谷子SiCIPK21的克隆及功能分析

doi:10.3864/j.issn.0578-1752.2024.22.003

摘要/Abstract

摘要：

【目的】Ca²⁺-CBL-CIPK信号系统在植物响应非生物逆境中具有重要功能。克隆谷子SiCIPK21，并研究其抗逆功能，为谷子抗逆分子育种提供关键候选基因和理论依据。【方法】利用生物信息学分析SiCIPK21启动子区域的顺式作用元件，预测其蛋白与拟南芥AtCBL的互作关系。通过PCR技术克隆SiCIPK21，构建融合表达载体在烟草中瞬时表达，确定亚细胞定位。从谷子品种豫谷1号叶片中特异性扩增SiCIPK21的部分片段，构建重组载体VIGS-pTRV2-SiCIPK21，以谷子八氢番茄红素脱氢酶（SiPDS）为指示基因，选取二叶期的谷子幼苗，通过子叶注射进行侵染，利用病毒诱导的基因沉默（virus-induced gene silencing，VIGS）技术研究盐（250 mmol·L^-1 NaCl）胁迫下SiCIPK21在谷子苗期的作用。在拟南芥中过表达SiCIPK21获得T₃代转基因株系。在不同浓度NaCl（150/175 mmol·L^-1）、甘露醇（300/400 mmol·L^-1）和ABA（0.25/0.5 μmol·L^-1）处理下对萌发期的表型进行分析，同时，对苗期的耐盐和耐旱表型进行分析。【结果】亚细胞定位显示，SiCIPK21位于细胞核。SiCIPK21可能与拟南芥AtCBL2、AtCBL3、AtCBL4、AtCBL9和AtCBL10互作。SiCIPK21启动子区域含有逆境应答元件，暗示SiCIPK21可能参与谷子的逆境应答。VIGS基因沉默试验表明，沉默谷子SiCIPK21植株对盐胁迫的敏感性增加。通过遗传转化获得3个独立的T₃代拟南芥过表达株系（2#、3#和6#）。在不同浓度NaCl（150/175 mmol·L^-1）、甘露醇（300/400 mmol·L^-1）和ABA（0.25/0.5 μmol·L^-1）条件下的萌发率、萌发速度、绿色子叶展开率、根系长度和鲜重均显著高于野生型植株（WT）；在拟南芥苗期，过表达株系的成活率和叶绿素含量显著提高，对盐胁迫和干旱的耐受性增强。【结论】SiCIPK21是植物响应盐和干旱胁迫的正调控因子，SiCIPK21可作为提高谷子抗逆分子育种的候选基因。

关键词: 谷子, SiCIPK21, 非生物胁迫, 表型分析, 基因沉默

Abstract:

【Objective】The Ca²⁺-CBL-CIPK signaling pathway has important functions in plant response to abiotic stresses. By cloning the SiCIPK21 gene and studying its function under stress conditions, we provide a key candidate gene and theoretical basis for molecular breeding of foxtail millet with stress tolerance.【Method】Bioinformatics was used to analyze the cis-acting elements in the promoter region of this gene and predict the interactions between this protein and AtCBLs in Arabidopsis thaliana. SiCIPK21 was cloned by PCR, and a fusion expression vector was constructed for transient expression in tobacco to determine the subcellular localization. foxtail millet cv. Yugu 1 was used as material, and specifically amplified part of the SiCIPK21 gene fragment from Yugu 1 leaves, and recombinant vector VIGS-pTRV2-SiCIPK21 was constructed, using the phytoene desaturase gene (SiPDS) as the indicator gene, and seedlings of foxtail millet at the two-leaf stage were selected and infiltrated by cotyledon injection to investigate the role of SiCIPK21 under salt stress (250 mmol·L^-1 NaCl) by using virus-induced gene silencing (VIGS) technology. T₃ generation transgenic lines were obtained by overexpressing SiCIPK21 in Arabidopsis thaliana. Phenotypes at germination were analyzed under different concentrations of NaCl (150/175 mmol·L^-1), mannitol (300/400 mmol·L^-1) and ABA (0.25/0.5 μmol·L^-1) treatments, and salt and drought tolerant phenotypes at seedling stage were also analyzed.【Result】Subcellular localization revealed that SiCIPK21 was located in the nucleus. The protein SiCIPK21 might interact with AtCBL2, AtCBL3, AtCBL4, AtCBL9, and AtCBL10 in Arabidopsis thaliana. The promoter region of SiCIPK21 contained adverse response elements, suggesting that SiCIPK21 may participate in the adverse responses. The VIGS gene silencing demonstrated that SiCIPK21-silenced foxtail millet plants had increased sensitivity to salt stress than the control plants. Three independent T₃ generation Arabidopsis thaliana overexpression lines (2#, 3# and 6#) were obtained by genetic transformation. Overexpression lines showed significantly higher germination rate, germination speed, green cotyledon unfolding rate, root length and fresh weight than the wild-type plants (WT) at different concentrations of NaCl (150/175 mmol·L^-1), mannitol (300/400 mmol·L^-1) and ABA (0.25/0.5 μmol·L^-1). Moreover, phenotypic analysis of salt and drought tolerance in Arabidopsis seedlings showed that overexpression lines had significantly higher survival rates and chlorophyll contents than WT.【Conclusion】SiCIPK21 is a positive regulator of plant response to salt and drought stresses, which makes it a candidate gene for improving stress tolerance by molecular breeding in foxtail millet.

Key words: foxtail millet, SiCIPK21, abiotic stress, phenotype analysis, gene silencing

杜艳伟, 阎晓光, 赵晋锋, 贾苏卿, 王高鸿, 余爱丽, 张鹏. 谷子SiCIPK21的克隆及功能分析[J]. 中国农业科学, 2024, 57(22): 4416-4430.

DU YanWei, YAN XiaoGuang, ZHAO JinFeng, JIA SuQing, WANG GaoHong, YU AiLi, ZHANG Peng. Cloning and Functional Verification of SiCIPK21 Gene in Foxtail Millet[J]. Scientia Agricultura Sinica, 2024, 57(22): 4416-4430.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2024.22.003

https://www.chinaagrisci.com/CN/Y2024/V57/I22/4416

图/表 10

表1

图1

表2

图2

图3

图4

图5

图6

图7

图8

参考文献 44

[1]	LI X K, GAO J H, SONG J Y, GUO K, HOU S Y, WANG X C, HE Q, ZHANG Y Y, ZHANG Y K, YANG Y L, TANG J Y, WANG H L, PERSSON S, HUANG M Q, XU L S, ZHONG L L, LI D Q, LIU Y M, WU H, DIAO X M, CHEN P, WANG X W, HAN Y H. Multi-omics analyses of 398 foxtail millet accessions reveal genomic regions associated with domestication, metabolite traits, and anti-inflammatory effects. Molecular Plant, 2022, 15(8): 1367-1383.
[2]	SANYAL S K, MAHIWAL S, NAMBIAR D M, PANDEY G K. CBL-CIPK module-mediated phosphoregulation: Facts and hypothesis. The Biochemical Journal, 2020, 477(5): 853-871.
[3]	DING X, LIU B W, SUN X Z, SUN X, ZHENG C S. New functions of CIPK gene family are continue to emerging. Molecular Biology Reports, 2022, 49(7): 6647-6658. doi: 10.1007/s11033-022-07255-x pmid: 35229240
[4]	VERMA P, SANYAL S K, PANDEY G K. Ca²⁺-CBL-CIPK: A modulator system for efficient nutrient acquisition. Plant Cell Reports, 2021, 40(11): 2111-2122.
[5]	PATRA N, HARIHARAN S, GAIN H, MAITI M K, DAS A, BANERJEE J. TypiCal but DeliCate Ca⁺⁺re: Dissecting the essence of calcium signaling network as a robust response coordinator of versatile abiotic and biotic stimuli in plants. Frontiers in Plant Science, 2021, 12: 752246.
[6]	ZENG H Q, ZHU Q Q, YUAN P G, YAN Y, YI K K, DU L Q. Calmodulin and calmodulin-like protein-mediated plant responses to biotic stresses. Plant, Cell & Environment, 2023, 46(12): 3680-3703.
[7]	KUDLA J, XU Q, HARTER K, GRUISSEM W, LUAN S. Genes for calcineurin B-like proteins in Arabidopsis are differentially regulated by stress signals. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(8): 4718-4723.
[8]	KOLUKISAOGLU U, WEINL S, BLAZEVIC D, BATISTIC O, KUDLA J. Calcium sensors and their interacting protein kinases: Genomics of the Arabidopsis and rice CBL-CIPK signaling networks. Plant Physiology, 2004, 134(1): 43-58.
[9]	DENG X M, HU W, WEI S Y, ZHOU S Y, ZHANG F, HAN J P, CHEN L H, LI Y, FENG J L, FANG B, LUO Q C, LI S S, LIU Y Y, YANG G X, HE G Y. TaCIPK29, a CBL-interacting protein kinase gene from wheat, confers salt stress tolerance in transgenic tobacco. PLoS ONE, 2013, 8(7): e69881.
[10]	CHEN X F, GU Z M, XIN D D, HAO L, LIU C J, HUANG J, MA B J, ZHANG H S. Identification and characterization of putative CIPK genes in maize. Journal of Genetics and Genomics, 2011, 38(2): 77-87. doi: 10.1016/j.jcg.2011.01.005 pmid: 21356527
[11]	CUI Y P, SU Y, WANG J J, JIA B, WU M, PEI W F, ZHANG J F, YU J W. Genome-wide characterization and analysis of CIPK gene family in two cultivated allopolyploid cotton species: Sequence variation, association with seed oil content, and the role of GhCIPK6. International Journal of Molecular Sciences, 2020, 21(3): 863.
[12]	REN H M, ZHANG Y T, ZHONG M Y, HUSSIAN J, TANG Y T, LIU S K, QI G N. Calcium signaling-mediated transcriptional reprogramming during abiotic stress response in plants. Theoretical and Applied Genetics, 2023, 136(10): 210.
[13]	TRIPATHI V, PARASURAMAN B, LAXMI A, CHATTOPADHYAY D. CIPK6, a CBL-interacting protein kinase is required for development and salt tolerance in plants. The Plant Journal, 2009, 58(5): 778-790. doi: 10.1111/j.1365-313X.2009.03812.x pmid: 19187042
[14]	陈小晶, 王东梅, 关红辉, 郭剑, 沙小茜, 李永祥, 张登峰, 刘旭洋, 何冠华, 石云素, 宋燕春, 王天宇, 黎裕, 刘颖慧, 李春辉. 玉米CIPK基因家族的鉴定及ZmCIPK3的抗旱性功能研究. 植物遗传资源学报, 2022, 23(4): 1064-1075. doi: 10.13430/j.cnki.jpgr.20220107006
	CHEN X J, WANG D M, GUAN H H, GUO J, SHA X Q, LI Y X, ZHANG D F, LIU X Y, HE G H, SHI Y S, SONG Y C, WANG T Y, LI Y, LIU Y H, LI C H. Identification of CIPK gene family members and investigation of the drought tolerance of ZmCIPK3 in maize. Journal of Plant Genetic Resources, 2022, 23(4): 1064-1075. (in Chinese)
[15]	XIANG Y, HUANG Y M, XIONG L Z. Characterization of stress-responsive CIPK genes in rice for stress tolerance improvement. Plant Physiology, 2007, 144(3): 1416-1428. doi: 10.1104/pp.107.101295 pmid: 17535819
[16]	RÓDENAS R, VERT G. Regulation of root nutrient transporters by CIPK23: 'One kinase to rule them all'. Plant & Cell Physiology, 2021, 62(4): 553-563.
[17]	ZHANG Y, ZHOU X N, LIU S Y, YU A Z, YANG C M, CHEN X L, LIU J Y, WANG A X. Identification and functional analysis of tomato CIPK gene family. International Journal of Molecular Sciences, 2019, 21(1): 110.
[18]	PODDAR N, DEEPIKA D, CHITKARA P, SINGH A, KUMAR S. Molecular and expression analysis indicate the role of CBL interacting protein kinases (CIPKs) in abiotic stress signaling and development in chickpea. Scientific Reports, 2022, 12(1): 16862.
[19]	LU L, WU X R, TANG Y, ZHU L M, HAO Z D, ZHANG J B, LI X L, SHI J S, CHEN J H, CHENG T L. Halophyte Nitraria billardieri CIPK25 promotes photosynthesis in Arabidopsis under salt stress. Frontiers in Plant Science, 2022, 13: 1052463.
[20]	YANG W Q, KONG Z S, OMO-IKERODAH E, XU W Y, LI Q, XUE Y B. Calcineurin B-like interacting protein kinase OsCIPK23 functions in pollination and drought stress responses in rice (Oryza sativa L.). Journal of Genetics and Genomics, 2008, 35(9): 531-543.
[21]	ZHAO J F, YU A L, DU Y W, WANG G H, LI Y F, ZHAO G Y, WANG X D, ZHANG W Z, CHENG K, LIU X, WANG Z H, WANG Y W. Foxtail millet (Setaria italica (L.) P. Beauv) CIPKs are responsive to ABA and abiotic stresses. PLoS ONE, 2019, 14(11): e0225091.
[22]	高俊凤. 植物生理学实验指导. 北京: 高等教育出版社, 2006.
	GAO J F. Laboratory Instruction of Plant Physiology. Beijing: Higher Education Press, 2006. (in Chinese)
[23]	KANWAR P, SANYAL S K, TOKAS I, YADAV A K, PANDEY A, KAPOOR S, PANDEY G K. Comprehensive structural, interaction and expression analysis of CBL and CIPK complement during abiotic stresses and development in rice. Cell Calcium, 2014, 56(2): 81-95. doi: 10.1016/j.ceca.2014.05.003 pmid: 24970010
[24]	ZHANG H F, YANG B, LIU W Z, LI H W, WANG L, WANG B Y, DENG M, LIANG W W, DEYHOLOS M K, JIANG Y Q. Identification and characterization of CBL and CIPK gene families in canola (Brassica napus L.). BMC Plant Biology, 2014, 14: 8.
[25]	LI J, JIANG M M, REN L, LIU Y, CHEN H Y. Identification and characterization of CBL and CIPK gene families in eggplant (Solanum melongena L.). Molecular Genetics and Genomics, 2016, 291(4): 1769-1781.
[26]	SUN T, WANG Y, WANG M, LI T T, ZHOU Y, WANG X T, WEI S Y, HE G Y, YANG G X. Identification and comprehensive analyses of the CBL and CIPK gene families in wheat (Triticum aestivum L.). BMC Plant Biology, 2015, 15: 269.
[27]	SHINOZAKI K, YAMAGUCHI-SHINOZAKI K. Molecular responses to dehydration and low temperature: Differences and cross-talk between two stress signaling pathways. Current Opinion in Plant Biology, 2000, 3(3): 217-223. pmid: 10837265
[28]	BASU S, ROYCHOUDHURY A, SENGUPTA D N. Deciphering the role of various cis-acting regulatory elements in controlling SamDC gene expression in rice. Plant Signaling & Behavior, 2014, 9(1): e28391.
[29]	KIM K N, CHEONG Y H, GRANT J J, PANDEY G K, LUAN S. CIPK3, a calcium sensor-associated protein kinase that regulates abscisic acid and cold signal transduction in Arabidopsis. The Plant Cell, 2003, 15(2): 411-423.
[30]	GAZZARRINI S, MCCOURT P. Genetic interactions between ABA, ethylene and sugar signaling pathways. Current Opinion in Plant Biology, 2001, 4(5): 387-391. pmid: 11597495
[31]	SPOLLEN W G, LENOBLE M E, SAMUELS T D, BERNSTEIN N, SHARP R E. Abscisic acid accumulation maintains maize primary root elongation at low water potentials by restricting ethylene production. Plant Physiology, 2000, 122(3): 967-976. pmid: 10712561
[32]	MAO J J, MANIK S M, SHI S J, CHAO J T, JIN Y R, WANG Q, LIU H B. Mechanisms and physiological roles of the CBL-CIPK networking system in Arabidopsis thaliana. Genes, 2016, 7(9): 62.
[33]	YADAV A K, JHA S K, SANYAL S K, LUAN S, PANDEY G K. Arabidopsis calcineurin B-like proteins differentially regulate phosphorylation activity of CBL-interacting protein kinase 9. The Biochemical Journal, 2018, 475(16): 2621-2636.
[34]	YU Q Y, AN L J, LI W L. The CBL-CIPK network mediates different signaling pathways in plants. Plant Cell Reports, 2014, 33(2): 203-214. doi: 10.1007/s00299-013-1507-1 pmid: 24097244
[35]	ZHU J K. Abiotic stress signaling and responses in plants. Cell, 2016, 167(2): 313-324.
[36]	KAYA C, UĞURLAR F, ADAMAKIS I D S. Molecular mechanisms of CBL-CIPK signaling pathway in plant abiotic stress tolerance and hormone crosstalk. International Journal of Molecular Sciences, 2024, 25(9): 5043.
[37]	HASEGAWA P M, BRESSAN R A, ZHU J K, BOHNERT H J. Plant cellular and molecular responses to high salinity. Annual Review of Plant Physiology and Plant Molecular Biology, 2000, 51: 463-499. pmid: 15012199
[38]	ZHU J K. Salt and drought stress signal transduction in plants. Annual Review of Plant Biology, 2002, 53: 247-273.
[39]	CUI X Y, DU Y T, FU J D, YU T F, WANG C T, CHEN M, CHEN J, MA Y Z, XU Z S. Wheat CBL-interacting protein kinase 23 positively regulates drought stress and ABA responses. BMC Plant Biology, 2018, 18(1): 93.
[40]	HUSSAIN Q, ASIM M, ZHANG R, KHAN R, FAROOQ S, WU J S. Transcription factors interact with ABA through gene expression and signaling pathways to mitigate drought and salinity stress. Biomolecules, 2021, 11(8): 1159.
[41]	SCHACHTMAN D P, GOODGER J Q D. Chemical root to shoot signaling under drought. Trends in Plant Science, 2008, 13(6): 281-287. doi: 10.1016/j.tplants.2008.04.003 pmid: 18467158
[42]	YAMAGUCHI-SHINOZAKI K, SHINOZAKI K. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annual Review of Plant Biology, 2006, 57: 781-803.
[43]	QIN F, SHINOZAKI K, YAMAGUCHI-SHINOZAKI K. Achievements and challenges in understanding plant abiotic stress responses and tolerance. Plant and Cell Physiology, 2011, 52(9): 1569-1582. doi: 10.1093/pcp/pcr106 pmid: 21828105
[44]	PIAO H L, XUAN Y H, PARK S H, JE B I, PARK S J, PARK S H, KIM C M, HUANG J, WANG G K, KIM M J, KANG S M, LEE I J, KWON T R, KIM Y H, YEO U S, YI G, SON D, HAN C D. OsCIPK31, a CBL-interacting protein kinase is involved in germination and seedling growth under abiotic stress conditions in rice plants. Molecules and Cells, 2010, 30(1): 19-27.

引物名称 Primer name	引物序列 Primer sequence (5′-3′)
SiCIPK21-GFP-F	ACTAGGGTCTCGCACCATGGCCATGGAGAAGAACCAAGACA
SiCIPK21-GFP-R	ACTAGGGTCTCTCGCCCGTGGTGGTCCGCCTCG
VIGS-pTRV2-SiCIPK21-F	AAGGTTACCGAATTCTCTAGGTTAGTGAAAAGGGGATCCTCATTTCCTC
VIGS-pTRV2-SiCIPK21-R	GAGACGCGTGAGCTCGGTACGAAATTCTCCAGGTGATCATGGGTGTG
pCMBIA3301-SiCIPK21-F	ACTAGGGTCTCGCACCATGGCCATGGAGAAGAACCAAGACA
pCMBIA3301-SiCIPK21-R	ACTAGGGTCTCTACCGCTACGTGGTGGTCCGCCTC
RT-qPCR-SiCIPK21-F	TAATCGCCAAGCACCTCCTA
RT-qPCR-SiCIPK21-R	CTTGGTTCTTCTCCATGGCC
PCR-SiCIPK21-F	ATGGCCATGGAGAAGAACCAAGACA
PCR-SiCIPK21-R	CCTGTAGTTGCTCTGCGTGA
β-Actin-F	CAGTGGACGCACAACAGGTAT
β-Actin-R	AGCAAGGTCAAGACGGAGAAT

顺式元件Cis-element	典型序列Typical sequence	特性Characteristic
TGA-element	AACGAC	生长素响应Auxin-responsive element
P-box	CCTTTTG	赤霉素响应Gibberellin-responsive element
CGTCA-motif	CGTCA	茉莉酸甲酯响应Cis-acting regulatory element involved in the MeJA-responsiveness
TGACG-motif	TGACG
LTR	CCGAAA	低温响应Cis-acting element involved in low-temperature responsiveness
MBS	CAACTG	干旱响应MYB binding site involved in drought-inducibility
ARE	AAACCA	厌氧响应Cis-acting regulatory element essential for the anaerobic induction
ATCT-motif	AATCTAATCC	光响应Part of a conserved DNA module involved in light responsiveness
Box 4	ATTAAT
GATA-motif	GATAGGA	光响应Part of a light responsive element
TCT-motif	TCTTAC
chs-CMA2a	TCACTTGA
AE-box	AGAAACAA
MSA-like	TCAAACGGT	细胞周期调控Cis-acting element involved in cell cycle regulation
TC-rich repeats	ATTCTCTAAC	防御和应激反应响应Cis-acting element involved in defense and stress responsiveness