,Plant Cell,25(1):38-55
Tunnacliffe A.,Hincha D.K.,Leprince O.,and Macherel D.,2010,LEA proteins:versatility of form and function,In:Lubzens E.,Cerda J.,and Clark M.(eds.),Dormancy and resistance in harsh environments,Springer,Berlin Heidelberg,Germany,pp.91-108
Van den Dries N.,Facchinelli F.,Giarola V.,Phillips J.R.,and Bartels D.,2011,Comparative analysis of LEA-like 11-24gene expression and regulation in related plant species within the linderniaceae that differ in desiccation tolerance,NewPhytol.,190(1):75-88
Wang H.Q.,Wu Y.C.,Yang X.B.,Guo X.R.,and Cao X.Y.,2017,Sm LEA2,a gene for late embryogenesis abundant protein isolated from Salvia miltiorrhiza,confers tolerance to drought and salt stress in Escherichia coli and S.miltiorrhiza,Protoplasma,254(2):685-696
Wise M.J.,and Tunnacliffe A.,2004,POPP the question:what do LEA proteins do?Trends Plant Sci.,9(1):13-17
Yang J.,Fan W.J.,Wang H.X.,Zhao S.S.,Wu Y.L.,and Zhang P.,2017,Advances in genome sequencing of Ipomoea plants,Zhiwu Shengli Xuebao(Plant Physiology Journal),53(5):768-771(杨俊,范维娟,王红霞,赵姗姗,吴银亮,张鹏,2017,番薯属植物基因组测序最新进展,植物生理学报,53(5):768-771)
Zhang Y.,Li Y.,Lai J.,Zhang H.,Liu Y.,Liang L.,and Xie Q.,2012,Ectopic expression of a LEA protein gene Ts LEA1from Thellungiella salsuginea confers salt-tolerance in yeast and Arabidopsis,Mol.Biol.Rep.,39(4):4627-4633
Zhou Y.,Yin X.C.,Duan R.J.,Hao G.P.,Guo J.,and Jiang X.Y.,2015,Sp AHA1 and Sp SOS1 coordinate in transgenic yeast to improve salt tolerance,PLo S One,10(9):e0137447