应用GST pull-down技术筛选番茄SIVQ6互作蛋白

【目的】含有VQ基序(VQ)的蛋白质是植物特异性蛋白质,具有保守的"FxxhVQxhTG"氨基酸序列,调节植物生长和发育。番茄SlMPK1在高温胁迫过程中发挥重要作用,酵母双杂交(Y2H)显示SlVQ6蛋白能够与SlMPK1相互作用,通过GST pull-down技术进一步验证SlMPK1与SlVQ6的互作以及SlVQ6的互作网络,为研究SlVQ6介导的高温应答信号通路奠定基础。【方法】通过pGEX-4T-1-PC-VQ6质粒构建,以含有目的基因SIVQ6的菌液为模板,设计特异性引物,扩增目的基因序列,将获得的目的基因VQ6克隆到载体pGEX-4T-1的Bam HⅠ和NotⅠ的酶切位点之间,将获得的重组质粒pGEX-4T-1-PC-VQ6转入TOP10克隆菌株经IPTG(异丙基硫代半乳糖苷)诱导表达,通过GST柱亲和纯化获得目标蛋白PC-VQ6,进而获得预期GST-SlVQ6融合蛋白,通过体外磷酸化进一步确定SlVQ6是否是SlMPK1的下游底物;以GST-SlVQ6为诱饵蛋白固定于GST Sepharose Beads上,与番茄叶片总蛋白孵育后洗脱,收集洗脱液进行SDS-PAGE凝胶...

Screening of Interacting Protein of Tomato SIVQ6 by GST Pull-Down

【Objective】The VQ motif-containing (VQ) proteins are plant-specific proteins with a conserved “FxxhVQxhTG” amino acid sequence, which regulate plant growth and development. SlMPK1 plays an important role in the process of high temperature stress, but its downstream target proteins are poorly understood. Although yeast double hybridization (Y2H) showed that SlVQ6 protein could interact with SlMPK1, there was no further experimental evidence. Therefore, it is especially important to verify the interaction between SlMPK1 and SlVQ6 and to study the interaction network of SIVQ6. 【Method】First, pGEX-4T-1-PC-VQ6 plasmid was constructed. Through design of specific primers based on the SlVQ6 gene sequence, and using the plasmid containing the target gene SIVQ6 as a template to amplify the target gene sequence, the obtained target gene VQ6 was cloned between the restriction sites of BamHⅠ and NotⅠ of the vector pGEX-4T-1, and the pGEX-4T-1-PC-VQ6 was transferred into a TOP10 clone strain. The target protein PC-VQ6 was obtained by IPTG (isopropyl thiogalactoside) inducing expression and the expected GST-SlVQ6 fusion protein was obtained by affinity purification by GST column. Phosphorylation was performed in vitro to further determine whether SlVQ6 is the substrate of SlMPK1. GST-SlVQ6 was used as the bait protein to fix on GST Sepharose Beads and incubated with tomato leaf total protein, and then the incubated fusion material was eluated. The eluate was collected and verified by SDS-PAGE gel electrophoresis. LC-MS/MS was used to detect the candidate proteins that could interact with SlVQ6, and the screened SIVQ6 interacting proteins were used for bioinformatics analysis by GO, KEGG, and protein interaction networks. 【Result】The results showed that we successfully constructed the recombinant gene expression plasmid pGEX4T-1-PC-SlVQ6 and obtained the GST-SlVQ6 fusion protein with a GST tag, of which the molecular weight is about 54 kD. GST-SlVQ6 and His-SlMPK1 were subjected to phosphorylation experiments in vitro. SlVQ6 could be phosphorylated by SlMPK1, however the GST could not be phosphorylated by SlMPK1 as a negative control and there was no autophosphorylation phenomenon in SlVQ6, indicating that SlVQ6 has an interaction with SlMPK1, and SlVQ6 is a downstream substrate of SlMPK1. GST-SlVQ6 fusion protein was used as bait protein (GST as negative control), and the protein bound to SlVQ6 protein in tomato leaf tissue protein was screened by pull-down test. Through SDS-PAGE, liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification, and Mascot protein database search, 37 SlVQ6-bound proteins were identified, including protein kinase Receptor for Activated C Kinase 1B (RACK1B). Bioinformatics analysis of GO, KEGG, and protein interaction networks indicated that these proteins are involved in a variety of signaling pathways, and eight of the ribosomal proteins may be closely related to high temperature stress. 【Conclusion】This research shows that SlVQ6 is a substrate protein of SlMPK1 and 37 proteins may interact with SlVQ6. These proteins are closely related to the stress response and may play an important role in the high temperature tolerance of tomato plants.