甘蓝型油菜CIPK2与CIPK16基因的克隆、表达分析与互作CBL蛋白的鉴定  

Cloning, Expression Analysis and Interacting Protein CBL Identification of CIPK2 and CIPK16 Genes in Oilseed Rape(Brassica napus)

作　　者：李燕飞 辛玉琼 高世东 赵培玉 燕敬利 陈芹芹 张翰风 刘舞贞 杨博 李竞[1] 江元清[1] LI Yan-Fei;XIN Yu-Qiong;GAO Shi-Dong;ZHAO Pei-Yu;YAN Jing-Li;CHEN Qin-Qin;ZHANG Han-Feng;LIU Wu-Zhen;YANG Bo;LI Jing;JIANG Yuan-Qing(State Key Laboratory of Crop Stress Biology for Arid Areas,College of Life Sciences,Northwest A&F University,Yangling 712100,China)

机构地区：[1]西北农林科技大学生命科学学院旱区作物逆境生物学国家重点实验室

出　　处：《农业生物技术学报》2020年第1期1-12,共12页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31301648;31471153)

摘　　要：类钙调磷酸酶B互作蛋白激酶(calcineurin B-like interacting protein kinase,CIPK)是植物中广泛存在的一类参与解码钙信号的丝氨酸/苏氨酸(Ser/Thr)蛋白激酶,在植物非生物与生物逆境信号转导中起着重要作用。本研究在甘蓝型油菜(Brassica napus)中通过逆转录PCR(reverse transcription PCR,RT-PCR)克隆获得BnaCIPK2(GenBank No.KF169730)与BnaCIPK16(GenBank No.MK571826)基因的cDNA序列。序列分析表明,BnaCIPK2和BnaCIPK16基因的开放阅读框分别为1389和1386 bp,编码氨基酸残基分别为463与461个,这2个BnaCIPK基因编码的蛋白含有保守的激酶结构域与天冬酰胺-丙氨酸-苯丙氨酸(asparagine-alanine-phenylalanine,NAF)基序。亚细胞定位结果显示,2个CIPK蛋白主要分布于细胞质与细胞核。qRT-PCR结果显示,茉莉酸(jasmonic acid,JA)、冷害以及聚乙二醇(PEG8000)显著诱导BnaCIPK2的表达,百草枯(methyl viologen,MV)处理24 h后极显著抑制BnaCIPK2的表达;水杨酸(salicylic acid,SA)、热害、冷害、双氧水(H2O2)和PEG8000可诱导BnaCIPK16的表达量上调,JA和脱落酸(abscisic acid,ABA)处理则导致BnaCIPK16的表达量下调。酵母双杂交(yeast two-hybrid,Y2H)与双分子荧光互补(bimolecular fluorescence complementation,BiFC)结果表明,BnaCIPK2与油菜钙调磷酸酶B4蛋白(Brassica napus calcineurin B-like protein 4,BnaCBL4)、BnaCIPK16与BnaCBL3存在互作。本研究为阐明BnaCIPK2与BnaCIPK16的功能与调控机制提供了基础资料。Calcineurin B-like interacting protein kinase(CIPK)is a class of serine/threonine(Ser/Thr)protein kinases widely existing in plants that are involved in decoding calcium signals.CIPKs play important roles in abiotic and biotic stress signal transduction.In this study,BnaCIPK2 and BnaCIPK16 genes were successfully cloned by reverse transcription-polymerase chain reaction(RT-PCR)from oilseed rape(Brassica napus).Sequence analysis showed that the 2 proteins encoded by the 2 BnaCIPK genes contained conserved kinase domain and asparagine-alanine-phenylalanine(NAF)motif.The subcellular localization analysis showed that both BnaCIPK2 and BnaCIPK16 were localized in both cytoplasm and nuclei.qRT-PCR profiling at 3 time points revealed that jasmonic acid(JA),cold and polyethylene glycol(PEG8000)significantly induced the expression of BnaCIPK2.After methyl viologen(MV)treatment for 24 h,the expression of BnaCIPK2 was extremely significantly inhibited.Salicylic acid(SA),heat,cold,hydrogen peroxide(H2 O2)and PEG8000 upregulated the expression of BnaCIPK16,while JA and abscisic acid(ABA)treatments resulted in the repression of BnaCIPK16 expression.Yeast two-hybrid(Y2 H)screening and bimolecular fluorescence complementation(BiFC)showed that BnaCIPK2 and BnaCIPK16 interacted with Brassica napus calcineurin B-like protein 4(BnaCBL4)and BnaCBL3,respectively.The present study provides basic information for elucidating the functions and regulatory mechanisms of BnaCIPK2 and BnaCIPK16.

关 键 词：油菜 类钙调磷酸酶B互作蛋白激酶(CIPK) 钙调磷酸酶B(CBL) 非生物胁迫 互作蛋白 

分 类 号：S184[农业科学—农业基础科学]