龙眼胚性愈伤组织Remorin家族成员的克隆及其在体胚发生过程中的表达分析
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摘要
以龙眼松散形胚性愈伤组织为材料,采用RT-PCR结合RACE法克隆Remorin家族5个成员的c DNA全长,Dl Remorin1全长2 127 bp,编码572个氨基酸;Dl Remorin2全长1 829 bp,编码444个氨基酸;Dl Remorin3全长1 937 bp,编码541个氨基酸;Dl Remorin4全长1 743 bp,编码466个氨基酸;Dl Remorin5全长1 043 bp,编码181个氨基酸。Dl Remorin1~5 DNA序列长度分别为:4 054、3 097、3 505、4 690和1 935 bp,所有内含子剪切位点均符合真核生物"GT-AG"规则。所有成员均是不稳定亲水性蛋白,都不具有信号肽,只有Dl Remorin1具有跨膜结构,都具有Remorin家族保守结构域,与其它植物的Remorin具有较高的同源性。系统进化树分析结果表明,Dl Remorin5为经典的Remorin蛋白,Dl Remorin1与Dl Remorin2属于长链Remorin。qRT-PCR结果显示,随着龙眼体胚的发育,Dl Remorin1的表达量呈类"N"状趋势,在心形胚时期和子叶形胚时期较高;Dl Remorin2在心形胚和子叶形胚时期迅速下降;Dl Remorin3在发育中后期迅速升高,并在子叶形胚时期最高;Dl Remorin4在球形胚和鱼雷形胚时期最低,而Dl Remorin5在不完全胚性紧实结构时期最低。说明Dl Remorin1~5在龙眼体胚发生过程的转录水平具有一定的组织特异性和时序性。ps RNA Target预测显示Dl Remorin还可能受到mi RNA家族的调控。
The RT-PCR combined with RACE method was used to clone the complete c DNA sequences of 5 members of Remorin family from embryogenic callus in Dimocarpus longan. The complete c DNA sequences of Dl Remorin1–5 were 2 127,1 829,1 937,1 743 and 1 043 bp,Dl Remorin1–5 encoding 572,444,541,466 and 181 amino acids,respectively. The DNA sequences of Dl Remorin1–5 were 4 054,3 097,3 505,4 690 and 1 935 bp,and all the splice sites of the introns contained were obeyed to the"GT-AG"rule. Dl Remorin1–5 belong to the instability and hydrophilous proteins,they all had the Remorin conserved domain,and had no signal peptide,only Dl Remorin1 had transmembrane structure. The sequences of both nucleotides and amino acids of the five members were high homologous with those of the known Remorin genes in other species. Anglicizing phylogenetic tree of Remorin in plants indicated that Dl Remorin5 belonged to the classical Remorin,Dl Remorin1 and Dl Remorin2 belonged to the long Remorin. The results of qRT-PCR indicated that Dl Remorin1 showed approximately an"N"curve,it expressed at the highest levels in the heart embryos and cotyledonary embryos cultures. Dl Remorin2 expressed at the lowest levels in heart embryos and cotyledonary embryos cultures. The expression of Dl Remorin3 increased rapidly at the middle and late developmental stages and peaked at the cotyledonary embryos cultures. Dl Remorin4 expressed at the lowest levels in globular embryos and topedo embryos cultures. Dl Remorin5 expressed at the lowest levels in incomplete compact pro-embryogenic cultures. Suggesting that Dl Remorin1–5 expressed with tissue specific and sequential characteristics during longan somatic embryogenesis. ps RNA Target projections show Dl Remorin may be regulated by mi RNAs.
The RT-PCR combined with RACE method was used to clone the complete c DNA sequences of 5 members of Remorin family from embryogenic callus in Dimocarpus longan. The complete c DNA sequences of Dl Remorin1–5 were 2 127,1 829,1 937,1 743 and 1 043 bp,Dl Remorin1–5 encoding 572,444,541,466 and 181 amino acids,respectively. The DNA sequences of Dl Remorin1–5 were 4 054,3 097,3 505,4 690 and 1 935 bp,and all the splice sites of the introns contained were obeyed to the"GT-AG"rule. Dl Remorin1–5 belong to the instability and hydrophilous proteins,they all had the Remorin conserved domain,and had no signal peptide,only Dl Remorin1 had transmembrane structure. The sequences of both nucleotides and amino acids of the five members were high homologous with those of the known Remorin genes in other species. Anglicizing phylogenetic tree of Remorin in plants indicated that Dl Remorin5 belonged to the classical Remorin,Dl Remorin1 and Dl Remorin2 belonged to the long Remorin. The results of qRT-PCR indicated that Dl Remorin1 showed approximately an"N"curve,it expressed at the highest levels in the heart embryos and cotyledonary embryos cultures. Dl Remorin2 expressed at the lowest levels in heart embryos and cotyledonary embryos cultures. The expression of Dl Remorin3 increased rapidly at the middle and late developmental stages and peaked at the cotyledonary embryos cultures. Dl Remorin4 expressed at the lowest levels in globular embryos and topedo embryos cultures. Dl Remorin5 expressed at the lowest levels in incomplete compact pro-embryogenic cultures. Suggesting that Dl Remorin1–5 expressed with tissue specific and sequential characteristics during longan somatic embryogenesis. ps RNA Target projections show Dl Remorin may be regulated by mi RNAs.
引文
Bariola P A,Retelska D,Stasiak A,Kammerer R A,Fleming A,Hijri M,Frank S,Farmer E E.2004.Remorins form a novel family of coiledcoil forming oligomeric and filamentous proteins associated with apical,vascular and embryonic tissues in plants.Plant Molecular Biology,55(4):579–594.
Chen Chun-ling,Lai Zhong-xiong.2002.Synchronization regulation of embryogenesis of embryogenic calli and their histological observations inlongan.Journal of Fujian Agriculture and Forestry University:Natural Science Edition,31(2):192–194.(in Chinese)陈春玲,赖钟雄.2002.龙眼胚性愈伤组织体胚发生同步化调控及组织细胞学观察.福建农林大学学报:自然科学版,31(2):192–194.
Chen K G,An Y Q.2006.Transcriptional responses to gibberellin and abscisic acid in barley aleurone.Journal of Integrative Plant Biology,48(5):591–612.
Chugh A,Khurana P.2002.Gene expression during somatic embryogenesis:Recent advances.Curr Sci,83:715–730.
Fu Zhen-fang,Yao Chun-chao,Zhang Chao-hong,Wang Yue-jin.2012.Cloning and functional analysis of Pbzs REMORIN gene related to Venturianashicola resistance in‘Zaosuli’pear.Acta Horticulturae Sinica,39(1):13–22.(in Chinese)付镇芳,姚春潮,张朝红,王跃进.2012.早酥梨抗黑星病相关基因Pbzs REMORIN的克隆及功能分析.园艺学报,39(1):13–22.
Fang Zhi-zhen,Lai Zhong-xiong.2009.Preliminary synchronization regulation at the middle developmental stage during longan somaticembryogenesis.Chinese Agricultural Science Bulletin,25(1):152–155.(in Chinese)方智振,赖钟雄.2009.龙眼体胚发生中期发育同步化的初步调控.中国农学通报,25(1):152–155.
Iakoucheva L M,Radivojac P,Brown C J,O'Connor T R,Sikes J G,Obradovic Z,Dunker A K.2004.The importance of intrinsic disorder for proteinphosphorylation.Nucleic Acids Res,32:1037–1049.
Iris K Jarsch,Thomas Ott.2011.Perspectives on remorin proteins,membrane rafts,and their role during plant-microbe interactions.MPMI,24(1):7–12.
Jacinto T,Farmer E E,Ryan C A.1993.Purification of potato leaf plasma membrane protein pp34,a protein phosphorylated in response tooligogalacturonide signals for defense and development.Plant Physiol,103(4):1393–1397.
Jiang Zheng,Wang Fang,He Xiang,Liu Da-wei,Chen Xuan-nan,Zhao Hong-qing,Huang Liu-yu,Yuan Jing.2009.Progress on protein/peptidephosphorylation.Lett Biotechnol,20(2):233–237.(in Chinese)姜铮,王芳,何湘,刘大伟,陈宣男,赵红庆,黄留玉,袁静.2009.蛋白质磷酸化修饰的研究进展.生物技术通讯,20(2):233–237.
Kreps J A,Wu Y,Chang H S,Zhu T,Wang X,Harper J F.2002.Transcriptome changes for Arabidopsis in response to salt,osmotic and cold stress.Plant Physiology,130(4):2129–2141.
Lai Cheng-chun,Lai Zhong-xiong,Fang Zhi-zhen,Lin Yu-ling,Jiang Shun-ri.2010.Cloning of mitochondrial F1-ATPase beta subunit gene fromembryogenic callus and its expression analysis by q-PCR during somatic embryogenesis in longan.Sci Agric Sin,43(16):3392–3401.(in Chinese)赖呈纯,赖钟雄,方智振,林玉玲,姜顺日.2010.龙眼胚性愈伤组织线粒体ATP合酶β亚基基因克隆及其在龙眼体胚发生过程中的表达分析.中国农业科学,43(16):3392–3401.
Lai Z X,Lin Y L.2013.Analysis of the global transcriptome of longan(Dimocarpus longan Lour.)embryogenic callus using Illumina paired-endsequencing.BMC Genomics,14:561.
Lefebvre B,Timmers T,Mbengue M,Moreau S,HervéC,Tóth K,Bittencourt-Silvestre J,Klaus D,Deslandes L,Godiard L,Murray J D,Udvardi M K,Raffaele S,Mongrand S,Cullimore J,Gamas P,Niebel A,Ott T.2010.A remorin protein interacts with symbiotic receptorsand regulates bacterial infection.Proceedings of the National Academy of Sciences of the United States of America,107(2):2343–2348.
Li Hui-hua,Lai Zhong-xiong,Lin Yu-ling,Su Ming-hua.2010.Cloning of ACO gene from embryogenic calli of longan(Dimocarpus longan Lour.)and its expression during longan somatic embryogenesis.Sci Agric Sin,43(18):3798–3808.(in Chinese)李惠华,赖钟雄,林玉玲,苏明华.2010.龙眼胚性愈伤组织ACC氧化酶基因的克隆及其在龙眼体胚发生过程中的表达分析.中国农业科学,43(18):3798–3808.
Lin Fang,Xu Shou-ling,Ni Wei-min,Chu Zhao-qing,Xu Zhi-hong,Xue Hong-wei.2003.Identification of ABA-responsive genes in rice shootsvia c DNA macroarray.Cell Res,13(1):59–68.
Lin Y L,Lai Z X.2010.Reference genes selection for q PCR analysis during somatic embryogenesis in longan tree.Plant Science,178(4):359–365.
Lin Y L,Lai Z X.2013.Comparative analysis reveals dynamic changes in mi RNAs and their targets and expression during somatic embryogenesis inlongan(Dimocarpus longan Lour.).PLo S ONE,8(4):e60337.
Malakshah S N,Rezaei M H,Heidary M,Salekdeh G H.2007.Proteomics reveals new salt responsive proteins associated with rice plasmamembrane.Bioscience,Biotechnology and Biochemistry,71(9):2144–2154.
Marín M,Ott T.2012.Phosphorylation of intrinsically disordered regions in Remorin proteins.Plant Sci,(3):86.
Marín M,Thallmair V,Ott T.2012.The intrinsically disordered N-terminal region of At REM1.3 remorin protein mediates protein-proteininteractions.Biol Chem,287:39982–39991.
Mongrand S,More L J,Laroche J,Claverol S,Carde J P,Hartmann M A,Bonneu M,Simon-Plas F,Lessire R,Bessoule J J.2004.Lipid raftsin higher plant cells:Purification and characterization of Triton X-100-insoluble micro domains from tobacco plasma membrane.Journal ofBiological Chemistry,279(35):36277–36286.
Perraki A,Cacas J L,Crowet J M,Lins L,Castroviejo M,Sylvie G R,Mongrand S,Raffaele S.2012.Plasma membrane localization of St REM1.3 Remorin is mediated by conformational changes in a novel C-terminal anchor and required for the restriction of PVX movement.PlantPhysiol,160:624–637.
Raffaele S,Bayer E,Mongrand S.2009.Upregulation of the plant protein remorin correlates with dehiscence and cell maturation:A link with thematuration of plasmodesmata?Plant Signal Behav,4(10):915–919.
Raffaele S,Mongrand S,Gamas P,Niebel A,Thomas Ott.2007.Genome-wide annotation of remorins,a plant-specific protein family:Evolutionaryand functional perspectives.Plant Physiol,145(3):593–600.
Raffaele S,Perraki A,Mongrand S.2013.The remorin C-terminal anchor was shaped by convergent evolution among membrane binding domains.Plant Signal Behav,8(3):e23207.
Reddy A R,Ramakrishna W,Sekhar A C,Ithal N,Babu P R,Bonaldo M F,Soares M B,Bennetzen J L.2002.Novel genes are enriched innormalized c DNA libraries from drought-stressed seedlings of rice(Oryza sativa L.subsp indica cv.Nagina 22).Genome,45(1):204–214.
Reymond P,Kunz B,Paul-pletzer K,Grimm R,Eckerskorn C,Farmer E E.1996.Cloning of a c DNA encoding a plasma membrane-associated,uronide binding phosphor-protein with physical properties similar to viral movement proteins.The Plant Cell Online,8(12):2265–2276.
Vandesompele J,De Preter K,Pattyn F,Poppe B,Nadine Van Roy,Paepe A D,Speleman F.2002.Accurate normalization of real time quantitativeRT-PCR data by geometric averaging of multiple internal control genes.Genome Biol,3(7):0034.1–0034.11.
Vibha G,Checker,Paramjit Khurana.2013.Molecular and functional characterization of mulberry EST encoding remorin(Mi REM)involved inabiotic stress.Plant Cell Rep,32:1729–1741.
Wang Da-hai.2008.Cloning and funtional analysis of wood properties candidate genes in Popilus deltoides[Ph.D.Dissertation].Beijing:TheChinese Academy of Forestry Sciences.(in Chinese)王大海.2008.美洲黑杨材性侯选基因的克隆和功能分析[博士论文].北京:中国林业科学研究院.
Wang Feng-hua,Lai Zhong-xiong,Zheng Jin-gui,LüLiu-xin.2003.Synchronization control of somatic embryogenesis from embryogenic calli andextraction of DNA and RNA in longan(Dimocarpus longan Lour.).Chinese Journal of Tropical Crops,24(3):33–37.(in Chinese)王凤华,赖钟雄,郑金贵,吕柳新.2003.龙眼胚性愈伤组织体胚发生的同步化调控及其DNA与RNA的提取方法.热带作物学报,24(3):33–37.
Wolf E,Kim P S,Berger B.1997.Multi Coil:A program for predicting two-and three-stranded coiled coils.Protein Science,6(6):1179–1189.
Chen Chun-ling,Lai Zhong-xiong.2002.Synchronization regulation of embryogenesis of embryogenic calli and their histological observations inlongan.Journal of Fujian Agriculture and Forestry University:Natural Science Edition,31(2):192–194.(in Chinese)陈春玲,赖钟雄.2002.龙眼胚性愈伤组织体胚发生同步化调控及组织细胞学观察.福建农林大学学报:自然科学版,31(2):192–194.
Chen K G,An Y Q.2006.Transcriptional responses to gibberellin and abscisic acid in barley aleurone.Journal of Integrative Plant Biology,48(5):591–612.
Chugh A,Khurana P.2002.Gene expression during somatic embryogenesis:Recent advances.Curr Sci,83:715–730.
Fu Zhen-fang,Yao Chun-chao,Zhang Chao-hong,Wang Yue-jin.2012.Cloning and functional analysis of Pbzs REMORIN gene related to Venturianashicola resistance in‘Zaosuli’pear.Acta Horticulturae Sinica,39(1):13–22.(in Chinese)付镇芳,姚春潮,张朝红,王跃进.2012.早酥梨抗黑星病相关基因Pbzs REMORIN的克隆及功能分析.园艺学报,39(1):13–22.
Fang Zhi-zhen,Lai Zhong-xiong.2009.Preliminary synchronization regulation at the middle developmental stage during longan somaticembryogenesis.Chinese Agricultural Science Bulletin,25(1):152–155.(in Chinese)方智振,赖钟雄.2009.龙眼体胚发生中期发育同步化的初步调控.中国农学通报,25(1):152–155.
Iakoucheva L M,Radivojac P,Brown C J,O'Connor T R,Sikes J G,Obradovic Z,Dunker A K.2004.The importance of intrinsic disorder for proteinphosphorylation.Nucleic Acids Res,32:1037–1049.
Iris K Jarsch,Thomas Ott.2011.Perspectives on remorin proteins,membrane rafts,and their role during plant-microbe interactions.MPMI,24(1):7–12.
Jacinto T,Farmer E E,Ryan C A.1993.Purification of potato leaf plasma membrane protein pp34,a protein phosphorylated in response tooligogalacturonide signals for defense and development.Plant Physiol,103(4):1393–1397.
Jiang Zheng,Wang Fang,He Xiang,Liu Da-wei,Chen Xuan-nan,Zhao Hong-qing,Huang Liu-yu,Yuan Jing.2009.Progress on protein/peptidephosphorylation.Lett Biotechnol,20(2):233–237.(in Chinese)姜铮,王芳,何湘,刘大伟,陈宣男,赵红庆,黄留玉,袁静.2009.蛋白质磷酸化修饰的研究进展.生物技术通讯,20(2):233–237.
Kreps J A,Wu Y,Chang H S,Zhu T,Wang X,Harper J F.2002.Transcriptome changes for Arabidopsis in response to salt,osmotic and cold stress.Plant Physiology,130(4):2129–2141.
Lai Cheng-chun,Lai Zhong-xiong,Fang Zhi-zhen,Lin Yu-ling,Jiang Shun-ri.2010.Cloning of mitochondrial F1-ATPase beta subunit gene fromembryogenic callus and its expression analysis by q-PCR during somatic embryogenesis in longan.Sci Agric Sin,43(16):3392–3401.(in Chinese)赖呈纯,赖钟雄,方智振,林玉玲,姜顺日.2010.龙眼胚性愈伤组织线粒体ATP合酶β亚基基因克隆及其在龙眼体胚发生过程中的表达分析.中国农业科学,43(16):3392–3401.
Lai Z X,Lin Y L.2013.Analysis of the global transcriptome of longan(Dimocarpus longan Lour.)embryogenic callus using Illumina paired-endsequencing.BMC Genomics,14:561.
Lefebvre B,Timmers T,Mbengue M,Moreau S,HervéC,Tóth K,Bittencourt-Silvestre J,Klaus D,Deslandes L,Godiard L,Murray J D,Udvardi M K,Raffaele S,Mongrand S,Cullimore J,Gamas P,Niebel A,Ott T.2010.A remorin protein interacts with symbiotic receptorsand regulates bacterial infection.Proceedings of the National Academy of Sciences of the United States of America,107(2):2343–2348.
Li Hui-hua,Lai Zhong-xiong,Lin Yu-ling,Su Ming-hua.2010.Cloning of ACO gene from embryogenic calli of longan(Dimocarpus longan Lour.)and its expression during longan somatic embryogenesis.Sci Agric Sin,43(18):3798–3808.(in Chinese)李惠华,赖钟雄,林玉玲,苏明华.2010.龙眼胚性愈伤组织ACC氧化酶基因的克隆及其在龙眼体胚发生过程中的表达分析.中国农业科学,43(18):3798–3808.
Lin Fang,Xu Shou-ling,Ni Wei-min,Chu Zhao-qing,Xu Zhi-hong,Xue Hong-wei.2003.Identification of ABA-responsive genes in rice shootsvia c DNA macroarray.Cell Res,13(1):59–68.
Lin Y L,Lai Z X.2010.Reference genes selection for q PCR analysis during somatic embryogenesis in longan tree.Plant Science,178(4):359–365.
Lin Y L,Lai Z X.2013.Comparative analysis reveals dynamic changes in mi RNAs and their targets and expression during somatic embryogenesis inlongan(Dimocarpus longan Lour.).PLo S ONE,8(4):e60337.
Malakshah S N,Rezaei M H,Heidary M,Salekdeh G H.2007.Proteomics reveals new salt responsive proteins associated with rice plasmamembrane.Bioscience,Biotechnology and Biochemistry,71(9):2144–2154.
Marín M,Ott T.2012.Phosphorylation of intrinsically disordered regions in Remorin proteins.Plant Sci,(3):86.
Marín M,Thallmair V,Ott T.2012.The intrinsically disordered N-terminal region of At REM1.3 remorin protein mediates protein-proteininteractions.Biol Chem,287:39982–39991.
Mongrand S,More L J,Laroche J,Claverol S,Carde J P,Hartmann M A,Bonneu M,Simon-Plas F,Lessire R,Bessoule J J.2004.Lipid raftsin higher plant cells:Purification and characterization of Triton X-100-insoluble micro domains from tobacco plasma membrane.Journal ofBiological Chemistry,279(35):36277–36286.
Perraki A,Cacas J L,Crowet J M,Lins L,Castroviejo M,Sylvie G R,Mongrand S,Raffaele S.2012.Plasma membrane localization of St REM1.3 Remorin is mediated by conformational changes in a novel C-terminal anchor and required for the restriction of PVX movement.PlantPhysiol,160:624–637.
Raffaele S,Bayer E,Mongrand S.2009.Upregulation of the plant protein remorin correlates with dehiscence and cell maturation:A link with thematuration of plasmodesmata?Plant Signal Behav,4(10):915–919.
Raffaele S,Mongrand S,Gamas P,Niebel A,Thomas Ott.2007.Genome-wide annotation of remorins,a plant-specific protein family:Evolutionaryand functional perspectives.Plant Physiol,145(3):593–600.
Raffaele S,Perraki A,Mongrand S.2013.The remorin C-terminal anchor was shaped by convergent evolution among membrane binding domains.Plant Signal Behav,8(3):e23207.
Reddy A R,Ramakrishna W,Sekhar A C,Ithal N,Babu P R,Bonaldo M F,Soares M B,Bennetzen J L.2002.Novel genes are enriched innormalized c DNA libraries from drought-stressed seedlings of rice(Oryza sativa L.subsp indica cv.Nagina 22).Genome,45(1):204–214.
Reymond P,Kunz B,Paul-pletzer K,Grimm R,Eckerskorn C,Farmer E E.1996.Cloning of a c DNA encoding a plasma membrane-associated,uronide binding phosphor-protein with physical properties similar to viral movement proteins.The Plant Cell Online,8(12):2265–2276.
Vandesompele J,De Preter K,Pattyn F,Poppe B,Nadine Van Roy,Paepe A D,Speleman F.2002.Accurate normalization of real time quantitativeRT-PCR data by geometric averaging of multiple internal control genes.Genome Biol,3(7):0034.1–0034.11.
Vibha G,Checker,Paramjit Khurana.2013.Molecular and functional characterization of mulberry EST encoding remorin(Mi REM)involved inabiotic stress.Plant Cell Rep,32:1729–1741.
Wang Da-hai.2008.Cloning and funtional analysis of wood properties candidate genes in Popilus deltoides[Ph.D.Dissertation].Beijing:TheChinese Academy of Forestry Sciences.(in Chinese)王大海.2008.美洲黑杨材性侯选基因的克隆和功能分析[博士论文].北京:中国林业科学研究院.
Wang Feng-hua,Lai Zhong-xiong,Zheng Jin-gui,LüLiu-xin.2003.Synchronization control of somatic embryogenesis from embryogenic calli andextraction of DNA and RNA in longan(Dimocarpus longan Lour.).Chinese Journal of Tropical Crops,24(3):33–37.(in Chinese)王凤华,赖钟雄,郑金贵,吕柳新.2003.龙眼胚性愈伤组织体胚发生的同步化调控及其DNA与RNA的提取方法.热带作物学报,24(3):33–37.
Wolf E,Kim P S,Berger B.1997.Multi Coil:A program for predicting two-and three-stranded coiled coils.Protein Science,6(6):1179–1189.