蓖麻RcTIP1;2与RcTIP1;3基因的克隆及低温胁迫下的表达分析
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摘要
植物水孔蛋白作为水分快速跨膜运输的通道蛋白,在保持细胞内外渗透平衡、调节气孔运动以及参与渗透胁迫应答等方面发挥着重要的作用。本研究以‘通蓖5号’叶片为材料,利用RT-PCR的方法成功克隆了两个液泡膜内源水孔蛋白基因的ORFs,将其命名为RcTIP1;2和RcTIP1;3。基因结构分析显示RcTIP1;2和RcTIP1;3均含有3个外显子和2个内含子。生物信息学分析表明2个基因编码的蛋白均含有6个跨膜螺旋以及MIP超家族典型的2个"NPA"保守结构域。多重序列比对发现RcTIP1;2与PtTIP1;2同源性高达84.92%,RcTIP1;3与AtTIP1;3同源性高达79.76%。进化树分析表明2个基因均属于TIP1类亚家族蛋白成员。qRT-PCR分析表明RcTIP1;2与RcTIP1;3基因受冷胁迫均下调表达,该基因的下调表达可能有助于抵抗和延缓冷冻诱导的细胞脱水。进一步对RcTIP1;2与RcTIP1;3调控冷响应的分子机理进行研究,从而为蓖麻分子辅助育种提供重要的理论依据。
Plant aq uaporins are water channels for rapid transmembrane transport of water, which play critical roles in maintaining intracellular and extracellular osmotic balance, regulating stomatal movement and osmotic stress responses. Here, we reported two tonoplast intrinsic proteins, RcTIP1;2 and RcTIP1;3, isolated from leaves of 'Tongbi5' castor bean using RT-PCR method. Gene structure analysis showed that the two genes both contained three exons and two introns. Bioinformatics analysis revealed that the two predicted proteins contained six transmembrane helices and two typical "NPA" motifs of MIP family. The multiple sequence alignment displayed that RcTIP1;2 and RcTIP1;3 had 84.92% and 79.76% amino acid identity with PtTIP1;2 and AtTIP1;3 separately.Phylogenetic analysis demonstrated that the two genes belonged to TIP1 subgroup. qRT-PCR analysis showed that expression of RcTIP1;2 and RcTIP1;3 was down-regulated by cold stress. Down-regulation of two genes might contribute to resist and/or delay freezing-induced cell dehydration. Furthermore, cold response molecular mechanism regulated by RcTIP1;2 and RcTIP1;3 will be studied, which can provide an important theoretical basis for castor molecular assisted breeding.
Plant aq uaporins are water channels for rapid transmembrane transport of water, which play critical roles in maintaining intracellular and extracellular osmotic balance, regulating stomatal movement and osmotic stress responses. Here, we reported two tonoplast intrinsic proteins, RcTIP1;2 and RcTIP1;3, isolated from leaves of 'Tongbi5' castor bean using RT-PCR method. Gene structure analysis showed that the two genes both contained three exons and two introns. Bioinformatics analysis revealed that the two predicted proteins contained six transmembrane helices and two typical "NPA" motifs of MIP family. The multiple sequence alignment displayed that RcTIP1;2 and RcTIP1;3 had 84.92% and 79.76% amino acid identity with PtTIP1;2 and AtTIP1;3 separately.Phylogenetic analysis demonstrated that the two genes belonged to TIP1 subgroup. qRT-PCR analysis showed that expression of RcTIP1;2 and RcTIP1;3 was down-regulated by cold stress. Down-regulation of two genes might contribute to resist and/or delay freezing-induced cell dehydration. Furthermore, cold response molecular mechanism regulated by RcTIP1;2 and RcTIP1;3 will be studied, which can provide an important theoretical basis for castor molecular assisted breeding.
引文
Abramson J.,and Vartanian A.S.,2013,Watch water flow,Science,340(6138):1294-1295
Aroca R.,Amodeo G.,Fernández-Illescas S.,Herman E.M.,Chaumont F.,and Chrispeels M.J.,2005,The role of aquaporins and membrane damage in chilling and hydrogen peroxide induced changes in the hydraulic conductance of maize roots,Plant Physiol.,137(1):341-353
Boursiac Y.,Chen S.,Luu D.T.,Sorieul M.,Dries N.,and Maurel C.,2005,Early effects of salinity on water transport in Arabidopsis roots.Molecular and cellular features of aquaporin expression,Plant Physiol.,139(2):790-805
Chinnusamy V.,Zhu J.,and Zhu J.K.,2006,Gene regulation during cold acclimation in plants,Physiol.Plant.,126(1):52-61
Danielson J.魡.H.,and Johanson U.,2008,Unexpected complexity of the aquaporin gene family in the moss Physcomitrella patens,BMC Plant Biol.,8(1):45
Huang G.T.,Ma S.L.,Bai L.P.,Zhang L.,Ma H.,Jia P.,Liu J.,Zhong M.,and Guo Z.F.,2012,Signal transduction during cold,salt,and drought stresses in plants,Mol.Biol.Rep.,39(2):969-987
Jang J.Y.,Kim D.G.,Kim Y.O.,Kim J.S.,and Kang H.,2004,An expression analysis of a gene family encoding plasma membrane aquaporins in response to abiotic stresses in Arabidopsis thaliana,Plant Mol.Biol.,54(5):713-725
Johanson U.,Karlsson M.,Johansson I.,Gustavsson S.,Sjovall S.,Fraysse L.,Weig A.R.,and Kjellbom P.,2001,The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants,Plant Physiol.,126(4):1358-1369
Khan K.,Agarwal P.,Shanware A.,and Sane V.A.,2015,Heterologous expression of two Jatropha aquaporins imparts drought and salt tolerance and improves seed viability in transgenic Arabidopsis thaliana,PLoS One,10(6):e0128866
Li D.D.,Tai F.J.,Zhang Z.T.,Li Y.,Zheng Y.,Wu Y.F.,and Li X.B.,2009,A cotton gene encodes a tonoplast aquaporin that is involved in cell tolerance to cold stress,Gene,438(1):26-32
Li J.Q.,Zhu G.L.,Wu G.L.,He Z.B.,Li J.X.,Zhang C.H.,Tian F.D.,and Jia J.X.,2004,Correlation and path analysis between seed oil content and major quantitative characters in castor,Zhongguo Youliao Zuowu Xuebao(Chinese Journal of Oil Crop Sciences),26(2):43-46(李金琴,朱国立,吴国林,何智彪,李靖霞,张春华,田福东,贾娟霞,2004,蓖麻种子含油量与主要数量性状的相关及通径分析,中国油料作物学报,26(2):43-46)
Maeshima M.,2001,Tonoplast transporters:organization and function,Ann.Rev.Plant Physiol.Plant Mol.Biol.,52(1):469-497
Maurel C.,Boursiac Y.,Luu D.T.,Santoni V.,Shahzad Z.,and Verdoucq L.,2015,Aquaporins in plants,Physiol.Rev.,95(4):1321-1358
Mei Y.,Li H.L.,Yang S.Y.,and Luo H.Y.,2007,Plant aquaporins function research,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),43(3):563-568(梅杨,李海蓝,杨尚元,罗红艺,2007,植物水孔蛋白的功能,植物生理学通讯,43(3):563-568)
Peng Y.,Arora R.,Li G.,Wang X.,and Fessehaie A.,2008,Rhododendron catawbiense plasma membrane intrinsic proteins are aquaporins,and their over-expression compromises constitutive freezing tolerance and cold acclimation ability of transgenic Arabidopsis plants,Plant Cell Environ.,31(9):1275-1289
Peng Y.,Lin W.,Cai W.,and Arora R.,2007,Overexpression of a panax ginseng tonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic Arabidopsis plants,Planta,226(3):729-740
Regon P.,Panda P.,Kshetrimayum E.,and Panda S.K.,2014,Genome-wide comparative analysis of tonoplast intrinsic protein(TIP)genes in plants,Funct.Integr.Genomics,14(4):617-629
Rodrigues M.I.,Bravo J.P.,Sassaki F.T.,Severino F.E.,and Maia I.G.,2013,The tonoplast intrinsic aquaporin(TIP)subfamily of Eucalyptus grandis:characterization of Eg TIP2,a rootspecific and osmotic stress-responsive gene,Plant Sci.,213:106-113
Sakurai J.,Ishikawa F.,Yamaguchi T.,Uemura M.,and Maeshima M.,2005,Identification of 33 rice aquaporin genes and analysis of their expression and function,Plant Cell Physiol.,46(9):1568-1577
Aroca R.,Amodeo G.,Fernández-Illescas S.,Herman E.M.,Chaumont F.,and Chrispeels M.J.,2005,The role of aquaporins and membrane damage in chilling and hydrogen peroxide induced changes in the hydraulic conductance of maize roots,Plant Physiol.,137(1):341-353
Boursiac Y.,Chen S.,Luu D.T.,Sorieul M.,Dries N.,and Maurel C.,2005,Early effects of salinity on water transport in Arabidopsis roots.Molecular and cellular features of aquaporin expression,Plant Physiol.,139(2):790-805
Chinnusamy V.,Zhu J.,and Zhu J.K.,2006,Gene regulation during cold acclimation in plants,Physiol.Plant.,126(1):52-61
Danielson J.魡.H.,and Johanson U.,2008,Unexpected complexity of the aquaporin gene family in the moss Physcomitrella patens,BMC Plant Biol.,8(1):45
Huang G.T.,Ma S.L.,Bai L.P.,Zhang L.,Ma H.,Jia P.,Liu J.,Zhong M.,and Guo Z.F.,2012,Signal transduction during cold,salt,and drought stresses in plants,Mol.Biol.Rep.,39(2):969-987
Jang J.Y.,Kim D.G.,Kim Y.O.,Kim J.S.,and Kang H.,2004,An expression analysis of a gene family encoding plasma membrane aquaporins in response to abiotic stresses in Arabidopsis thaliana,Plant Mol.Biol.,54(5):713-725
Johanson U.,Karlsson M.,Johansson I.,Gustavsson S.,Sjovall S.,Fraysse L.,Weig A.R.,and Kjellbom P.,2001,The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants,Plant Physiol.,126(4):1358-1369
Khan K.,Agarwal P.,Shanware A.,and Sane V.A.,2015,Heterologous expression of two Jatropha aquaporins imparts drought and salt tolerance and improves seed viability in transgenic Arabidopsis thaliana,PLoS One,10(6):e0128866
Li D.D.,Tai F.J.,Zhang Z.T.,Li Y.,Zheng Y.,Wu Y.F.,and Li X.B.,2009,A cotton gene encodes a tonoplast aquaporin that is involved in cell tolerance to cold stress,Gene,438(1):26-32
Li J.Q.,Zhu G.L.,Wu G.L.,He Z.B.,Li J.X.,Zhang C.H.,Tian F.D.,and Jia J.X.,2004,Correlation and path analysis between seed oil content and major quantitative characters in castor,Zhongguo Youliao Zuowu Xuebao(Chinese Journal of Oil Crop Sciences),26(2):43-46(李金琴,朱国立,吴国林,何智彪,李靖霞,张春华,田福东,贾娟霞,2004,蓖麻种子含油量与主要数量性状的相关及通径分析,中国油料作物学报,26(2):43-46)
Maeshima M.,2001,Tonoplast transporters:organization and function,Ann.Rev.Plant Physiol.Plant Mol.Biol.,52(1):469-497
Maurel C.,Boursiac Y.,Luu D.T.,Santoni V.,Shahzad Z.,and Verdoucq L.,2015,Aquaporins in plants,Physiol.Rev.,95(4):1321-1358
Mei Y.,Li H.L.,Yang S.Y.,and Luo H.Y.,2007,Plant aquaporins function research,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),43(3):563-568(梅杨,李海蓝,杨尚元,罗红艺,2007,植物水孔蛋白的功能,植物生理学通讯,43(3):563-568)
Peng Y.,Arora R.,Li G.,Wang X.,and Fessehaie A.,2008,Rhododendron catawbiense plasma membrane intrinsic proteins are aquaporins,and their over-expression compromises constitutive freezing tolerance and cold acclimation ability of transgenic Arabidopsis plants,Plant Cell Environ.,31(9):1275-1289
Peng Y.,Lin W.,Cai W.,and Arora R.,2007,Overexpression of a panax ginseng tonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic Arabidopsis plants,Planta,226(3):729-740
Regon P.,Panda P.,Kshetrimayum E.,and Panda S.K.,2014,Genome-wide comparative analysis of tonoplast intrinsic protein(TIP)genes in plants,Funct.Integr.Genomics,14(4):617-629
Rodrigues M.I.,Bravo J.P.,Sassaki F.T.,Severino F.E.,and Maia I.G.,2013,The tonoplast intrinsic aquaporin(TIP)subfamily of Eucalyptus grandis:characterization of Eg TIP2,a rootspecific and osmotic stress-responsive gene,Plant Sci.,213:106-113
Sakurai J.,Ishikawa F.,Yamaguchi T.,Uemura M.,and Maeshima M.,2005,Identification of 33 rice aquaporin genes and analysis of their expression and function,Plant Cell Physiol.,46(9):1568-1577