Abstract Calcineurin B-like interacting protein kinase (CIPK) is a class of serine/threonine (Ser/Thr) protein kinases widely existing in plants that are involved in decoding calcium signals. CIPKs play important roles in abiotic and biotic stress signal transduction. In this study, BnaCIPK2 and BnaCIPK16 genes were successfully cloned by reverse transcription-polymerase chain reaction (RT-PCR) from oilseed rape (Brassica napus). Sequence analysis showed that the 2 proteins encoded by the 2 BnaCIPK genes contained conserved kinase domain and asparagine-alanine-phenylalanine (NAF) motif. The subcellular localization analysis showed that both BnaCIPK2 and BnaCIPK16 were localized in both cytoplasm and nuclei. qRT-PCR profiling at 3 time points revealed that jasmonic acid (JA), cold and polyethylene glycol (PEG8000) significantly induced the expression of BnaCIPK2. After methyl viologen (MV) treatment for 24 h, the expression of BnaCIPK2 was inhibited. Salicylic acid (SA), heat, cold, hydrogen peroxide (H2O2) and PEG8000 up-regulated the expression of BnaCIPK16, while JA and abscisic acid (ABA) treatments resulted in the repression of BnaCIPK16 expression. Yeast two-hybrid (Y2H) screening and bimolecular fluorescence complementation (BiFC) showed that BnaCIPK2 and BnaCIPK16 interacted with Brassica napus calcineurin B-like protein 4 (BnaCBL4) and BnaCBL3, respectively. The present study provides basic information for elucidating the functions and regulatory mechanisms of BnaCIPK2 and BnaCIPK16.
LI Yan-Fei,XIN Yu-Qiong,GAO Shi-Dong, et al. Cloning, Expression Analysis and Interacting Protein CBL Identification of CIPK2 and CIPK16 Genes in Oilseed Rape (Brassica napus)[J]. 农业生物技术学报, 2020, 28(1): 1-12.
[1] 李琪, 李烨, 牛芳芳, 等. 2019. 拟南芥转录因子基因WRKY72的特性分析及其抗逆功能鉴定[J]. 农业生物技术学报, 27(2): 191-203. (Li Q, Li Y, Niu F F, et al.2019. Characterization and stress resistance identification of WRKY72 gene in Arabidopsis thaliana[J]. Journal of Agricultural Biotechnology, 27(2): 191-203.) [2] 申威, 姚文孔, 郑巧玲, 等. 2019. 山葡萄VaCBL01基因克隆及其与VaCIPKs蛋白互作分析[J]. 农业生物技术学报, 27(7): 1149-1160. (Shen W, Yao W K, Zheng Q L, et al.2019. Cloning of VaCBL01 gene from Vitis amurensis and analysis of its interaction with VaCIPKs proteins[J]. Journal of Agricultural Biotechnology, 27(7): 1149-1160.) [3] 王汉中, 殷艳. 2014. 我国油料产业形势分析与发展对策建议[J]. 中国油料作物学报, 36(3): 414-421. (Wang H Z, Yin Y.2014. Analysis and strategy for oil crop industry in China[J]. Chinese Journal of Oil Crop Sciences, 36(3): 2414-2421.) [4] 肖振, 赵琪, 张川芳. 2016. 蛋白质组学研究揭示的甘蓝型油菜非生物胁迫应答机制[J]. 植物科学学报, 34(6): 949-961. (Xiao Z, Zhao Q, Zhang C F.2016. Abiotic stress response mechanism of oilseed rape (Brassica napus L.) revealed from proteomics[J]. Plant Science Journal, 34(6): 2949-2961.) [5] 朱婷婷, 王彦霞, 裴丽丽, 等. 2017. 植物蛋白激酶与作物非生物胁迫抗性的研究[J]. 植物遗传资源学报, 18(4): 763-770. (Zhu T T, Wang Y X, Pei L L, et al.2017. Research progress of plant protein kinase and abiotic stress resistance[J]. Journal of Plant Genetic Resources, 18(2014): 763-770.) [6] Albrecht V, Ritz O, Linder S, et al.2001. The NAF domain defines a novel protein-protein interaction module conserved in Ca2+-regulated kinases[J]. The Embo Journal, 20(5): 1051-1063. [7] Amarasinghe S, Watson-Haigh N S, Gilliham M, et al.2016. The evolutionary origin of CIPK16: A gene involved in enhanced salt tolerance[J]. Molecular Phylogenetics and Evolution, 100: 135-147. [8] Batistic O, Kudla J.2004. Integration and channeling of calcium signaling through the CBL calcium sensor/CIPK protein kinase network[J]. Planta, 219(6): 915-924. [9] Batistic O, Kudla J.2012. Analysis of calcium signaling pathways in plants[J]. Biochimica et Biophysica Acta, 1820(8): 1283-1293. [10] Chen L, Ren F, Zhou L, et al.2012. 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, 63(17): 6211-6222. [11] Cheong Y H, Pandey G K, Grant J J, et al.2007. Two Calcineurin B-like calcium sensors, interacting with protein kinase CIPK23, regulate leaf transpiration and root potassium uptake in Arabidopsis[J]. The Plant Journal, 52: 223-239. [12] Guo Y, Huang Y, Gao J, et al.2018. CIPK9 is involved in seed oil regulation in Brassica napus L. and Arabidopsis thaliana (L.) Heynh[J]. Biotechnology for Biofuels, 11: 124. [13] Ho C, Lin S, Hu H, et al.2009. CHL1 functions as a nitrate sensor in plants[J]. Cell, 138(6): 1184-1194. [14] Kim B G, Waadt R, Cheong Y H, et al.2007. The calcium sensor CBL10 mediates salt tolerance by regulating ion homeostasis in Arabidopsis[J]. The Plant Journal, 52(3): 473-484. [15] Kolukisaoglu U, Weinl S, Blazevic D, et al.2004. Calcium sensors and their interacting protein kinases: Genomics of the Arabidopsis and rice CBL-CIPK signaling networks[J]. Plant Physiology, 134(1): 43-58. [16] Kudla J, Xu Q, Harter K, et al.1999. Genes for calcineurin B-like proteins in Arabidopsis are differentially regulated by stress signals[J]. Proceedings of the National Academy of Sciences of the USA, 96(8): 4718-4723. [17] Lan W Z, Lee S C, Che Y F, et al.2011. Mechanistic analysis of AKT1 regulation by the CBL-CIPK-PP2CA interactions[J]. Molecular Plant, 4(3): 527-536. [18] Lee S C, Lan W Z, Kim B G, et al.2007. A protein phosphorylation/dephosphorylation network regulates a plant potassium channel[J]. Proceedings of the National Academy of Sciences of the USA, 104(40): 15959-15964. [19] Liu P, Guo J, Zhang R, et al.2019. TaCIPK10 interacts with and phosphorylates TaNH2 to activate wheat defense responses to stripe rust[J]. Plant Biotechnology Journal, 17(5): 956-968. [20] Liu W Z, Deng M, Li L, et al.2015. Rapeseed calcineurin B-like protein CBL4, interacting with CBL-interacting protein kinase CIPK24, modulates salt tolerance in plants[J]. Biochemical Biophysical Research Communications, 467(3): 467-471. [21] Luan S, Lan W, Chul Lee S.2009. Potassium nutrition, sodium toxicity, and calcium signaling: Connections through the CBL-CIPK network[J]. Current Opinion in Plant Biology, 12(3): 339-346. [22] Ma Q J, Sun M H, Kang H, et al.2019. A CIPK protein kinase targets sucrose transporter MdSUT2.2 at Ser254 for phosphorylation to enhance salt tolerance[J]. Plant, Cell & Environment, 42(3): 918-930. [23] Ma Q J, Sun M H, Lu J, et al.2017. An apple CIPK protein kinase targets a novel residue of AREB transcription factor for ABA-dependent phosphorylation[J]. Plant, Cell & Environment, 40(10): 2207-2219. [24] Pfaffl M W.2001. A new mathematical model for relative quantification in real-time RT-PCR[J]. Nucleic Acids Research, 29(9): e45. [25] Qiu Q S, Guo Y, Quintero F J, et al.2004. Regulation of vacuolar Na+/H+ exchange in Arabidopsis thaliana by the salt-overly-sensitive (SOS) pathway[J]. Journal of Biological Chemistry, 279(1): 207-215. [26] Quan R, Lin H, Mendoza I, et al.2007. SCABP8/CBL10, a putative calcium sensor, interacts with the protein kinase SOS2 to protect Arabidopsis shoots from salt stress[J]. The Plant Cell, 19(4): 1415-1431. [27] Roy S J, Huang W, Wang X J, et al.2013. A novel protein kinase involved in Na(+) exclusion revealed from positional cloning[J]. Plant Cell & Environment, 36(3): 553-568. [28] Sanyal S K, Kanwar P, Yadav A K, et al.2017. Arabidopsis CBL interacting protein kinase 3 interacts with ABR1, an APETALA2 domain transcription factor, to regulate ABA responses[J]. Plant Science, 254: 48-59. [29] Shi J, Kim K N, Ritz O, et al.1999. Novel protein kinases associated with calcineurin B-like calcium sensors in Arabidopsis[J]. The Plant Cell, 11(12): 2393-2405. [30] Shigetaka Y, Shoki A, Yoko H, et al.2017. Arabidopsis CIPKs regulate carbon nitrogen-nutrient response by phosphorylating ubiquitin ligase ATL31[J]. Molecular Plant, 10: 618. [31] Weinl S, Kudla J.2009. The CBL-CIPK Ca(2+)-decoding signaling network: Function and perspectives[J]. New Phytologist, 184(3): 517-528. [32] Xiang Y, Huang Y, Xiong L.2007. Characterization of stress-responsive CIPK genes in rice for stress tolerance improvement[J]. Plant Physiology, 144(3): 1416-1428. [33] Yan J, Tong T, Li X, et al.2018. A novel NAC-type transcription factor, NAC87, from oilseed rape modulates reactive oxygen species accumulation and cell death[J]. Plant and Cell Physiology, 59(2): 290-303. [34] Zhang H, Yang B, Liu W Z, et al.2014. Identification and characterization of CBL and CIPK gene families in canola (Brassica napus L.)[J]. BMC Plant Biology, 14: 8.
