烟草中蛋白激酶基因NtCIPK3的表达载体构建及表达分析

Construction of Expression Vector and Expression Analysis of Tobacco Protein Kinase Gene NtCIPK3

为了研究烟草中蛋白激酶CIPK3在植物非生物胁迫应答中的功能,通过同源克隆的方法,在普通烟草品种K326中克隆到1个CIPK家族基因NtCIPK3。利用生物信息学软件对该基因编码蛋白的结构和功能进行了预测分析。该基因包含一个1 272 bp的开放阅读框,编码423个氨基酸残基。蛋白序列分析表明,该蛋白含有一个跨膜结构域,且平均疏水性系数小于0,属于亲水性膜蛋白。蛋白比对分析发现,NtCIPK3含有高度保守的N端激酶区、连接区以及C端调控区,与野生烟草CIPK3的同源性最高,达99%。亚细胞定位预测显示,该蛋白主要定位于细胞核,且具有双分型的核定位信号序列。运用qRT-PCR技术,对该基因的表达特性进行了分析,组织表达分析发现,该基因在烟草的根、茎、叶、花中均有表达,但在叶中表达水平明显高于其他组织,推测可能主要在叶中行使功能。NtCIPK3基因在不同程度上受低钾、高盐、干旱、ABA、H_2O_2、低温处理的诱导,推测NtCIPK3基因在烟草的非生物胁迫应答反应中发挥了重要作用。并成功构建pBI121-NtCIPK3过表达载体,为今后该基因在非生物胁迫下的功能研究奠定了基础。

In order to study the function of CIPK3in abiotic stress response,a CIPK family gene NtCIPK3was cloned from common tobacco K326by homologous cloning.The gene contained a1272bp ORF encoding423amino acid.Protein sequence analysis indicated that the protein contained a transmembrane domain and belonged to a hydrophilic membrane protein.Protein alignment analysis revealed that NtCIPK3contained a highly conserved N-terminal kinase region,a junction region,and a C-terminal regulatory region,with the highest homology to wild tobacco CIPK3,reaching99%.Subcellular localization predictions showed that the protein was mainly located in the nucleus and had a bipartite typing nuclear localization signal sequence.The expression characteristics of this gene were analyzed by qRT-PCR.Analysis of tissue expression revealed that the gene was expressed in the roots,stems,leaves and flowers of tobacco and had tissue-specific expression and the expression level in leaves was significantly higher than that in other tissues,and it was speculated that it might function mainly in leaves.Meanwhile,NtCIPK3gene was induced by low potassium,high salt,drought,ABA,H2O2,and low temperature treatment.It is speculated that NtCIPK3gene played an important role in tobacco abiotic stress response.And successfully constructed the pBI121-NtCIPK3overexpression vector,which laid the foundation for the future research of the gene under abiotic stress.