杜氏盐藻LYCB基因克隆及在盐胁迫下的表达分析
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摘要
[目的]为探究杜氏盐藻(Dunaliella Salina)富集β-胡萝卜素的分子机理。[方法]本文选用从运城盐湖分离的杜氏盐藻株系Ds-YC011为试材,克隆编码番茄红素-β-环化酶(lycopene b-cyclase,LYCB)的基因(DsLYCB)并分析其功能。[结果]DsLYCB基因cDNA全长1 910bp,开放阅读框(ORF)长1 769bp,编码584个氨基酸。生物信息学分析显示,DsLYCB为不稳定亲水性蛋白,无跨膜结构,定位于叶绿体。蛋白二级结构中α-螺旋结构占39.9%,杜氏盐藻DsLYCB蛋白与雨生红球藻亲缘关系最近。qRT-PCR分析表明,在高盐(3mol·L~(-1))胁迫下,DsLYCB基因表达量显著上升,胁迫4h时表达量达峰值,为正常对照(1.5mol·L~(-1))条件下的2倍。高盐(3mol·L~(-1))胁迫下,DsLYCB基因上调表达模式与藻细胞类β-胡萝卜素积累增加相一致,预示着DsLYCB在杜氏盐藻类β-胡萝卜素合成积累中起重要作用。[结论]这为解析微藻类胡萝卜素合成调控机制及遗传修饰提供了科学依据。
[Objective]Present study was proposed in order to understand the molecular mechanism underlying the biosynthesis and accumulation ofβ-carotene in Dunaliella salina.[Methods]A D.salinastrain Ds-YC011 isolated from Yuncheng Salt Lake was selected as genotype to clone lycopeneβ-cyclase(LYCB)gene(DsLYCB)and used as material to analyze its function.[Results]The full-length cDNA sequence of DsLYCBwas determined as 1 910 bp with 1 769 bp ORF(open reading frame)encoding 584 amino acids.The bioinformatics analysis showed that DsLYCB located in chloroplast was an unstable hydrophilic protein with no transmembrane structure,andα-helix secondary structure was predominated(36.3%).Phylogenetic analysis indicated that DsLYCB was closely related to LYCB protein in Haematococcus pluvialis.The results of qRT-PCR demonstrated that DsLYCBexpression was increased significantly under high salt stress(3 mol·L~(-1) NaCl)with its peak value shown at 4 hpost treatment,which was two times higher than that of the normal growth condition(1.5 mol·L~(-1) NaCl).Under the high salt stress,the upregulated expression pattern of DsLYCBgene was in accordance with the accumulation pattern of carotenoids in microalgal cells which indicated that DsLYCB may play an important role in the biosynthesis and accumulation of carotenoids in Dunaliella salina.[Conclusion]The present data provided a solid evidence for better understanding of carotenoid biosynthesis-regulation mechanism and genetic modification on this pathway.
[Objective]Present study was proposed in order to understand the molecular mechanism underlying the biosynthesis and accumulation ofβ-carotene in Dunaliella salina.[Methods]A D.salinastrain Ds-YC011 isolated from Yuncheng Salt Lake was selected as genotype to clone lycopeneβ-cyclase(LYCB)gene(DsLYCB)and used as material to analyze its function.[Results]The full-length cDNA sequence of DsLYCBwas determined as 1 910 bp with 1 769 bp ORF(open reading frame)encoding 584 amino acids.The bioinformatics analysis showed that DsLYCB located in chloroplast was an unstable hydrophilic protein with no transmembrane structure,andα-helix secondary structure was predominated(36.3%).Phylogenetic analysis indicated that DsLYCB was closely related to LYCB protein in Haematococcus pluvialis.The results of qRT-PCR demonstrated that DsLYCBexpression was increased significantly under high salt stress(3 mol·L~(-1) NaCl)with its peak value shown at 4 hpost treatment,which was two times higher than that of the normal growth condition(1.5 mol·L~(-1) NaCl).Under the high salt stress,the upregulated expression pattern of DsLYCBgene was in accordance with the accumulation pattern of carotenoids in microalgal cells which indicated that DsLYCB may play an important role in the biosynthesis and accumulation of carotenoids in Dunaliella salina.[Conclusion]The present data provided a solid evidence for better understanding of carotenoid biosynthesis-regulation mechanism and genetic modification on this pathway.
引文
[1]姬翔.杜氏盐藻(Dunaliella salina)14-3-3蛋白cDNA全长的克隆与序列分析[D].郑州:郑州大学,2006.
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[8]Zhan A Y,You X L,Zhan Y G.Biosynthetic pathway and applications of plant terpenoid isoprenoid[J].Letters in Biotechnology,2010,21(1):131-135.
[9]Zhu C,Yamamura S,Nishihara M,et al.cDNAs for the synthesis of cyclic carotenoids in petals of Gentiana lutea and their regulation during flower development[J].Biochimica et Biophysica Acta(BBA)-Gene Structure and Expression,2003,1625(3):305-308.
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[11]Stickforth P,Steiger S,Hess W R,et al.A novel type of lycopeneε-cyclase in the marine cyanobacterium Prochlorococcus marinus MED4[J].Archives of Microbiology,2003,179(6):409-415.
[12]Moreno J C,Pizarro L,Fuentes P,et al.Levels of lycopeneβ-cyclase 1modulate carotenoid gene expression and accumulation in Daucus carota[J].Plos One,2013,8(3):e58144.
[13]Xu Q,Yu K,Zhu A,et al.Comparative transcripts profiling reveals new insight into molecular processes regulating lycopene accumulation in a sweet orange(Citrus sinensis)red-flesh mutant[J].BMC Genomics,2009,10(1):540.
[14]Xu Q,Liu Y,Zhu A,et al.Discovery and comparative profiling of microRNAs in a sweet orange red-flesh mutant and its wild type[J].BMC Genomics,2010,11(1):246.
[15]Yu K,Xu Q,Da X,et al.Transcriptome changes during fruit development and ripening of sweet orange(Citrus sinensis)[J].BMC Genomics,2012,13(1):10.
[16]吕军,张颖,冯立芹,等.生物信息学工具BLAST的使用简介[J].内蒙古大学学报(自然科学版),2003,34(2):179-187.
[17]薛飞,柴晓杰,余祝君,等.盐藻新基因DsSTPK的克隆及生物信息学分析[J].中国生物化学与分子生物学报,2012,28(3):289-295.
[18]Qu X,Chen Y,Qiao S.Predicting the subcellular localization of proteins with multiple sites based on multiple features fusion[C]//Proceedings of the 10th International Conference Intelligent Computing in Bioinformatics.Berlin:Springer,2014:456-465.
[19]Iliyas S,Ansari S N.A comparative study of different properties provided by protein structure visualization tools[J].International Journal of Basic and Applied Chemical Sciences,2013,3(1):1-6.
[20]Guex N,Peitsch M C.SWISS-MODEL and the Swiss-PdbViewer:An environment for comparative protein modeling[J].Electrophoresis,1997,18(15):2714-2723.
[21]Lei G,Qiao D,Bai L,et al.Isolation and characterization of a mitogen-activated protein kinase gene in the halotolerant alga Dunaliella salina[J].Journal of Applied Phycology,2008,20(1):13-17.
[22]Livak K J,Schmittgen T D.Analysis of relative gene expression datausing real-time quantitative PCR and the 2(-Delta Delta C(T))Method[J].Methods,2001,25(4):402-408.
[23]周静,龚一富,马颖瑞,等.绿色杜氏藻DvGGPS生物信息学及转录差异研究[J].核农学报,2017,31(4):635-642.
[24]陶俊,刘兴满.枇杷番茄红素β-环化酶基因片段的克隆和序列分析[J].苏州科技学院学报,2006,23(4):43-46.
[25]张建成,陶能国,周文静,等.红肉脐橙番茄红素β-环化酶基因的克隆及其在大肠杆菌中的功能表达[J].园艺学报,2008,35(9):1269-1276.
[26]梁燕,王鸣,陈杭,等.茄红素-β-环化酶反义RNA基因对烟草的遗传转化[J].西北农林科技大学学报(自然科学版),2003,31(3):73-76.
[27]晏露,姜建国,刘冠楠,等.盐藻番茄红素β-环化酶全长cDNA的克隆及结构分析[J].现代食品科技,2010,26(3):218-221.
[2]刘红涛,冯书营,陈涛,等.杜氏盐藻分子生物学最新进展及展望[J].中国生物工程杂志,2007,27(10):113-118.
[3]冯书营,刘红涛,李杰,等.杜氏盐藻基因工程研究现状及应用前景[J].中国生物工程杂志,2007,27(2):108-112.
[4]Britton G,Liaaen-Jensen S,Pfander H,et al.Carotenoids.Handbook[J].Carotenoids Handbook,2004.
[5]朱长甫,陈星,王英典.植物类胡萝卜素生物合成及其相关基因在基因工程中的应用[J].植物生理及分子生物学学报,2004,30(6):609-618.
[6]Eisenreich W,Rohdich F,Bacher A.Deoxyxylulose phosphate pathway to terpenoids[J].Trends in Plant Science,2001,6(2):78-84.
[7]Rodrguez-Concepción M,Boronat A.Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids.A metabolic milestone achieved through genomics[J].Plant Physiology,2002,130(3):1079-1089.
[8]Zhan A Y,You X L,Zhan Y G.Biosynthetic pathway and applications of plant terpenoid isoprenoid[J].Letters in Biotechnology,2010,21(1):131-135.
[9]Zhu C,Yamamura S,Nishihara M,et al.cDNAs for the synthesis of cyclic carotenoids in petals of Gentiana lutea and their regulation during flower development[J].Biochimica et Biophysica Acta(BBA)-Gene Structure and Expression,2003,1625(3):305-308.
[10]Cunningham F X,Gantt E.One ring or two?Determination of ring number in carotenoids by lycopene-cyclases[J].Proceedings of the National Academy of Sciences of the United States of America,2001,98(5):2905-2910.
[11]Stickforth P,Steiger S,Hess W R,et al.A novel type of lycopeneε-cyclase in the marine cyanobacterium Prochlorococcus marinus MED4[J].Archives of Microbiology,2003,179(6):409-415.
[12]Moreno J C,Pizarro L,Fuentes P,et al.Levels of lycopeneβ-cyclase 1modulate carotenoid gene expression and accumulation in Daucus carota[J].Plos One,2013,8(3):e58144.
[13]Xu Q,Yu K,Zhu A,et al.Comparative transcripts profiling reveals new insight into molecular processes regulating lycopene accumulation in a sweet orange(Citrus sinensis)red-flesh mutant[J].BMC Genomics,2009,10(1):540.
[14]Xu Q,Liu Y,Zhu A,et al.Discovery and comparative profiling of microRNAs in a sweet orange red-flesh mutant and its wild type[J].BMC Genomics,2010,11(1):246.
[15]Yu K,Xu Q,Da X,et al.Transcriptome changes during fruit development and ripening of sweet orange(Citrus sinensis)[J].BMC Genomics,2012,13(1):10.
[16]吕军,张颖,冯立芹,等.生物信息学工具BLAST的使用简介[J].内蒙古大学学报(自然科学版),2003,34(2):179-187.
[17]薛飞,柴晓杰,余祝君,等.盐藻新基因DsSTPK的克隆及生物信息学分析[J].中国生物化学与分子生物学报,2012,28(3):289-295.
[18]Qu X,Chen Y,Qiao S.Predicting the subcellular localization of proteins with multiple sites based on multiple features fusion[C]//Proceedings of the 10th International Conference Intelligent Computing in Bioinformatics.Berlin:Springer,2014:456-465.
[19]Iliyas S,Ansari S N.A comparative study of different properties provided by protein structure visualization tools[J].International Journal of Basic and Applied Chemical Sciences,2013,3(1):1-6.
[20]Guex N,Peitsch M C.SWISS-MODEL and the Swiss-PdbViewer:An environment for comparative protein modeling[J].Electrophoresis,1997,18(15):2714-2723.
[21]Lei G,Qiao D,Bai L,et al.Isolation and characterization of a mitogen-activated protein kinase gene in the halotolerant alga Dunaliella salina[J].Journal of Applied Phycology,2008,20(1):13-17.
[22]Livak K J,Schmittgen T D.Analysis of relative gene expression datausing real-time quantitative PCR and the 2(-Delta Delta C(T))Method[J].Methods,2001,25(4):402-408.
[23]周静,龚一富,马颖瑞,等.绿色杜氏藻DvGGPS生物信息学及转录差异研究[J].核农学报,2017,31(4):635-642.
[24]陶俊,刘兴满.枇杷番茄红素β-环化酶基因片段的克隆和序列分析[J].苏州科技学院学报,2006,23(4):43-46.
[25]张建成,陶能国,周文静,等.红肉脐橙番茄红素β-环化酶基因的克隆及其在大肠杆菌中的功能表达[J].园艺学报,2008,35(9):1269-1276.
[26]梁燕,王鸣,陈杭,等.茄红素-β-环化酶反义RNA基因对烟草的遗传转化[J].西北农林科技大学学报(自然科学版),2003,31(3):73-76.
[27]晏露,姜建国,刘冠楠,等.盐藻番茄红素β-环化酶全长cDNA的克隆及结构分析[J].现代食品科技,2010,26(3):218-221.