水稻OsCPK2、OsCPK15和OsCPK29的功能鉴定
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摘要
胞质Ca~(2+)是重要的第二信使,通过Ca~(2+)结合蛋白产生磷酸化信号级联,调控下游基因表达。钙依赖而钙调素不依赖的蛋白激酶(calcium-dependent/calmodulin-independentprotein kinases,CDPKs)是一类仅在植物和部分原生生物中存在的丝氨酸/苏氨酸蛋白激酶,在钙信号转导中具有重要功能。越来越多的证据表明,CDPKs广泛参与植物生长发育、病原防御、非生物环境胁迫等生理反应的信号传递过程。目前在水稻中已发现31个CDPKs基因,但功能明确的只有少数几个基因,本研究的目的就是利用超量表达和RNA干涉(RNA interference,RNAi)技术分析水稻OsCPK2、OsCPK15和OsCPK29的功能,为利用基因工程技术培育水稻新品种提供新的基因资源和理论指导。本研究的主要试验结果如下:
1.通过RT-PCR方法检测了OsCPK2、OsCPK15和OsCPK29三个基因在粳稻品种日本晴分蘖期的根、成熟叶片、穗尖至剑叶叶枕距离分别为0 cm、0-1cm、1-3 cm、3-5 cm、5-8 cm、8-12 cm、12-16 cm、16-20 cm、20-25 cm、25-30 cm、灌浆期稻穗(命名为P1-P12)中的表达谱。试验结果显示:OsCPK2和OsCPK29在P2-P11时期稻穗中表达量较高,在成熟叶片和P12时期稻穗中表达量很低,在分蘖期的根和幼穗中不表达;而OsCPK15在所检测的各个组织和时期中表达量均很高。表达谱结果说明OsCPK2和OsCPK29的表达具有组织时空特异性,而OsCPK15是组成型表达。
2.构建了OsCPK2超量表达载体p1300S-CPK2,通过根癌农杆菌介导法转化日本晴愈伤组织,得到42株再生植株。通过Southern blot和Northern blot检测,共得到16株单拷贝植株、4株单拷贝超量表达植株。
3.构建了OsCPK15超量表达载体p1300S-CPK15,通过根癌农杆菌介导法转化日本晴愈伤组织,得到43株再生植株。通过Southern blot和Northern blot检测,共得到11株单拷贝植株、2株单拷贝超量表达植株。
4.构建了OsCPK29超量表达载体p1300S-CPK29,通过根癌农杆菌介导法转化日本晴愈伤组织,得到74株再生植株。PCR检测结果显示阳性率为92%。通过Southern blot和Northern blot检测,共得到11株单拷贝植株、4株单拷贝超量表达植株。
5.对OsCPK29 T_2代转基因家系进行花粉育性考察,结果显示转基因植株花粉育性与野生型相比没有明显降低。
6.对OsCPK29 T_2代转基因家系进行干旱、高盐、低温3种非生物逆境胁迫处理,通过表型观察发现转基因植株的抗旱性、耐盐性、抗寒性与对照相比没有明显差异。同时还对OsCPK29 T_2代转基因家系进行甘露醇胁迫和ABA敏感性试验,结果显示处理后转基因植株与对照相比没有明显差异。
7.对OsCPK29 T_2代转基因家系进行叶片衰老进程检测,结果显示,OsCPK29超量表达植株叶片衰老延迟。同时测量了处理前后转基因植株和对照的离体剑叶叶绿素含量,结果显示,处理前转基因植株和对照离体剑叶叶绿素含量没有明显差异,而在处理后转基因植株离体剑叶叶绿素含量极显著地高于对照。
8.构建了OsCPK2、OsCPK15和OsCPK29 RNAi载体pDS1301-CPK2、pDS1301-CPK15和pDS1301-CPK29,通过根癌农杆菌介导法转化日本晴愈伤组织,分别得到39株、38株、43株再生植株。通过PCR检测,阳性率均在90%以上。
9.通过RT-RCR方法分别检测了OsCPK2、OsCPK15和OsCPK29抑制表达植株中相应基因的表达量。结果显示与日本晴野生型相比,这三个基因均未被抑制。
10.构建了OsCPK2、OsCPK15和OsCPK29亚细胞定位载体p1391aGFP-CPK2、p1391aGFP-CPK15和p1391bGFP-CPK29。利用基因枪将包被载体质粒的金粉分别导入洋葱表皮细胞,通过激光共聚焦显微镜观察到融合蛋白OsCPK2-GFP、OsCPK15-GFP和OsCPK29-GFP均定位在细胞质。
Cytoplasmic Ca~(2+) is an important second messenger.Phosphorylation signal cascades are generated through Ca~(2+) binding proteins,regulating the expression of downstream genes.Calcium-dependent/calmodulin-independent protein kinases(CDPKs) are a Ser/Thr protein kinase family which exist only in the plant and parts of protista,and play a very important role in Ca~(2+) signal transduction.More and more evidences indicate that CDPKs participate widely in singnal transduction processes of physiological responses,including plant development,pathogen defense and abiotic stress response etc. Thirty one CDPK genes have been found in rice so far,however only few of them have been well characterized.The objective of this research is to reveal the functions of OsCPK2,OsCPK15 and OsCPK29 through over-expression and RNA interference technology,which can provide new gene resources and theoretical guidances for rice breeding using genetic engineering.Main research results of this study are as follows:
1.The expression profiles of OsCPK2,OsCPK15 and OsCPK29 in the root at tillering stage,the mature leaf,the distance from panicle tip to flag leaf pulvinus is 0 cm, 0-1cm,1-3 cm,3-5 cm,5-8 cm,8-12cm,12-16 cm,16-20 cm,20-25cm,25-30 cm,the panicle at filling stage(named P1 to P12 stages) of Oryza sativa cv.Nipponbare have been analysised by RT-PCR.The results show that OsCPK2 and OsCPK29 have high expression level in the panicle at P2 to P11 stages,and weak expression level in the mature leaf and the panicle at P12 stage,while no expression in the root at tillering stage and the young panicle.OsCPK15 has high expression level in all detected tissue stages. These results indicate that the expression of OsCPK2 and OsCPK29 is tissue-specific and temporal-spatial,while OsCPK15 expresses constitutively.
2.The OsCPK2-overexpressing vector(p1300S-CPK2) was constructed and transformed by Agrobacterium tumefaciens-mediated transformation using Nipponbare as the recipient material.Fourty two regenerated plants have generated through transformation.Southern blot and Northern blot analysis indicate that 16 among them are single-copy inserted and 4 are over-expression plants within 16 single-copy inserted transgenic plants.
3.The OsCPK15-overexpressing vector(p1300S-CPK15) was constructed and transformed by Agrobacterium tumefaciens-mediated transformation using Nipponbare. Fourty three regenerated plants have generated through transformation.Southern blot and Northern blot analysis indicate that 11 among them are single-copy inserted and 2 are over-expression plants within 11 single-copy inserted transgenic plants.
4.The OsCPK29-overexpressing vector(p1300S-CPK29) was constructed and transformed by Agrobacterium tumefaciens-mediated transformation using Nipponbare. Seventy four regenerated plants have generated through transformation.The positive rate of transgenic plants is 92%by PCR analysis.Southern blot and Northern blot analysis indicate that 11 among them are single-copy inserted and 4 are over-expression plants within 11 single-copy inserted transgenic plants.
5.The pollen fertility was observed in T_2 families of OsCPK29-overexpressing plants.The results show that there is no difference on pollen fertility between transgenic plants and wild type.
6.T_2 families of OsCPK29-overexpressing plants were treated under three different abiotic stresses(drought,salt and cold).The results showed that there is no significant difference on drought,salt and cold tolerence between transgenic plants and the control based on phenotypic observation.Mannitol and ABA treatments were also conducted on T_2 families of OsCPK29-overexpressing plants and the transgenic plants did not show significant differences from the control.
7.Leaf senescence process of T_2 families of OsCPK29-overexpressing plants were also detected,and the results showed that the leaf senescence process of OsCPK29-overexpressing plants delayed compared with the control.Meanwhile, chlorophyll content of the flag leaf were determined in vitro before and after treatments, and the result showed that the chlorophyll content of transgenic plants is significantly higher than that of the control after the treatment but no difference before that.
8.The OsCPK2,OsCPK15 and OsCPK29 RNAi vectors(pDS1301-CPK2, pDS1301-CPK15 and pDS1301-CPK29) were constructed and transformed by Agrobacterium tumefaciens-mediated method.Thirty nine,thirty eight and fourty three regenerated plants were generated respectively.The positive rate of transgenic plants is more than 90%by PCR analysis.
9.The expression level of OsCPK2,OsCPK15 and OsCPK29 in the corresponding RNAi transgenetic plants have been detected by RT-PCR,and the results showed that all of three target genes were not suppressed.
10.The OsCPK2,OsCPK15 and OsCPK29 subcellular location vectors (p1391aGFP-CPK2,p1391aGFP-CPK15 and p1391bGFP-CPK29) were constructed,and coated on gold particles respectively,and then bombarded into onion epidermal cells.All of the fusion proteins are localized in the cytoplasm by laser scanning confocal microscope.
1.通过RT-PCR方法检测了OsCPK2、OsCPK15和OsCPK29三个基因在粳稻品种日本晴分蘖期的根、成熟叶片、穗尖至剑叶叶枕距离分别为0 cm、0-1cm、1-3 cm、3-5 cm、5-8 cm、8-12 cm、12-16 cm、16-20 cm、20-25 cm、25-30 cm、灌浆期稻穗(命名为P1-P12)中的表达谱。试验结果显示:OsCPK2和OsCPK29在P2-P11时期稻穗中表达量较高,在成熟叶片和P12时期稻穗中表达量很低,在分蘖期的根和幼穗中不表达;而OsCPK15在所检测的各个组织和时期中表达量均很高。表达谱结果说明OsCPK2和OsCPK29的表达具有组织时空特异性,而OsCPK15是组成型表达。
2.构建了OsCPK2超量表达载体p1300S-CPK2,通过根癌农杆菌介导法转化日本晴愈伤组织,得到42株再生植株。通过Southern blot和Northern blot检测,共得到16株单拷贝植株、4株单拷贝超量表达植株。
3.构建了OsCPK15超量表达载体p1300S-CPK15,通过根癌农杆菌介导法转化日本晴愈伤组织,得到43株再生植株。通过Southern blot和Northern blot检测,共得到11株单拷贝植株、2株单拷贝超量表达植株。
4.构建了OsCPK29超量表达载体p1300S-CPK29,通过根癌农杆菌介导法转化日本晴愈伤组织,得到74株再生植株。PCR检测结果显示阳性率为92%。通过Southern blot和Northern blot检测,共得到11株单拷贝植株、4株单拷贝超量表达植株。
5.对OsCPK29 T_2代转基因家系进行花粉育性考察,结果显示转基因植株花粉育性与野生型相比没有明显降低。
6.对OsCPK29 T_2代转基因家系进行干旱、高盐、低温3种非生物逆境胁迫处理,通过表型观察发现转基因植株的抗旱性、耐盐性、抗寒性与对照相比没有明显差异。同时还对OsCPK29 T_2代转基因家系进行甘露醇胁迫和ABA敏感性试验,结果显示处理后转基因植株与对照相比没有明显差异。
7.对OsCPK29 T_2代转基因家系进行叶片衰老进程检测,结果显示,OsCPK29超量表达植株叶片衰老延迟。同时测量了处理前后转基因植株和对照的离体剑叶叶绿素含量,结果显示,处理前转基因植株和对照离体剑叶叶绿素含量没有明显差异,而在处理后转基因植株离体剑叶叶绿素含量极显著地高于对照。
8.构建了OsCPK2、OsCPK15和OsCPK29 RNAi载体pDS1301-CPK2、pDS1301-CPK15和pDS1301-CPK29,通过根癌农杆菌介导法转化日本晴愈伤组织,分别得到39株、38株、43株再生植株。通过PCR检测,阳性率均在90%以上。
9.通过RT-RCR方法分别检测了OsCPK2、OsCPK15和OsCPK29抑制表达植株中相应基因的表达量。结果显示与日本晴野生型相比,这三个基因均未被抑制。
10.构建了OsCPK2、OsCPK15和OsCPK29亚细胞定位载体p1391aGFP-CPK2、p1391aGFP-CPK15和p1391bGFP-CPK29。利用基因枪将包被载体质粒的金粉分别导入洋葱表皮细胞,通过激光共聚焦显微镜观察到融合蛋白OsCPK2-GFP、OsCPK15-GFP和OsCPK29-GFP均定位在细胞质。
Cytoplasmic Ca~(2+) is an important second messenger.Phosphorylation signal cascades are generated through Ca~(2+) binding proteins,regulating the expression of downstream genes.Calcium-dependent/calmodulin-independent protein kinases(CDPKs) are a Ser/Thr protein kinase family which exist only in the plant and parts of protista,and play a very important role in Ca~(2+) signal transduction.More and more evidences indicate that CDPKs participate widely in singnal transduction processes of physiological responses,including plant development,pathogen defense and abiotic stress response etc. Thirty one CDPK genes have been found in rice so far,however only few of them have been well characterized.The objective of this research is to reveal the functions of OsCPK2,OsCPK15 and OsCPK29 through over-expression and RNA interference technology,which can provide new gene resources and theoretical guidances for rice breeding using genetic engineering.Main research results of this study are as follows:
1.The expression profiles of OsCPK2,OsCPK15 and OsCPK29 in the root at tillering stage,the mature leaf,the distance from panicle tip to flag leaf pulvinus is 0 cm, 0-1cm,1-3 cm,3-5 cm,5-8 cm,8-12cm,12-16 cm,16-20 cm,20-25cm,25-30 cm,the panicle at filling stage(named P1 to P12 stages) of Oryza sativa cv.Nipponbare have been analysised by RT-PCR.The results show that OsCPK2 and OsCPK29 have high expression level in the panicle at P2 to P11 stages,and weak expression level in the mature leaf and the panicle at P12 stage,while no expression in the root at tillering stage and the young panicle.OsCPK15 has high expression level in all detected tissue stages. These results indicate that the expression of OsCPK2 and OsCPK29 is tissue-specific and temporal-spatial,while OsCPK15 expresses constitutively.
2.The OsCPK2-overexpressing vector(p1300S-CPK2) was constructed and transformed by Agrobacterium tumefaciens-mediated transformation using Nipponbare as the recipient material.Fourty two regenerated plants have generated through transformation.Southern blot and Northern blot analysis indicate that 16 among them are single-copy inserted and 4 are over-expression plants within 16 single-copy inserted transgenic plants.
3.The OsCPK15-overexpressing vector(p1300S-CPK15) was constructed and transformed by Agrobacterium tumefaciens-mediated transformation using Nipponbare. Fourty three regenerated plants have generated through transformation.Southern blot and Northern blot analysis indicate that 11 among them are single-copy inserted and 2 are over-expression plants within 11 single-copy inserted transgenic plants.
4.The OsCPK29-overexpressing vector(p1300S-CPK29) was constructed and transformed by Agrobacterium tumefaciens-mediated transformation using Nipponbare. Seventy four regenerated plants have generated through transformation.The positive rate of transgenic plants is 92%by PCR analysis.Southern blot and Northern blot analysis indicate that 11 among them are single-copy inserted and 4 are over-expression plants within 11 single-copy inserted transgenic plants.
5.The pollen fertility was observed in T_2 families of OsCPK29-overexpressing plants.The results show that there is no difference on pollen fertility between transgenic plants and wild type.
6.T_2 families of OsCPK29-overexpressing plants were treated under three different abiotic stresses(drought,salt and cold).The results showed that there is no significant difference on drought,salt and cold tolerence between transgenic plants and the control based on phenotypic observation.Mannitol and ABA treatments were also conducted on T_2 families of OsCPK29-overexpressing plants and the transgenic plants did not show significant differences from the control.
7.Leaf senescence process of T_2 families of OsCPK29-overexpressing plants were also detected,and the results showed that the leaf senescence process of OsCPK29-overexpressing plants delayed compared with the control.Meanwhile, chlorophyll content of the flag leaf were determined in vitro before and after treatments, and the result showed that the chlorophyll content of transgenic plants is significantly higher than that of the control after the treatment but no difference before that.
8.The OsCPK2,OsCPK15 and OsCPK29 RNAi vectors(pDS1301-CPK2, pDS1301-CPK15 and pDS1301-CPK29) were constructed and transformed by Agrobacterium tumefaciens-mediated method.Thirty nine,thirty eight and fourty three regenerated plants were generated respectively.The positive rate of transgenic plants is more than 90%by PCR analysis.
9.The expression level of OsCPK2,OsCPK15 and OsCPK29 in the corresponding RNAi transgenetic plants have been detected by RT-PCR,and the results showed that all of three target genes were not suppressed.
10.The OsCPK2,OsCPK15 and OsCPK29 subcellular location vectors (p1391aGFP-CPK2,p1391aGFP-CPK15 and p1391bGFP-CPK29) were constructed,and coated on gold particles respectively,and then bombarded into onion epidermal cells.All of the fusion proteins are localized in the cytoplasm by laser scanning confocal microscope.
引文
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35. Harmon A C, Putnam-Evans C, Cormier M J. A Calcium-dependent but calmodulin-independent protein kinase from soybean. Plant Physiol, 1987, 83: 830-837
36. Harper J F, Breton G, Harmon A. Decoding Ca~(2+) signals through plant protein kinases. Annu Rev Plant Biol, 2004, 55: 263-288
37. Harper J F, Huang J F, Lloyd S J. Genetic identification of an autoinhibitor in CDPK, a protein kinase with a calmodulin-like domain. Biochemistry, 1994, 33: 7267-7277
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39. Hepler P K, Vidali L, Cheung AY. Polarized cell growth in higher plants. Annu Rev Cell Dev Biol, 2001, 17: 159-187
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43. Kawasaki T, Hayashida N, Baba T, Shinozaki K, Shimada H. The gene encoding a calcium-dependent protein kinase located near the sbel gene encoding starch branching enzyme I is specifically expressed in developing rice seeds. Gene, 1993, 129: 183-189
44. Komatsu S, Yang G, Khan M, Onodera H, Toki S, Yamaguchi M. Over-expression of calcium-dependent protein kinase 13 and calreticulin interacting protein 1 confers cold tolerance on rice plants. Mol Genet Genomics, 2007, 277: 713-723
45. Kwak S H, Lee S H. The requirements for Ca~(2+), protein phosphorylation, and dephosphorylation for ethylene signal transduction in Pisum sativum L. Plant Cell Physiol, 1997, 38: 1142-1149
46. Lee J Y, Yoo B C, Harmon A C. Kinetic and calcium-binding properties of three calcium-dependent protein kinase isoenzymes from soybean. Biochemistry, 1998, 37: 6801-6809
47. Lee S S, Cho H S, Yoon G M, Ann J W, Kim H H, Pai H S. Interaction of NtCDPKl calcium-dependent protein kinase with NtRpn3 regulatory subunit of the 26S proteasome in Nicotiana tabacum. Plant J, 2003, 33: 825-840
48. Leshem Y Y, Sridhara S, Thompson J E. Involvement of calcium and calmodulin in membrane deterioration during senescence of pea foliage. Plant Physiol, 1984, 75: 329-335
49. Li A L, Zhu Y F, Tan X M, Wang X, Wei B, Guo H Z, Zhang Z L, Chen X B, Zhao G Y, Kong X Y, Jia J Z, Mao L. Evolutionary and functional study of the CDPK gene family in wheat (Triticum aestivum L.). Plant Mol Biol, 2008, 66: 429-443
50. Li R J, Hua W, Lu Y T. Arabidopsis cytosolic glutamine synthetase AtGLNl ;1 is a potential substrate of AtCRK3 involved in leaf senescence. Biochem Biophys Res Commun, 2006, 342: 119-126
51. Liu K D, Wang J, Li H B, Xu C G, Liu A M, Li X H, Zhang Q. A genome-wide analysis of wide compatibility in rice and the precise location of the S_5 locus in the molecular map. TAG Theoretical and Applied Genetics, 1997, 95: 809-814
52. Lu S X, Hrabak E M. An Arabidopsis calcium-dependent protein kinase is associated with the endoplasmic reticulum. Plant Physiol, 2002, 128: 1008-1021
53.Ludwig A A,Romeis T,Jones J D.CDPK-mediated signalling pathways:specificity and cross-talk.J Exp Bot,2004,55:181-188
54.Mackinney G.Absorption of light by chlorophyll solutions.J.Biol.Chem.,1941,140:315-322
55.Martinez-Noel G,Nagaraj V J,Calo G,Wiemken A,Pontis H G.Sucrose regulated expression of a Ca~(2+)-dependent protein kinase(TaCDPK1) gene in excised leaves of wheat.Plant Physiol Biochem,2007,45:410-419
56.Morello L,Frattini M,Giani S,Christou P,Breviario D.Overexpression of the calcium-dependent protein kinase OsCDPK2 in transgenic rice is repressed by light in leaves and disrupts seed development.Transgenic Res,2000,9:453-462
57.Mori I C,Murata Y,Yang Y,Munemasa S,Wang Y F,Andreoli S,Tiriac H,Alonso J M,Harper J F,Ecker J R,Kwak J M,Schroeder J I.CDPKs CPK6 and CPK3function in ABA regulation of guard cell S-type anion- and Ca~(2+)-permeable channels and stomatal closure.PLoS Biol,2006,4:e327
58.Moutinho A,Trewavas A J,Malho R.Relocation of a Ca~(2+)-dependent protein kinase activity during pollen tube reorientation.Plant Cell,1998,10:1499-1510
59.Murray M G,Thompson W F.Rapid isolation of high molecular weight plant DNA.Nucleic Acids Res,1980,8:4321-4325
60.Osuna L,Coursol S,Pierre J N,Vidal J.A Ca~(2+)-dependent protein kinase with characteristics of protein kinase C in leaves and mesophyll cell protoplasts from Digitaria sanguinalis:possible involvement in the C_4-phosphoenolpyruvate carboxylase phosphorylation cascade.Biochem Biophys Res Commun,2004,314:428-433
61.Patharkar O R,Cushman J C.A stress-induced calcium-dependent protein kinase from Mesembryanthemum crystallinum phosphorylates a two-component pseudo-response regulator.Plant J,2000,24:679-691
62.Patharkar O R,Cushman J C.A novel coiled-coil protein co-localizes and interacts with a calcium-dependent protein kinase in the common ice plant during low-humidity stress.Planta,2006,225:57-73
63.Pei Z M,Ward J M,Harper J F.Schroeder J I.A novel chloride channel in Vicia faba guard cell vacuoles activated by the serine/threonine kinase,CDPK.Embo J.1996,15: 6564-6574
64.Poovaiah B W.Molecular and cellular aspects of calcium action in plants.HortScience,1988,23:267-271
65.Poovaiah B W,Leopold A C.Deferral of leaf senescence with calcium.Plant Physiol,1973,52:236-239
66.Ramachandiran S,Takezawa D,Wang W,Poovaiah B W.Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase:regulation by autoinhibitory and visinin-like domains.J Biochem,1997,121:984-990
67.Ray S,Agarwal P,Arora R,Kapoor S,Tyagi A K.Expression analysis of calcium-dependent protein kinase gene family during reproductive development and abiotic stress conditions in rice(Oryza sativa L.ssp.indica).Mol Genet Genomics,2007,278:493-505
68.Reddy V S,Reddy A S.Proteomics of calcium-signaling components in plants.Phytochemistry,2004,65:1745-1776
69.Romeis T,Ludwig A A,Martin R,Jones J D.Calcium-dependent protein kinases play an essential role in a plant defence response.Embo J,2001,20:5556-5567
70.Romeis T,Piedras P,Jones J D.Resistance gene-dependent activation of a calcium-dependent protein kinase in the plant defense response.Plant Cell,2000,12:803-816
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