小麦蔗糖非发酵相关蛋白激酶SnRK3基因的克隆与分析

Cloning and analysis of wheat sucrose non-ferment-1-related protein kinase(SnRK3)gene

为明确小麦蔗糖非发酵-1相关蛋白激酶(sucrose non-ferment-1-related protein kinase,SnRK)在协调植物体内多种信号通路之间的作用,通过普通PCR方法从扬麦20中克隆SnRK基因,利用生物信息学方法对其进行分析,使用实时荧光定量PCR技术分析其在激素、干旱、盐和病菌胁迫下的表达模式,并测定瞬时过表达该基因对烟草叶片抗致病疫霉Phytophthora infestans侵染能力的影响。结果显示,从扬麦20中克隆获得SnRK3基因,命名为TaSnRK3.16-D;该基因编码蛋白可能通过与多种蛋白互作参与多种形式的调控;过表达TaSnRK3.16-D增强了小麦对脱落酸、NaCl、PEG、白粉病菌Blumeria graminis f.sp.tritici和赤霉病菌Fusarium graminearum胁迫的抗性,经亚细胞定位和根癌农杆菌Agrobacterium tumefaciens瞬时表达试验验证表明TaSnRK3.16-D定位于细胞膜上,且其在烟草叶片上过表达部位的致病疫霉侵染病斑颜色较对照病斑颜色浅,一定程度上抑制了致病疫霉的侵染。表明SnRK基因在小麦抗逆过程中发挥了一定作用。

To clarify the role of sucrose non-ferment 1-related protein kinase(SnRK)in coordinating multiple signaling pathways in wheat,SnRK gene was cloned from Yangmai 20 by ordinary PCR.Bioinformatics method was used for systematic analysis.Real-time PCR was used to analyze the expression pattern of the gene under hormone,drought,salt and pathogen stresses,and to determine the resistance to Phytophthora infestans in the tobacco leaves with transient overexpression of the gene.The results showed that TaSnRK3.16-D gene might interact with multiple proteins and participate in various forms of regulation.Overexpression of TaSnRK3.16-D enhanced wheat resistance to ABA,NaCl,PEG,Blumeria graminis f.sp.tritici and Fusarium graminearum stresses.Subcellular localization experiment showed that TaSnRK3.16-D was located on the cell membrane.Transient overexpression experiments with Agrobacterium tumefaciens showed that the color of the infected spots on the overexpressed parts of Nicotiana benthamiana leaves was lighter than that of the control,suggesting it inhibited the infection of P.infestation to a certain extent.These results provided data supporting for further research on the mechanisms of SnRK gene in wheat stress resistance.