Calcium-dependent protein kinases (CDPKs or CPKs) are important calcium sensors in higher plants, which are extensively involved in plant development and stress responding. In this study, one sequence that contained a complete ORF of 1 611 bp encoding a 568 amino acids protein was cloned from Camellia sinensis cv.
Longjing 43. Sequence alignments revealed that this protein was a typical plant CDPK possesses N-terminus myristoylation site and protein kinases domain and showed the highest similarity with Arabidopsis AtCDPK17. Thus, the gene was defined as CsCDPK17 base on further phylogenetic analysis (Genbank accession No. MK238482). Basic protein character analysis shows that CsCDPK17 was a hydrophilic membrane-binding protein with molecularweight of 59.9 kD and PI of 5.43. Further subcellular localization assay using transient CsCDPK17-GFP expression in rice protoplasts and tobacco leaves proved that CsCDPK17 was localized in plasma membrane and nucleus. A series of gene transcription, light and hormone (such as ABA, SA, MeJA, etc) responding related cis-elements were detected in CsCDPK17 2 000 bp promoter regions. Tissue-specific expression analysis found that high expressions of CsCDPK17 were in the mature leaves and seeds, while the lowest transcription in roots. The transcription of CsCDPK17 was increased during cold acclimation and decreased during de-acclimation procedures in four cultivars with different cold resistance abilities. Moreover, stress induced expression indicated that CsCDPK17 could be induced by cold, drought and osmotic stresses with the highest induction levels of 5.1, 2.3 and 2.4 folds, respectively. Overall, our results suggest that the newly cloned CsCDPK17 might be involve in the regulation of both development and abiotic stress responses (such as cold, drought and osmotic stress) in tea plants.
[1] Valmonte G R, Arthur K, Higgins C M, et al. Calcium-dependent protein kinases in plants: evolution, expression and function [J]. Plant Cell Physiol, 2014, 55(3): 551-569.
[2] Bagur R, Hajnoczky G. Intracellular Ca2+ sensing: its role in calcium homeostasis and signaling [J]. Mol Cell, 2017, 66(6): 780-788.
[3] Dupont G, Sneyd J. Recent developments in models of calcium signalling [J]. Current Opinion in Systems Biology, 2017, 3: 15-22.
[4] Schulz P, Herde M, Romeis T. Calcium-dependent protein kinases: hubs in plant stress signaling and development [J]. Plant Physiol, 2013, 163(2): 523-530.
[5] Shi S, Li S, Asim M, et al. The Arabidopsis Calcium-dependent protein kinases (CDPKs) and their roles in plant growth regulation and abiotic stress responses [J]. International Journal of Molecular Sciences 2018, 19(7). Doi: 10.3390/ijms19071900.
[6] Cheng S H, Willmann M R, Chen H C, et al. Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family [J]. Plant Physiol, 2002, 129(2): 469-485.
[7] Ray S, Agarwal P, Arora R, et al. Expression analysis of calcium-dependent protein kinase gene family during reproductive development and abiotic stress conditions in rice (Oryza sativa L. ssp. indica) [J]. Mol Genet Genomics, 2007, 278(5): 493-505.
[8] Zuo R, Hu R, Chai G, et al. Genome-wide identification, classification, and expression analysis of CDPK and its closely related gene families in poplar (Populus trichocarpa) [J]. Mol Biol Rep, 2013, 40(3): 2645-2662.
[9] Mittal S, Mallikarjuna M G, Rao A R, et al. Comparative Analysis of CDPK Family in Maize, Arabidopsis, rice, and sorghum revealed potential targets for drought tolerance improvement [J]. Front Chem, 2017, 5: 115. Doi: 10.3389/fchem.2017.00115.
[10] 张成才. 茶树育性相关基因的克隆与表达研究[D]. 武汉: 华中农业大学, 2017.
[11] Wang M, Li Q, Sun K, et al. Involvement of CsCDPK20 and CsCDPK26 in regulation of thermotolerance in tea plant (Camellia sinensis) [J]. Plant Molecular Biology Reporter, 2018, 36(2): 176-187.
[12] Li A L, Zhu Y F, Tan X M, et al. Evolutionary and functional study of the CDPK gene family in wheat (Triticum aestivum L.) [J]. Plant Molecular Biology, 2008, 66(4): 429-443.
[13] Komatsu S, Yang G, Khan M, et al. Over-expression of calcium-dependent protein kinase 13 and calreticulin interacting protein 1 confers cold tolerance on rice plants [J]. Molecular Genetics & Genomics, 2007, 277(6): 713-723.
[14] Jiang S, Zhang D, Wang L, et al. A maize calcium-dependent protein kinase gene, ZmCPK4, positively regulated abscisic acid signaling and enhanced drought stress tolerance in transgenic Arabidopsis [J]. Plant Physiology and Biochemistry, 2013, 71: 112-120.
[15] Franz S, Ehlert B, Liese A, et al. Calcium-dependent protein kinase CPK21 functions in abiotic stress response in Arabidopsis thaliana [J]. Mol Plant, 2011, 4(1): 83-96.
[16] 林郑和, 钟秋生, 游小妹, 等. 低温胁迫对茶树抗氧化酶活性的影响[J]. 茶叶科学, 2018, 38(4): 363-371.
[17] 孙海伟, 曹德航, 尚涛, 等. 茶树抗寒育种及转基因研究进展[J]. 山东农业科学, 2013, 45(6): 119-122.
[18] Wei C, Yang H, Wang S, et al. Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality [J]. Proceedings of the National Academy of Sciences, 2018, 115(18): E4151-E4158.
[19] Hao X, Horvath D, Chao W, et al. Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze) [J]. International Journal of Molecular Sciences, 2014, 15(12): 22155-22172.
[20] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2–Δ?Ct method [J]. Methods, 2001, 25(4): 402-408.
[21] Liu Y, Xu C, ZhuY, et al. The calcium-dependent kinase OsCPK24 functions in cold stress responses in rice [J]. J Integr Plant Biol, 2018, 60(2): 173-188.
[22] Wang X, Zhao Q, Ma C, et al. Global transcriptome profiles of Camellia sinensis during cold acclimation [J]. Bmc Genomics, 2013, 14(1): 415. https://doi.org/10.1186/1471-2164-14-415.
[23] Rutschmann F, Stalder U, Piotrowski M, et al. LeCPK1, a calcium-dependent protein kinase from tomato. Plasma membrane targeting and biochemical characterization [J]. Plant Physiol, 2002, 129(1): 156-168.
[24] 丁玉娇, 韩颖颖, 周婧雯. 棕榈酰化蛋白及蛋白质的棕榈酰化研究进展[J]. 亚热带植物科学, 2018, 47(4): 43-50.
[25] Mart??n M L, Busconi L. Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated by myristoylation and palmitoylation [J]. The Plant Journal, 2000, 24(4): 429-435.
[26] T Gutermuth, R Lassig, MT Porteset, et al. Pollen tube growth regulation by free anions depends on the interaction between the anion channel SLAH3 and calcium-dependent protein kinases CPK2 and CPK20 [J]. Plant Cell, 2013, 25(11): 4525-4543.
[27] Xu X, Liu M, Lu L, et al. Genome-wide analysis and expression of the calcium-dependent protein kinase gene family in cucumber [J]. Mol Genet Genomics, 2015, 290(4): 1403-1414.
[28] Zhao L N, Shen L K, Zhang W Z, et al. Ca2+-dependent protein kinase11 and 24 modulate the activity of the inward rectifying K+ channels in Arabidopsis pollen tubes [J]. Plant Cell, 2013, 25(2): 649-661.
[29] Zou J J, Wei F J, Wang C, et al. Arabidopsis calcium-dependent protein kinase CPK10 functions in abscisic acid- and Ca2+-mediated stomatal regulation in response to drought stress [J]. Plant Physiol, 2010, 154(3): 1232-1243.
[30] Kobayashi M, Yoshioka M, Asai S, et al. StCDPK5 confers resistance to late blight pathogen but increases susceptibility to early blight pathogen in potato via reactive oxygen species burst [J]. New Phytol, 2012, 196(1): 223-237.
[31] Asano T, Hayashi N, Kobayashi M, et al. A rice calcium-dependent protein kinase OsCPK12 oppositely modulates salt-stress tolerance and blast disease resistance [J]. Plant J, 2012, 69(1): 26-36.
浙公网安备 33019902000101号 
