CDPK4亚细胞定位、超表达和RNAi载体构建与辣椒转化
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摘要
CDPKs (calcium-dependent protein kinases)是在植物中首先发现的一种钙依赖型蛋白激酶,属于丝氨酸/苏氨酸激酶,可以不经钙调素的作用而直接被钙信号激活,在植物生长发育和适应逆境过程中起重要的调节作用。但其作用机制仍不很清楚。本研究在前期分离获得应答多种逆境的辣椒CaCDPK4全长cDNA的基础上,进一步进行其亚细胞定位以及超表达和RNAi的载体构建和遗传转化,获得辣椒转基因植株,为进一步开展CaCDPK4在辣椒中的功能分析奠定基础。主要结果如下:
1、通过构建CaCDPK4与YFP融合蛋白的瞬间表达载体,并利用基因枪轰击洋葱表皮进行亚细胞定位,结果表明,CaCDPK4定位于细胞质中。
2、分别构建了辣椒CaCDPK4的超表达pK7WG2-CaCDPK4、RNAi载体pHELLSGATE12-CaCDPK4,并进行了测序验证。开展了辣椒农杆菌介导的遗传转化,在所获得的转基因植株中,PCR鉴定出3株pK7WG2-CaCDPK4和2株pHELLSGATE12-CaCDPK4的转基因T0代植株。
以上研究结果为进一步通过超表达和RNAi研究辣椒CaCDPK4候选基因的功能奠定了基础。
CDPKs (calcium-dependent protein kinases), firstly discovered in plants as a calcium-dependent protein kinases, are serine / threonine kinases that can be directly activated by calcium signal independent of calmodulin. The functional identification of CDPKs so far suggested important regulatory roles of CDPKs in plant growth, development and plants stresses responses, but the underlying mechanism remains largely unknown. Our previous work cloned a full length cDNA(CaCDPK4) of CDPKs from pepper cDNA library. In this study, the subcellular localization, the construction of over-expression and RNAi vectors of CaCDPK4 are carried out, and their corresponding transgenic pepper plants are acquired. The main results are as followings:
1. To identify the cellular localization of CaCDPK4, the transient expression vector of CaCDPK4 fused to YFP protein was constructed, and subcellular localization of CaCDPK4 was determined in onion epidermal cell through gene gun bombardment. The result showed that the CaCDPK4 locate in the cytoplasm.
2. The over-expression vector pK7WG2-CaCDPK4 and RNAi vector pHELLSGATE12-CaCDPK4 were constructed and verified by sequencing, and their corresponding transgenic pepper plants was acquired by Agrobacterium-mediated transformation method. The result turned among the regenerating plants, 3 plants were pK7WG2-CaCDPK4 positive and 2 plants were pHELLSGATE12-CaCDPK4 positive.
The results of this study laid foundation for further functional identification of CaCDPK4 by overexpression and RNAi analysis.
1、通过构建CaCDPK4与YFP融合蛋白的瞬间表达载体,并利用基因枪轰击洋葱表皮进行亚细胞定位,结果表明,CaCDPK4定位于细胞质中。
2、分别构建了辣椒CaCDPK4的超表达pK7WG2-CaCDPK4、RNAi载体pHELLSGATE12-CaCDPK4,并进行了测序验证。开展了辣椒农杆菌介导的遗传转化,在所获得的转基因植株中,PCR鉴定出3株pK7WG2-CaCDPK4和2株pHELLSGATE12-CaCDPK4的转基因T0代植株。
以上研究结果为进一步通过超表达和RNAi研究辣椒CaCDPK4候选基因的功能奠定了基础。
CDPKs (calcium-dependent protein kinases), firstly discovered in plants as a calcium-dependent protein kinases, are serine / threonine kinases that can be directly activated by calcium signal independent of calmodulin. The functional identification of CDPKs so far suggested important regulatory roles of CDPKs in plant growth, development and plants stresses responses, but the underlying mechanism remains largely unknown. Our previous work cloned a full length cDNA(CaCDPK4) of CDPKs from pepper cDNA library. In this study, the subcellular localization, the construction of over-expression and RNAi vectors of CaCDPK4 are carried out, and their corresponding transgenic pepper plants are acquired. The main results are as followings:
1. To identify the cellular localization of CaCDPK4, the transient expression vector of CaCDPK4 fused to YFP protein was constructed, and subcellular localization of CaCDPK4 was determined in onion epidermal cell through gene gun bombardment. The result showed that the CaCDPK4 locate in the cytoplasm.
2. The over-expression vector pK7WG2-CaCDPK4 and RNAi vector pHELLSGATE12-CaCDPK4 were constructed and verified by sequencing, and their corresponding transgenic pepper plants was acquired by Agrobacterium-mediated transformation method. The result turned among the regenerating plants, 3 plants were pK7WG2-CaCDPK4 positive and 2 plants were pHELLSGATE12-CaCDPK4 positive.
The results of this study laid foundation for further functional identification of CaCDPK4 by overexpression and RNAi analysis.
引文
[1]章文华,陈亚华,刘有良.钙在植物盐胁迫信号转导中的作用[J].植物生理学通讯, 2000, 36(2):146~153.
[2]Bush DS(.1995).Calcium regulation in plant cells and its role in signaling[J].Annu Rev Plant Physiol Plant Mol Biol, 46:95~122.
[3]刘贯山,陈珈.钙依赖蛋白激酶(CDPKs)在植物钙信号转导中的作用[J].植物学通报,2003,(20):160~167.
[4]Stone J M,Walker J C.(1995).Plant protein kinase families and signal transduction[J].Plant Physiol,108(2):451~457.
[5]Weljie A M ,Clarke T E ,J uffer A H ,et al.(2000).Roles of Calcium-dependent Protein Kinases ( CDPKs ) in Plant Calcium Signal Transduction [ J ] . Proteins,39 (4) :3431.
[6]Li J,Lee YRJ,Assmann SM(1998).Guard cells possess a calcium-dependent protein kinase that phosphorylates the KAT1 potassium channel[J].Plant Physiology,116:785~795.
[7]Nishiyama R,Mizuno H,et a1.(1999).Two mRNA species encoding calcium-dependent protein kinases are differentially expressed in sexual organs of Marchantia polymorpha through alternative splicing[J].Plant Cell Physiol,40(2):205~212.
[8]Yahalom A,Lando R,et a1.(1998).A calcium-dependent protein kinase is associated with maize mesocotyl plasmodesmata[J].plant physiol,153:354~362.
[9]Schenk PW,Snaar Jagalska BE. (1999).Signal perception and transduction:the role of protein kinases[J].Biochim Biophys Acta,1449(1):1~24.
[10] Cloning and Expression of Calcium-Dependent Protein Kinase (CDPK) GeneFamily in Common Tobacco (Nicotiana tabacum) Agricultural Sciences in China2009, 8(12): 1448~1457.
[11]Ludwig AA, Romeis T, Jones JDG .(2004). CDPK-mediated signalling pathways: specificity and cross-talk. J Exp Bot, 55 (395):181~188.
[12]Lecourieux D, Ranjeva R, Pugin A .(2006). Calcium in plant defence-signalling pathways. New Phytol, 171: 249~269.
[13]Cheng SH, Willmann MR, Chen HC, Sheen J. (2002). Calcium-signalling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family. Plant Physiol, 129:469~485.
[14]Yang G, Komat su S.(2000). Involvement of calcium-dependent protein kinase in rice (Ory z a sativa L ) lamina inclination caused by brassinolide[J ]1 Plant Cell Physiology , 41 ,1243~1250
[15] Rudd JJ, Franklin-Tong VE .(2001). Unravelling response-specificity in Ca2+ signallingpathways in plant cells. New Phytol, 151:733~749
[16]Lee SS, Cho HS, Yoon GM, Ahn JW, Kim HH, Pai HS (2003).Interaction of NtCDPK1 calcium-dependent protein kinasewith NtRpn3 regulatory subunit of the 26S proteasome in Nicotiana tabacum. Plant J, 33: 825~840
[17]Zhu SY, Yu XC, Wang XJ, Zhao R, Li Y, Fan RC, Shang Y, Du SY,Wang XF, Wu FQ et al. (2007). Two calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic acid signal transduction in Arabidopsis. Plant Cell, 19: 3019~3036
[18]Lanteri ML, Pagnussat GC, Lamattina L .(2006). Calcium and calcium-dependent protein kinases are involved in nitricoxide- and auxin-induced adventitious root formation in cucumber. J Exp Bot, 57 (6): 1341~1351
[19]Anil VS,Harmon AC,Rao KS.(2000).Spatio—temporal accumulation and activity of calcium-dependent protein kinases during embryogenesis,seed development,and germ ination in sandalwood[J].Plant Physiol,122:1035~1043.
[20]Morello L, Frattini M, GianìS, Christou P, Breviario D.(2000). Overexpression of the calcium-dependent protein kinase OsCDPK2 in transgenic rice is repressed by light in leaves and disrupts seed development. Trans Res, 9: 453~462
[21] Frattini M,Morello L and Breviario D.(1993).Rice calcium-dependent protein kinase isoforms OsCDPK2 and OsCDPK11 show different responses to light and different expression patterns during seed development[J].Plant Mol Bio1,41:753~764.
[22]Gyeong Mee Yoon , Peter E Dowd , Simon Gilroy , et al.(2006) . Calcium-dependent protein kinase isoforms in petunia have distinct functions in pollen tube growt h , Including regulating polarity[J ]. The Plant Cell , 18 : 867~878.
[23]Estruch JJ, Kadwell S, Merlin E, Crossland L .(1994). Cloning and characterization of a maize pollen-specific calcium-dependent calmodulin-independent protein kinase. Proc Natl Acad Sci USA, 91: 8837~8841
[24]Raíces M, Chico JM, Téllez-I?ón MT, Ulloa RM .(2001). Molecular characterization of StCDPK1, a calcium-dependent protein kinase from Solanum tuberosum that is induced at the onsetof tuber development. Plant Mol Biol, 46: 591~601
[25] White PJ, Broadley MR.(2003). Calcium in plants. Ann Bot, 92:487~511
[26]Saijo Y,Hata S,et a1.Over-expression of a single Ca2+ -dependent protein kinase confer both cold and salt/drought tolerance on rice plants[J].Plant Journal,2000,3:319~327.
[27]Lee J,Rudd J J.(2002).Calcium -dependent protein kinases:versatile plant signalling components necessary for pathogen defence [J]. Trends in Plant Science .7:97~98.
[28]Romeis T , Piedras P , Jones JDG.( 2000). Resistance gene dependent activation of a calcium-dependent protein kinase in the plant defense response[J ] . Plant Cell , 12 : 803~815.
[29]Romeis T , Ludwig A A , Martin R , et al.(2001). Calcium-dependent protein kinases play an essential role in a plant defence response[J ].EMBO J,20 : 5556~5567.
[30]余叔文,汤章城.植物生理与分子生物学[M].北京:科学出版社, 1998,54-76.
[31]别之龙,刘佩英.辣椒的离体培养与遗传转化[J].长江蔬菜,1996,(8);1~5.
[32]唐亮,刘文轩.辣椒再生体系的建立及基因工程研究进展[J].中国蔬菜,2003,(6):59~62.
[33]Liu W,Parroll W.A,Hildbebrand.D.F.et a1.(1990).Agrobacterium incluced crown gall formation in bell pepper(Capsicum annuum L) and formation of shoot-like structures expressing introduced genes[J].Plant Cell Report, 9(7);360~364.
[34]王玉文,杨美珠,潘乃隧等.甜椒的离体再生及基因转化[J]植物学报.1991.33(10):780~786.
[35]叶志彪,李汉霞,张健等.辣椒转基因植株再生[J].植物学报,1993,35(增刊):88~93.
[36]王慧中,赵培洁,韩献忠.转基因植株再生的研究[J].科技通报,l994,l0(1):30~32.
[37] Jeanne Curry, Maneesha Aluru, Marcus Mendoza, Jacob Nevarez, Martin Melendrez, Mary A.O’Connell.(1999).Transcripts for possible capsaicinoid biosynthetic genes are differentially accumulated in pungent and non-pungent Capsicum spp[J]. Plant Science, Volume 148, Issue 1, 15 October Pages 47~57
[38] Prasad B.C.N. Gururaj H. B. and Kumar V. Influence of 8-Methyl-nonenoic acid on capsaicin biosynthesis in in-Vivo and in-Vitro cell cultures of Capsicum Spp. Food Chem,54(5): 1854~1859.
[39]罗欢,段承杰,陈保善,唐纪良,冯家勋.辣椒CaNPR1-RNAi表达载体的构建及其对辣椒的转化[J].广西植物,2008,28(1):107~112.
[40]王立浩,王萱,张宝玺,毛胜利,郭家珍.利用CAPS标记辅助辣椒PVY抗性基因PVr4转育的研究[J].辣椒杂志,2006,4:1~3.
[41]黄国存,朱生伟,董越梅等.绿色荧光蛋白及其在植物研究中的应用[J].植物学通报,1998,15(5):24~30.
[42]Robert J G,Elizabeth P, Georgina M L .et a1.(1997).Green fluorescent protein fusions foreficient characterization of nuclear targeting[J].The Plant Journal. ll(3):573~586.
[43]Sheen J. (1996). Ca2+-dependent protein kinases and stress signal transduction in plants[J].Science, 274:1900~1902.
[2]Bush DS(.1995).Calcium regulation in plant cells and its role in signaling[J].Annu Rev Plant Physiol Plant Mol Biol, 46:95~122.
[3]刘贯山,陈珈.钙依赖蛋白激酶(CDPKs)在植物钙信号转导中的作用[J].植物学通报,2003,(20):160~167.
[4]Stone J M,Walker J C.(1995).Plant protein kinase families and signal transduction[J].Plant Physiol,108(2):451~457.
[5]Weljie A M ,Clarke T E ,J uffer A H ,et al.(2000).Roles of Calcium-dependent Protein Kinases ( CDPKs ) in Plant Calcium Signal Transduction [ J ] . Proteins,39 (4) :3431.
[6]Li J,Lee YRJ,Assmann SM(1998).Guard cells possess a calcium-dependent protein kinase that phosphorylates the KAT1 potassium channel[J].Plant Physiology,116:785~795.
[7]Nishiyama R,Mizuno H,et a1.(1999).Two mRNA species encoding calcium-dependent protein kinases are differentially expressed in sexual organs of Marchantia polymorpha through alternative splicing[J].Plant Cell Physiol,40(2):205~212.
[8]Yahalom A,Lando R,et a1.(1998).A calcium-dependent protein kinase is associated with maize mesocotyl plasmodesmata[J].plant physiol,153:354~362.
[9]Schenk PW,Snaar Jagalska BE. (1999).Signal perception and transduction:the role of protein kinases[J].Biochim Biophys Acta,1449(1):1~24.
[10] Cloning and Expression of Calcium-Dependent Protein Kinase (CDPK) GeneFamily in Common Tobacco (Nicotiana tabacum) Agricultural Sciences in China2009, 8(12): 1448~1457.
[11]Ludwig AA, Romeis T, Jones JDG .(2004). CDPK-mediated signalling pathways: specificity and cross-talk. J Exp Bot, 55 (395):181~188.
[12]Lecourieux D, Ranjeva R, Pugin A .(2006). Calcium in plant defence-signalling pathways. New Phytol, 171: 249~269.
[13]Cheng SH, Willmann MR, Chen HC, Sheen J. (2002). Calcium-signalling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family. Plant Physiol, 129:469~485.
[14]Yang G, Komat su S.(2000). Involvement of calcium-dependent protein kinase in rice (Ory z a sativa L ) lamina inclination caused by brassinolide[J ]1 Plant Cell Physiology , 41 ,1243~1250
[15] Rudd JJ, Franklin-Tong VE .(2001). Unravelling response-specificity in Ca2+ signallingpathways in plant cells. New Phytol, 151:733~749
[16]Lee SS, Cho HS, Yoon GM, Ahn JW, Kim HH, Pai HS (2003).Interaction of NtCDPK1 calcium-dependent protein kinasewith NtRpn3 regulatory subunit of the 26S proteasome in Nicotiana tabacum. Plant J, 33: 825~840
[17]Zhu SY, Yu XC, Wang XJ, Zhao R, Li Y, Fan RC, Shang Y, Du SY,Wang XF, Wu FQ et al. (2007). Two calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic acid signal transduction in Arabidopsis. Plant Cell, 19: 3019~3036
[18]Lanteri ML, Pagnussat GC, Lamattina L .(2006). Calcium and calcium-dependent protein kinases are involved in nitricoxide- and auxin-induced adventitious root formation in cucumber. J Exp Bot, 57 (6): 1341~1351
[19]Anil VS,Harmon AC,Rao KS.(2000).Spatio—temporal accumulation and activity of calcium-dependent protein kinases during embryogenesis,seed development,and germ ination in sandalwood[J].Plant Physiol,122:1035~1043.
[20]Morello L, Frattini M, GianìS, Christou P, Breviario D.(2000). Overexpression of the calcium-dependent protein kinase OsCDPK2 in transgenic rice is repressed by light in leaves and disrupts seed development. Trans Res, 9: 453~462
[21] Frattini M,Morello L and Breviario D.(1993).Rice calcium-dependent protein kinase isoforms OsCDPK2 and OsCDPK11 show different responses to light and different expression patterns during seed development[J].Plant Mol Bio1,41:753~764.
[22]Gyeong Mee Yoon , Peter E Dowd , Simon Gilroy , et al.(2006) . Calcium-dependent protein kinase isoforms in petunia have distinct functions in pollen tube growt h , Including regulating polarity[J ]. The Plant Cell , 18 : 867~878.
[23]Estruch JJ, Kadwell S, Merlin E, Crossland L .(1994). Cloning and characterization of a maize pollen-specific calcium-dependent calmodulin-independent protein kinase. Proc Natl Acad Sci USA, 91: 8837~8841
[24]Raíces M, Chico JM, Téllez-I?ón MT, Ulloa RM .(2001). Molecular characterization of StCDPK1, a calcium-dependent protein kinase from Solanum tuberosum that is induced at the onsetof tuber development. Plant Mol Biol, 46: 591~601
[25] White PJ, Broadley MR.(2003). Calcium in plants. Ann Bot, 92:487~511
[26]Saijo Y,Hata S,et a1.Over-expression of a single Ca2+ -dependent protein kinase confer both cold and salt/drought tolerance on rice plants[J].Plant Journal,2000,3:319~327.
[27]Lee J,Rudd J J.(2002).Calcium -dependent protein kinases:versatile plant signalling components necessary for pathogen defence [J]. Trends in Plant Science .7:97~98.
[28]Romeis T , Piedras P , Jones JDG.( 2000). Resistance gene dependent activation of a calcium-dependent protein kinase in the plant defense response[J ] . Plant Cell , 12 : 803~815.
[29]Romeis T , Ludwig A A , Martin R , et al.(2001). Calcium-dependent protein kinases play an essential role in a plant defence response[J ].EMBO J,20 : 5556~5567.
[30]余叔文,汤章城.植物生理与分子生物学[M].北京:科学出版社, 1998,54-76.
[31]别之龙,刘佩英.辣椒的离体培养与遗传转化[J].长江蔬菜,1996,(8);1~5.
[32]唐亮,刘文轩.辣椒再生体系的建立及基因工程研究进展[J].中国蔬菜,2003,(6):59~62.
[33]Liu W,Parroll W.A,Hildbebrand.D.F.et a1.(1990).Agrobacterium incluced crown gall formation in bell pepper(Capsicum annuum L) and formation of shoot-like structures expressing introduced genes[J].Plant Cell Report, 9(7);360~364.
[34]王玉文,杨美珠,潘乃隧等.甜椒的离体再生及基因转化[J]植物学报.1991.33(10):780~786.
[35]叶志彪,李汉霞,张健等.辣椒转基因植株再生[J].植物学报,1993,35(增刊):88~93.
[36]王慧中,赵培洁,韩献忠.转基因植株再生的研究[J].科技通报,l994,l0(1):30~32.
[37] Jeanne Curry, Maneesha Aluru, Marcus Mendoza, Jacob Nevarez, Martin Melendrez, Mary A.O’Connell.(1999).Transcripts for possible capsaicinoid biosynthetic genes are differentially accumulated in pungent and non-pungent Capsicum spp[J]. Plant Science, Volume 148, Issue 1, 15 October Pages 47~57
[38] Prasad B.C.N. Gururaj H. B. and Kumar V. Influence of 8-Methyl-nonenoic acid on capsaicin biosynthesis in in-Vivo and in-Vitro cell cultures of Capsicum Spp. Food Chem,54(5): 1854~1859.
[39]罗欢,段承杰,陈保善,唐纪良,冯家勋.辣椒CaNPR1-RNAi表达载体的构建及其对辣椒的转化[J].广西植物,2008,28(1):107~112.
[40]王立浩,王萱,张宝玺,毛胜利,郭家珍.利用CAPS标记辅助辣椒PVY抗性基因PVr4转育的研究[J].辣椒杂志,2006,4:1~3.
[41]黄国存,朱生伟,董越梅等.绿色荧光蛋白及其在植物研究中的应用[J].植物学通报,1998,15(5):24~30.
[42]Robert J G,Elizabeth P, Georgina M L .et a1.(1997).Green fluorescent protein fusions foreficient characterization of nuclear targeting[J].The Plant Journal. ll(3):573~586.
[43]Sheen J. (1996). Ca2+-dependent protein kinases and stress signal transduction in plants[J].Science, 274:1900~1902.