拟南芥蛋白激酶SnRK1.1对转录因子FD的磷酸化分析

doi:10.7668/hbnxb.2017.04.006

摘要/Abstract

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 kDa的GST-SnRK1.1蛋白以及58 kDa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。

关键词: SnRK1.1, FD, 蛋白激酶, 转录因子, 磷酸化

Abstract: This study aimed at obtaining purified SnRK1.1 and FD proteins,and analyzing whether SnRK1.1 could phosphorylate transcription factor FD in flowering regulation pathway. 1 608 bp SnRK1.1 gene and 858 bp FD/FD-T282A genes were cloned successfully and linked into PGEX-4T-3 vector respectively. GST-SnRK1.1,GST-FD and GST-FD-T282A 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-FD-T282A proteins. 84 kDa GST-SnRK1.1 and 58 kDa GST-FD or mutated GST-FD-T282A proteins were purified using GST tag protein purification system. The purified GST-SnRK1.1 recombinant protein could phosphorylate GST-FD proteins but not mutated GST-FD-T282A protein. This result would provide a good basis to study how SnRK1.1 mediates flowering through phosphorylating FD.

Key words: SnRK1.1, FD, Protein kinase, Transcription factor, Phosphorylation

中图分类号:

袁敏, 葛伟娜, 王莉, 张岚, 张家琦. 拟南芥蛋白激酶SnRK1.1对转录因子FD的磷酸化分析[J]. 华北农学报, 2017, 32(4): 37-41. doi: 10.7668/hbnxb.2017.04.006.

YUAN Min, GE Weina, WANG Li, ZHANG Lan, ZHANG Jiaqi. Protein Kinase SnRK1.1 Phosphorylates Transcriptional Factor FD in Arabidopsis thaliana[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2017, 32(4): 37-41. doi: 10.7668/hbnxb.2017.04.006.

http://www.hbnxb.net/CN/Y2017/V32/I4/37

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