马铃薯stuProT1.2基因的克隆与非生物胁迫的表达分析
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摘要
为了探究马铃薯脯氨酸转运蛋白在逆境下的功能,以马铃薯GS393 (Solanum commersonii-LZ3.4-Wisconsin, United States)为试验材料,采用PCR的方法,克隆得到2个脯氨酸转运蛋白基因分别命名为stuProT1和stuProT2,它们的开放阅读框长度分别为504 bp和600 bp,分别编码189个和199个氨基酸。系统进化树分析结果表明,这2个蛋白和番茄ProT蛋白具有高度同源性。实时荧光定量PCR表明,stuProT1和stuProT2基因在GS393不同组织(根,茎,叶,匍匐茎和块茎)中均有表达,其中根和茎的表达量较高。q PCR分析这2个基因在不同激素、重金属、低温、干旱和盐胁迫下的表达情况。分析结果表明:ABA抑制stuProT1和stuProT2基因表达(36 h内);2个基因分别在GA3、IAA、6-BA处理的2 h、8 h、24 h出现诱导表达峰值。供试重金属都能诱导2个基因的表达,其中Al、Cd、Hg处理48 h内始终处于上调水平,不同处理诱导表达峰值出现时间不同;Cu处理下2 h时出现表达峰值,其他时间回调。stuProT1和stuProT2基因对PEG-6000处理十分敏感,处理时间内始终高水平表达。NaCl处理2～8 h内出现高水平表达,之后回调。4℃和-4℃处理下2个基因都表现为先下调后回升的特点。综合分析表明stuProT2比stuProT1在各种胁迫条件下的响应更强烈。本研究弄清了在非生物胁迫下,ProT是脯氨酸高度选择的运输的转运体。
To identified the function of potato proline transporter under stress conditions, potato GS393(Solanum commersonii-LZ3.4-Wisconsin, United States) was used as the experimental material, and two proline transporter genes were cloned for stuProT1 and stuProT2 by PCR. stuProT1 open reading frame is 504 bp in length and encodes 189 amino acids; stuProT2 open reading frame is 600 bp in length and encodes 199 amino acids.Phylogenetic tree analysis showed that the two proteins and tomato ProT protein have high homology. Real-time PCR showed that stuProT1 and stuProT2 genes were expressed in different tissues(roots, stems, leaves, stolons and tubers) of GS393, and the expression of roots and stems was higher. The results of qPCR analysis for stuProT1 and stuProT2 genes under different hormones, heavy metals, low temperature, drought and salt stress, showed that:ABA inhibits expression of stuProT1 and stuProT2 gene(within 36 h). The absorption peaks of two genes were observed at 2 h, 8 h, and 24 h of GA3, IAA, 6-BA induced treatment. The heavy metals could induce the expression of stuProT1 and stuProT2 gene, among which Al, Cd and Hg were always in the up-regulated level within 48 h, and the peak of expression were distinct in different treatments. The peak of expression was observed at 2 h after Cu treatment, and the other time was below. The stuProT1 and stuProT2 genes are sensitive to PEG-6000 treatment and are always expressed at high levels. High levels of expression within 2～8 hours of NaCl treatment and callback followed. Both genes under the treatment of 4℃ and-4℃ showed the characteristics of down-regulation and then callback. Comprehensive analysis showed that stuProT2 responded more strongly than stuProT1 under various stress conditions. This study identified ProT as a highly selective transporter of proline under abiotic stress.
To identified the function of potato proline transporter under stress conditions, potato GS393(Solanum commersonii-LZ3.4-Wisconsin, United States) was used as the experimental material, and two proline transporter genes were cloned for stuProT1 and stuProT2 by PCR. stuProT1 open reading frame is 504 bp in length and encodes 189 amino acids; stuProT2 open reading frame is 600 bp in length and encodes 199 amino acids.Phylogenetic tree analysis showed that the two proteins and tomato ProT protein have high homology. Real-time PCR showed that stuProT1 and stuProT2 genes were expressed in different tissues(roots, stems, leaves, stolons and tubers) of GS393, and the expression of roots and stems was higher. The results of qPCR analysis for stuProT1 and stuProT2 genes under different hormones, heavy metals, low temperature, drought and salt stress, showed that:ABA inhibits expression of stuProT1 and stuProT2 gene(within 36 h). The absorption peaks of two genes were observed at 2 h, 8 h, and 24 h of GA3, IAA, 6-BA induced treatment. The heavy metals could induce the expression of stuProT1 and stuProT2 gene, among which Al, Cd and Hg were always in the up-regulated level within 48 h, and the peak of expression were distinct in different treatments. The peak of expression was observed at 2 h after Cu treatment, and the other time was below. The stuProT1 and stuProT2 genes are sensitive to PEG-6000 treatment and are always expressed at high levels. High levels of expression within 2～8 hours of NaCl treatment and callback followed. Both genes under the treatment of 4℃ and-4℃ showed the characteristics of down-regulation and then callback. Comprehensive analysis showed that stuProT2 responded more strongly than stuProT1 under various stress conditions. This study identified ProT as a highly selective transporter of proline under abiotic stress.
引文
An Y.,Zhang M.,Liu G.,Han R.,and Liang Z.,2013,Proline accumulation in leaves of Periploca sepium via both biosynthesis up-regulation and transport during recovery from severe drought,PLoS One,8(7):1-10
Breitkreuz K.E.,Shelp B.J.,Fischer W.N.,Schwacke R.,and Rentsch D.,1999,Identification and characterization of GA-BA,proline and quaternary ammonium compound transporters from Arabidopsis thaliana,Febs Lett.,450(3):280-284
Chen J.,Wu J.,Lu Y.,Cao Y.,Zeng H.,Zhang Z.,Wang L.,and Wang S.,2016,Molecular cloning and characterization of a gene encoding the proline transporter protein in common bean(Phaseolus vulgaris L.),Crop J.,4(5):384-390
Fujiwara T.,Mitsuya S.,Miyake H.,Hattori T.,and Takabe T.,2010,Characterization of a novel glycinebetaine/proline transporter gene expressed in the mestome sheath and lateral root cap cells in barley,Planta,232(1):133-143
Grallath S.,Meyer A.,Neuhaus J.M.,and Rentsch D.,2005,The atprot family,compatible solute transporters with similar substrate specificity but differential expression patterns,Plant Physiol.,137(1):117-126
Hare P.D.,and Cress W.A.,1997,Metabolic implications of stress-induced proline accumulation in plants,Plant Growth Regul.,21(2):79-102
Igarashi Y.,Yoshiba Y.,Takeshita T.,Nomura S.,Otomo J.,Yamaguchi-Shinozaki K.,and Shinozaki K.,2000,Molecular cloning and characterization of a cDNA encoding proline transporter in rice?Plant Cell Physiol.,41(6):750-756
Kavi-Kishor P.B.,and Sreenivasulu N.,2014,Is proline accumulation per se correlated with stress tolerance or is proline homeostasis a more critical issue,Plant Cell Environ.,37(2):300-311
Kishor P.B.K.,1989,Salt stress in cultured rice cells:effects of proline and abscisic acid,Plant Cell Environ.,12(6):629-633
Lei P.,Xu Z.,Liang J.,Luo X.,Zhang Y.,Feng X.,and Xu H.,2016,Poly(γ-glutamic acid)enhanced tolerance to salt stress by promoting proline accumulation in Brassica napus l,Plant Growth Regul.,78(2):1-9
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the2(-ΔΔCT)method,Methods,25(4):402-408
Raba M.,Dunkel S.,Hilger D.,Lipiszko K.,Polyhach Y.,Jeschke G.,Bracher S.,Klare J.P.,Quick M.,Jung H.,and Steinhoff H.J.,2014,Extracellular loop 4 of the proline transporter putp controls the periplasmic entrance to ligand binding sites,Structure,22(5):769-780
Schwacke R.,Grallath S.,Breitkreuz K.E.,Stransky E.,Stransky H.,Frommer W.B.,and Rentsch D.,1999,LeProT1,a transporter for proline,glycine betaine,andγ-amino butyric acid in tomato pollen,Plant Cell,11(3):377-392
Shen Y.G.,zhang W.K.,Yan D.Q.,She B.X.,Zhang J.S.,and Chen S.Y.,2002,Overexpression of proline transporter gene isolated from halophyte confers salt tolerance in Arabidopsis,Acta Botanica Sinica,44(8):956-962
Siripornadulsil S.,Traina S.,Verma D.P.S.,and Sayre R.T.,2002,Molecular mechanisms of proline-mediated tolerance to toxic heavy metals in transgenic microalgae,Plant Cell,14(11):2837-2847
Ueda A.,Yamamoto-Yamane Y.,and Takabe T.,2007,Salt stress enhances proline utilization in the apical region of barley roots,Biochem.Biophys.Res.Commun.,355(1):61-66
Verbruggen N.,and Hermans C.,2008,Proline accumulation in plants:a review,Amino Acids,35(4):753-759
Verslues P.E.,and Sharma S.,2010,Proline metabolism and its implications for plant-environment interaction,Arabidopsis Book,doi:10.1199/tab.0140
Wu Z.,Liang J.,Wang C.,Zhao X.,Zhong X.,Cao X.,Li G.,He J.,and Yi M.,2018,Overexpression of lily Hsf A3s in Arabidopsis confers increased thermotolerance and salt sensitivity via alterations in proline catabolism,J.Exp.Bot.,69(8):2005-2021
Yang T.,and Zhang W.,2013,Transformation of mangrove betaine/proline transporter gene Bet/ProT2 into rice and salttolerance study in transgenic plants,Nanjing Nongye Daxue Xuebao(Journal of Nanjing Agricultural University),36(5):51-58(杨婷婷,张炜,2013,红树甜菜碱/脯氨酸转运蛋白基因Bet/ProT2转化水稻及耐盐性的研究,南京农业大学学报,36(5):51-58)
Zhang M.,Huang H.,and Dai S.,2014,Isolation and expression analysis of proline metabolism-related genes in Chrysanthemum lavandulifolium,Gene,537(2):203-213
Breitkreuz K.E.,Shelp B.J.,Fischer W.N.,Schwacke R.,and Rentsch D.,1999,Identification and characterization of GA-BA,proline and quaternary ammonium compound transporters from Arabidopsis thaliana,Febs Lett.,450(3):280-284
Chen J.,Wu J.,Lu Y.,Cao Y.,Zeng H.,Zhang Z.,Wang L.,and Wang S.,2016,Molecular cloning and characterization of a gene encoding the proline transporter protein in common bean(Phaseolus vulgaris L.),Crop J.,4(5):384-390
Fujiwara T.,Mitsuya S.,Miyake H.,Hattori T.,and Takabe T.,2010,Characterization of a novel glycinebetaine/proline transporter gene expressed in the mestome sheath and lateral root cap cells in barley,Planta,232(1):133-143
Grallath S.,Meyer A.,Neuhaus J.M.,and Rentsch D.,2005,The atprot family,compatible solute transporters with similar substrate specificity but differential expression patterns,Plant Physiol.,137(1):117-126
Hare P.D.,and Cress W.A.,1997,Metabolic implications of stress-induced proline accumulation in plants,Plant Growth Regul.,21(2):79-102
Igarashi Y.,Yoshiba Y.,Takeshita T.,Nomura S.,Otomo J.,Yamaguchi-Shinozaki K.,and Shinozaki K.,2000,Molecular cloning and characterization of a cDNA encoding proline transporter in rice?Plant Cell Physiol.,41(6):750-756
Kavi-Kishor P.B.,and Sreenivasulu N.,2014,Is proline accumulation per se correlated with stress tolerance or is proline homeostasis a more critical issue,Plant Cell Environ.,37(2):300-311
Kishor P.B.K.,1989,Salt stress in cultured rice cells:effects of proline and abscisic acid,Plant Cell Environ.,12(6):629-633
Lei P.,Xu Z.,Liang J.,Luo X.,Zhang Y.,Feng X.,and Xu H.,2016,Poly(γ-glutamic acid)enhanced tolerance to salt stress by promoting proline accumulation in Brassica napus l,Plant Growth Regul.,78(2):1-9
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the2(-ΔΔCT)method,Methods,25(4):402-408
Raba M.,Dunkel S.,Hilger D.,Lipiszko K.,Polyhach Y.,Jeschke G.,Bracher S.,Klare J.P.,Quick M.,Jung H.,and Steinhoff H.J.,2014,Extracellular loop 4 of the proline transporter putp controls the periplasmic entrance to ligand binding sites,Structure,22(5):769-780
Schwacke R.,Grallath S.,Breitkreuz K.E.,Stransky E.,Stransky H.,Frommer W.B.,and Rentsch D.,1999,LeProT1,a transporter for proline,glycine betaine,andγ-amino butyric acid in tomato pollen,Plant Cell,11(3):377-392
Shen Y.G.,zhang W.K.,Yan D.Q.,She B.X.,Zhang J.S.,and Chen S.Y.,2002,Overexpression of proline transporter gene isolated from halophyte confers salt tolerance in Arabidopsis,Acta Botanica Sinica,44(8):956-962
Siripornadulsil S.,Traina S.,Verma D.P.S.,and Sayre R.T.,2002,Molecular mechanisms of proline-mediated tolerance to toxic heavy metals in transgenic microalgae,Plant Cell,14(11):2837-2847
Ueda A.,Yamamoto-Yamane Y.,and Takabe T.,2007,Salt stress enhances proline utilization in the apical region of barley roots,Biochem.Biophys.Res.Commun.,355(1):61-66
Verbruggen N.,and Hermans C.,2008,Proline accumulation in plants:a review,Amino Acids,35(4):753-759
Verslues P.E.,and Sharma S.,2010,Proline metabolism and its implications for plant-environment interaction,Arabidopsis Book,doi:10.1199/tab.0140
Wu Z.,Liang J.,Wang C.,Zhao X.,Zhong X.,Cao X.,Li G.,He J.,and Yi M.,2018,Overexpression of lily Hsf A3s in Arabidopsis confers increased thermotolerance and salt sensitivity via alterations in proline catabolism,J.Exp.Bot.,69(8):2005-2021
Yang T.,and Zhang W.,2013,Transformation of mangrove betaine/proline transporter gene Bet/ProT2 into rice and salttolerance study in transgenic plants,Nanjing Nongye Daxue Xuebao(Journal of Nanjing Agricultural University),36(5):51-58(杨婷婷,张炜,2013,红树甜菜碱/脯氨酸转运蛋白基因Bet/ProT2转化水稻及耐盐性的研究,南京农业大学学报,36(5):51-58)
Zhang M.,Huang H.,and Dai S.,2014,Isolation and expression analysis of proline metabolism-related genes in Chrysanthemum lavandulifolium,Gene,537(2):203-213