茶树钙依赖性蛋白激酶基因CsCDPK17的鉴定及表达分析

LEI Lei; WANG Lu; YAO Lina; HAO Xinyuan; ZENG Jianming; DING Changqing*; WANG Xinchao*; YANG Yajun

doi:10.13305/j.cnki.jts.2019.03.004

PDF(2055 KB)

茶叶科学 ›› 2019, Vol. 39 ›› Issue (3) : 267-279. DOI: 10.13305/j.cnki.jts.2019.03.004

茶树钙依赖性蛋白激酶基因CsCDPK17的鉴定及表达分析

雷蕾，王璐，姚丽娜，郝心愿，曾建明，丁长庆*，王新超*，杨亚军

作者信息 +

Identification and Expression Analysis of Calcium-dependent Protein Kinase CsCDPK17 in Tea Plant (Camellia sinensis)

LEI Lei, WANG Lu, YAO Lina, HAO Xinyuan, ZENG Jianming, DING Changqing*, WANG Xinchao*, YANG Yajun

Author information +

文章历史 +

摘要

钙依赖型蛋白激酶（Calcium-dependent protein kinases，CDPKs或CPKs）是植物细胞中重要的钙离子信号受体，普遍参与了植物生长发育和胁迫响应等调控过程。本研究从茶树龙井43中克隆到1条CDPK基因，经测序验证该序列包含1 611 bp的完整ORF，编码536个氨基酸。结合序列比对和进化分析发现该蛋白N-末端具有豆蔻酰化位点，具备钙依赖性蛋白激酶的保守结构域，并且与拟南芥AtCDPK17的同源关系最近，因此命名为CsCDPK17（Genbank登录号为：MK238482）。蛋白质理化特性分析发现其蛋白分子量为59.9 kD，理论等电点pI为5.43，属无跨膜结构域的亲水性蛋白。使用水稻原生质体和烟草叶片两种瞬时表达的方法均证明CsCDPK17是细胞质膜和细胞核双定位蛋白。对克隆到的CsCDPK17上游2 000 bp的启动子区分析发现了多个与基因转录、光、激素（ABA、SA、MeJA等）相关的顺式作用元件。表达分析显示，CsCDPK17在成熟叶和种子中表达水平较高，而在根中的表达水平最低；在龙井43、大面白等4个品种冷驯化过程中，该基因的表达随着冷驯化过程显著上调，随着脱驯化过程下调；同时还发现CsCDPK17能够不同程度地响应低温（最高5.1倍）、干旱（最高2.3倍）、渗透（最高2.4倍）胁迫。本研究的结果表明CsCDPK17在茶树的生长发育和低温、干旱、渗透等非生物胁迫响应的过程中均发挥一定的调控作用。

Abstract

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.

导出引用

雷蕾, 王璐, 姚丽娜, 郝心愿, 曾建明, 丁长庆, 王新超, 杨亚军. 茶树钙依赖性蛋白激酶基因CsCDPK17的鉴定及表达分析[J]. 茶叶科学. 2019, 39(3): 267-279 https://doi.org/10.13305/j.cnki.jts.2019.03.004

LEI Lei, WANG Lu, YAO Lina, HAO Xinyuan, ZENG Jianming, DING Changqing, WANG Xinchao, YANG Yajun. Identification and Expression Analysis of Calcium-dependent Protein Kinase CsCDPK17 in Tea Plant (Camellia sinensis)[J]. Journal of Tea Science. 2019, 39(3): 267-279 https://doi.org/10.13305/j.cnki.jts.2019.03.004

中图分类号： S571.1 Q52

参考文献

[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.