水稻PP2Ac类蛋白磷酸酶在盐胁迫下的表达研究
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摘要
背景:水稻是世界上最重要的粮食作物之一,也是基础研究的模式植物之一。盐胁迫造成的影响几乎涉及所有的生理和生化过程,严重时导致水稻不能正常生长、减产甚至绝收。对抗盐逆境基因介导的分子机理研究对水稻生产具有重要的意义。PP2Ac类蛋白磷酸酶(phosphatase 2A catalytic subunits,PP2Ac)在动植物中广泛存在,已有研究表明,水稻PP2Ac家族成员在生长发育和抗逆过程中发挥着重要作用。蛋白磷酸酶与蛋白激酶作为一对开关调控细胞内的一系列生化反应,蛋白磷酸酶可以催化去磷酸化反应,使磷酸化的蛋白质脱磷酸化,终止信号分子的传递,从而调控细胞信号转导通路。
研究目的:制备针对水稻OsPP2Ac基因编码的蛋白质的特异性抗体,了解重要蛋白质的表达模式进而探讨水稻耐盐的分子机理,为其功能研究累计数据。
材料与方法:对候选基因编码的蛋白质进行抗原决定簇的预测,选出抗原决定簇峰值较高的片段后,合成多肽偶联后免疫兔子制备多克隆抗体。用Western blotting检测OsPP2Ac蛋白质在水稻品种93-11与抗盐品种兰胜在苗期盐处理后不同时间点的表达变化以及在耐、受及对照反应中的差异表达,并与水稻MPSS数据库中叶片苗期的转录信息进行比较分析。
结果:发现在耐盐水稻品种兰胜中,OsPP2Ac-4的表达上调,在盐敏感的水稻品种93-11中,OsPP2Ac-2、OsPP2Ac-3和OsPP2Ac-5的表达也发生了上调,但OsPP2Ac-4的表达下调,比较二个品种间PP2Ac蛋白质的表达,发现在正常生长条件下,OsPP2Ac蛋白质的表达没有显著区别且基本保持恒定,其表达变化仅发生在盐胁迫条件下。分析水稻MPSS数据库提供的苗期盐胁迫的转录数据,发现OsPP2Ac-2、OsPP2Ac-3和OsPP2Ac-5在盐胁迫条件下转录水平下调。
结论:通过对OsPP2Ac在水稻品种兰胜及93-11盐胁迫处理过程中的表达及表型分析,发现大部分OsPP2Ac家族在耐盐材料兰胜中没有修饰表达,在93-11中盐处理后0.5h内出现高丰度表达,且随着处理时间的延长表达量逐渐增加,在48h达到峰值,在8天后植物死亡,说明PP2Ac的表达与盐胁迫导致的细胞程序性死亡具有关联性。通过比较耐、受反应的蛋白质差异表达,发现蛋白质的存在与表达量的变化与其功能密切相关。同时OsPP2Ac-4在兰胜与93-11中的差异表达,可能为揭示兰胜的耐盐通路提供线索。本实验发现了OsPP2Ac-2、OsPP2Ac-3、OsPP2Ac-4和OsPP2Ac-5,4个盐胁迫条件下表达发生变化的PP2Ac蛋白质,为了解PP2Ac类蛋白磷酸酶蛋白质在水稻盐胁迫过程中的功能及耐盐机理提供了有价值的线索。
Background: Rice is one of the most important food crops in world, one of the basic research model plants. Of large-scale production of rice caused by salt damage, the impact of salt stress, involving almost all plant physiological and biochemical processes, leading to serious plant can not grow normally, cuts or even crops. Against salt stress mediated salt adversity molecular mechanism of rice production is of great significance. The PP2Ac class of protein phosphatase (phosphatase 2A catalytic subunits, PP2Ac) is widespread in plants and animals have been shown that rice PP2Ac family members play an important role in the process of growth and development and resilience. Protein phosphatase catalyzed dephosphorylation reaction, so that protein phosphorylation and dephosphorylation, terminate the transfer of signaling molecules, and thereby regulate cell signaling signal transduction pathway. There is evidence that protein kinase and protein phosphatase as a pair of switch regulation of cell within the range of biochemical reactions.
Objective: Preparation of the corresponding specific antibodies against rice PP2Ac gene encodes a protein, and then explore the molecular mechanism of salt tolerance of rice for the understanding of an important protein expression patterns, as function of accumulated data.
Materials and Methods: The candidate gene encodes a protein epitope prediction, to elect the antigenic determinants of the fragment cluster peak higher, synthetic peptides coupled immune rabbit polyclonal antibody. Detected by Western Blotting PP2Ac genes encoding proteins in rice super hybrid rice parent varieties 93-11 and salt varieties Lan Sheng expression of seedling salt treatment at different time points after the change in resistance by the control reaction in the expression of differentially expressed and transcription of leaf seedling stage in rice MPSS database for comparative analysis.
Results: Found in the salt-tolerant rice varieties Lan OsPP2Ac-4 up regulated in salt-sensitive rice varieties 93-11 OsPP2Ac-2, OsPP2Ac-3 and OsPP2Ac-5 expression also occurred in the increase, but OsPP2Ac-4 down regulation of PP2Ac protein expression, comparison between the two species and found that PP2Ac protein expression in normal growth conditions, no significant difference remained constant, the expression change occurs only under salt stress. Seedling salt stress transcription data provided by the analysis of the rice MPSS database found OsPP2Ac-2, OsPP2Ac-3 and OsPP2Ac-5 transcription under salt stress levels down.
Conclusion: The expression and phenotypic analysis of rice varieties Lan Sheng and 93-11 salt stress on OsPP2Ac process, most OsPP2Ac family in no modification of the expression of salt-tolerant material Lan Sheng, 93-11 in Salt highly expressed within 0.5h, and with the treatment time was gradually increased, peaked at 48h, then the plants die, PP2Ac expression and non-salt-tolerant plant tolerance to salt stress has relevance. More resistant to protein differentially expressed by the reaction and found that the presence of protein is closely related to the expression changes its function. At the same time differentially expressed OsPP2Ac-4 Lan Sheng and 93-11, may provide clues to reveal salt pathway. This experiment found that the four PP2Ac protein expression changes in salt stress, and provide valuable clues for the understanding of the PP2Ac class phosphatase protein function in salt stress in rice and salt-tolerance mechanism.
研究目的:制备针对水稻OsPP2Ac基因编码的蛋白质的特异性抗体,了解重要蛋白质的表达模式进而探讨水稻耐盐的分子机理,为其功能研究累计数据。
材料与方法:对候选基因编码的蛋白质进行抗原决定簇的预测,选出抗原决定簇峰值较高的片段后,合成多肽偶联后免疫兔子制备多克隆抗体。用Western blotting检测OsPP2Ac蛋白质在水稻品种93-11与抗盐品种兰胜在苗期盐处理后不同时间点的表达变化以及在耐、受及对照反应中的差异表达,并与水稻MPSS数据库中叶片苗期的转录信息进行比较分析。
结果:发现在耐盐水稻品种兰胜中,OsPP2Ac-4的表达上调,在盐敏感的水稻品种93-11中,OsPP2Ac-2、OsPP2Ac-3和OsPP2Ac-5的表达也发生了上调,但OsPP2Ac-4的表达下调,比较二个品种间PP2Ac蛋白质的表达,发现在正常生长条件下,OsPP2Ac蛋白质的表达没有显著区别且基本保持恒定,其表达变化仅发生在盐胁迫条件下。分析水稻MPSS数据库提供的苗期盐胁迫的转录数据,发现OsPP2Ac-2、OsPP2Ac-3和OsPP2Ac-5在盐胁迫条件下转录水平下调。
结论:通过对OsPP2Ac在水稻品种兰胜及93-11盐胁迫处理过程中的表达及表型分析,发现大部分OsPP2Ac家族在耐盐材料兰胜中没有修饰表达,在93-11中盐处理后0.5h内出现高丰度表达,且随着处理时间的延长表达量逐渐增加,在48h达到峰值,在8天后植物死亡,说明PP2Ac的表达与盐胁迫导致的细胞程序性死亡具有关联性。通过比较耐、受反应的蛋白质差异表达,发现蛋白质的存在与表达量的变化与其功能密切相关。同时OsPP2Ac-4在兰胜与93-11中的差异表达,可能为揭示兰胜的耐盐通路提供线索。本实验发现了OsPP2Ac-2、OsPP2Ac-3、OsPP2Ac-4和OsPP2Ac-5,4个盐胁迫条件下表达发生变化的PP2Ac蛋白质,为了解PP2Ac类蛋白磷酸酶蛋白质在水稻盐胁迫过程中的功能及耐盐机理提供了有价值的线索。
Background: Rice is one of the most important food crops in world, one of the basic research model plants. Of large-scale production of rice caused by salt damage, the impact of salt stress, involving almost all plant physiological and biochemical processes, leading to serious plant can not grow normally, cuts or even crops. Against salt stress mediated salt adversity molecular mechanism of rice production is of great significance. The PP2Ac class of protein phosphatase (phosphatase 2A catalytic subunits, PP2Ac) is widespread in plants and animals have been shown that rice PP2Ac family members play an important role in the process of growth and development and resilience. Protein phosphatase catalyzed dephosphorylation reaction, so that protein phosphorylation and dephosphorylation, terminate the transfer of signaling molecules, and thereby regulate cell signaling signal transduction pathway. There is evidence that protein kinase and protein phosphatase as a pair of switch regulation of cell within the range of biochemical reactions.
Objective: Preparation of the corresponding specific antibodies against rice PP2Ac gene encodes a protein, and then explore the molecular mechanism of salt tolerance of rice for the understanding of an important protein expression patterns, as function of accumulated data.
Materials and Methods: The candidate gene encodes a protein epitope prediction, to elect the antigenic determinants of the fragment cluster peak higher, synthetic peptides coupled immune rabbit polyclonal antibody. Detected by Western Blotting PP2Ac genes encoding proteins in rice super hybrid rice parent varieties 93-11 and salt varieties Lan Sheng expression of seedling salt treatment at different time points after the change in resistance by the control reaction in the expression of differentially expressed and transcription of leaf seedling stage in rice MPSS database for comparative analysis.
Results: Found in the salt-tolerant rice varieties Lan OsPP2Ac-4 up regulated in salt-sensitive rice varieties 93-11 OsPP2Ac-2, OsPP2Ac-3 and OsPP2Ac-5 expression also occurred in the increase, but OsPP2Ac-4 down regulation of PP2Ac protein expression, comparison between the two species and found that PP2Ac protein expression in normal growth conditions, no significant difference remained constant, the expression change occurs only under salt stress. Seedling salt stress transcription data provided by the analysis of the rice MPSS database found OsPP2Ac-2, OsPP2Ac-3 and OsPP2Ac-5 transcription under salt stress levels down.
Conclusion: The expression and phenotypic analysis of rice varieties Lan Sheng and 93-11 salt stress on OsPP2Ac process, most OsPP2Ac family in no modification of the expression of salt-tolerant material Lan Sheng, 93-11 in Salt highly expressed within 0.5h, and with the treatment time was gradually increased, peaked at 48h, then the plants die, PP2Ac expression and non-salt-tolerant plant tolerance to salt stress has relevance. More resistant to protein differentially expressed by the reaction and found that the presence of protein is closely related to the expression changes its function. At the same time differentially expressed OsPP2Ac-4 Lan Sheng and 93-11, may provide clues to reveal salt pathway. This experiment found that the four PP2Ac protein expression changes in salt stress, and provide valuable clues for the understanding of the PP2Ac class phosphatase protein function in salt stress in rice and salt-tolerance mechanism.
引文
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[2] Blakeslee J J, Zhou H W, Heath J T, et al. Specificity of RCN1-Mediated Protein Phosphatase 2A Regulation in Meristem Organization and Stress Response in Roots[J]. Plant Physiology, 2007, 146(2): 539-553.
[3]何新建,陈建权,张志刚,等.通过cDNA微列阵鉴定水稻(Oryza sativa L.)盐胁迫应答基因[J].中国科学(C辑), 2002, 32(6): 488-493.
[4] Li X J, Yang M F, Chen H, et al. Abscisic acid pretreatment enhances salt tolerance of rice seedlings: proteomic evidence[J]. Biochim Biophys Acta, 2010, 1804(4): 929-940.
[5] Ouyang S Q, Liu Y F, Liu P, et al. Receptor-like kinase OsSIK1 improves drought and salt stress tolerance in rice (Oryza sativa) plants[J]. Plant J, 2010, 62(2): 316-329.
[6] Cohen P. The structure and regulation of protein phosphatases[J]. Annu Rev Biochem, 1989, 58: 453-508.
[7] Kloeker S. Parallel purification of three catalytic subunits of the protein serine/threonine phosphatase 2A family (PP2AC, PP4C, and PP6C) and analysis of the interaction of PP2AC with alpha4 protein[J]. Protein Expression and Purification, 2003, 31(1): 19-33.
[8] Luan S. Proteinphosphatases Inplants[J]. Annual Review of Plant Biology, 2003, 54(1): 63-92.
[9] Barford D. Molecular mechanisms of the protein serine/threonine phosphatases[J]. Trends Biochem Sci, 1996, 21(11): 407-412.
[10] Cohen P T. Novel protein serine/threonine phosphatases: variety is the spice of life[J]. Trends Biochem Sci, 1997, 22(7): 245-251.
[11] Terol J, Bargues M, Carrasco P, et al. Molecular characterization and evolution of the protein phosphatase 2A B' regulatory subunit family in plants[J]. Plant Physiol, 2002, 129(2): 808-822.
[12] Yu S, Lei H, Chang W, et al. Protein phosphatase 2A: identification in Oryza sativa of the gene encoding the regulatory A subunit[J]. Plant Mol Biol, 2001, 45(1): 107-112.
[13] Arino J, Perez-Callejon E, Cunillera N, et al. Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana[J]. Plant Mol Biol, 1993, 21(3): 475-485.
[14] Casamayor A, Perez-Callejon E, Pujol G, et al. Molecular characterization of a fourth isoform of the catalytic subunit of protein phosphatase 2A from Arabidopsis thaliana[J]. Plant Mol Biol, 1994, 26(1): 523-528.
[15] Yu R M K, Wong M M L, Jack R W, et al. Structure, evolution and expression of a second subfamily of protein phosphatase 2A catalytic subunit genes in the rice plant (Oryza sativa L.) [J]. Planta, 2005, 222(5): 757-768.
[16] Chang M, Wang B, Chen X, et al. Molecular characterization of catalytic-subunit cDNA sequences encoding protein phosphatases 1 and 2A and study of their roles in the gibberellin-dependent Osamy-c expression in rice[J]. Plant Mol Biol, 1999, 39(1): 105-115.
[17] Yu R M, Zhou Y, Xu Z F, et al. Two genes encoding protein phosphatase 2A catalytic subunits are differentially expressed in rice[J]. Plant Mol Biol, 2003, 51(3): 295-311.
[18] Papdi C, Abraham E, Joseph M P, et al. Functional identification of Arabidopsis stress regulatory genes using the controlled cDNA overexpression system[J]. Plant Physiol, 2008, 147(2): 528-542.
[19] He X, Anderson J C, del Pozo O, et al. Silencing of subfamily I of protein phosphatase 2A catalytic subunits results in activation of plant defense responses and localized cell death[J]. Plant J, 2004, 38(4): 563-577.
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