应用GST pull-down技术筛选番茄SIVQ6互作蛋白  

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

作　　者：原贵波 莫双榕 钱莹 臧栋楠 杨帆 蒋红亮 武媛 丁海东[1] YUAN GuiBo;MO ShuangRong;QIAN Ying;ZANG DongNan;YANG Fan;JIANG HongLiang;WU Yuan;DING HaiDong(College of Bioscience and Biotechnology,Yangzhou University,Yangzhou 225009,Jiangsu)

机构地区：[1]扬州大学生物科学与技术学院,江苏扬州225009

出　　处：《中国农业科学》2020年第15期3146-3157,共12页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31101092);江苏省自然科学基金(BK20191437);扬州大学大学生科创基金(X20180673,X20180674和X20190706)。

摘　　要：【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BamHⅠ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凝胶电泳验证,通过LC-MS/MS检测SlVQ6可能互作的候选蛋白,并对筛选的SIVQ6互作蛋白通过GO、KEGG和蛋白互作网络进行生物信息学分析。【结果】成功构建pGEX-4T-1-PC-SlVQ6基因重组表达质粒,并获得带有GST标签的GST-SlVQ6融合蛋白,其分子量大小约为54 kD;将GST-SlVQ6和His-SlMPK1进行体外磷酸化试验,SlMPK1能够磷酸化SlVQ6,而作为阴性对照的GST与SlMPK1无相互作用,且SlVQ6不存在自磷酸化的现象,表明GST-SlVQ6和His-SlMPK1具有相互作用,且SlVQ6是SlMPK1的下游底物;以GST-SlVQ6融合蛋白为诱饵蛋白(空GST为阴性对照),利用pull-down试验筛选番茄叶片组织蛋白中与SlVQ6蛋白结合的蛋白,经SDS-PAGE分离、液相色谱-串联质谱(LC-MS/MS)鉴定以及Mascot与蛋白数据库检索,共鉴定出37个与SlVQ6结合的蛋白,包括蛋白激酶Receptor for Activated C Kinase 1B(RACK1B),通过GO、KEGG和蛋白互作网络的生物信息学分析,表明这些蛋白参�【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 BamHⅠand NotⅠof 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 phosphoryl

关 键 词：番茄 SlVQ6 SlMPK1 pull-down 互作蛋白 

分 类 号：S641.2[农业科学—蔬菜学]