柑橘抗溃疡病转录因子CsAP2-09互作蛋白筛选与分析

Interacting Protein Screening and Analysis of CsAP2-09-A Citrus Bacterial Canker Related Transcription Factor

以前期获得的柑橘抗溃疡病的正调控转录因子基因CsAP2-09超表达植株为材料,利用GST融合蛋白沉降技术(GST pull-down)联合液相色谱串联质谱(LC–MS/MS)方法筛选并鉴定CsAP2-09的互作蛋白。首先原核表达并纯化GST-CsAP2-09作为诱饵蛋白,然后与CsAP2-09超表达植株叶片总蛋白孵育、洗脱后进行SDS-PAGE验证,最后进行LC–MS/MS鉴定。得到17个互作蛋白,将蛋白进行注释、GO分析、KEGG分析,发现其中5个可能与植物抗病相关(如过氧化氢酶Cs3g27280)。构建这17个蛋白的互作网络,其中14个蛋白的互作关系得到已有数据的支持。对CsAP2-09与互作蛋白的共表达分析发现CsAP2-09超表达引起了16个蛋白表达上调和1个蛋白表达下调。

CsAP2-09 is a positive transcription factor involved in the citrus bacterial canker resistance.In this study,the GST pull-down and LC–MS/MS strategies were used for the screening and network analysis of the interacting proteins of CsAP2-09.Firstly,the fusion proteins GST-CsAP2-09 were expressed and purified for the next GST pull-down.Then GST-CsAP2-09 was fixed on the GST-beads and incubated with total proteins extracted from CsAP2-09 over-expression plant.The interacting proteins of CsAP2-09 were detected by LC–MS/MS.From the annotation,GO and KEGG,17 interacting proteins were obtained,5 of which were reported to be involved in bacterial resistance,e.g.Cs3 g27280(CAT)-a catalase.The interacting network was constructed,among which 14 of 17 interacting proteins were involved.Sixteen proteins were positively regulated by CsAP2-09 in the transgenic plant while 1 protein was negatively regulated.