湖北海棠植物络合素合酶MhPCS基因克隆及表达分析Isolation and expression analysis of a phytochelatin synthase gene (MhPCS) from Malus hupehensis
姜倩倩;孙晓莉;曹慧;邹岩梅;束怀瑞;
摘要(Abstract):
【目的】为了探明植物络合素酶参与植物解毒重金属的作用,【方法】以湖北海棠(Malus hupehensis)为试材,克隆其植物络合素合酶基因(MhPCS)编码区序列,并研究该基因的表达特点。【结果】结果表明MhPCS基因编码区全长为1 494 bp,编码497个氨基酸。序列分析表明,MhPCS编码的氨基酸序列由2个典型的植物络合素合酶亚家族结构域组成,具有3个相邻的Cys-Cys元件(331-332、351-352和369-370位氨基酸)和植物络合素合酶蛋白的特征位点(Cys-56、Cys-90/91和Cys-109)。【结论】湖北海棠MhPCS与杜梨PbPCS蛋白同源性最高(94%),属于系统发生树的同一分支。MhPCS基因为组成型表达,各器官表达水平存在差异,其中根的表达量最高;100μmol.L-1CdSO4处理48 h内,MhPCS基因的表达量迅速上升;不同金属离子对其上调表达的诱导能力为:Cd2+>Cu2+>Pb2+。这为揭示重金属胁迫下湖北海棠环境适应的分子机制奠定了基础。
关键词(KeyWords): 湖北海棠;植物络合素合酶基因(MhPCS);克隆;表达
基金项目(Foundation): 国家现代苹果产业技术体系;; 山东省自然科学基金(Y2007D60);; 潍坊市科技发展计划(20121303);; 潍坊学院博士基金(2012BS18)
作者(Author): 姜倩倩;孙晓莉;曹慧;邹岩梅;束怀瑞;
Email:
DOI: 10.13925/j.cnki.gsxb.2013.03.006
参考文献(References):
- [1]RAMOA J,NAYA L,GAY,ABIAN J,BECANA M.Functionalcharacterization of an unusual phytochelatin synthase,LjPCS3,ofLotus japonicus[J].Plant Physiology,2008,148:536-545.
- [2]STEPHAN C.Evolution and function of phytochelatin synthases[J].Journal of Plant Physiology,2006,163:319-332.
- [3]RALPH B,ANDREAS B,ARTHUR K,ANNA S,THOMAS L,KLAUS L,ERWIN G.Function of phytochelatin synthase incatabolism of glutathione-conjugates[J].The Plant Journal,2007,49:740-749.
- [4]GRILL E,LOFFLER S,WIMMACKER E L,ZENK M H.Phy-tochelatins,the heavy-metal-binding peptides of plants,are syn-thesized from glutathione by a specific gamma-glutamylcysteinedipeptidyl transpeptidase(phytochelatin synthase)[J].Proceedingsof the National Academy of Sciences of the United States of Ameri-ca,1989,86:6838-6842.
- [5]HA S,SMITH A P,HOWDEN R,DIETRICH W M,BUGG S,O’CONNELL M J,GOLDSBROUGH P B,C0BBRTT C S.Phy-tochelatin synthase genes from Arabidopsis and the yeast Schizosac-charomyces pombe[J].The Plant Cell,1999,11:1153-1163.
- [6]VATAMANIUK O K,MARI S,LU Y P,REA P A.AtPCS1,a phy-tochelatin synthase from Arabidopsis:isolation and in vitro reconsti-tution[J].Proceedings of the National Academy of Sciences of the U-nited States of America,1999,96:7110-7115.
- [7]CLEMENS S,KIM E J,NEUMANN D,SCHROEDER J I.Toler-ance to toxic metals by a gene family of phytochelatin synthases fromplants and yeast[J].EMBO Journal,1999,18:3325-3333.
- [8]HEISS S,WACHTER A,BOGS J,COBBETT C,RAUSCH T.Phy-tochelatin synthase(PCS)protein is induced in Brassica juncea leavesafter prolonged Cd exposure[J].Journal of Experimental Botany,2003,54:1833-1839.
- [9]JIANG Ying-nan,FENG Bao-min,ZHANG Hai-yan,MA Mi.Im-proving heavy metal tolerance of yeast by transferring a phytochelatinsynthase gene from garlic[J].Acta Phytoecologica Sinica,2005,29:659-664.姜瑛楠,冯保民,张海燕,麻密.大蒜植物络合素合酶基因转化对酵母重金属抗性的提高[J].植物生态学报,2005,29:659-664.
- [10]HE Z,LI J,ZHANG H,MA M.Different effects of calcium and lan-thanum on the expression of phytochelatin synthase gene and cadmi-um absorption in Lactuca sativa[J].Plant Science,2005,168:309-318.
- [11]LI Hui,CONG Yu,WANG Hong-wei,SHENG Bao-long,LINJing,CHANG You-hong.Molecular cloning and expression analysisof a phytochelatin synthase gene,PcPCS1,from Pyrus calleryanaDcne[J].Acta Horticulturae Sinica,2010,37(6):880-890.李慧,丛郁,王宏伟,盛宝龙,蔺经,常有宏.豆梨植物络合素合酶PcPCS1基因克隆及其表达分析[J].园艺学报,2010,37(6):880-890.
- [12]LIU Yu-xia,WANG Xiao-tong,SU Xu-dong,DAI Liang,ZHANGZhi-yi,XU Ji-chen.Cloning and expression analysis of a phy-tochelatin synthase gene(PtPCS)in Populus tomentosa Carr[J].Molecular Plant Breeding,2012,10(2):174-183.柳玉霞,王晓桐,苏旭东,代亮,张志毅,徐吉臣.毛白杨植物络合素合酶(PtPCS)基因克隆及其表达研究[J].分子植物育种,2012,10(2):174-183.
- [13]POMPNI M,CENSI V,DI GIROLAMO V,DE PAOLIS A,DIT0PPI L S,ARO MOLO R,COSTANTINO P,CARDARELLI M.Overexpression of Arabidopsis phytochelatin synthase in tobaccoplants enhances Cd2+tolerance and accumulation but not transloca-tion to the shoot[J].Planta,2006,223:180-190.
- [14]GASIC K,KORBAN S S.Expression of Arabidopsis phytochelatinsynthase in Indian mustard(Brassica juncea)plants enhances toler-ance for Cd and Zn[J].Planta,2007,225:1277-1285.
- [15]GASIC K,KORBAN S S.Transgenic Indian mustard(Brassicajuncea)plants expressing an Arabidopsis phytochelatin synthase(AtPCS1)exhibit enhanced As and Cd tolerance[J].Plant MolecularBiology,2007,64:361-369.
- [16]WU Cai-bin,LEI Heng-yi,NING Ping.Introduction to Environ-mental Studies[M].Beijing:China Environmental Science Pub-lishing House,2011:376-378.吴彩斌,雷恒毅,宁平.环境学概论[M].北京:中国环境科学出版社,2011:376-378.
- [17]KANG Ming,HUANG Hong-wen.Allozymic variation and geneticdiversity in Malus hupehensis(Rosaceae)[J].Biodiversity Science,2002,10(4):376-385.康明,黄宏文.湖北海棠的等位酶变异和遗传多样性研究[J].生物多样性,2002,10(4):376-385.
- [18]LU Qiu-nong,JIA Ding-xian,WANG Zhong-ying,LI Yu-nong,YANG Bin,SHENG Bin-cheng.Apple volume of China fruit trees[M].China Forestry Publishing House,1999.陆秋农,贾定贤,王中英,李育农,杨彬,盛炳成.《中国果树志苹果卷》[M].中国林业出版社,1999.
- [19]RAN Kun.Genome-wide analysis of the caspase-like gene familiesin apple and functional characterization of MhVPEγgene in Malushupehensis[D].Tai’an:Shandong Agricultural University,2012.冉昆.苹果caspase-like基因家族的全基因组分析及平邑甜茶MhVPEγ基因的功能鉴定[D].泰安:山东农业大学,2012.
- [20]CLEMENS S.Evolution and function of phytochelatin synthases[J].Journal of Plant Physiology,2006,163:319-332.
- [21]RUOTOLO R,PERACCHI A,BOLCHI A,INFUSINI G,AMORE-SANO A,OTTONELLO S.Domain organization of phytochelatinsynthase[J].The Journal of Biological Chemistry,2004,279:14686-14693.
- [22]REA P A.Phytochelatin synthase,papain’s cousin,in stereo[J].Proceedings of the National Academy of Sciences of the UnitedStates of America,2006,103:507-508.
- [23]RAMOS J,CLEMENTE M R,NAYA L,LOSCOS J,PEREZ-RONTOME C,SATO S,TABATA S,BECANA M.Phytochelatinsynthases of the model legume Lotus japonicus.A small multigenefamily with differential response to cadmium and alternativelyspliced variants[J].Plant Physiology,2007,143:1110-1118.
- [24]HA S B,SMITH A P,HOWDEN R,DIETRICH W M,BUGG S,O'CONNELL M J,GOLDSBROUGH P B,COBBETTA C S.Phy-tochelatin synthase genes from Arabidopsis and the yeast Schizosac-charomyces pombe[J].Plant Cell,1999,11(6):1153-1164.
- [25]HEISS S,WACHTER A,BOGS J,COBBETT C,RAUSCH T.Phy-tochelatin synthase(PCS)protein is induced in Brassica juncealeaves after prolonged Cd exposure[J].The Journal of Biological Chem-istry,2003,54(389):1833-1839.
- [26]HE Z Y,LI J C,ZHANG H Y,MA M.Different effects of calciumand lanthanum on the expression of phytochelatin synthase gene andcadmium absorption in Lactuca sativa[J].Plant Science,2005,168(2):309-318.
- [27]LI Hui,CONG Yu,CHANG You-hong.Cloning of a phytochelatinsynthase gene(LePCS1)from tomato seedlings and its expressioncharacteristics under heavy metal stress[J].Jiangsu Journal of Agri-cultural Sciences,2010,26(1):136-142.李慧,丛郁,常有宏.番茄植物络合素合酶基因全长cDNA的克隆及其表达特点[J].江苏农业学报,2010,26(1):136-142.