甘蓝型油菜BnaCIPK15基因的表达分析与功能鉴定
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摘要
CBL-CIPK是高等植物中广泛存在的一类解析Ca~(2+)信号的蛋白。该研究在前期工作基础上,对甘蓝型油菜(Brassica napus L.)的BnaCIPK15基因进行了亚细胞定位、双分子荧光互补(BiFC)、酵母双杂交和qRT-PCR检测等一系列分析,以探究BnaCIPK15蛋白在ABA激素响应中的作用。结果显示:(1)亚细胞定位发现,BnaCIPK15蛋白定位于细胞质和细胞核中; BiFC分析发现,BnaCIPK15蛋白与BnaCBL1/3/4/9蛋白之间的互作较强,与BnaCBL10仅有微弱互作。(2)qRT-PCR检测发现,BnaCIPK15基因受ABA和冷胁迫的诱导极显著上调表达,而对百草枯(Paraquat)、活性氧(H_2O_2)和热胁迫的诱导较弱,表明BnaCIPK15基因很可能参与ABA和冷胁迫的调控过程。(3)酵母滴定实验结果显示,BnaCIPK15蛋白与脱落酸(ABA)信号通路中的BnaHAB1蛋白(属于蛋白磷酸酶PP2C家族)存在明显的互作,而与BnaABFs/AREB3/ABI5转录因子无明显互作;BiFC验证显示,BnaCIPK15与BnaHAB1蛋白之间存在互作信号,而BnaCIPK15与BnaHAB2组合没有观察到信号,证明BnaCIPK15与BnaHAB1磷酸酶具有特异互作特征,推测BnaCIPK15可能参与调控ABA信号转导。研究认为,甘蓝型油菜中可能存在基于BnaCIPK15-BnaHAB1的互作模块,并参与ABA的信号转导和网络调控。
CBL-CIPKs are universal proteins in higher plants that decode Ca~(2+) signals. Base on the previous work, a series of investigations of BnaCIPK15 gene in Brassica napus L., including subcellular localization, bimolecular fluorescence complementation(BiFC), yeast two-hybrid(Y2 H) and qRT-PCR, were carried out to explore its role in ABA hormone response.(1) Subcellular localization result showed that BnaCIPK15 protein was localized in the cytoplasm and nucleus. BiFC assay demonstrated that BnaCIPK15 interacted strongly with BnaCBL1/3/4/9 proteins, and interacted weakly with BnaCBL10 protein as well.(2) Using qRT-PCR, the expression pattern of BnaCIPK15 gene was examined under various phytohormone and abiotic stress treatments, and the results revealed that abscisic acid(ABA) and cold stress significantly induced its expression. Moreover, a mild up-regulation of BnaCIPK15 gene was also observed by paraquat, reactive oxygen species(H_2O_2) burst and heat stress treatments, suggesting that BnaCIPK15 gene may be involved in the regulation of ABA and cold stress.(3) Y2 H assay discovered that BnaCIPK15 protein interacted with BnaHAB1, which belongs to the protein phosphatase type 2 C family negatively regulating ABA signaling pathway, and did not show any evident interaction with BnaABFs/AREB3/ABI5 transcription factors. At the same time, BiFC assay also showed that there were interaction signals between BnaCIPK15 and BnaHAB1 proteins, rather than BnaHAB2 protein, which proved that they had specific interaction, suggesting that BnaCIPK15 protein might be involved in regulating ABA signal transduction. In conclusion, this study identified that BnaCIPK15-BnaHAB1 module may be involved in ABA signal transduction and regulatory network in Brassica napus.
CBL-CIPKs are universal proteins in higher plants that decode Ca~(2+) signals. Base on the previous work, a series of investigations of BnaCIPK15 gene in Brassica napus L., including subcellular localization, bimolecular fluorescence complementation(BiFC), yeast two-hybrid(Y2 H) and qRT-PCR, were carried out to explore its role in ABA hormone response.(1) Subcellular localization result showed that BnaCIPK15 protein was localized in the cytoplasm and nucleus. BiFC assay demonstrated that BnaCIPK15 interacted strongly with BnaCBL1/3/4/9 proteins, and interacted weakly with BnaCBL10 protein as well.(2) Using qRT-PCR, the expression pattern of BnaCIPK15 gene was examined under various phytohormone and abiotic stress treatments, and the results revealed that abscisic acid(ABA) and cold stress significantly induced its expression. Moreover, a mild up-regulation of BnaCIPK15 gene was also observed by paraquat, reactive oxygen species(H_2O_2) burst and heat stress treatments, suggesting that BnaCIPK15 gene may be involved in the regulation of ABA and cold stress.(3) Y2 H assay discovered that BnaCIPK15 protein interacted with BnaHAB1, which belongs to the protein phosphatase type 2 C family negatively regulating ABA signaling pathway, and did not show any evident interaction with BnaABFs/AREB3/ABI5 transcription factors. At the same time, BiFC assay also showed that there were interaction signals between BnaCIPK15 and BnaHAB1 proteins, rather than BnaHAB2 protein, which proved that they had specific interaction, suggesting that BnaCIPK15 protein might be involved in regulating ABA signal transduction. In conclusion, this study identified that BnaCIPK15-BnaHAB1 module may be involved in ABA signal transduction and regulatory network in Brassica napus.
引文
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[14] HUANG C L,DING S,ZHANG H,et al.CIPK7 is involved in cold response by interacting with CBL1 in Arabidopsis thaliana[J].Plant Science,2011,181(1):57-64.
[15] XU J,LI H D,CHEN L Q,et al.A protein kinase,interacting with two calcineurin B-like proteins,regulates K+ transporter AKT1 in Arabidopsis[J].Cell,2006,125(7):1 347-1 360.
[16] LI L,KIM B G,CHEONG Y H,et al.A Ca2+ signaling pathway regulates a K+ channel for low-K response in Arabidopsis[J].Proceedings of the National Academy of Sciences,2006,103(33):12 625-12 630.
[17] CHEN L,REN F,ZHOU L,et al.The Brassica napus calcineurin B-Like 1/CBL-interacting protein kinase 6 (CBL1/CIPK6) component is involved in the plant response to abiotic stress and ABA signalling[J].Journal of Experimental Botany,2012,63(17):6 211-6 222.
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[20] ZHOU X N,HAO H M,ZHANG Y G,et al.SOS2-LIKE PROTEIN KINASE5,an SNF1-RELATED PROTEIN KINASE3-type protein kinase,is important for abscisic acid responses in Arabidopsis through phosphorylation of ABSCISIC ACID-INSENSITIVE5[J].Plant Physiology,2015,168(2):659-676.
[21] MA Q J,SUN M H,LU J,et al.An apple CIPK protein kinase targets a novel residue of AREB transcription factor for ABA-dependent phosphorylation[J].Plant,Cell & Environment,2017,40(10):2 207-2 219.
[22] HIRAYAMA T,SHINOZAKI K.Perception and transduction of abscisic acid signals:keys to the function of the versatile plant hormone ABA[J].Trends in Plant Science,2007,12(8):343-351.
[23] MYO T,刘芃,马微,等.小麦CBL结合蛋白激酶基因TaCIPK16的克隆及特征分析[J].西北植物学报,2016,36(6):1 073-1 079.MYO T,LIU P,MA W,et al.Characterization of CBL-interacting protein kinase gene TaCIPK16 in wheat[J].Acta Botanica Boreali-Occidentalia Sinica,2016,36(6):1 073-1 079.
[24] 范晓江,郭小华,牛芳芳,等.拟南芥WRKY61转录因子的转录活性与互作蛋白分析[J].西北植物学报,2018,38(1):1-8.FAN X J,GUO X H,NIU F F,et al.Exploring the transcriptional activity and interacting proteins of WRKY61 transcriptional factor in Arabidopsis thaliana[J].Acta Botanica Boreali-Occidentalia Sinica,2018,38(1):1-8.
[25] SANYAL S K,PANDEY A,PANDEY G K.The CBL-CIPK signaling module in plants:A mechanistic perspective[J].Physiologia Plantarum,2015,155(2):89-108.
[2] MACROBBIE E A.ABA activates multiple Ca2+ fluxes in stomatal guard cells,triggering vacuolar K+ (Rb+) release[J].Proceedings of the National Academy of Sciences,2000,97(22):12 361-12 368.
[3] SANDERS D,BROWNLEE C,HARPER J F.Communicating with calcium[J].The Plant Cell,1999,11(4):691-706.
[4] EVANS N H,MCAINSH M R,HETHERINGTON A M.Calcium oscillations in higher plants[J].Current Opinion in Plant Biology,2001,4(5):415-420.
[5] HARMON A C,GRIBSKOV M,GUBRIUM E,et al.The CDPK superfamily of protein kinases[J].New Phytologist,2001,151(1):175-183.
[6] LUAN S,KUDLA J,RODRIGUEZ-CONCEPCION M,et al.Calmodulins and calcineurin B-like proteins:Calcium sensors for specific signal response coupling in plants[J].The Plant Cell,2002,14(Suppl):S389-S400.
[7] CHENG S H.Calcium signaling through protein kinases.The Arabidopsis calcium-dependent protein kinase gene family[J].Plant Physiology,2002,129(2):469-485.
[8] SHI J,KIM K N,RITZ O,et al.Novel protein kinases associated with calcineurin B-like calcium sensors in Arabidopsis[J].The Plant Cell,1999,11(12):2 393-2 405.
[9] ALBRECHT V,RITZ O,LINDER S,et al.The NAF domain defines a novel protein-protein interaction module conserved in Ca2+ -regulated kinases[J].The EMBO Journal,2001,20(5):1 051-1 063.
[10] ZHANG H F,YANG B,LIU W Z,et al.Identification and characterization of CBL and CIPK gene families in canola (Brassica napus L.)[J].BMC Plant Biology,2014,14:8.
[11] KOLUKISAOGLU U,WEINL S,BLAZEVIC D,et al.Calcium sensors and their interacting protein kinases:Genomics of the Arabidopsis and rice CBL-CIPK signaling networks[J].Plant Physiology,2004,134(1):43-58.
[12] SONG C P,AGARWAL M,OHTA M,et al.Role of an Arabidopsis AP2/EREBP-type transcriptional repressor in abscisic acid and drought stress responses[J].The Plant Cell,2005,17(8):2 384-2 396.
[13] LIU J,ISHITANI M,HALFTER U,et al.The Arabidopsis thaliana SOS2 gene encodes a protein kinase that is required for salt tolerance[J].Proceedings of the National Academy of Sciences,2000,97(7):3 730-3 734.
[14] HUANG C L,DING S,ZHANG H,et al.CIPK7 is involved in cold response by interacting with CBL1 in Arabidopsis thaliana[J].Plant Science,2011,181(1):57-64.
[15] XU J,LI H D,CHEN L Q,et al.A protein kinase,interacting with two calcineurin B-like proteins,regulates K+ transporter AKT1 in Arabidopsis[J].Cell,2006,125(7):1 347-1 360.
[16] LI L,KIM B G,CHEONG Y H,et al.A Ca2+ signaling pathway regulates a K+ channel for low-K response in Arabidopsis[J].Proceedings of the National Academy of Sciences,2006,103(33):12 625-12 630.
[17] CHEN L,REN F,ZHOU L,et al.The Brassica napus calcineurin B-Like 1/CBL-interacting protein kinase 6 (CBL1/CIPK6) component is involved in the plant response to abiotic stress and ABA signalling[J].Journal of Experimental Botany,2012,63(17):6 211-6 222.
[18] CUTLER S R,RODRIGUEZ P L,FINKELSTEIN R R,et al.Abscisic acid:emergence of a core signaling network[J].Annual Review of Plant Biology,2010,61:651-679.
[19] PANDEY G K,GRANT J J,CHEONG Y H,et al.Calcineurin-B-like protein CBL9 interacts with target kinase CIPK3 in the regulation of ABA response in seed germination[J].Molecular Plant,2008,1(2):238-248.
[20] ZHOU X N,HAO H M,ZHANG Y G,et al.SOS2-LIKE PROTEIN KINASE5,an SNF1-RELATED PROTEIN KINASE3-type protein kinase,is important for abscisic acid responses in Arabidopsis through phosphorylation of ABSCISIC ACID-INSENSITIVE5[J].Plant Physiology,2015,168(2):659-676.
[21] MA Q J,SUN M H,LU J,et al.An apple CIPK protein kinase targets a novel residue of AREB transcription factor for ABA-dependent phosphorylation[J].Plant,Cell & Environment,2017,40(10):2 207-2 219.
[22] HIRAYAMA T,SHINOZAKI K.Perception and transduction of abscisic acid signals:keys to the function of the versatile plant hormone ABA[J].Trends in Plant Science,2007,12(8):343-351.
[23] MYO T,刘芃,马微,等.小麦CBL结合蛋白激酶基因TaCIPK16的克隆及特征分析[J].西北植物学报,2016,36(6):1 073-1 079.MYO T,LIU P,MA W,et al.Characterization of CBL-interacting protein kinase gene TaCIPK16 in wheat[J].Acta Botanica Boreali-Occidentalia Sinica,2016,36(6):1 073-1 079.
[24] 范晓江,郭小华,牛芳芳,等.拟南芥WRKY61转录因子的转录活性与互作蛋白分析[J].西北植物学报,2018,38(1):1-8.FAN X J,GUO X H,NIU F F,et al.Exploring the transcriptional activity and interacting proteins of WRKY61 transcriptional factor in Arabidopsis thaliana[J].Acta Botanica Boreali-Occidentalia Sinica,2018,38(1):1-8.
[25] SANYAL S K,PANDEY A,PANDEY G K.The CBL-CIPK signaling module in plants:A mechanistic perspective[J].Physiologia Plantarum,2015,155(2):89-108.