雪莲SikCDPK1基因的表达特征和蛋白激酶活性分析  

Expression characteristics and protein kinase activity analysis of the SikCDPK1 gene in Saussurea involucrata

作　　者：刘玉玲 朱新霞[1] 吕新华[1] 孙辉[1] LIU Yu-ling;ZHU Xin-xia;LYU Xin-hua;SUN Hui(School of Life Sciences,Shihezi University,Key Laboratory of Oasis Town and Mountain Basin System Ecology Corps,Shihezi 832003,China;Urumqi Vocational University,Urumqi 830000,China)

机构地区：[1]石河子大学生命科学学院,绿洲城镇与山盆系统生态兵团重点实验室,新疆石河子832003 [2]乌鲁木齐职业大学,新疆乌鲁木齐830000

出　　处：《草业学报》2023年第9期213-221,共9页Acta Prataculturae Sinica

基　　金：国家自然科学基金项目(31760066)资助。

摘　　要：极端生境草本植物天山雪莲中的Sik CDPK1基因可明显提高转基因烟草的低温、干旱耐受性。为探究Sik CDPK1基因的表达特征和蛋白激酶活性,本研究利用实时荧光定量PCR技术检测了Sik CDPK1基因在雪莲不同组织器官的表达水平以及对信号分子CaCl_(2)、脱落酸(ABA)、赤霉素(GA_(3))、水杨酸(SA)、H_(2)O_(2)的诱导响应模式,采用Kinase-GloTM技术检测了SIKCDPK1蛋白激酶活性,利用绿色荧光蛋白(GFP)基因融合蛋白表达法进行了SIKCDPK1蛋白的亚细胞定位。结果显示:Sik CDPK1基因在雪莲根、茎、叶、种子、幼茎、幼叶中均表达,Sik CDPK1基因受CaCl_(2)、ABA、GA_(3)、SA、H_(2)O_(2)的诱导表达但响应模式有差异;激酶活性分析发现Ca^(2+)存在时,SIKCDPK1具有激酶催化活性,加入Ca^(2+)螯合剂EGTA后,SIKCDPK1几乎没有激酶活性。SIKCDPK1激酶活性随Ca^(2+)浓度增加而增加,Ca^(2+)浓度(K0.5)为48.7 nmol·L^(-1)时,SIKCDPK1激酶活性可达到最大活性的1/2;以HisⅢ为底物时,SIKCDPK1的Km值可达到43.8μg·m L^(-1),SIKCDPK1蛋白定位于细胞核。上述结果表明Sik CDPK1基因表达无明显组织特异性,可响应信号分子Ca^(2+)、ABA、GA_(3)、SA、H_(2)O_(2)的诱导;SIKCDPK1发挥蛋白激酶活性需要Ca^(2+),SIKCDPK1主要在细胞核里发挥作用。The SikCDPK1 gene in the extreme habitat plant Saussurea involucrata can significantly improve the low temperature and drought tolerance of genetically modified tobacco.In order to further explore the expression characteristics and protein kinase activity of SikCDPK1 gene,real-time quantitative PCR was used to detect the expression level of SikCDPK1 gene in different tissues and organs of S.involucrata and the induced response mode of signal molecules CaCl_(2),abscisic acid,gibberellin,salicylic acid and hydrogen peroxide,while Kinase-Glo™technology was used to detect the activity of SIKCDPK1 protein kinase,and the green fluorescent protein(GFP)fusion protein expression method was used to carry out the subcellular localization of SIKCDPK1 protein.The SikCDPK1 gene was found to be expressed in roots,stems,leaves,seeds,young stems and young leaves of S.involucrata and expression was induced by CaCl_(2),abscisic acid,gibberellin,salicylic acid,hydrogen peroxide,but the response mode was different.Kinase activity analysis showed that SIKCDPK1 had kinase catalytic activity when Ca^(2+)was present,and SIKCDPK1 had almost no kinase activity after adding the Ca^(2+)chelating agent ethylene glycol tetraacetic acid.SIKCDPK1 kinase activity increased with increase in Ca^(2+)concentration,and SIKCDPK1 kinase activity reached 1/2 of the maximum activity when Ca^(2+)concentration(K0.5)was 48.7 nm.When HisⅢwas used as the substrate,the Km value of SIKCDPK1 reached 43.8μg·mL^(-1),and SIkCDPK1 protein was localized to the nucleus.In summary,the expression of the SikCDPK1 gene had no obvious tissue specificity,and could respond to the induction of signaling molecules Ca^(2+),abscisic acid,gibberellin,salicylic acid,hydrogen peroxide.Ca^(2+)is required for SIKCDPK1 to exert protein kinase activity,and SIKCDPK1 mainly functions in the nucleus.

关 键 词：SikCDPK1基因 表达 蛋白激酶活性 

分 类 号：Q943.2[生物学—植物学]