Cloning of StCIPK11 Gene and Analysis of Its Response to Drought Stress in Solanum tuberosum

doi:10.13560/j.cnki.biotech.bull.1985.2023-0062

Abstract

Abstract:

This study is to clarify the function and mechanism of StCIPK11 in potato（Solanum tuberosum）in the signal transduction in responses to drought stress, aiming to provide theoretical basis for deeply understanding the molecular mechanism responding to drought stress. Homologous recombination and artificial microRNA technology were used to construct the overexpression vector and the interference vector of potato StCIPK11, and they were transferred into the potato cultivar ‘Atlantic’ via Agrobacterium. According to the RT-qPCR data, StCIPK11 was expressed 11.59 and 21.76 times more in the overexpression plants than in the non-transgenic plants, and the the interference degree of StCIPK11 in the interference plants reached 78%. Under drought stress via PEG, the proline content and antioxidant enzymes（superoxide dismutase and peroxidase）activities of the overexpression plants were lower than those of the non-transgenic plants, and the malondialdehyde content in their leaves was significantly higher than that of the non-transgenic plants. The StCIPK11-interfering plants, however, presented the opposite trend. StCIPK11 was involved in the drought stress response process, and StCIPK11 interference expression could lessen the sensitivity of potato plants to water stress.

Key words: potato, StCIPK11, drought stress, physiologic index

LIU Wen-jin, MA Rui, LIU Sheng-yan, YANG Jiang-wei, ZHANG Ning, SI Huai-jun. Cloning of StCIPK11 Gene and Analysis of Its Response to Drought Stress in Solanum tuberosum[J]. Biotechnology Bulletin, 2023, 39(9): 147-155.

Figures/Tables 7

Table 1 Primer sequences used in the study

引物名称Primer name	引物序列Primer sequence（5'-3'）	用途Purpose
StCIPK11-F	CGGGGGACGAGCTCGGTACCATGGCCTTATTCTCTTCTTCGG	PCR扩增 PCR amplification
StCIPK11-R	CCATGTCGACTCTAGATTATGGATCCACCTCTGTTGAAATC	PCR扩增 PCR amplification
StCIPK11-I	GATTCTATTTCCGCGCTAGCCTCTCTCTCTTTTGTATTCC	amiR-StCIPK11 PCR扩增 amiR-StCIPK11 for PCR amplification
StCIPK11-II	GAGAGGCTAGCGCGGAAATAGAATCAAAGAGAATCAATGA
StCIPK11-III	GAGAAGCTAGCGCGGTAATAGATTCACAGGTCGTGATATG
StCIPK11-IV	GAATCTATTACCGCGCTAGCTTCTCTACATATATATTCCT
StCIPK11-A	CTGCAAGGCGATTAAGTTGGGTAAC
StCIPK11-B	GCGGATAACAATTTCACACAGGAAACAG
Q- StCIPK11-F	TACGACACCCACACATCGTC	实时荧光定量PCR Quantitative real-time PCR
Q-StCIPK11-R	TTTGCGAACAATTCGCCTCC	实时荧光定量PCR Quantitative real-time PCR
StEf1a-F	CAAGGATGACCCAGCCAAG	内参基因Reference gene
StEf1a-R	TTCCTTACCTGAACGCCTGT	内参基因Reference gene
HYG-F	GTGATTTCATATGCGCGATTGCTG	HYG基因PCR扩增PCR amplification for HYG gene
HYG-R	ACGAGTGCTGGGGCGTCGGTTTCC	HYG基因PCR扩增PCR amplification for HYG gene
NPT II-F	GCTATGACTGGGCACAACAG	NPT II基因PCR扩增PCR amplification for NPT II gene
NPT II-R	ATACCGTAAAGCACGAGGAA	NPT II基因PCR扩增PCR amplification for NPT II gene

Fig. 1 StCIPK11 amplified product bands and amiR-StCIPK11 precursor fragments amplification A: StCIPK11 amplification product bands. B: a, b and c are small fragments. C: d fragment. M: DNA marker DL2000. 1-2: Gene fragment. 3-4: d fragment

Fig. 2 Multiple alignments of homologous sequences between StCIPK11 protein and other species Black: Completely identitical sequence. Pink: Sequence identity above 75%. Blue: Sequence identity above 50%. White: Sequence identity below 30%

Fig. 3 Phylogenetic tree analysis of StCIPK11 protein

Fig. 4 Double-digestion verification of recombinant plasmid A: Double-digestion verification of p1300-StCIPK11. B: Double-digestion verification of pBI121-amiR-StCIPK11. M: DNA marker DL2000. 1-2: Digestion fragment

Fig. 5 RT-qPCR detection of the transgenic plants OE-1, OE-2: The overexpressed plants. Ri-1, Ri-2: The interfering expression plants. NT: Non-transgenic plants. Data are mean ±SD（n=3）. LSD post hoc test were used for significant analysis. *: P< 0.05; **: P< 0.01. The same below

Fig. 6 Analysis of physiological indices of transgenic and NT potato plants under PEG6000 treatment A: MDA content; B: proline content; C: SOD activity; D: POD activity

References 37

[1]	陈玉珍, 唐广彬, 马宪新, 等. 马铃薯块茎发育的四大调控途径[J]. 作物杂志, 2022(4): 9-13.
	Chen YZ, Tang GB, Ma XX, et al. Four major regulatory pathways of potato tuber development[J]. Crops, 2022(4): 9-13.
[2]	The Potato Genome Sequencing Consortium. Genome sequence and analysis of the tuber crop potato[J]. Nature, 2011, 475(7355): 189-195. doi: 10.1038/nature10158
[3]	贾立国, 陈玉珍, 樊明寿, 等. 干旱对马铃薯光合特性及块茎形成的影响[J]. 干旱区资源与环境, 2018, 32(2): 188-193.
	Jia LG, Chen YZ, Fan MS, et al. Influence of drought stress on potato photosynthetic characteristics and tuberization and its possible mechanism[J]. J Arid Land Resour Environ, 2018, 32(2): 188-193.
[4]	吴明阳. 转AtCIPK23基因马铃薯耐旱性的初步研究[D]. 雅安: 四川农业大学, 2012.
	Wu MY. The preliminary study on the drought tolerance of transgenic ArCIPK23 potato[D]. Ya'an: Sichuan Agricultural University, 2012.
[5]	孙慧生. 马铃薯育种学[M]. 北京: 中国农业出版社, 2003: 242-254.
	Sun HS. Potato breeding[M]. Beijing: China Agriculture Press, 2003: 242-254.
[6]	Лetyxob CН, 陶金萍, 张相英. 马铃薯抗旱性状的遗传[J]. 杂粮作物, 2000, 20(6): 11-13.
	Лetyxob CH, Tao JP, Zhang XY. Inheritance of drought-resistant traits in potatoes[J]. Rain Fed Crops, 2000, 20(6): 11-13.
[7]	Ranty B, Aldon D, Cotelle V, et al. Calcium sensors as key hubs in plant responses to biotic and abiotic stresses[J]. Front Plant Sci, 2016, 7: 327. doi: 10.3389/fpls.2016.00327 pmid: 27014336
[8]	Rudd JJ, Franklin-Tong VE. Unravelling response-specificity in Ca²⁺ signaling pathways in plant cells[J]. New Phytol, 2001, 151(1): 7-33. doi: 10.1046/j.1469-8137.2001.00173.x URL
[9]	Boudsocq M, Sheen J. CDPKs in immune and stress signaling[J]. Trends Plant Sci, 2013, 18(1): 30-40. doi: 10.1016/j.tplants.2012.08.008 pmid: 22974587
[10]	Luan S, Lan WZ, Lee SC. Potassium nutrition, sodium toxicity, and calcium signaling: connections through the CBL-CIPK network[J]. Curr Opin Plant Biol, 2009, 12(3): 339-346. doi: 10.1016/j.pbi.2009.05.003 pmid: 19501014
[11]	Tang RJ, Zhao FG, Garcia VJ, et al. Tonoplast CBL-CIPK calcium signaling network regulates magnesium homeostasis in Arabidopsis[J]. Proc Natl Acad Sci USA, 2015, 112(10): 3134-3139. doi: 10.1073/pnas.1420944112 URL
[12]	刘砚璞, 肖雪, 赵建明, 等. 中国野生燕山葡萄VyCIPK9基因的克隆与功能分析[J]. 果树学报, 2022, 39(10): 1748-1758.
	Liu YP, Xiao X, Zhao JM, et al. Cloning and functional analysis of VyCIPK9 gene in Chinese wild grape(Vitis yeshanensis ‘Yanshan’)[J]. J Fruit Sci, 2022, 39(10): 1748-1758.
[13]	Kim KN, Cheong YH, Grant JJ, et al. CIPK3, a calcium sensor-associated protein kinase that regulates abscisic acid and cold signal transduction in Arabidopsis[J]. Plant Cell, 2003, 15(2): 411-423. doi: 10.1105/tpc.006858 URL
[14]	Kudla J, Xu QA, Harter K, et al. Genes for calcineurin B-like proteins in Arabidopsis are differentially regulated by stress signals[J]. Proc Natl Acad Sci USA, 1999, 96(8): 4718-4723. doi: 10.1073/pnas.96.8.4718 pmid: 10200328
[15]	Albrecht V, Ritz O, Linder S, et al. The NAF domain defines a novel protein-protein interaction module conserved in Ca²⁺-regulated kinases[J]. EMBO J, 2001, 20(5): 1051-1063. doi: 10.1093/emboj/20.5.1051 pmid: 11230129
[16]	Bose J, Rodrigo-Moreno A, Shabala S. ROS homeostasis in halophytes in the context of salinity stress tolerance[J]. J Exp Bot, 2014, 65(5): 1241-1257. doi: 10.1093/jxb/ert430 pmid: 24368505
[17]	Qi XH, Tang X, Liu WG, et al. A potato RING-finger protein gene StRFP2 is involved in drought tolerance[J]. Plant Physiol Biochem, 2020, 146: 438-446. doi: 10.1016/j.plaphy.2019.11.042 URL
[18]	Armengaud P, Thiery L, Buhot N, et al. Transcriptional regulation of proline biosynthesis in Medicago truncatula reveals developmental and environmental specific features[J]. Physiol Plant, 2004, 120(3): 442-450. doi: 10.1111/j.0031-9317.2004.00251.x pmid: 15032841
[19]	Zhang HY, Mao XG, Jing RL, et al. Characterization of a common wheat(Triticum aestivum L.) TaSnRK2.7 gene involved in abiotic stress responses[J]. J Exp Bot, 2011, 62(3): 975-988. doi: 10.1093/jxb/erq328 URL
[20]	Tsou PL, Lee SY, Allen NS, et al. An ER-targeted calcium-binding peptide confers salt and drought tolerance mediated by CIPK6 in Arabidopsis[J]. Planta, 2012, 235(3): 539-552. doi: 10.1007/s00425-011-1522-9 URL
[21]	Xiang Y, Huang YM, Xiong LZ. Characterization of stress-responsive CIPK genes in rice for stress tolerance improvement[J]. Plant Physiol, 2007, 144(3): 1416-1428. doi: 10.1104/pp.107.101295 pmid: 17535819
[22]	Tripathi V, Parasuraman B, Laxmi A, et al. CIPK6, a CBL-interacting protein kinase is required for development and salt tolerance in plants[J]. Plant J, 2009, 58(5): 778-790. doi: 10.1111/tpj.2009.58.issue-5 URL
[23]	Abdula SE, Lee HJ, Ryu H, et al. Overexpression of BrCIPK1 gene enhances abiotic stress tolerance by increasing proline biosynthesis in rice[J]. Plant Mol Biol Rep, 2016, 34(2): 501-511. doi: 10.1007/s11105-015-0939-x URL
[24]	柴畅. 番茄CIPK8基因在低温、盐和干旱胁迫下功能研究[D]. 哈尔滨: 东北农业大学, 2021.
	Chai C. Function of CIPK8 gene under low temperature, salt and drought stress in tomato[D]. Harbin:Northeast Agricultural University, 2021.
[25]	Ma X, Li Y, Gai WX, et al. The CaCIPK3 gene positively regulates drought tolerance in pepper[J]. Hortic Res, 2021, 8(1): 216. doi: 10.1038/s41438-021-00651-7
[26]	Ma R, Liu WG, Li SG, et al. Genome-wide identification, characterization and expression analysis of the CIPK gene family in potato(Solanum tuberosum L.) and the role of StCIPK10 in response to drought and osmotic stress[J]. Int J Mol Sci, 2021, 22(24): 13535. doi: 10.3390/ijms222413535 URL
[27]	Huai S. An efficient protocol for Agrobacterium-mediated transformation with microtuber and the introduction of an antisense class I patatin gene into potato[J]. Acta Agronomica Sinica, 2003(6): 801-805.
[28]	Osmolovskaya N, Shumilina J, Kim A, et al. Methodology of drought stress research: experimental setup and physiological characterization[J]. Int J Mol Sci, 2018, 19(12): 4089. doi: 10.3390/ijms19124089 URL
[29]	Ravikumar G, Manimaran P, Voleti SR, et al. Stress-inducible expression of AtDREB1A transcription factor greatly improves drought stress tolerance in transgenic indica rice[J]. Transgenic Res, 2014, 23(3): 421-439. doi: 10.1007/s11248-013-9776-6 pmid: 24398893
[30]	陈刚, 李胜. 植物生理学实验[M]. 北京: 高等教育出版社, 2016.
	Chen G, Li S. Plant physiology experiment[M]. Beijing: Higher Education Press, 2016.
[31]	Lu L, Chen XY, Wang PK, et al. CIPK11: a calcineurin B-like protein-interacting protein kinase from Nitraria tangutorum, confers tolerance to salt and drought in Arabidopsis[J]. BMC Plant Biol, 2021, 21(1): 123. doi: 10.1186/s12870-021-02878-x pmid: 33648456
[32]	苏倩, 杜文宣, 马琳, 等. 紫花苜蓿MsCIPK2的克隆及功能分析[J]. 中国农业科学, 2022, 55(19): 3697-3709. doi: 10.3864/j.issn.0578-1752.2022.19.002
	Su Q, Du WX, Ma L, et al. Cloning and functional analyses of MsCIPK2 in Medicago sativa[J]. Sci Agric Sin, 2022, 55(19): 3697-3709.
[33]	洪鼎立, 安畅, 徐如宏, 等. 小麦GzCIPK19基因的克隆及表达分析[J]. 分子植物育种, 2022, 20(12): 3837-3846.
	Hong DL, An C, Xu RH, et al. Cloning and expression analysis of wheat GzCIPK19 gene[J]. Mol Plant Breed, 2022, 20(12): 3837-3846.
[34]	Ma YL, Cao J, Chen QQ, et al. The kinase CIPK11 functions as a negative regulator in drought stress response in Arabidopsis[J]. Int J Mol Sci, 2019, 20(10): 2422. doi: 10.3390/ijms20102422 URL
[35]	Gratz R, Manishankar P, Ivanov R, et al. CIPK11-dependent phosphorylation modulates FIT activity to promote Arabidopsis iron acquisition in response to calcium signaling[J]. Dev Cell, 2019, 48(5): 726-740.e10. doi: 10.1016/j.devcel.2019.01.006 URL
[36]	陈小晶, 王东梅, 关红辉, 等. 玉米CIPK基因家族的鉴定及ZmCIPK3的抗旱性功能研究[J]. 植物遗传资源学报, 2022, 23(4): 1064-1075. doi: 10.13430/j.cnki.jpgr.20220107006
	Chen XJ, Wang DM, Guan HH, et al. Identification of CIPK gene family members and investigation of the drought tolerance of ZmCIPK3 in maize[J]. J Plant Genet Resour, 2022, 23(4): 1064-1075.
[37]	杨宏羽, 李瑞瑞, 何雷, 等. 马铃薯StCIPK基因家族鉴定及表达特征分析[J]. 植物生理学报, 2022, 58(10): 1919-1934.
	Yang HY, Li RR, He L, et al. Identification and expression characterization of StCIPK gene family in potato(Solanum tuberosum)[J]. Plant Physiol J, 2022, 58(10): 1919-1934.