西藏野生垂穗披碱草EnCBL10基因的克隆与功能研究

doi:10.11733/j.issn.1007-0435.2022.09.011

草地学报 ›› 2022, Vol. 30 ›› Issue (9): 2315-2324.DOI: 10.11733/j.issn.1007-0435.2022.09.011

西藏野生垂穗披碱草EnCBL10基因的克隆与功能研究

张慎彤¹, 高浩¹, 张金鹏¹, 任智慧¹, 姜惠娜¹, 其美拉姆², 普布卓玛³, 付娟娟¹

1. 西北农林科技大学草业与草原学院, 陕西杨凌 712100;
2. 西藏自治区拉萨市曲水县自然资源局, 西藏拉萨 850000;
3. 西藏自治区农科院草业科学研究所, 西藏拉萨 85000

收稿日期:2022-03-03 修回日期:2022-04-06 出版日期:2022-09-15 发布日期:2022-09-30
通讯作者: 付娟娟,E-mail:fujuanjuan1234@126.com
作者简介:张慎彤(2000-),女,汉族,山东泰安人,硕士研究生,主要从事植物逆境生物学研究,E-mail:Zhang.st@nwafu.edu.cn
基金资助:
国家自然科学基金项目（31901382；U21A20240）；国家自然科学面上项目（31872411）；国家自然科学基金-区域联合发展重点基金（U20A2050）；大学生创新创业训练计划创新训练项目（202110712056）共同资助

Cloning and Function Identification of EnCBL10 Gene from Tibetan Wild Elymus nutans Griseb.

ZHANG Shen-tong¹, GAO Hao¹, ZHANG Jin-peng¹, REN Zhi-hui¹, JIANG Hui-na¹, MEI La-mu², BU Zhuo-ma³, FU Juan-juan¹

1. Department of Grassland Science, College of Grassland Agriculture, Northwest A&F University, Yangling, Shaanxi Province 712100, China;
2. Natural Resources Bureau of Qushui County, Lhasa, Tibet 850000, China;
3. College of Animal Science, Agricultural and Animal Husbandry College of Tibet University, Lhasa, Tibet 850000, China

Received:2022-03-03 Revised:2022-04-06 Online:2022-09-15 Published:2022-09-30

摘要/Abstract

摘要： 钙调磷酸酶B类蛋白（Calcineurin B-Like Proteins，CBLs）是钙信号通路中重要成员，在植物应答多种非生物胁迫中具有重要的作用。本研究基于前期转录组数据，在西藏野生垂穗披碱草（Elymus nutans Griseb.）中筛选EnCBL10基因，将其异源表达于烟草（Nicotiana tabacum）中，分析转基因植株EnCBL10在低温和干旱胁迫下的生长及生理响应。结果表明：EnCBL10开放阅读框为792 bp，编码263个氨基酸。EnCBL10蛋白的理论等电点为5.17，为疏水蛋白和非分泌蛋白。低温和干旱胁迫下，对比于野生型烟草，转基因烟草叶片的细胞膜稳定性显著增加，过氧化物酶（Peroxidase，POD），抗坏血酸过氧化物酶（Aseorbateperoxidase，APX）和谷胱甘肽还原酶（Gluathione reductase，GR）活性显著升高。低温胁迫下，两个转基因株系的脯氨酸含量分别提高了79.7%和70.8%（P<0.05）；在干旱胁迫下，野生型植株（WT）和过表达植株中脯氨酸含量均有所降低，但转基因植株比WT的下降幅度小。因此，EnCBL10基因可能在垂穗披碱草的耐寒和耐旱调控中发挥重要功能。

关键词: 垂穗披碱草, EnCBL10, 基因克隆, 低温, 干旱, 基因调控, 生理机制

Abstract: Calcineurin B-Like Proteins (CBLs) are important members of the calcium signaling pathway and play an important role in plant response to various abiotic stresses. Based on the previous transcriptome data, EnCBL10 gene was screened from wild Elymus nutans Griseb. in Tibet, and its heterologous expression in tobacco was performed to analyze the growth and physiological response of transgenic plants EnCBL10 in low temperature and drought. The results showed that the open reading frame of EnCBL10 gene was 792 bp, encoding 263 amino acids. The theoretical isoelectric point of EnCBL10 protein was 5.17, which was a hydrophobic and nonsecretory protein. Under cold and drought stress, the cell membrane stability of transgenic tobacco leaves was significantly higher than that of wild-type tobacco leaves. Furthermore, the activities of peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR) in EnCBL10 transgenic plants increased significantly under cold and drought stress. Under cold stress, the proline content in the leaves of the two transgenic lines increased by 79.7% and 70.8% (P<0.05) compared with their respective controls. Under drought stress, the proline content decreased in WT and overexpressed plants, but the decrease of transgenic plants was less than in WT. Therefore, EnCBL10 may play an important role in low temperature and drought tolerance regulation in Elymus nutans.

Key words: Elymus nutans Griseb., EnCBL10, Gene cloning, Cold stress, Dought stress, Gene regulatipon, Physiological mechanism

中图分类号:

张慎彤, 高浩, 张金鹏, 任智慧, 姜惠娜, 其美拉姆, 普布卓玛, 付娟娟. 西藏野生垂穗披碱草EnCBL10基因的克隆与功能研究[J]. 草地学报, 2022, 30(9): 2315-2324.

ZHANG Shen-tong, GAO Hao, ZHANG Jin-peng, REN Zhi-hui, JIANG Hui-na, MEI La-mu, BU Zhuo-ma, FU Juan-juan. Cloning and Function Identification of EnCBL10 Gene from Tibetan Wild Elymus nutans Griseb.[J]. Acta Agrestia Sinica, 2022, 30(9): 2315-2324.

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参考文献

[1] 呼天明, 苗彦军. 西藏野生优质牧草繁育技术研究述评[J]. 草学, 2020(2):75-79
[2] BATISTI O, KUDLA J. Analysis of calcium signaling pathways in plants[J]. Biochimica et Biophysica Acta, 2012, 820(8):1283-1293
[3] ZHU J K. Abiotic stress signaling and responses in plants[J]. Cell, 2016(167):313-324
[4] KIM B, WAADT R, CHEONG Y, et al. The calcium sensor CBL10 mediates salt tolerance by regulating ion homeostasis in Arabidopsis[J]. The Plant Journal, 2007, 52(3):473-484
[5] YANG Y, HANG C, TANG R, et al. Calcineurin B-Like proteins CBL4 and CBL10 mediate two independent salt tolerance pathways in Arabidopsis[J]. International Journal of Molecular Sciences, 2019, 20(10):2421
[6] EGEA I, PINEDA B, ORTIZ A A, et al. The SlCBL10 calcineurin B-Like protein ensures plant growth under salt stress by regulating Na⁺ and Ca²⁺ homeostasis[J]. Plant physiology, 2018, 176(2):1676-1693
[7] KANG H, NAM K. Reverse function of ROS-induced CBL10 during salt and drought stress responses[J]. Plant Science, 2016(243):49-55
[8] FU J J, MIAO Y J, SHAO L H, et al. De novo transcriptome sequencing and gene expression profiling of Elymus nutans under cold stress[J]. BMC Genomics, 2016, 17(1):870
[9] 姜惠娜, 敬松, 李晗玉, 等. 西藏野生垂穗披碱草EnPLA1基因克隆与表达分析[J]. 草地学报, 2021, 29(10):2141-2148
[10] 其美拉姆, 普布卓玛, 崔彤彤, 等. 外源钙对低温胁迫下西藏野生垂穗披碱草生理及相关基因表达的影响[J]. 草地学报, 2021, 29(5):919-928
[11] KENNETH J. L, THOMAS D. S. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4):402-408
[12] SONG L, DING W, ZHAO M, et al. Nitric oxide protects against oxidative stress under heat stress in the calluses from two ecotypes of reed[J]. Plant Science, 2006, 171(4):449-458
[13] 李合生. 植物生理生化实验原理和技术[M]. 北京:高等教育出版社, 2000:279-298
[14] ORMANCEY M, THULEAU P, MAZARS C, et al. CDPKs and 14-3-3 Proteins:emerging duo in signaling[J]. Trends in Plant Science, 2017, 22(3):263-272
[15] TANG R, WANG C, LI K, et al. The CBL-CIPK calcium signaling network:unified paradigm from 20 years of discoveries[J]. Trends in Plant Science, 2020, 25(6):604-617
[16] SANYAL S. Plant stress responses mediated by CBL-CIPK phosphorylation network Enzymes[J]. 2016(40):31-64
[17] WEINL S, KUDLA J. The CBL-CIPK Ca²⁺-Decoding Signaling Network:Function and Perspectives[J]. New Phytologist, 2009, 184(3):517-528
[18] OLIVER B, RAINER W, LEONIE S, et al. CBL-mediated targeting of CIPKs facilitates the decoding of calcium signals emanating from distinct cellular stores[J]. The Plant Journal, 2010, 61(2):211-222
[19] PLASENCIA F A, ESTEADA Y, FLORES F B, et al. The Ca²⁺ sensor calcineurin B-Like protein 10 in plants:Emerging new crucial roles for plant abiotic stress tolerance[J]. Frontiers in Plant Science, 2021(11):599944
[20] 孙玉珺, 闫冬, 朱晶桓, 等. 外源油菜素内酯对低温胁迫下玉米发芽及幼苗生理特性的影响[J]. 华北农学报, 2019, (3):119-128
[21] 陈汇林, 侯伟. 低温对豇豆幼苗抗氧化酶系统的影响[J]. 气象与环境科学, 2018, 41(3):47-50
[22] CARVALHO M H C D. Drought stress and reactive oxygen species[J]. Plant Signal Behavior, 2008, 3(3):156-165
[23] FU J J, SUN P Y, LUO Y L, et al. Brassinosteroids enhance cold tolerance in Elymus nutans via mediating redox homeostasis and proline biosynthesis[J]. Environmental and Experimental Botany, 2019, 167(11):103831
[24] KAMEL A T, SAHAR A. Stimulation of ROS-scavenging systems in squash (Cucurbita pepo L.) plants by compost supplementation under normal and low temperature conditions[J]. Scientia Horticulturae, 2011, 130(4):862-868
[25] 郭燕, 张树航, 李颖, 等. 板栗抗寒性相关指标筛选与评价方法建立[J]. 西北农林科技大学学报, 2018, 46(10):40-48
[26] 项洪涛, 齐德强, 李琬, 等. 低温胁迫下外源ABA对开花期水稻叶鞘激素含量及抗寒生理的影响[J]. 草业学报, 2019, 28(4):81-94
[27] MAROK M A, TARRAGO L, KSAS B, et al. A drought-sensitive barleyvariety displays oxidative stress and strongly increased contents in low-molecular weight antioxidant compounds during water deficit compared to a tolerant variety[J]. Journal of Plant Physiology, 2013, 170(7):633-645
[28] WANG W B, KIM Y H, LEE H S, et al. Analysis of antioxidant enzyme activity during germination of alfalfa under salt and drought stresses[J]. Plant Physiology and Biochemistry, 2009, 47(7):570-577
[29] KAUL S, SHARMA S, MEHTA I. Free radical scavenging potential of L-proline:evidence from in vitro assays[J]. Amino Acids, 2008, 34(2):315-320
[30] 韩忠明, 胥苗苗, 王云贺, 等. 干旱胁迫对防风叶片保护酶活性、渗透调节物质含量及药材品质的影响[J]. 华南农业大学学报, 2016, 37(6):91-97
[31] CHEN X X, DING Y L, YANG Y Q, et al. Protein kinases in plant responses to drought, salt, and cold stress[J]. Journal of Integrative Plant Biology, 2021, 63(1):53-78
[32] LIU Y, XU C J, ZHU Y F, et al.The calcium-dependent kinase OsCPK24 functions in cold stress responses in rice[J]. Journal of Integrative Plant Biology, 2018, 60(2):173-188
[33] ZOU J J, LI X D, RATNASEKERA D, et al. Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE8 and CATALASE3 function in abscisic acid-Mediated signaling and H₂O₂ homeostasis in stomatal guard cells under drought stress[J]. Plant Cell, 2015, 27(5):1445-1460

西藏野生垂穗披碱草EnCBL10基因的克隆与功能研究

Cloning and Function Identification of EnCBL10 Gene from Tibetan Wild Elymus nutans Griseb.

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