Cloning and Expression Analysis of CsCML16 in Tea Plants (Camellia sinensis) under Low Temperature Stress

CHEN Siwen; KANG Rui; GUO Zhiyuan; ZHOU Qiongqiong; FENG Jiancan

doi:10.13305/j.cnki.jts.20210514.001

PDF(1933 KB)

Journal of Tea Science ›› 2021, Vol. 41 ›› Issue (3) : 315-326. DOI: 10.13305/j.cnki.jts.20210514.001

Research Paper

Cloning and Expression Analysis of CsCML16 in Tea Plants (Camellia sinensis) under Low Temperature Stress

CHEN Siwen, KANG Rui, GUO Zhiyuan, ZHOU Qiongqiong^*, FENG Jiancan^*

Author information +

History +

Abstract

Calmodulin-like proteins (CMLs) are important calcium signaling proteins in plants, which play a pivotal role in abiotic stress. In this study, one-year-old tea cutting seedlings of ‘Longjing43’, ‘Fuding Dabai’ and ‘Huangjinya’ were used as research materials. The physiological changes of the three tea cultivars were explored to preliminarily screen the cold-resistance tea cultivar. The CsCML16 gene was cloned to analyze its expression patterns in different cold-tolerance tea cultivars under low temperature stress. The results show that ‘Longjing43’ had the highest cold tolerance under low temperature stress, followed by ‘Fuding Dabai’ and ‘Huangjinya’. CsCML16 gene was cloned from ‘Longjing43’. Sequence analysis shows that the CDS length of CsCML16 is 480 bp, encodes 160 amino acids, contains EF-hand conserved domains with molecular weight of 17.58 kDa. Subcellular localization assay indicates that CsCML16 localized in the nucleus and plasma membrane. Quantitative real-time PCR analysis reveals that CsCML16 gene was involved in the regulation of tea plants in response to low temperature stress, and its expression levels varied with tea cultivars with different cold tolerance. This study provided a basis for further study on the biological function of CsCML16 gene.

Key words

Camellia sinensis / calmodulin-like protein / expression analysis / low temperature stress / physiological analysis

Cite this article

EndNote

Ris (Procite)

Bibtex

Download Citations

CHEN Siwen, KANG Rui, GUO Zhiyuan, ZHOU Qiongqiong, FENG Jiancan. Cloning and Expression Analysis of CsCML16 in Tea Plants (Camellia sinensis) under Low Temperature Stress[J]. Journal of Tea Science. 2021, 41(3): 315-326 https://doi.org/10.13305/j.cnki.jts.20210514.001

References

[1] Yang T, Chaudhuri S, Yang L, et al.A calcium/calmodulin-regulated member of the receptor-like kinase family confers cold tolerance in plants[J]. Biological Chemistry, 2010, 285(10): 7119-26.
[2] Sanders D, Pelloux J, Brownlee C, et al.Calcium at the crossroads of signaling[J]. The Plant Cell, 2002, 14: S401-S417.
[3] Batisti O, Kudla J.Analysis of calcium signaling pathways in plants[J]. Biochimica Et Biophysica Acta, 2012, 8: 1283-1293.
[4] Wilkins K A, Matthus E, Swarbreck S M, et al.Calcium-mediated abiotic stress signaling in roots[J]. Frontiers in Plant Science, 2016, 8: 245. doi: 10.3389/fpls.2016.01296.
[5] 杨俊. OsCaM1-1和OsCML16调控水稻耐逆性机制的研究[D]. 武汉: 华中农业大学, 2018.
Yang J.Study on the mechanisms of OsCaM1-1 and OsCML16 to regulate stress tolerance in rice [D]. Wuhan: Huazhong Agricultural University, 2018.
[6] Xu G Y, Rocha P S, Wang M L, et al.A novel rice calmodulin-like gene, OsMSR2, enhances drought and salt tolerance and increases ABA sensitivity in Arabidopsis[J]. Planta, 2011, 234(1): 47-59.
[7] Zhu X Y, Robe E, Jomat L, et al.CML8, an Arabidopsis calmodulin-like protein, plays a role in Pseudomonas syringae plant immunity[J]. Plant and Cell Physiology, 2016, 58(2): 307-319.
[8] Delk N A, Johnson K A, Chowdhury N I, et al.CML24, regulated in expression by diverse stimuli, encodes a potential Ca²+ sensor that functions in responses to abscisic acid, day length, and ion stress[J]. Plant Physiology, 2005, 139(1): 240-253.
[9] Park H C, Chan Y P, Koo S C, et al.AtCML8, a calmodulin-like protein, differentially activating CaM-dependent enzymes in Arabidopsis thaliana[J]. Plant Cell Reports, 2010, 29(11): 1297-1304.
[10] Scholz S S, Vadassery J, Heyer M, et al.Mutation of the Arabidopsis calmodulin-Like protein CML37 deregulates the Jasmonate pathway and enhances susceptibility to herbivory[J]. Molecular Breeding, 2014, 7(12): 1712-1726.
[11] Munir S, Hui L, Xing Y, et al.Overexpession of calmodulin-like (ShCML44) stesresponsive gene from Solanum habrochaites enhances tolerance to multiple abiotic sresses[J]. Scientifc Reports, 2016, 8: 31772. doi: 10.1038/srep31772.
[12] 蒋芯. 低温胁迫下茶树花粉管相关基因的分离与表达分析[D]. 南京: 南京农业大学, 2013.
Jiang X.Isolation and expression of genes in tea (Camellia Sinensis (L.) O.Kuntze) pollen tube under cold stress [D]. Nanjing: Nanjing Agricultural University, 2013.
[13] 杜昱林. 茶树花粉CsE1α、CsCML21基因的亚细胞定位及启动子克隆与功能验证[D]. 南京: 南京农业大学, 2015.
Du Y L.Subcellular localization of CsE1α and CsCML21 and cloning and expression of the promoters from the pollen of Camellia Sinensis [D]. Nanjing: Nanjing Agricultural University, 2015.
[14] Li Y Y, Wang X W, Ban Q Y, et al.Comparative transcriptomic analysis reveals gene expression associated with cold adaptation in the tea plant Camellia sinenis[J]. BMC Genomics, 2019, 20(1): 624. doi: 10.1186/s12864-019-5988-3.
[15] Wang X C, Zhao Q Y, Ma C L, et a1. Global transcriptome profiles of Camellia sinensis during cold acclimation[J]. BMC Genomics, 2013, 14: 415. doi: 10.1186/1471-2164-14-415.
[16] Ma Q P, Zhou Q Q, Chen C, et al.Isolation and expression analysis of CsCML genes in response to abiotic stresses in the tea plant (Camellia sinensis)[J]. Scientific Reports, 2019, 9(1): 8211. doi: 10.1038/s41598-019-44681-7.
[17] Ding C Q, Lei L, Yao L N, et al.The involvements of calcium-dependent protein kinases and catechins in tea plant [Camellia sinensis (L.) O. Kuntze] cold responses[J]. Plant Physiology and Biochemistry, 2019, 143: 190-202.
[18] 杨亚军, 郑雷英, 王新超. 冷驯化和ABA对茶树抗寒力及其体内脯氨酸含量的影响[J]. 茶叶科学, 2004, 24(3): 177-182.
Yang Y J, Zheng L Y, Wang X C.Effect of cold acclimation and ABA on cold hardiness, contents of proline in tea plants[J]. Journal of Tea Science, 2004, 24(3): 177-182.
[19] 王玉, 王会, 丁兆堂. 茶树黄山种自然杂交后代抗寒性研究[J]. 山东农业科学, 2012(5): 28-32.
Wang Y, Wang H, Ding Z T.Research on cold resistance of natural hybrid progenies of Camellia sinensis cv. Huangshanzhong[J]. Shandong Agricultural Sciences, 2012(5): 28-32.
[20] 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.
Lin Z H, Zhong Q S, You X M, et al.Antioxidant enzyme activity of tea plant (Camellia sinensis) in response to low temperature stress[J]. Journal of Tea Science, 2018, 38(4): 363-371.
[21] 刘宇鹏, 陈芳, 胡家敏, 等. 低温对茶树叶片生理生化指标的影响[J]. 浙江农业科学, 2018, 59(7): 1120-1122, 1126.
Liu Y P, Chen F, Hu J M, et al.Effects of low temperature on physiological and biochemical indexes of tea leaves[J]. Zhejiang Agricultural Science, 2018, 59(7): 1120-1122, 1126.
[22] 黄海涛, 余继忠, 张伟, 等. 电导法配合Logistic方程鉴定茶树抗寒性的探讨[J]. 浙江农业科学, 2009(3): 577-579.
Huang H T, Yu J Z, Zhang W, et al.Determination of cold tolerance based on electrical conductivity method combined with logistic equation in tea plant[J]. Journal of Zhejiang Agricultural Sciences, 2009(3): 577-579.
[23] Hao X Y, Horvath, Chao W S, 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.
[24] Livak K J, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the method[J]. Methods, 2001, 25(4): 402-408.
[25] Maxwell K, Johnson G N.Chlorophyll fluorescence-a practical guide[J]. Journal of Experimental Botany, 2000, 51: 659-668.
[26] 李叶云, 庞磊, 陈启文, 等. 低温胁迫对茶树叶片生理特性的影响[J]. 西北农林科技大学学报, 2012, 40(4): 134-139.
Li Y Y, Pang L, Chen Q W, et al.Effects of low temperature stress on physiological characteristics of tea leaves (Camellia sinensis L.)[J]. Journal of Northwest A&F University, 2012, 40(4): 134-139.
[27] 张玉翠, 王连翠. 低温对茶树叶片膜透性和保护酶活性的影响[J]. 北方园艺, 2010(9): 38-40.
Zhang Y C, Wang L C.Effect of low temperature stress on membrane permeability and protection of activity in tea leaves[J]. Northern Horticulture, 2010(9): 38-40.
[28] 罗军武, 唐和平. 茶树不同抗寒性品种间保护酶类活性的差异[J]. 湖南农业大学学报(自然科学版), 2001, 27(2): 94-96.
Luo J W, Tang H P.Differences of activities of protective enzymes of tea plant varieties with different cold resistant abilities[J]. Journal of Hunan Agricultural University (Natural Sciences), 2001, 27(2): 94-96.
[29] 朱政, 蒋家月, 江昌俊, 等. 低温胁迫对茶树叶片SOD可溶性蛋白和可溶性糖含量的影响[J]. 安徽农业大学学报, 2011, 38(1): 24-26.
Zhu Z, Jiang J Y, Jiang C J, et al.Effects of low temperature stress on SOD activity, soluble protein content and soluble sugar content in Camellia sinensis leaves[J]. Journal of Anhui Agricultural University, 2011, 38(1): 24-26.
[30] 李志博, 魏亦农, 杨敏, 等. 低温胁迫对棉花幼苗叶绿素荧光特性的影响初探[J]. 棉花学报, 2006(4): 65-67.
Li Z B, Wei Y N, Yang M, et al.Primary study on effects of low temperature on chlorophyll fluorescence characteristics of cotton seedling[J]. Cotton Science, 2006(4): 65-67.
[31] 朱政. 茶树抗寒性生理指标的筛选及抗寒性鉴定方法的建立[D]. 合肥: 安徽农业大学, 2011.
Zhu Z.Screening of physiological indicators of cold resistance of tea tree and establishment of cold resistance identification method [D]. Hefei: Anhui Agricultural University, 2011.
[32] Zeng H, Xu L, Singh A, et al.Involvement of calmodulin and calmodulin-like proteins in plant responses to abiotic stresses[J]. Frontiers in Plant Science, 2015, 6: 600. doi: 10.3389/fpls.2015.00600.
[33] Gifford J, Walsh M, Vogel H.Structures and metal-ion-binding properties of the Ca²+ -binding helix-loop-helix EF-hand motifs[J]. Biochemical Journal, 2007, 405: 199-221.
[34] Mccormack E, Braam J.Calmodulins and related potential calcium sensors of Arabidopsis[J]. New Phytologist, 2003, 159(3): 585-598.
[35] Boonburapong B, Buaboocha T.Genome-wide identification and analyses of the rice calmodulin and related potential calcium sensor proteins[J]. BMC Plant Biology, 2007, 7: 4. doi: 10.1186/1471-2229-7-4.
[36] Munir S, Hui L, Xing Y, et al.Genome-wide identification, characterization and expression analysis of calmodulin-like (CML) proteins in tomato (Solanum lycopersicum)[J]. Plant Physiology and Biochemistry, 2016, 102: 167-179.
[37] Yin X M, Huang L F, Zhang X, et al.OsCML4 improves drought tolerance through scavenging of reactive oxygen species in rice[J]. Journal of Plant Biology, 2015, 58(1): 68-73.
[38] 刘伟, 滕腾, 赵懿琛, 等. 杜仲类钙调蛋白基因EuCML5的克隆及表达分析[J]. 园艺学报, 2020, 47(3): 590-600.
Liu W, Teng T, Zhao Y C, et al.Cloning and expression analysis of EuCML5 gene in Eucommia ulmoides[J]. Acta Horticulturae Sinica, 2020, 47(3): 590-600.
[39] Li C L, Meng D, Zhang J H, et al.Genome-wide identification and expression analysis of calmodulin and calmodulin-like genes in apple (Malus × domestica)[J]. Plant Physiology and Biochemistry, 2019, 139: 600-612.
[40] Zhang X, Wang T, Liu M, et al.Calmodulin-like gene MtCML40 is involved in salt tolerance by regulating MtHKTs transporters in Medicago truncatula[J]. Environmental and Experimental Botany, 2018, 157: 79-90.