林烟草蛋白激酶NsylCIPK3基因的克隆与功能分析

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,与类钙调磷酸酶B亚基CBL(calcineurin B-like protein)蛋白共同形成CBL-CIPK网络,在植物的生长发育和逆境胁迫响应过程中发挥重要作用。烟草中该家族的研究还比较少,本研究从林烟草(Nicotiana sylvestris)中获得一个CIPK家族基因,该基因与拟南芥和杨树中的CIPK3同源性分别为68.4%和87.5%,将其命名为Nsyl CIPK3。氨基酸序列分析表明,Nsyl CIPK3具有CIPK蛋白家族的典型结构特征,在N端和C端分别具有典型的激活环结构域和NAF结构域。进化树分析显示,Nsyl CIPK3属于CIPK蛋白亚家族Ⅱ。表达模式研究表明,该基因在林烟草的叶和腋芽中的表达量相对较高,在主根中的表达量次之,在侧根、茎、花瓣和萼片中的表达量相对较低,并且在烟草成熟期的叶中表达量明显升高。在高盐、紫外光和低钾胁迫下该基因的表达发生不同程度的上调。酵母双杂交结果显示,Nsyl CIPK3可与Nsyl CBL9互作。推测Nsyl CIPK3可能通过与Nsyl CBL9互作形成信号通路,激活下游靶蛋白,参与烟草响应非生物逆境胁迫的信号转导过程。

Cloning and Functional Analysis of NsylCIPK3 Gene from Nicotiana sylvestris

As a serine/threonine protein kinase family, CBL-interacting protein kinase (CIPK) is one of the most populated families. CIPKs are activated by calcineurin B-like protein CBLs and form the CBL-CIPK network which can play an important role with response to different adversity stress and impact on the process of plant growth and development. Although many research have done in Arabidopsis, but seldom studies have done in model plant tobacco. We have identified NsylCIPK3, one CIPK gene from Nicotiana sylvestris genome which is highly homolog with PtCIPK3 and AtCIPK3 up to 87.5% and 68.4% accordingly. Amino acid sequence analysis showed that, the NsylCIPK3 protein encoded a typical activation loop domains at N-terminal, and C-terminal has a highly conserved NAF domain. Phylogeny revealed that it belonged to number II subfamily of CIPK family with rich in introns. Expression pattern analysis of different tissue demonstrated that NsylCIPK3 can express in both root and leaf, especially highly expressed in the leaves of mature stage. Real-time quantitative PCR results displayed that it can response to high salt, high temperature, UV, low potassium and chilling treatments. The results of protein-protein interaction by yeast two hybrid illustrated that NsylCIPK3 interacts with NsylCBL9. It assumes that NsylCIPK3 can be activated by NsylCBL9 and perform as NsylCBL9-NsylCIPK3complex, this complex might be involved in abiotic stress signal transductions pathways in tobacco.