割手密2C型蛋白磷酸酶ScPP2C基因的克隆与表达分析
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摘要
割手密由于具有抗病、抗虫、抗逆等潜在的有利基因,历来被用于甘蔗的抗性遗传改良。在割手密中克隆编码2C型蛋白磷酸酶(protein phosphatase 2C, PP2C)的基因,并分析其在干旱胁迫下的差异表达。本研究利用RT-PCR方法从割手密叶片中克隆得到PP2C基因的CDS全长序列,利用生物信息学在线软件对PP2C蛋白的理化性质、蛋白结构进行预测分析,并采用实时荧光定量的方法分析该基因在不同干旱处理下的差异表达。结果显示从割手密中克隆到一个全长951 bp编码316个氨基酸的2C型蛋白磷酸酶基因,命名为ScPP2C,GenBank登录号为MG322120。荧光定量分析表明,随着干旱胁迫时间的延长,其表达量呈先上调后下调的表达模式,说明该基因是干旱胁迫诱导型基因。本研究通过挖掘甘蔗野生种割手密中的抗旱新基因ScPP2C,为甘蔗野生种质资源开发利用、转基因抗旱甘蔗新品种的选育提供了参考依据。
S. spontaneum L. has always been an important germplasm resource for sugarcane genetic improvement due to its potential beneficial genes such as disease resistance, insect resistance and stress resistance, etc. In this study, a PP2 C gene designated as ScPP2 C(GenBank Accession: MG322120), which including an ORF of 951 bp coding sequence and coding 316 amino acids, was amplified from Saccharum spontaneum L., Realtime-PCR analysis revealed that the expression level of ScPP2 C was up-down-regulated under different drought conditions,indicating that the gene is a drought stress-inducible gene. In this study, a novel gene of drought-resistance was found in S. spontaneum L., which was to develope the excellent resources of wild species, as well as to explore the drought resistance of these resources, so as to provide the basis for the selection of the parents of the fine and drought resistance in the future.
S. spontaneum L. has always been an important germplasm resource for sugarcane genetic improvement due to its potential beneficial genes such as disease resistance, insect resistance and stress resistance, etc. In this study, a PP2 C gene designated as ScPP2 C(GenBank Accession: MG322120), which including an ORF of 951 bp coding sequence and coding 316 amino acids, was amplified from Saccharum spontaneum L., Realtime-PCR analysis revealed that the expression level of ScPP2 C was up-down-regulated under different drought conditions,indicating that the gene is a drought stress-inducible gene. In this study, a novel gene of drought-resistance was found in S. spontaneum L., which was to develope the excellent resources of wild species, as well as to explore the drought resistance of these resources, so as to provide the basis for the selection of the parents of the fine and drought resistance in the future.
引文
Cheng Z.Y.,2015,Research es on sugarcane drought-tolerant gene and its clone and analysis based on transcriptome sequencing,Dissertation for Ph.D.,Yunnan Agriculture Uni versity,Supervisor:Li F.S.,pp.75-79(程志远,2015,基于转录组测序的甘蔗抗旱基因研究及其克隆研究及其克隆与分析博士学位论文,云南农业大学,导师:李富生,pp.75-79)
Guo P.,Zhang S.G.,Xing X.,and Jiang J.,2015,Cloning and function analysis of PdPP2C from Populus deltoids,Beijing Linye Daxue Xuebao(Journal of Beijing Forestry University),37(2):100-106(郭鹏,张士刚,邢鑫,姜健,2015,欧美杨PdPP2C基因的克隆与功能分析,北京林业大学学报,37(2):100-106)
Hu X.L.,and Li D.Q.,2007,Protein phosphatase 2C in plants and its functions of signal transduction,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),43(3):407-412(胡晓丽,李德全,2007,植物蛋白磷酸酶2C(PP2C)及其在信号转导中的作用,植物生理学通讯,43(3):407-412)
Hu X.W.,Yao Y.L.,Xing S.L.,Xu L.,and Liu Y.,2015,Isolation and comparative analysis of classⅢperoxidases gene DNAon Saccharum officinarum,Saccharum spontaneum and Sac-charum arundinaceum,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(10):2340-2347(胡小文,姚艳丽,邢淑莲,徐磊,刘洋,2015,甘蔗、斑茅及割手密Ⅲ型过氧化物酶基因克隆及比较分析,分子植物育种,13(10):2340-2347)
Huang Z.X.,Zhou F.,Wang Q.N.,Jin Y.F.,Fu C.,Hu H.X.,Zhang C.M.,Chang H.L.,Ji J.L.,Wu Q.W.,Qi Y.W.,and Liu S.M.,2012,Genetic diversity assessment of Saccharum spontanuem L.native of domestic and overseas with phenotype agronomic traits,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),13(5):825-829(黄忠兴,周峰,王勤南,金玉峰,符成,胡后祥,张垂明,常海龙,吉家乐,吴其卫,齐永文,刘少谋,2012,国内外割手密资源农艺性状表型遗传多样性分析,植物遗传资源学报,13(5):825-829)
Kerk D.,Bulgrien J.,Smith D.W.,Barsam B.,Veretnik S.,and Gribskov M.,2002,The complement of protein phosphatase catalytic subunits encoded in the genome of Arabidopsis,Plant Physiol.,129(2):908-925
Li Q.W.,and Deng H.H.,2011,Major problems and strategy in sugarcane breeding and variety extension in China,Ganzhe Tangye(Sugarcane and Canesugar),(4):70-76(李奇伟,邓海华,2011,当前我国甘蔗品种选育与推广中存在的突出问题及对策,甘蔗糖业,(4):70-76)
Luan S.,2003,Protein phosphatases in plants,Annu.Rev.Plant Biol.,54(1):63-92
Mihindukulasuriya K.A.,Zhou G.,Qin J.,and Tan T.H.,2004,Protein phosphatase 4 interacts with and down-regulates insulin receptor substrate 4 following tumor necrosis factor-alpha stimulation,J.Biol.Chem.,279(45):46588-46594
Millward T.A.,Zolnierowicz S.,and Hemmings B.A.,1999,Regulation of protein kinase cascades by protein phosphatase 2a,Trends Biochem.Sci.,24(5):186-191
Pan Y.,Zhao N.,Liu K.,Zhang X.J.,and Wang R.G.,2017,Cloning and expression analysis of Ci PP2C8-like gene from Caragana intermedia,Jiyinzuxue Yu Yingyong Shengwuxe(Genomics and Applied Biology),36(9):3848-3854(潘雨,赵娜,刘坤,张秀娟,王瑞刚,2017,中间锦鸡儿Ci PP2C8-like基因的克隆及表达分析,基因组学与应用生物学,36(9):3848-3854)
Saijo Y.,Kinoshita N.,Ishiyama K.,Hata S.,Kyozuka J.Hayakawa T.,Nakamura T.,Shimamoto K.,Yamaya T.,and Katsura L.,2001,A Ca2-dependent protein kinase that endows rice plants with cold-and salt-stress tolerance functions in vascular bundles,Plant Cell Physiol.,42(11)1228-1233
Schwarz S.,Hufnagel B.,Dworak M.,Klumpp S.,and Krieglstein J.,2006,Protein phosphatase type 2Calpha and 2Cbeta are involved in fatty acid-induced apoptosis of neuronal and endothelial cells,Apoptosis,11(7):1111-1119
Sheng M.M.,Cheng Z.Y.,Chen S.Y.,Wang X.H.,He L.L.,and Li F.S.,2017,Cloning and expression analysis on the Sc ALDH gene of Saccharum spontaneum,Guangxi Zhiwu(Guihaia),37(3):380-387(申明明,程志远,陈疏影,王先宏,何丽莲,李富生,2017,割手密醛脱氢酶Sc ALDH基因的克隆与表达分析,广西植物,37(3):380-387)
Xu W.H.,and Yang Q.H.,2005,Drought resistance of S.spontaneum L.clones,Yaredai Nongye Yanjiu(Subtropical Agriculture Research),1(1):22-26(许文花,杨清辉,2005,甘蔗割手密无性系抗旱性鉴定,亚热带农业研究,1(1):22-26)
Zhang J.H.,and Tao N.G.,2015,Research progress of plant PP2C-type protein phosphatase in ABA signal transduction and adversity stress regulation mechanism,Guangxi Zhiwu(Guihaia),(6):935-941(张继红,陶能国,2015,植物PP2C蛋白磷酸酶ABA信号转导及逆境胁迫调控机制研究进展,广西植物,(6):935-941)
Guo P.,Zhang S.G.,Xing X.,and Jiang J.,2015,Cloning and function analysis of PdPP2C from Populus deltoids,Beijing Linye Daxue Xuebao(Journal of Beijing Forestry University),37(2):100-106(郭鹏,张士刚,邢鑫,姜健,2015,欧美杨PdPP2C基因的克隆与功能分析,北京林业大学学报,37(2):100-106)
Hu X.L.,and Li D.Q.,2007,Protein phosphatase 2C in plants and its functions of signal transduction,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),43(3):407-412(胡晓丽,李德全,2007,植物蛋白磷酸酶2C(PP2C)及其在信号转导中的作用,植物生理学通讯,43(3):407-412)
Hu X.W.,Yao Y.L.,Xing S.L.,Xu L.,and Liu Y.,2015,Isolation and comparative analysis of classⅢperoxidases gene DNAon Saccharum officinarum,Saccharum spontaneum and Sac-charum arundinaceum,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(10):2340-2347(胡小文,姚艳丽,邢淑莲,徐磊,刘洋,2015,甘蔗、斑茅及割手密Ⅲ型过氧化物酶基因克隆及比较分析,分子植物育种,13(10):2340-2347)
Huang Z.X.,Zhou F.,Wang Q.N.,Jin Y.F.,Fu C.,Hu H.X.,Zhang C.M.,Chang H.L.,Ji J.L.,Wu Q.W.,Qi Y.W.,and Liu S.M.,2012,Genetic diversity assessment of Saccharum spontanuem L.native of domestic and overseas with phenotype agronomic traits,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),13(5):825-829(黄忠兴,周峰,王勤南,金玉峰,符成,胡后祥,张垂明,常海龙,吉家乐,吴其卫,齐永文,刘少谋,2012,国内外割手密资源农艺性状表型遗传多样性分析,植物遗传资源学报,13(5):825-829)
Kerk D.,Bulgrien J.,Smith D.W.,Barsam B.,Veretnik S.,and Gribskov M.,2002,The complement of protein phosphatase catalytic subunits encoded in the genome of Arabidopsis,Plant Physiol.,129(2):908-925
Li Q.W.,and Deng H.H.,2011,Major problems and strategy in sugarcane breeding and variety extension in China,Ganzhe Tangye(Sugarcane and Canesugar),(4):70-76(李奇伟,邓海华,2011,当前我国甘蔗品种选育与推广中存在的突出问题及对策,甘蔗糖业,(4):70-76)
Luan S.,2003,Protein phosphatases in plants,Annu.Rev.Plant Biol.,54(1):63-92
Mihindukulasuriya K.A.,Zhou G.,Qin J.,and Tan T.H.,2004,Protein phosphatase 4 interacts with and down-regulates insulin receptor substrate 4 following tumor necrosis factor-alpha stimulation,J.Biol.Chem.,279(45):46588-46594
Millward T.A.,Zolnierowicz S.,and Hemmings B.A.,1999,Regulation of protein kinase cascades by protein phosphatase 2a,Trends Biochem.Sci.,24(5):186-191
Pan Y.,Zhao N.,Liu K.,Zhang X.J.,and Wang R.G.,2017,Cloning and expression analysis of Ci PP2C8-like gene from Caragana intermedia,Jiyinzuxue Yu Yingyong Shengwuxe(Genomics and Applied Biology),36(9):3848-3854(潘雨,赵娜,刘坤,张秀娟,王瑞刚,2017,中间锦鸡儿Ci PP2C8-like基因的克隆及表达分析,基因组学与应用生物学,36(9):3848-3854)
Saijo Y.,Kinoshita N.,Ishiyama K.,Hata S.,Kyozuka J.Hayakawa T.,Nakamura T.,Shimamoto K.,Yamaya T.,and Katsura L.,2001,A Ca2-dependent protein kinase that endows rice plants with cold-and salt-stress tolerance functions in vascular bundles,Plant Cell Physiol.,42(11)1228-1233
Schwarz S.,Hufnagel B.,Dworak M.,Klumpp S.,and Krieglstein J.,2006,Protein phosphatase type 2Calpha and 2Cbeta are involved in fatty acid-induced apoptosis of neuronal and endothelial cells,Apoptosis,11(7):1111-1119
Sheng M.M.,Cheng Z.Y.,Chen S.Y.,Wang X.H.,He L.L.,and Li F.S.,2017,Cloning and expression analysis on the Sc ALDH gene of Saccharum spontaneum,Guangxi Zhiwu(Guihaia),37(3):380-387(申明明,程志远,陈疏影,王先宏,何丽莲,李富生,2017,割手密醛脱氢酶Sc ALDH基因的克隆与表达分析,广西植物,37(3):380-387)
Xu W.H.,and Yang Q.H.,2005,Drought resistance of S.spontaneum L.clones,Yaredai Nongye Yanjiu(Subtropical Agriculture Research),1(1):22-26(许文花,杨清辉,2005,甘蔗割手密无性系抗旱性鉴定,亚热带农业研究,1(1):22-26)
Zhang J.H.,and Tao N.G.,2015,Research progress of plant PP2C-type protein phosphatase in ABA signal transduction and adversity stress regulation mechanism,Guangxi Zhiwu(Guihaia),(6):935-941(张继红,陶能国,2015,植物PP2C蛋白磷酸酶ABA信号转导及逆境胁迫调控机制研究进展,广西植物,(6):935-941)