林烟草中与蛋白激酶NsylCIPK24a互作的NsylCBL蛋白家族成员的筛选鉴定

Screening and Identification of NsylCBL Family Members Interacting with Protein Kinase NsylCIPK24a in Nicotiana sylvestris

植物CIPK蛋白激酶家族(CBL-interacting protein kinase)是一类丝氨酸/苏氨酸蛋白激酶家族,该家族成员通过与上游的CBL蛋白(Calcineurin B-like protein)互作,形成CBL-CIPK信号系统,参与调控植物的生长发育和逆境胁迫响应过程。早期研究发现,拟南芥AtCIPK24分别通过与AtCBL4和AtCBL10互作,激活下游靶蛋白的活性,应对高盐胁迫。本研究前期从林烟草中获得了1个与拟南芥AtCIPK24同源的基因NsylCIPK24a,但与其互作的CBL家族成员及该激酶的具体功能尚不清楚。因此,本研究对林烟草CBL家族成员进行了全基因组预测;利用RT-PCR方法克隆CBL家族成员,并对其基因结构、蛋白保守结构域及组织表达情况进行了分析;通过酵母双杂交试验鉴定与NsylCIPK24a互作的林烟草CBL家族成员。结果表明,林烟草中共预测并克隆获得12个CBL家族成员;NsylCBL4、NsylCBL5、NsylCBL9和NsylCBL10 4个成员可与NsylCIPK24a在酵母中产生互作。推测烟草中可能也存在与拟南芥类似的应对盐胁迫的CBL-CIPK响应路径。本研究为解析林烟草NsylCIPK24a的功能、加深人们对烟草中CBL-CIPK调控通路的理解提供了研究数据。

The CIPK(CBL-interacting protein kinase) kinase family in plants is a type of serine/threonine protein kinase family. The members of this family interact with the upstream CBL protein(Calcineurin B-like protein) to form the CBL-CIPK signal system, involved in regulating plant growth and development as well as stress response processes. Early research found that AtCIPK24 interacts with AtCBL4 and AtCBL10, respectively, to activate downstream target proteins to respond to high salt stress. Earlier in this study, NsylCIPK24 a homologous to AtCIPK24 from Arabidopsis thaliana was obtained from Nicotiana sylvestris, but CBL family members interacting with NsylCIPK24 a and its specific function are unclear. Therefore, a genome-wide prediction of CBL family members of N.sylvestris was carried out;predicted NsylCBL genes were cloned by RT-PCR, and the analyses of gene structure,protein conserved domain and expression pattern were then conducted. The NsylCBL members interacting with Nsyl CIPK24 a were screened by the yeast two-hybrid system. Results showed that there are 12 potential NsylCBL genes in N. sylvestris, and all of them were cloned successfully. Four members including NsylCBL4, NsylCBL5,Nsyl CBL9 and NsylCBL10 could interact with NsylCIPK24 a in yeast. There might be some similar CBL-CIPK pathways in tobacco plants, compared with Arabidopsis. The study provides experimental data for the functional identification of Nsyl CIPK24 a, and increases understanding of CBL-CIPK network in N. sylvestris.