欢迎访问作物学报,今天是

作物学报 ›› 2014, Vol. 40 ›› Issue (03): 405-415.doi: 10.3724/SP.J.1006.2014.00405

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

花生AhSOS2基因的克隆及功能初探

张国嘉1,2,侯蕾2,王庆国2,李臻2,戴绍军1,刘炜2,*   

  1. 1东北林业大学盐碱地生物资源环境研究中心 / 东北油田盐碱植被恢复与重建教育部重点实验室, 黑龙江哈尔滨150040; 2山东省农业科学院生物技术研究中心 / 山东省作物遗传改良与生态生理重点实验室, 山东济南 250100
  • 收稿日期:2013-06-20 修回日期:2013-12-14 出版日期:2014-03-12 网络出版日期:2014-01-16
  • 通讯作者: 刘炜, E-mail: wheiliu@163.com, Tel: 0531-83179572
  • 基金资助:

    本研究由国家转基因生物新品种培育重大专项(2009ZX08001-010B)资助。

Cloning and Functional Characterization of Peanut Gene AhSOS2

ZHANG Guo-Jia1,2,HOU Lei2,WANG Qing-Guo2,LI Zhen2,DAI Shao-Jun1,LIU Wei2,*   

  1. 1Alkali Soil Natural Environmental Science Center, Northeast Forestry University / Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field, Ministry of Education, Harbin 150040, China; 2 Biotechnology Research Center, Shandong Academy of Agricultural Sciences / Key Laboratory of Genetic Improvement, Ecology and Physiology of Crops, Shandong Province, Jinan 250100, China
  • Received:2013-06-20 Revised:2013-12-14 Published:2014-03-12 Published online:2014-01-16
  • Contact: 刘炜, E-mail: wheiliu@163.com, Tel: 0531-83179572

摘要:

SOS2 (Salt Overly Sensitive 2)作为植物中一类重要的耐盐相关基因, 在调控细胞内离子平衡及参与植物对盐害的响应及适应过程中具有重要作用。本研究通过RACE的方法, 从花生叶片中分离到一长1462 bp、包含1341 bp开放阅读框(ORF)cDNA片段, 生物信息学分析显示, 该基因属SOS2类基因, 被命名为AhSOS2(GenBank登录号为HG797656), 编码446个氨基酸, 为丝氨酸/苏氨酸蛋白激酶。对基因表达特性的荧光定量PCR分析显示, AhSOS2在花生中为组成型表达; 且受盐胁迫及干旱诱导。经250 mmol L-1 NaCl处理后, 该基因在花生幼苗茎中被诱导表达, 表达量约是对照茎中的30; 而在30% PEG-6000模拟干旱处理下, 该基因在花生幼苗叶中表达量也明显升高。综合以上结果, 显示AhSOS2可能参与并调控花生对逆境的抗性及耐受性。目前, 已成功构建了AhSOS2的植物双元表达载体pCAMBIA1301P-AhSOS2并获得转基因植株, 初步功能分析显示, 过表达AhSOS2基因的转基因水稻对盐胁迫的耐受性提高。预期这方面的工作对于解析花生对盐害、干旱等逆境的适应及防御机制, 进而指导花生抗性育种及品质改良具有重要意义。

关键词: 花生, SOS2类基因AhSOS2, 荧光定量PCR, 表达特性, 抗逆, 品质改良

Abstract:

SOS2 (Salt Overly Sensitive 2) is a kind of important salt tolerance genes, which is involved in mediating the intracellular ion balance and plays an important role in the response of plants to salt damage and adaptation. In this study, a 1462 bp full-length cDNA, including a 1341 bp open reading frame, was isolated and harvested by RACE method. As the gene showed more than 70% homologous to members of SOS2 subfamily in other plants by bioinformatics analysis, it is then named as AhSOS2 (GenBank accession numberHG797656). Its coding protein AhSOS2 has 446 amino acids, and is a member of serine/threonine protein kinases. Quantitative real-time PCR (qRT-PCR) analysis showed that AhSOS2 was expressed constitutively in peanut tissues, and could be induced to express higher under salt and drought treatments. When treated with 250 mmol L-1 NaCl, the gene expression level increased dramatically to about 30 times higher than that of control in the stems of peanut seedlings, while the expression level of AhSOS2 increased markedly in the leaves of peanut seedlings when treated by drought stress that imitated by 30% PEG-6000.AhSOS2 is supposed to participate in stress resistance and tolerance in peanut. The expression binary vector of pCAMBIA1301P-AhSOS2 was constructed and the transgenic rice plants were obtained. The primary functional verification showed that the ability of salt tolerance and adaptation was enhanced in AhSOS2 overexpression rice. The above research is expected to uncover the adversity defense mechanisms of peanut grown under stress conditions, and further guide the resistance breeding and quality improvement of peanut.

Key words: Peanut, SOS2 gene AhSOS2, Quantitative real-time PCR, Expression pattern, Stress resistance, Quality improvement

[1]Greenway H, Munns R. Mechanisms of salt tolerance in nonhalophytes. Annu Rev Plant Physiol, 1980, 31: 149–190



[2]Zhu J K. Plant salt tolerance. Trends Plant Sci, 2001, 6: 66–71



[3]Bohnert H J, Nelson D E, Jensen R G. Adaptation to environmental stresses. Plant Cell, 1995, 7: 1099–1111



[4]Katiyar-Agarwal S, Zhu J, Kim K, Agarwal M, Fu X, Huang A, Zhu J K. The plasmamembrane Na+/H+ antiporter SOS1 interacts with RCD1 and functions in oxidative stress tolerance in Arabidopsis. Proc Natl Acad Sci USA, 2006, 103: 18816–18821



[5]Ji H, Pardo J M, Batelli G, Van Oosten M J, Bressan R A, Li X. The salt overly sensitive (SOS) pathway: established and emerging roles. Mol Plant, 2013, 6: 275–186



[6]Shi H, Pardo Q, Zhu J K. The putative plasma membrane Na+/H+ antiporter SOS1 controls long distance Na+ transport in plants. Plant Cell, 2002, 14: 465–477



[7]Gong D, Guo Y, Schumaker K S, Zhu J K. The SOS3 family of calcium sensors and SOS2 family of protein kinases in Arabidopsis. Plant Physiol, 2004, 134: 919–926



[8]Wu S J, Ding L, Zhu J K. SOS1, a genetic locus essential for salt tolerance and potassium acquisition. Plant Cell, 1996, 8: 617–627



[9]Liu J, Ishitani M, Halfter U, Kim C S, Zhu J K. The arabidopsis thaliana SOS2 gene encodes a protein kinase that is required for salt tolerance. Proc Natl Acad Sci USA, 2000, 97: 3735–3740



[10]Shi H, Zhu J K. SOS4, a pyridoxal kinase gene, is required for root hair development in arabidopsis. Plant Physiol, 2002, 129: 585–593



[11]Shi H, Kin Y S, Guo Y, Stevenson B, Zhu J K. The Arabidopsis SOS5 locus encodes a putative cell surface adhesion protein and required for normal cell expansion. Plant Cell, 2003, 15: 19–32



[12]Gong D, Guo Y, Jagendorf A T, Zhu J K. Biochemical characterization of the Arabidopsis protein kinase SOS2 that functions in salt tolerance. Plant Physiol, 2002, 130: 256–264



[13]Guo Y, Halfter U, Ishitani M, Zhu J K. Molecular characterization of functional domains in the protein kinase SOS2 that is required for plant salt tolerance. Plant Cell, 2001, 13: 1383–1399



[14]Liu J, Halfter U, Kim C S, Shi W, Zhu J K. SOS3 function in plant salt tolerance requires N-myristoylation and calcium binding. Proc Natl Acad Sci USA, 2000, 97: 3730–3734



[15]Qiu Q S, Guo Y, Dietrich M A, Schumaker K S, Zhu J K. Regulation of SOS1, a plasma membrane Na+/H+ exchanger in Arabidopsis thaliana, by SOS2 and SOS3. Proc Natl Acad Sci USA, 2002, 99: 8436–8441



[16]Hu D G, Li M, Luo H, Dong Q L, Yao Y X, You C X, Hao Y J. Molecular cloning and functional characterization of MdSOS2 reveals its involvement in salt tolerance in apple callus and Arabidopsis. Plant Cell, 2012, 31: 713–722



[17]Quan R, Lin H, Mendoza I, Zhang Y, Cao W, Yang Y, Shang M, Chen S, Pardo J M, Guo Y. SCABP8/CBL10, a putative calcium sensor, interacts with the protein kinase SOS2 to protect arabidopsis shoots from salt stress. Plant Cell, 2007, 19: 1415–1431



[18]张吉民, 苗华荣, 李正超, 郝素美, 闫强. 花生加工利用、贸易现状与展望. 武汉工业学院学报, 2002, (2): 104–106



Zhang J M, Miao H R, Li Z C, Hao S M , Yan Q, Zhu E J. Status and prospectsfor the processing, utilization and trade of peanut. Wuhan Polytech Univ, 2002, (2): 104–106 (in Chinese with English abstract)



[19]Moretzsohn Mde C, Hopkins M S, Mitchell S E, Kresovich S, Valls J F, Ferreira M E. Genetic diversity of Peanut (Arachis hypogaea L.) and its wild relatives based on the analysis of hypervariable regions of the genome. BMC Plant Biol, 2004, 4: 11



[20]姜慧芳, 任小平. 干旱胁迫对花生叶片SOD 活性和蛋白质的影响. 作物学报, 2004, 30: 169–174



Jing H F, Ren X P. Effect on SOD activity and protein content in groundnut leaves by drought stress. Acta Agron Sin, 2004, 30: 169–174 (in Chinese with English abstract)



[21]Bi Y P, Liu W, Xia H, Su L, Zhao C Z, Wan S B, Wang X J. EST sequencing and gene expression profiling of cultivated peanut (Arachis hypogaea L.). Genome, 2010, 53: 832–839



[22]Huang C, Picimbona J F, Li H Q, Li Z, Liu, Q, Liu W. An efficient method for total RNA extraction from peanut seeds. Russ J Plant Physiol, 2012, 59: 129–133



[23]Liu W, Xu Z H, Luo D, Xue H W. OsCKI1, a rice casein kinase I, plays significant roles in auxin related root development and functions of plant hormones. Plant J, 2003, 36, 189–202



[24]Hofgen R, Willmitzer L. Storage of competent cell for agrobacterium transformation. Nucl Acids Res, 1988, 16: 9877



[25]刘巧泉, 张景六, 王宗阳, 洪孟民, 顾铭洪. 根癌农杆菌介导的水稻高效转化系统的建立. 植物生理学报, 1998, 24: 259–271



Liu Q Q, Zhang J L, Wang Z Y, Hong M M, Gu M H. A highly efficient transformation system mediated by Agrobacterium tumefaciens in rice (Oryza sativa L.). Plant Physiol Mol Biol, 1998, 24: 259–271 (in Chinese with English abstract)



[26]Jefferson R A. Assaying chimeric genes in plants: the GLJS gene fusion system. Plant Mol Biol Rep, 1987, 5: 387−405



[27]Murray M G, Thompson W F. Rapid isolation of high molecular weight plant DNA. Nucl Acids Res, 1980, 8: 4321−4325



[28]Zhu J K. Genetic analysis of plant salt tolerance using Arabidopsis. Plant Physion, 2000, 124: 941–948



[29]Chinnusamy V, Jagendorf A, Zhu J K. Understanding and improving salt tolerance in plants. Crop Sci, 2005, 45: 437–448



[30]Li L, Jia Z Q, Zhu Y J, Qi Y l. Research advances on drought resistance mechanism of plant species in arid area of China. J Desert Res, 2010, 30: 1053–1060



[31]Shi H, Ishitani M, Kim C, Zhu J K. The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na+/H+ antiporter. Proc Natl Acad Sci USA, 2000, 97: 6896–6901



[32]Ishitani M, Liu J, Halfter U, Kim C S, Shi W, Zhu J K. SOS3 function in plant salt tolerance requires N-myristoylation and calcium-binding. Plant Cell, 2000, 12: 1667–1677



[33]Zhu J K, Liu J, Xiong L. Genetic analysis of salt tolerance in Arabidopsis: evidence for a critical role of potassium nutrition. Plant Cell, 1998, 10, 1181–1191



[34]Liu J, Zhu J K. An Arabidopsis mutant that requires increased calcium for potassium nutrition and salt tolerance. Proc Natl Acad Sci USA, 1997, 94, 14960–14964



[35]Chen X F, Gu Z M, Liu F, Ma B J, Zhang H S. Molecular analysis of rice CIPKs involved in both biotic and abiotic stress responses. Rice Sci, 2011, 18: 1–9



[36]Halfter U, Ishitani M, Zhu J K. The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3. Proc Natl Acad Sci USA, 2000, 97: 3735–3740



[37]Jeong H J, Jwa N S, Kim K N. Identification and characterization of protein kinases that interact with the CBL3 calcium sensor in Arabidopsis. Plant Sci, 2005, 169: 1125–1135



[38]Hwang Y S, Bethke P C, Cheong Y H, Chang H S, Zhu T, Jones R L. A gibberellin-regulated calcineurin B in rice localizes to the tonoplast and is implicated in vacuole function. Plant Physiol, 2005, 138: 1347–1358



[39]Mahajan S, Sopory S K, Tuteja N. Cloning and characterization of CBL-CIPK signaling components from a legume (Pisum sativum). FEBS J, 2006, 273: 907–925



[40]Hrabak E M, Chan C W, Gribskow M, Harper J F, Choi J H, Halford N, Kudla J, Luan S, Nimmo H G, Sussman M R, Thomas M, Walker-Simmons K, Zhu J K, Harmon A C. Arabidopsis CDPK-SnRK super family of protein kinases. Plant Physiol, 2003, 132, 666–680



[41]Weinl S, Kudla J. The CBL-CIPK Ca2+-decoding signaling network: function and perspectives. New Phytol, 2009, 184: 517–528



[42]Chenk P W, Snaar-Jagalska B E. Signal perception and transduction: the role of protein kinases. Biochim Biophys Acta, 1999, 1449: 1–24



[43]Stone J M, Walker J C. Plant protein kinase families and signal transduction. Plant Physiol, 1995, 108: 451–457



[44]Pauly N, Knight M R, Thuleau P, vander Luit A H, Moreau M, Trewavas A J, Ranjeva R, Mazars C. Control of free calcium in plant cell nuclei. Nature, 2000, 405: 754–755



[45]Knight H. Calcium signaling during abiotic stress in plants. Int Rev Cytol, 1999, 195: 269–324



[46]Zhu J K, Liu J P, Xiong L M. Genetic analysis of salt tolerance in Arabidopsis thaliana: evidence of a critical role for potassium nutrition. Plant Cell, 1998, 10:1181–1192

 


 
[1] 杨欢, 周颖, 陈平, 杜青, 郑本川, 蒲甜, 温晶, 杨文钰, 雍太文. 玉米-豆科作物带状间套作对养分吸收利用及产量优势的影响[J]. 作物学报, 2022, 48(6): 1476-1487.
[2] 李海芬, 魏浩, 温世杰, 鲁清, 刘浩, 李少雄, 洪彦彬, 陈小平, 梁炫强. 花生电压依赖性阴离子通道基因(AhVDAC)的克隆及在果针向地性反应中表达分析[J]. 作物学报, 2022, 48(6): 1558-1565.
[3] 刘嘉欣, 兰玉, 徐倩玉, 李红叶, 周新宇, 赵璇, 甘毅, 刘宏波, 郑月萍, 詹仪花, 张刚, 郑志富. 耐三唑并嘧啶类除草剂花生种质创制与鉴定[J]. 作物学报, 2022, 48(4): 1027-1034.
[4] 丁红, 徐扬, 张冠初, 秦斐斐, 戴良香, 张智猛. 不同生育期干旱与氮肥施用对花生氮素吸收利用的影响[J]. 作物学报, 2022, 48(3): 695-703.
[5] 黄莉, 陈玉宁, 罗怀勇, 周小静, 刘念, 陈伟刚, 雷永, 廖伯寿, 姜慧芳. 花生种子大小相关性状QTL定位研究进展[J]. 作物学报, 2022, 48(2): 280-291.
[6] 汪颖, 高芳, 刘兆新, 赵继浩, 赖华江, 潘小怡, 毕晨, 李向东, 杨东清. 利用WGCNA鉴定花生主茎生长基因共表达模块[J]. 作物学报, 2021, 47(9): 1639-1653.
[7] 王建国, 张佳蕾, 郭峰, 唐朝辉, 杨莎, 彭振英, 孟静静, 崔利, 李新国, 万书波. 钙与氮肥互作对花生干物质和氮素积累分配及产量的影响[J]. 作物学报, 2021, 47(9): 1666-1679.
[8] 石磊, 苗利娟, 黄冰艳, 高伟, 张忠信, 齐飞艳, 刘娟, 董文召, 张新友. 花生AhFAD2-1基因启动子及5'-UTR内含子功能验证及其低温胁迫应答[J]. 作物学报, 2021, 47(9): 1703-1711.
[9] 高芳, 刘兆新, 赵继浩, 汪颖, 潘小怡, 赖华江, 李向东, 杨东清. 北方主栽花生品种的源库特征及其分类[J]. 作物学报, 2021, 47(9): 1712-1723.
[10] 张鹤, 蒋春姬, 殷冬梅, 董佳乐, 任婧瑶, 赵新华, 钟超, 王晓光, 于海秋. 花生耐冷综合评价体系构建及耐冷种质筛选[J]. 作物学报, 2021, 47(9): 1753-1767.
[11] 薛晓梦, 吴洁, 王欣, 白冬梅, 胡美玲, 晏立英, 陈玉宁, 康彦平, 王志慧, 淮东欣, 雷永, 廖伯寿. 低温胁迫对普通和高油酸花生种子萌发的影响[J]. 作物学报, 2021, 47(9): 1768-1778.
[12] 郝西, 崔亚男, 张俊, 刘娟, 臧秀旺, 高伟, 刘兵, 董文召, 汤丰收. 过氧化氢浸种对花生种子发芽及生理代谢的影响[J]. 作物学报, 2021, 47(9): 1834-1840.
[13] 张旺, 冼俊霖, 孙超, 王春明, 石丽, 于为常. CRISPR/Cas9编辑花生FAD2基因研究[J]. 作物学报, 2021, 47(8): 1481-1490.
[14] 戴良香, 徐扬, 张冠初, 史晓龙, 秦斐斐, 丁红, 张智猛. 花生根际土壤细菌群落多样性对盐胁迫的响应[J]. 作物学报, 2021, 47(8): 1581-1592.
[15] 李增强, 丁鑫超, 卢海, 胡亚丽, 岳娇, 黄震, 莫良玉, 陈立, 陈涛, 陈鹏. 铅胁迫下红麻生理特性及DNA甲基化分析[J]. 作物学报, 2021, 47(6): 1031-1042.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!