拟南芥蛋白激酶SnRK1.1对转录因子FD的磷酸化分析  

Protein Kinase SnRK1.1 Phosphorylates Transcriptional Factor FD in Arabidopsis thaliana

作　　者：袁敏 葛伟娜 王莉 张岚 张家琦 

机构地区：[1]华北理工大学生命科学学院基因组学与计算生物学研究中心,河北唐山063000

出　　处：《华北农学报》2017年第4期37-41,共5页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金项目(31401212);河北省自然科学基金项目(C2014209134);唐山市科技局项目(15140202a)

摘　　要：为了获得纯化的SnRK1.1与FD蛋白,分析蛋白激酶SnRK1.1是否能够磷酸化开花调控途径中的转录因子FD,成功克隆了1 608 bp的SnRK1.1基因以及858 bp的FD与FD-T282A基因,分别与PGEX-4T-3载体连接,获得原核表达载体GST-SnRK1.1、GST-FD和GST-FD-T282A,并分别转化大肠杆菌BL21菌株;SDS-PAGE电泳和考马斯亮蓝染色(CBB)表明,0.5 mmol/L IPTG能够成功诱导出GST-SnRK1.1、GST-FD和GST-FD-T282A蛋白。利用GST纯化介质纯化获得了84 k Da的GST-SnRK1.1蛋白以及58 k Da的GST-FD与GST-FD-T282A融合蛋白;利用体外磷酸化体系证实SnRK1.1能够磷酸化FD,不能磷酸化突变的FD-T282A。研究结果为进一步解析SnRK1.1通过磷酸化转录因子FD参与开花途径调控的机制奠定了良好的基础。This study aimed at obtaining purified SnRK1. 1 and FD proteins,and analyzing whether SnRK1. 1could phosphorylate transcription factor FD in flowering regulation pathway. 1 608 bp SnRK1. 1 gene and 858 bp FD/FD-T282 A genes were cloned successfully and linked into PGEX-4T-3 vector respectively. GST-SnRK1. 1,GST-FD and GST-FD-T282 A expression vectors were made and transformed into E. coli strain BL21. SDS-PAGE and CBB staining showed that 0. 5 mmol/L IPTG could successfully induce GST-SnRK1. 1,GST-FD and GST-FDT282 A proteins. 84 k Da GST-SnRK1. 1 and 58 k Da GST-FD or mutated GST-FD-T282 A proteins were purified using GST tag protein purification system. The purified GST-SnRK1. 1 recombinant protein could phosphorylate GSTFD proteins but not mutated GST-FD-T282 A protein. This result would provide a good basis to study how SnRK1. 1mediates flowering through phosphorylating FD.

关 键 词：SnRK1.1 FD 蛋白激酶 转录因子 磷酸化 

分 类 号：Q71[生物学—分子生物学] Q78