‘西伯利亚’百合钙依赖型蛋白激酶基因的克隆及表达分析
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摘要
钙依赖型蛋白激酶(calcium-dependent protein kinase, CDPK)是一类Ser/Thr型蛋白激酶,在植物体内钙信号的传递中起着重要的作用。本研究以东方百合‘西伯利亚’(Oriental Lilium'siberia')为材料,通过RACE方法克隆了LiCDPK基因,并进行了时空表达分析。结果表明,百合LiCDPK基因全长2 231 bp,开放阅读框长1 551 bp,编码516个氨基酸,其蛋白序列与其他物种的CDPK蛋白相似性在80%以上。LiCDPK基因在不同花期和9个不同组织中的表达存在差异。不同花期的qRT-PCR表明,花蕾期LiCDPK基因的表达量最高;盛花期的不同组织器官中,花药LiCDPK基因的表达量最高。本研究为进一步揭示LiCDPK基因在百合花香释放过程中的信号作用提供了帮助。
Calcium-dependent protein kinase(CDPK) is a type of Ser/Thr protein kinases, which plays an important role in signaling transduction of calcium in plants. In this study, the LiCDPK gene was cloned by RACE technique using oriental Lilium 'Siberia' as materials, and the temporal and spatial expression were analysed. The results showed that the whole length of LiCDPK was 2 231 bp, ORF was 1 551 bp, encoding 516 amino acids, and its protein sequence was more than 80% similar to that of other species. The expression of LiCDPK presented different levels in different flowering stages and nine different tissues. The qRT-PCR of different flowering stages revealed that the expression level of LiCDPK was the highest at the bud stage. Among the different tissues of full bloom stage, the highest expression level of LiCDPK was found in anther. This study could be helpful for further studies on the signal function of LiCDPK in the process of fragrance release of Lilium.
Calcium-dependent protein kinase(CDPK) is a type of Ser/Thr protein kinases, which plays an important role in signaling transduction of calcium in plants. In this study, the LiCDPK gene was cloned by RACE technique using oriental Lilium 'Siberia' as materials, and the temporal and spatial expression were analysed. The results showed that the whole length of LiCDPK was 2 231 bp, ORF was 1 551 bp, encoding 516 amino acids, and its protein sequence was more than 80% similar to that of other species. The expression of LiCDPK presented different levels in different flowering stages and nine different tissues. The qRT-PCR of different flowering stages revealed that the expression level of LiCDPK was the highest at the bud stage. Among the different tissues of full bloom stage, the highest expression level of LiCDPK was found in anther. This study could be helpful for further studies on the signal function of LiCDPK in the process of fragrance release of Lilium.
引文
Batisti O.,and Kudla J.,2012,Analysis of calcium signaling pathways in plants,Biochim.Biophys.Acta,1820(8):1283-1293
Cai H.,Zhu Y.M.,Li J.,Bai X.,and Li Y.,2006,Clone the conserved sequence of CDPK from Glycine soja and analysis the sequence,Dongbei Nongye Daxue Xuebao(Journal of Northeast Agricultural University),37(2):159-164(才华,朱延明,李杰,柏锡,李勇,2006,野生大豆CDPK基因保守区的克隆及序列分析,东北农业大学学报,37(2):159-164)
Cheng S.H.,Willmann M.R.,Chen H.C.,and Sheen J.,2002,Calcium signaling through protein kinases.The Arabidopsis calcium-dependent protein kinase gene family,Plant Physiol.,129(2):469-485
Feng J.Y.,2012,Isolation,expression and functional analysis of a calcium-dependent protein kinase gene FvCDPK1 from strawberry(Fragaria vesca),Dissertation for Ph.D.,Northwest A&F University,Supervisor:Zou Z.R.,pp.11-21(冯嘉玥,2012,草莓钙依赖蛋白激酶基因FvCDPK1克隆,表达及功能分析,博士学位论文,西北农林科技大学,导师:邹志荣,pp.11-21)
Harmon A.C.,Gribskov M.,Gubrium E.,and Harper J.F.,2001,The CDPK superfamily of protein kinases,New Phytol.,151(1):5859-5867
Hetherington A.,and Trewavas A.,1982,Calcium-dependent protein kinase in pea shoot membranes,FEBS Letters,145(1):67-71
Ray S.,Agarwal P.,Arora R.,Kapoor S.,and Tyagi A.K.,2007,Expression analysis of calcium-dependent protein kinase gene famliy during reproductive development and abiotic stress conditions in rice(Oryza sativa L.ssp.indica),Mol.Genet.Genomics,278(5):493-505
Sheng K.,Gao Y.H.,Si J.P.,and Zhu Y.Q.,2016,Cloning and expression analysis of DoCDPK genes in Dendrobium officinale,Yuanyi Xuebao(Acta Horticulturae Sinica),43(12):2412-2422(盛况,高燕会,斯金平,朱玉球,2016,铁皮石斛DoC-DPK基因的克隆与表达分析,园艺学报,43(12):2412-2422)
Tai S.S.,Liu G.S.,Sun Y.H.,and Chen J.,2009,Cloning and expression of calcium-dependent protein kinase(CDPK)gene family in Nicotiana tabacum,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),42(10):3600-3608(太帅帅,刘贯山,孙玉合,陈珈,2009,普通烟草CDPK基因家族的克隆及表达分析,中国农业科学,42(10):3600-3608)
Wei S.Y.,Zhang Y.Y.,Zhao X.D.,Liu X.D.,Luo Q.C.,Wei Q.H.,Chen P.,He G.Y.,and Yang G.X.,2013,Construction and genetic transformation of RNA interference vectors for OsCPK9 gene in rice,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),32(5):581-588(韦淑亚,张莹莹,赵旭东,刘小东,罗青晨,卫秋慧,陈鹏,何光源,杨广笑,2013,水稻钙依赖型蛋白激酶OsCPK9基因RNAi表达载体的构建及遗传转化,基因组学与应用生物学,32(5):581-588)
Wei S.Y.,Zhao X.D.,Yang G.X.,and He G.Y.,2017,Clone and expression of calcium-dependent protein kinase BdCDPK14gene from Brachypodium distachyon,Nanfang Nongye Xuebao(Journal of Southern Agriculture),48(5):761-767(韦淑亚,赵旭东,杨广笑,何光源,2017,二穗短柄草钙依赖型蛋白激酶基因BdCDPK14克隆及表达分析,南方农业学报,48(5):761-767)
Zhang Q.P.,Wen L.,Wang F.,Liao Z.Q.,Li H.,Liu R.Y.,and Guan C.Y.,2014,Molecular cloning and expression analysis of calcium-dependent protein kinase BnCDPK1 in Brassica napus,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),15(6):1320-1326(张秋平,文李,王峰,廖志强,李慧,刘睿洋,官春云,2014,油菜钙依赖蛋白激酶BnCDPK1的克隆和表达分析,植物遗传资源学报,15(6):1320-1326)
Zheng Z.Z.,Shen J.Q.,Pan W.H.,and Pan J.W.,2013,Calcium sensors and their stress signaling pathways in plants,Yichuan(Hereditas),35(7):875-884(郑仲仲,沈金秋,潘伟槐,潘建伟,2013,植物钙感受器及其介导的逆境信号途径,遗传,35(7):875-884)
Cai H.,Zhu Y.M.,Li J.,Bai X.,and Li Y.,2006,Clone the conserved sequence of CDPK from Glycine soja and analysis the sequence,Dongbei Nongye Daxue Xuebao(Journal of Northeast Agricultural University),37(2):159-164(才华,朱延明,李杰,柏锡,李勇,2006,野生大豆CDPK基因保守区的克隆及序列分析,东北农业大学学报,37(2):159-164)
Cheng S.H.,Willmann M.R.,Chen H.C.,and Sheen J.,2002,Calcium signaling through protein kinases.The Arabidopsis calcium-dependent protein kinase gene family,Plant Physiol.,129(2):469-485
Feng J.Y.,2012,Isolation,expression and functional analysis of a calcium-dependent protein kinase gene FvCDPK1 from strawberry(Fragaria vesca),Dissertation for Ph.D.,Northwest A&F University,Supervisor:Zou Z.R.,pp.11-21(冯嘉玥,2012,草莓钙依赖蛋白激酶基因FvCDPK1克隆,表达及功能分析,博士学位论文,西北农林科技大学,导师:邹志荣,pp.11-21)
Harmon A.C.,Gribskov M.,Gubrium E.,and Harper J.F.,2001,The CDPK superfamily of protein kinases,New Phytol.,151(1):5859-5867
Hetherington A.,and Trewavas A.,1982,Calcium-dependent protein kinase in pea shoot membranes,FEBS Letters,145(1):67-71
Ray S.,Agarwal P.,Arora R.,Kapoor S.,and Tyagi A.K.,2007,Expression analysis of calcium-dependent protein kinase gene famliy during reproductive development and abiotic stress conditions in rice(Oryza sativa L.ssp.indica),Mol.Genet.Genomics,278(5):493-505
Sheng K.,Gao Y.H.,Si J.P.,and Zhu Y.Q.,2016,Cloning and expression analysis of DoCDPK genes in Dendrobium officinale,Yuanyi Xuebao(Acta Horticulturae Sinica),43(12):2412-2422(盛况,高燕会,斯金平,朱玉球,2016,铁皮石斛DoC-DPK基因的克隆与表达分析,园艺学报,43(12):2412-2422)
Tai S.S.,Liu G.S.,Sun Y.H.,and Chen J.,2009,Cloning and expression of calcium-dependent protein kinase(CDPK)gene family in Nicotiana tabacum,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),42(10):3600-3608(太帅帅,刘贯山,孙玉合,陈珈,2009,普通烟草CDPK基因家族的克隆及表达分析,中国农业科学,42(10):3600-3608)
Wei S.Y.,Zhang Y.Y.,Zhao X.D.,Liu X.D.,Luo Q.C.,Wei Q.H.,Chen P.,He G.Y.,and Yang G.X.,2013,Construction and genetic transformation of RNA interference vectors for OsCPK9 gene in rice,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),32(5):581-588(韦淑亚,张莹莹,赵旭东,刘小东,罗青晨,卫秋慧,陈鹏,何光源,杨广笑,2013,水稻钙依赖型蛋白激酶OsCPK9基因RNAi表达载体的构建及遗传转化,基因组学与应用生物学,32(5):581-588)
Wei S.Y.,Zhao X.D.,Yang G.X.,and He G.Y.,2017,Clone and expression of calcium-dependent protein kinase BdCDPK14gene from Brachypodium distachyon,Nanfang Nongye Xuebao(Journal of Southern Agriculture),48(5):761-767(韦淑亚,赵旭东,杨广笑,何光源,2017,二穗短柄草钙依赖型蛋白激酶基因BdCDPK14克隆及表达分析,南方农业学报,48(5):761-767)
Zhang Q.P.,Wen L.,Wang F.,Liao Z.Q.,Li H.,Liu R.Y.,and Guan C.Y.,2014,Molecular cloning and expression analysis of calcium-dependent protein kinase BnCDPK1 in Brassica napus,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),15(6):1320-1326(张秋平,文李,王峰,廖志强,李慧,刘睿洋,官春云,2014,油菜钙依赖蛋白激酶BnCDPK1的克隆和表达分析,植物遗传资源学报,15(6):1320-1326)
Zheng Z.Z.,Shen J.Q.,Pan W.H.,and Pan J.W.,2013,Calcium sensors and their stress signaling pathways in plants,Yichuan(Hereditas),35(7):875-884(郑仲仲,沈金秋,潘伟槐,潘建伟,2013,植物钙感受器及其介导的逆境信号途径,遗传,35(7):875-884)