林烟草NsylCBL10互作蛋白激酶NsylCIPK的鉴定与分析

Identification and Analysis of Interacted Protein Kinase NsylCIPK of NsylCBL10 in Nicotiana sylvestris

植物中的类钙调神经素B亚基蛋白CBL (calcineurin B-like protein)家族成员与其互作蛋白激酶CIPK (CBL-interacting kinase protein)家族成员形成了复杂精细的CBL-CIPK信号调控网络,在解码生物和非生物胁迫激发的钙信号及信号的进一步传导中发挥重要作用。CBL家族成员CBL10参与植物抵抗高盐胁迫、应对病菌侵染、参与分生组织和花器官发育、调节离子平衡等多个过程,对其下游互作CIPK家族成员的筛选与鉴定对于明确上述调节机制至关重要。为了鉴定林烟草(Nicotiana sylvestris) NsylCBL10下游互作NsylCIPK家族成员,本研究首先以拟南芥(Arabidopsis thaliana) AtCIPK家族成员和水稻(Oryza sativa) OsCIPK家族成员基因的CDS序列为检索序列,利用NCBI和中国烟草基因组数据库对林烟草中可能存在的NsylCIPK家族成员进行了预测;然后通过RT-PCR方法对预测到的NsylCIPK家族成员进行了克隆并利用生物学分析软件对其基因结构和蛋白保守结构域进行了分析;最后通过酵母双杂交实验鉴定了与Nsyl CBL10互作的NsylCIPK家族成员。结果表明,在林烟草中可能存在28个NsylCIPK家族成员,实际克隆获得了20个NsylCIPK家族成员,在酵母双杂交体系中与NsylCBL10互作的只有1个NsylCIPK家族成员Nsyl CIPK14a。本研究为解析林烟草及其他物种中CBL-CIPK信号通路的调节功能提供了试验数据。

Calcineurin B-like protein （CBL） family members interact with CBL-interacting kinase protein （CIPK） family members in plant to establish an exquisite and complicated CBL-CIPK network which plays a vital role in the process of decoding and further conducting calcium signals triggered by biotic and abiotic stresses. CBL10 of CBL family participates in multiple processes including tolerating high salinity, resisting bacteria pathogen invasion, taking part in the meristem and floral organ development, and regulating on homeostasis. Thus, the screening and identification of downstream CIPK family members is quite important for uncovering the above regulatory mechanism. In order to identify NsyICIPK family members which interact with NsylCBLIO in Nicotiana sylvestris, potential NsylCIPK members were firstly predicted in NCBI and China Tobacco Genome Database based on CDS sequences of Arabidopsis thaliana AtCIPK family members and Oryza sativa OsCIPK family members. Then the predicted NsylCIPK family members were cloned by RT-PCR, followed by the analysis of their gene structures and conserved protein domains. Finally, NsylCIPK family members interacting with Nsyl- CBL10 were identified in yeast two-hybird system. Results showed that there might be 28 potential NsylCIPK family members in Nicotiana sylvestris, of which 20 members were successfully cloned and 1 member, NsylCIPK14a, interacted with NsylCBL10 in yeast two-hybird system. This research could provide experimental data for the functional identification of CBL-CIPK pathways in Nicotiana sylvestris and other species.