【摘要】 磷酸核酮糖激酶(phosphoribulokinase, PRK)是光合反应卡尔文-本森循环中的关键酶，影响植物的生长发育。但在水稻(Oryza sativa)中对PRK在逆境胁迫响应中的功能研究报道较少。本研究开展了OsPRK (GenBank No. LOC4330413)蛋白亚细胞定位、胁迫(干旱,高盐,高温)与激素(脱落酸,茉莉酸,水杨酸)处理后基因的表达分析及互作蛋白筛选，研究OsPRK在水稻逆境胁迫响应中的功能。瞬时表达实验表明，OsPRK定位于水稻原生质体叶绿体基质中。qPCR结果发现，水稻幼苗在干旱胁迫处理0.5与3 h时及高盐、高温处理1与6 h时，OsPRK的转录与表达受抑制。外源脱落酸和茉莉酸处理1 h时，幼苗OsPRK转录水平未受明显影响，但处理4 h后，随处理时间的延长OsPRK的转录水平持续降低；水杨酸处理过程中，OsPRK的转录水平在0～8 h持续急剧下降，其后缓慢下降趋势一直持续至24 h。利用蛋白互作MBP标签融合蛋白沉降(MBP pull-down)技术筛选到了82个与OsPRK存在互作关系的候选蛋白，其中67个候选蛋白为未知功能蛋白，15个为已知功能蛋白，包括糖酵解关键酶甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)、抗病相关R蛋白(resistance proteins)和WRKY转录因子(WRKY transcription factor)。本研究从转录水平明确了OsPRK参与逆境胁迫与激素应答，并鉴定到多个与OsPRK可能互作的调控蛋白，为后续深入解析该基因在逆境胁迫中的生物学功能提供理论依据。更多还原

【Abstract】 Phosphoribulokinase(PRK) is a key enzyme in Calvin-Benson cycle which influences on plant growth and development. Nuclear-encoded plastid-localized PRK, coupling with other regulatory enzyme,involved in carbon assimilation in functional leaves. However, the research on RPK functions in stress response was still limited, especially in rice(Oryza sativa). In this study, target protein subcellular localization,expression characteristics analyses of rice phosphoribulokinase(OsPRK, GenBank No. LOC4330413) under abiotic stresses(drought, high salt and high temperature) and exogenous hormone treatments(abscisic acid(ABA), jasmonic acid(JA) and salicylic acid(SA)), as well as MBP pull-down protein interaction screening were conducted to investigate the function of OsPRK involving in stress responses. By detecting the GFP report using laser confocal microscopy, the transient expression analysis demonstrated that OsPRK was strictly localized in chloroplast stroma of transformed rice protoplasts after 16 h protoplast culture, contrasting to control native GFP only found around the cytoplasm of protoplast. To characterize the change of the transcript abundance of OsPRK, quantitative realtime PCR(qPCR) was performed and found that the transcriptional expression of OsPRK was significantly inhibited at 0.5 and 3 h drought treatments, 1 and 6 h high salt or high temperature treatments, which implicated that OsPRK inclined to reduce transcriptional expression in drought,high salt and high temperature conditions. Beyond that, qPCR results also showed that the OsPRK transcription levels of seedlings under ABA and JA treatments were not significantly changed within 1 h,while continuously decreased when treated more than 4 h. Unlike ABA and JA treatment, the transcription level of OsPRK of SA treatment had a dramatical reduction from 0 to 8 h, and then gradually declined until 24h. To investigate the potential OsPRK interacting proteins, OsPRK was expressed with MBP-Tag using recombinant plasmid pMAL-c5x and purified from BL21(DE3)plys prokaryotic expression system for pulldown protein interaction screening. The protein interaction experiment totally obtained 82 candidate proteins that might interact with OsPRK using MBP pull-down method. Of these, the functions of 67 candidate proteins were unknown while 15 candidate proteins were with known functions, including GAPDH(glyceraldehyde-3-phosphate dehydrogenase) and WRKY transcription factor. This study found that OsPRK should be involved in abiotic stress and hormone response of rice seedlings. In addition, the study identified numbers of potential OsPRK interacting proteins, which can be used for further biological functions of OsPRK in stress avoidance and tolerance. All these findings accelerated the understanding of OsPRK roles in response to developmental requirements and environmental constraints.更多还原