蓖麻RcSEC14p基因的克隆及低温胁迫下的表达分析
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摘要
Sec14-like蛋白参与细胞的基本生物学过程,如磷脂代谢、膜运输、信号转导及逆境响应。本研究采用RT-PCR的方法在蓖麻叶片中成功克隆了一个磷脂酰肌醇转运蛋白基因RcSEC14p (Accession No.MK205181)。RcSEC14p基因全长包含一个1 752 bp的完整开放阅读框,推测编码583个氨基酸。生物信息分析发现RcSEC14p为不稳定的亲水性蛋白;蛋白质二级结构元件多为α螺旋和无规卷曲。三级结构分析表明RcSEC14p结构内部含有一个潜在的磷脂结合口袋,可能在脂质代谢和磷酸肌醇信号转导等方面发挥重要的作用。多重序列比对表明蓖麻RcSEC14p与麻风树同源蛋白的相似性高达77.03%。系统进化树分析表明RcSEC14p属于Sec-like蛋白家族的SFH亚家族成员,N端包含一个SEC结构域,C端包含一个Nodulin结构域。q RT-PCR分析发现RcSEC14p受冷胁迫下调表达。该基因的下调表达可能会降低蓖麻的抗冷性。该研究为进一步探讨RcSEC14p是否通过调节磷脂信号转导途径参与冷胁迫响应提供依据。
Sec14-like protein participated in the basic biological process of cells, such as phospholipid metabolism,membrane traffic, signal transduction and stress response. In this study, a phosphatidylinositol transfer protein gene RcSEC14 p(Accession No.MK205181) was successfully cloned from castor leaves by RT-PCR method. Full length of RcSEC14 p gene consists of a complete open reading frame(1 752 bp) encoded a putative polypeptide of 583 amino acids. Bioinformatic analysis showed that RcSEC14 p was an unstable hydrophilic protein. Most of the protein secondary structural elements were α-helices and random coil. Tertiary structure analysis revealed that there was a potential phospholipid binding pocket in the structure of RcSEC14 p that may play a critical role in lipid metabolism and phosphoinositide signaling transduction. Multiple sequence alignment displayed that the similarity between castor RcSEC14 p and Jatropha carcas L. homologous protein was as high as 77.03%. Phylogenetic tree analysis showed that RcSEC14 p belongs to the SFH subfamily of Sec-like protein family, whose N-terminal contained a SEC domain and C-terminal contained a Nodulin domain. qRT-PCR analysis showed that the expression of RcSEC14 p was down-regulated by cold stress. Down-regulation of the gene expression might reduce the cold tolerance of castor. This study laid the foundation to test whether RcSEC14 p is involved in cold stress response by modulating phospholipid signaling transduction which deserves further investigation.
Sec14-like protein participated in the basic biological process of cells, such as phospholipid metabolism,membrane traffic, signal transduction and stress response. In this study, a phosphatidylinositol transfer protein gene RcSEC14 p(Accession No.MK205181) was successfully cloned from castor leaves by RT-PCR method. Full length of RcSEC14 p gene consists of a complete open reading frame(1 752 bp) encoded a putative polypeptide of 583 amino acids. Bioinformatic analysis showed that RcSEC14 p was an unstable hydrophilic protein. Most of the protein secondary structural elements were α-helices and random coil. Tertiary structure analysis revealed that there was a potential phospholipid binding pocket in the structure of RcSEC14 p that may play a critical role in lipid metabolism and phosphoinositide signaling transduction. Multiple sequence alignment displayed that the similarity between castor RcSEC14 p and Jatropha carcas L. homologous protein was as high as 77.03%. Phylogenetic tree analysis showed that RcSEC14 p belongs to the SFH subfamily of Sec-like protein family, whose N-terminal contained a SEC domain and C-terminal contained a Nodulin domain. qRT-PCR analysis showed that the expression of RcSEC14 p was down-regulated by cold stress. Down-regulation of the gene expression might reduce the cold tolerance of castor. This study laid the foundation to test whether RcSEC14 p is involved in cold stress response by modulating phospholipid signaling transduction which deserves further investigation.
引文
Agarwal P.K.,Agarwal P.,Reddy M.K.,and Sopory S.K.,2006,Role of DREB transcription factors in abiotic and biotic stress tolerance in plants,Plant Cell Rep.,25(12):1263-1274
Bankaitis V.A.,Vincent P.,Merkulova M.,Tyeryar K.,and Liu Y.,2007,Phosphatidylinositol transfer proteins and functional specification of lipid signaling pools,Adv.Enzyme Regul.,47(1):27-40
Cha M.K.,Hong S.K.,Oh Y.M.,and Kim I.H.,2003,The protein interaction of Saccharomyces cerevisiae cytoplasmic thiol peroxidaseⅡwith Sfh2p and its in vivo function,J.Biol.Chem.,278(37):34952-34958
Ding Y.,Li H.,Zhang X.,Xie Q.,Gong Z.,and Yang S.,2015,OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis,Dev.Cell,32(3):278-289
Gelli M.,Duo Y.,Konda A.R.,Zhang C.,Holding D.,and Dweikat I.,2014,Identification of differentially expressed genes between sorghum genotypes with contrasting nitrogen stress tolerance by genome-wide transcriptional profiling,BMC Genomics,15(1):179
Giansanti M.G.,Bonaccorsi S.,Kurek R.,Farkas R.M.,Dimitri P.,Fuller M.T.,and Gatti M.,2006,The class I PITP giotto is required for drosophila cytokinesis,Curr.Biol.,16(2):195-201
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
Huang J.,Kim C.M.,Xuan Y.,Park S.J.,Piao H.L.,Je B.,Liu J.,Kim T.H.,Kim B.K.,and Han C.D.,2013,Os SNDP1,a Sec14-nodulin domain-containing protein,plays a critical role in root hair elongation in rice,Plant Mol.Biol.,82(1-2):39-50
Kiba A.,Nakano M.,Vincent-Pope P.,Takahashi H.,Sawasaki T.,Endo Y.,Ohnishi K.,Yoshioka H.,and Hikichi Y.,2012,A novel Sec14 phospholipid transfer protein from Nicotiana benthamiana is up-regulated in response to Ralstonia solanacearum infection,pathogen associated molecular patterns and effector molecules and involved in plant immunity,J.Plant Physiol.,169(10):1017-1022
Lee J.,Lim Y.P.,Han C.K.,Nou I.S.,and Hur Y.,2013,Genomewide expression profiles of contrasting inbred lines of Chinese cabbage,Chiifu and Kenshin under temperature stress,Genes Genom.,35(3):273-288
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the2-△△Ctmethod,Methods,25(4):402-408
Monks D.E.,Aghoram K.,Courtney P.D.,DeWald D.B.,and Dewey R.E.,2001,Hyperosmotic stress induces the rapid phosphorylation of a soybean phosphatidylinositol transfer protein homolog through activation of the protein kinases SPK1 and SPK2,Plant Cell,13(5):1205-1219
Phillips S.E.,Vincent P.,Rizzieri K.E.,Schaaf G.,and Bankaitis V.A.,2006,The diverse biological functions of phosphatidylinositol transfer proteins in eukaryotes,Crit.Rev.Biochem.Mol.Biol.,41(1):21-49
Saito K.,Tautz L.,and Mustelin T.,2007,The lipid-binding SEC14 domain,Biochim.Biophys.Acta,1771(6):719-726
Wang X.,Shan X.,Wu Y.,Su S.,Li S.,Liu H.,Han J.,Xue C.,and Yuan Y.,2016,i TRAQ-based quantitative proteomics analysis reveals new metabolic pathways responding to chilling stress in maize seedlings,J.Proteomics,146:14-24
Wang X.Y.,2016,Isolation and functional characterization of chilling responsive genes and quantitative proteomic analysis responding to chilling stress in maize,Dissertation for Ph.D.,Jilin University,Supervisor:Yuan Y.P.,pp.66-67(王晓宇,2016,玉米冷响应相关基因的克隆、功能鉴定及定量蛋白质组学研究,博士学位论文,吉林大学,导师:原亚萍,pp.66-67)
Yamaguchi-Shinozaki K.,and Shinozaki K.,2006,Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses,Annu.Rev.Plant Biol.,57:781-803
Bankaitis V.A.,Vincent P.,Merkulova M.,Tyeryar K.,and Liu Y.,2007,Phosphatidylinositol transfer proteins and functional specification of lipid signaling pools,Adv.Enzyme Regul.,47(1):27-40
Cha M.K.,Hong S.K.,Oh Y.M.,and Kim I.H.,2003,The protein interaction of Saccharomyces cerevisiae cytoplasmic thiol peroxidaseⅡwith Sfh2p and its in vivo function,J.Biol.Chem.,278(37):34952-34958
Ding Y.,Li H.,Zhang X.,Xie Q.,Gong Z.,and Yang S.,2015,OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis,Dev.Cell,32(3):278-289
Gelli M.,Duo Y.,Konda A.R.,Zhang C.,Holding D.,and Dweikat I.,2014,Identification of differentially expressed genes between sorghum genotypes with contrasting nitrogen stress tolerance by genome-wide transcriptional profiling,BMC Genomics,15(1):179
Giansanti M.G.,Bonaccorsi S.,Kurek R.,Farkas R.M.,Dimitri P.,Fuller M.T.,and Gatti M.,2006,The class I PITP giotto is required for drosophila cytokinesis,Curr.Biol.,16(2):195-201
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
Huang J.,Kim C.M.,Xuan Y.,Park S.J.,Piao H.L.,Je B.,Liu J.,Kim T.H.,Kim B.K.,and Han C.D.,2013,Os SNDP1,a Sec14-nodulin domain-containing protein,plays a critical role in root hair elongation in rice,Plant Mol.Biol.,82(1-2):39-50
Kiba A.,Nakano M.,Vincent-Pope P.,Takahashi H.,Sawasaki T.,Endo Y.,Ohnishi K.,Yoshioka H.,and Hikichi Y.,2012,A novel Sec14 phospholipid transfer protein from Nicotiana benthamiana is up-regulated in response to Ralstonia solanacearum infection,pathogen associated molecular patterns and effector molecules and involved in plant immunity,J.Plant Physiol.,169(10):1017-1022
Lee J.,Lim Y.P.,Han C.K.,Nou I.S.,and Hur Y.,2013,Genomewide expression profiles of contrasting inbred lines of Chinese cabbage,Chiifu and Kenshin under temperature stress,Genes Genom.,35(3):273-288
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the2-△△Ctmethod,Methods,25(4):402-408
Monks D.E.,Aghoram K.,Courtney P.D.,DeWald D.B.,and Dewey R.E.,2001,Hyperosmotic stress induces the rapid phosphorylation of a soybean phosphatidylinositol transfer protein homolog through activation of the protein kinases SPK1 and SPK2,Plant Cell,13(5):1205-1219
Phillips S.E.,Vincent P.,Rizzieri K.E.,Schaaf G.,and Bankaitis V.A.,2006,The diverse biological functions of phosphatidylinositol transfer proteins in eukaryotes,Crit.Rev.Biochem.Mol.Biol.,41(1):21-49
Saito K.,Tautz L.,and Mustelin T.,2007,The lipid-binding SEC14 domain,Biochim.Biophys.Acta,1771(6):719-726
Wang X.,Shan X.,Wu Y.,Su S.,Li S.,Liu H.,Han J.,Xue C.,and Yuan Y.,2016,i TRAQ-based quantitative proteomics analysis reveals new metabolic pathways responding to chilling stress in maize seedlings,J.Proteomics,146:14-24
Wang X.Y.,2016,Isolation and functional characterization of chilling responsive genes and quantitative proteomic analysis responding to chilling stress in maize,Dissertation for Ph.D.,Jilin University,Supervisor:Yuan Y.P.,pp.66-67(王晓宇,2016,玉米冷响应相关基因的克隆、功能鉴定及定量蛋白质组学研究,博士学位论文,吉林大学,导师:原亚萍,pp.66-67)
Yamaguchi-Shinozaki K.,and Shinozaki K.,2006,Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses,Annu.Rev.Plant Biol.,57:781-803
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