茶树CsCIPK12与CsKIN10的互作鉴定及其表达分析

蔗糖非酵解-1相关蛋白激酶（Sucrose non-fermenting 1-related protein kinase,SnRK）在代谢调控和胁迫信号传递中发挥重要的作用。本文以茶树SnRK3亚家族的CsCIPK12基因和SnRK1亚家族的CsKIN10基因为研究对象,通过序列比对和进化分析,发现CsCIPK12和CsKIN10都具有N端激酶结构域和C端调节域;CsCIPK12具有与CBLs结合的NAF/FISL结构域,与拟南芥AtCIPK12,杨树PtCIPK17、18、19同源关系最近;而CsKIN10具有泛素相关的UBA结构域,与杨树PtSnRK1同源关系最近。通过酵母双杂交系统,证实了CsCIPK12和CsKIN10蛋白存在相互作用。表达分析发现,在自然冷驯化过程中,CsKIN10的表达模式与前期对CsCIPK12的研究结果一致,在龙井43、浙农12、大面白3个茶树品种中受低温不同程度地诱导;4℃短时低温处理发现,CsCIPK12和CsKIN10在成熟叶中受低温显著诱导（最高诱导水平分别为4倍和2.3倍）,而在新梢中,二者对低温的响应并不显著;ABA、葡萄糖以及蔗糖处理发现,CsCIPK12和CsKIN10在成熟叶中受这3种处理的显著诱导。结果表明,CsCIPK12与CsKIN10蛋白相互作用,参与ABA和糖信号途径,在茶树低温胁迫响应中可能发挥重要作用。

Interaction Identification and Expression Analysis of CsCIPK12 and CsKIN10 in Tea Plant

Sucrose non-fermenting 1-related protein kinase (SnRK) plays important roles in metabolic regulation and stress signal transduction. In this study, CsCIPK12 gene of the SnRK3 subfamily and CsKIN10 gene of the SnRK1 subfamily in tea plant were used as research objects. Protein sequence alignment and phylogenetic tree analysis showed that both CsCIPK12 and CsKIN10 have an N-terminal kinase domain and a C-terminal regulatory domain; CsCIPK12 has a NAF/FISL domain that binds to CBLs, and is homologous with AtCIPK12 in Arabidopsis thaliana and PtCIPK17, 18, and 19 in poplar; CsKIN10 has the ubiquitin-related UBA domain and is homologous with PtSnRK1 in poplar. Yeast two-hybrid analysis showed that CsCIPK12 could interact with CsKIN10. Expression analysis found that the expression of CsKIN10 was induced by natural cold acclimation to varying degrees in the three tea cultivars ‘Longjing 43’, ‘Zhenong 12’ and ‘Damianbai’, which was consistent with the results of the previous study on CsCIPK12. Under 4℃ cold stress, the expressions of CsCIPK12 and CsKIN10 were significantly up-regulated in mature leaves (the highest induction levels were 4-fold and 2.3-fold, respectively), while in the shoots, the responses of CsCIPK12 and CsKIN10 to the cold stress were not significant. Under ABA, glucose and sucrose treatments, the expression levels of CsCIPK12 and CsKIN10 were significantly up-regulated in mature leaves. These results suggest that CsCIPK12 interacts with CsKIN10, which are involved in ABA and sugar signaling pathways and may play an important role in cold response in tea plants.