河北师范大学学报—

期刊信息

刊名：河北师范大学学报（自然科学版）Journal of Hebei Normal University (Natural Science)
主办：河北师范大学
ISSN： 1000-5854
CN： 13-1061/N
中国科技核心期刊
中国期刊方阵入选期刊
中国高校优秀科技期刊
华北优秀期刊
河北省优秀科技期刊

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拟南芥蛋白激酶CDPK11融合蛋白的纯化及鉴定

袁敏¹

邢继红²

王莉¹

张岚¹

葛伟娜¹

张家琦¹

1. 华北理工大学生命科学学院, 基因组学与计算生物学研究中心, 河北唐山 063000;
2. 河北农业大学真菌毒素与植物分子病理学实验室, 河北保定 071001
DOI： 10.13763/j.cnki.jhebnu.nse.2017.06.012

Recombinant Purification and Identification of Protein Kinase CDPK11 of Arabidopsis thaliana

YUAN Min¹, XING Jihong², WANG Li¹, ZHANG Lan¹, GE Weina¹, ZHANG Jiaqi¹

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摘要/Abstract

摘要：

CDPK11是钙依赖蛋白激酶家族的重要成员，为了获得纯化的His-CDPK11融合蛋白，首先克隆了1488bp的 CDPK11 全长基因，酶切连接构建到pGEX-4T-3载体上，获得His-CDPK11原核表达载体并转化大肠杆菌表达菌株BL21：经IPTG诱导和His镍柱体系纯化获得分子量为50ku的His-CDPK11融合蛋白：Western blot免疫印迹技术检测证实该蛋白能被Anti-His抗体识别，表明获得了正确融合的His-CDPK11蛋白，为进一步解析CDPK11参与植物开花调控的分子机制提供了有力的工具.

Abstract：

CDPK11 is an important member of Ca²⁺ dependent kinases.In order to obtain His-CDPK11 recombinant protein,the full length of 1488 bp CDPK11 gene was cloned,digested and constructed into pGEX-4T-3 vector to obtain His-CDPK11 prokaryotic expression construct.The recombinant His-CDPK11 plasmid was transformed into Escherichia coli strain BL21.The 50 ku recombinant His-CDPK11 protein was expressed induction with IPTG and purified by Ni-NTA affinity column.The purified protein could be detected by Anti-His antibody in western blot assay,indicating that the purified protein was correct recombinant His-CDPK11 protein.This would provide a powerful tool for further illustration of the molecular mechanism of CDPK11 mediated flowering in plants.

关键词

关键词： CDPK11 ; 原核表达 ; 融合蛋白 ; 免疫印迹

Key words： CDPK11 ; prokaryotic expression ; recombinant protein ; western blot

参考文献 18

[1] 张春宝,赵丽梅,赵洪锟,等.植物蛋白激酶研究进展[J].生物技术通报,2011,27(10):17-22.doi:10.13560/j.cnki.biotech.bull.1985.2011.10.019
[2] SIMEUNOVIC A,MAIR A,WURZINGER B,et al.Subcellular Localization and Targets of Calcium-dependent Protein Kinases[J].Journal of Experimental Botany,2016,67(13):3855-3872.doi:10.1093/jxb/erw157
[3] 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
[4] 袁敏,邢继红,王莉,等.拟南芥蛋白激酶SnRK1.1的激酶活性分析[J].分子植物育种,2017,15(8):3003-3009.doi:10.13271/j.mpb.015.003003
[5] 高安礼,吴晴阳,张宇,等.拟南芥钙依赖的蛋白激酶CPK28正向参与渗透胁迫应答反应[J].科学通报,2014,59(1):50-58.doi:10.1007/s11434-013-0062-Z
[6] 曾后清,张夏俊,张亚仙,等.植物类钙调素生理功能的研究进展[J].中国科学,2016,46(6):705-715.doi:10.1360/N052015-00351
[7] VALMONTE G R,ARTHUR K,HIGGINS C M,et al.Calcium-dependent Protein Kinases in Plants:Evolution,Expression and Function[J].Plant and Cell Physiology,2014,55(3):551-569.doi:10.1093/pcp/pct200
[8] 袁敏,邢继红,王莉,等.拟南芥开花抑制因子TFL1与GRFs蛋白的相互作用[J].中国农业科学,2017,50(10):1772-1780.doi:10.3864/j.issn.0578-1752.2017.10.003
[9] SAIJO Y,KINOSHITA N,LSHIYAMA K,et al.A Ca²⁺-dependent Protein Kinase That Endows Rice Plants with Cold-and Salt-stress Tolerance Functions in Vascular Bundles[J].Plant and Cell Physiology,2001,42(11):1228-1233.doi:10.1093/pcp/pce158
[10] ASANO T,HAYASHI N,KOBAYASHI M,et al.A Rice Calcium-dependent Protein Kinase OsCPK12 Oppositely Modulates Salt-stress Tolerance and Blast Disease Resistance[J].Plant Journal,2012,69(1):26-36.doi:10.1111/j.1365-313X.2011.04766.x
[11] SZCZEGIELNIAK J,BORKIEWICZ L,SZURMAK B,et al.Maize Calcium-dependent Protein Kinase(ZmCPK11):Local and Systemic Response to Wounding,Regulation by Touch and Components of Jasmonate Signaling[J].Physiol Plantarum,2012,146(1):1-14.doi:10.1111/j.1399-3054.2012.01587.x
[12] JIANG S,ZHANG D,WANG L,et al.A Maize Calcium-dependent Protein Kinase Gene,ZmCPK4,Positively Regulated Abscisic Acid Signaling and Enhanced Drought Stress Tolerance in Transgenic Arabidopsis[J].Plant Physiology and Biochemistry,2013,71:112-120.doi:10.1016/j.plaphy.2013.07.004
[13] WANG Y,ZHANG M,KE K,et al.Cellular Localization and Biochemical Characterization of a Novel Calcium-dependent Protein Kinase from Tobacco[J].Cell Research,2005,15(8):604-612.doi:10.1038/sj.cr.7290330
[14] MA S Y,WU W H.AtCPK23 Functions in Arabidopsis Responses to Drought and Salt Stresses[J].Plant Molecular Biology,2007,65(4):511-518.doi:10.1007/s11103-007-9187-2
[15] CHOI H I,PARK H J,PARK J H,et al.Arabidopsis Calcium-dependent Protein Kinase AtCPK32 Interacts with ABF4,a Transcriptional Regulator of Abscisic Acid-responsive Gene Expression,and Modulates Its Activity[J].Plant Physiol,2005,139:1750-1761.doi:10.1104/pp.105.069757
[16] MATSCHI S,WERNER S,SCHULZE W X,et al.Function of Calcium-dependent Protein Kinase CPK28 of Arabidopsis thaliana in Plant Stem Elongation and Vascular Development[J].Plant Journal,2013,73:883-896.doi:10.1111/tpj.12090
[17] 崔超,呼延霆,尹大川.重组标签蛋白在蛋白质纯化中的研究进展[J].现代生物医学进展,2014,14(32):6372-6378.doi:10.13241/j.cnki.pmb.2014.32.043
[18] 袁敏,邢朝斌,葛伟娜,等.拟南芥开花诱导基因FT的蛋白表达及纯化[J].基因组学与应用生物学,2017,36(8):3053-3056.doi:10.13417/j.gab.036.003053