玉米大斑病菌转录调节因子StFlo8互作蛋白的筛选与鉴定  

Screening and Identification of the Interaction Proteins of the Transcriptional Regulator StFlo8 in Setosphaeria turcica

作　　者：孙贺贺 王程泽 贾京哲 申珅[1] 郝志敏[1] 董金皋[1] SUN He-He;WANG Cheng-Ze;JIA Jing-Zhe;SHEN Shen;HAO Zhi-Min;DONG Jin-Gao(State Key Laboratory of North China Crop Improvement and Regulation/Hebei Bioinformatic Utilization and Technological Innovation Center for Agricultural Microbes/Hebei Key Laboratory of Plant Physiology and Molecular Pathology,Hebei Agricultural University,Baoding 071000,China)

机构地区：[1]河北农业大学华北作物改良与调控国家重点实验室/河北省农业微生物生物信息利用技术创新中心/河北省植物生理与分子病理学重点实验室,保定071000

出　　处：《农业生物技术学报》2024年第6期1385-1392,共8页Journal of Agricultural Biotechnology

基　　金：河北省自然科学基金(C2021204112,C2020204172);国家玉米产业技术体系(CARS-02)。

摘　　要：Flo8(flocculation 8)基因是cAMP途径下游的关键转录调控因子,在真菌生长发育调控中发挥重要作用。本课题组前期初步明确了玉米大斑病菌(Setosphaeria turcica)StFlo8基因参与调控其附着胞发育及侵染丝形成。本研究利用大肠杆菌(Escherichia coli)表达系统对该基因与谷胱甘肽S-转移酶(glutathione S-transferase,GST)标签进行异源表达,进一步通过GST融合蛋白沉降技术(GST pull-down)从玉米大斑病菌菌丝蛋白中筛选可能与StFlo8有互作关系的蛋白,并通过酵母双杂交对其中部分蛋白进行互作关系的验证。结果表明,原核表达获得大小为116 kD的目标产物,与GST-StFlo8大小一致。将成功纯化的表达产物经GST pull-down,共获得41种和StFlo8可能有互作关系的蛋白。挑选其中部分蛋白进行酵母双杂交验证,发现Flo8与FAD依赖性氧化还原酶(FAD-dependent oxidoreductase)(GenBank No.165285)有互作关系。本研究初步明确了StFlo8的互作蛋白,为进一步解析玉米大斑病菌StFlo8活性调控分子机制提供了有效参考。Flo8(flocculation 8)gene,a key transcription regulatory factor downstream of the cAMP pathway,plays an important role in the regulation of fungal growth and development.In previous study,the involvement of StFlo8 in regulating the appressorium development and the formation of infection filaments in the early stage of Setosphaeria turcica was identified.In the present study,StFlo8 fused with glutathione S-transferase(GST)tag were heterologously expressed via the Escherichia coli expression system.Further,the GST pull down technology was used to screen the proteins that may interact with StFlo8 from the mycelial proteins of S.turcica,and the yeast two hybrid technology was used to verify the interaction of some of the proteins.The results showed that the target product with a size of 116 kD obtained by prokaryotic expression was consistent with the size of GST-StFlo8,41 proteins that may interact with StFlo8 were obtained from the successfully purified expression product through GST pull down.After validating a subset of proteins using the yeast two-hybrid technique,it was revealed that Flo8 interacts with FAD-dependent oxidoreductase(GenBank No.165285).This study preliminarily clarified the interaction protein of StFlo8,providing informative references for further elucidating the regulatory molecular mechanism of the activity of StFlo8 in S.turcica.

关 键 词：玉米大斑病菌 Flocculation 8(Flo8) 谷胱甘肽-S-转移酶融合蛋白沉降技术 互作蛋白 

分 类 号：S453.131.4[农业科学—植物保护]