玉米大斑病菌蛋白激酶A催化亚基StPKA-C1/C2的表达与互作蛋白筛选  

Expression and Interacting Protein Screening of the Catalytic Subunit of Protein Kinase A StPKA-C1/C2 from Setosphaeria turcica

作　　者：武建颖 张燕 孙贺贺 赵玉兰 董金皋 申珅[1,2] 郝志敏 WU Jian-Ying;ZHANG Yan;SUN He-He;ZHAO Yu-Lan;DONG Jin-Gao;SHEN Shen;HAO Zhi-Min(State Key Laboratory of Crop Improvement and Regulation in North China,Baoding 071001,China;College of Life Sciences/Key Laboratory of Hebei Province for Plant Physiology and Molecular Pathology,Hebei Agricultural University,Baoding 071001,China;College of Plant Protection,Hebei Agricultural University,Baoding 071001,China)

机构地区：[1]华北作物改良与调控国家重点实验室,保定071001 [2]河北农业大学生命科学学院/河北省植物生理与分子病理学重点实验室,保定071001 [3]河北农业大学植物保护学院,保定071001

出　　处：《农业生物技术学报》2022年第10期1976-1986,共11页Journal of Agricultural Biotechnology

基　　金：河北省自然科学基金(C2020204172;C2021204112);国家现代农业产业技术体系(CARS-02-25);河北省省属高等学校基本科研业务费研究项目(KY202001)。

摘　　要：蛋白激酶A(protein kinase A,PKA)是真核细胞cAMP信号转导途径的核心元件,通过磷酸化多种活性蛋白调节丝状真菌的生长、发育及致病等生理过程。为明确玉米大斑病菌(Setosphaeria turcica)PKA可能的互作蛋白,进一步解析其作用的分子机制,本研究以玉米大斑病菌cDNA作为模板,扩增StPKA-C1/2基因编码区的完整序列,构建pGS21T-StPKA-C1/2原核表达重组载体。利用大肠杆菌(Escherichia coli)表达系统以异丙基β-D-硫代腺苷(isopropylβ-D-thiogalactopyranoside,IPTG)作为诱导物诱导表达并通过亲和层析法纯化获得融合蛋白GST-StPKA-C1/2。通过GST pull-down实验筛选玉米大斑病菌中与StPKA-C1/C2有互作关系的蛋白质,利用液相色谱-串联质谱法(LC-MS/MS)对所得蛋白质进行分析。结果表明,与StPKA-C1/2互作的蛋白中已有功能注释的蛋白包括催化活性蛋白、酶调节活性蛋白、运输活性蛋白、电子载体蛋白、分子伴侣蛋白、结构分子蛋白及代谢酶等。Venn分析发现,与StPKAC1与StPKA-C2共有互作蛋白52个,仅与StPKA-C1互作的蛋白有17个,仅与StPKA-C2互作的蛋白有18个。其中,靶蛋白SETTUDRAFT_168881作为延伸因子(elongation factor G,EFG)家族中的一员,可能调控菌丝生长。靶蛋白SETTUDRAF_37808属于丝束蛋白(Fimbrin)家族,可能参与调控肌动蛋白细胞骨架、氧化应激反应和形态发生,推测StPKA-C1/2通过与这些靶蛋白相互作用发挥重要生物学功能。本研究初步明确了玉米大斑病菌StPKA-C1/2的潜在互作蛋白,为进一步解析玉米大斑病菌PKA调控病菌致病性的分子机制提供了理论基础。Protein kinase A(PKA)is the core element of c AMP signal transduction pathway in eukaryotie,which regulates the growth and development of filamentous fungus through phosphorylation of a variety of active proteins.In order to clarify the possible interaction protein of PKA in Setosphaeria turcica,and further analyze the molecular mechanism of its acts,in this study,the complete coding region of StPKA-C1/2 was amplified from the total complementary DNA of S.turcica to construct the expression vector p GST-StPKA-C1/2,which was introduced into Eschaeria coli.The fusion protein GST-StPKA-C1/C2 was induced in E.coli by isopropylβ-D-thiogalactopyranoside and then purfied by affinity chromatography.The GST pull-down strategy was used to screen the interacting proteins of StPKA-C1/2.The interacting proteins were detected by liquid chromatography-tandem mass spectrometry(LC-MS/MS).The results showed that 69 StPKA-C1interacting proteins and 70 StPKA-C2 interacting proteins were identified.The functional annotations of proteins included catalytically active protein,enzyme regulating active protein,transport electron carrier protein,molecular chaperone protein,structural molecular protein,metabolic enzyme,etc.Venn analysis found that StPKA-C1 shared 52 interacting proteins with StPKA-C2,with only 17 proteins interacting with StPKA-C1,and only 18 proteins interacting with StPKA-C2.The target protein SETTUDRAFT_168881,as a member of the elongation factor G(EFG)family,may regulate hyphal growth.The target protein SETTUDRAF_37808 belonged to the Fimbrin protein family of microfilament-binding proteins and may be involved in the regulation of the actin cytoskeleton,oxidative stress response,and morphogenesis.It was suggested that StPKA-C1/2 might play important biological functions by interacting with these target proteins.The above results preliminarily clarified the potential interaction protein of StPKA-C1/2.This study provides a theoretical basis for further analyzing the molecular mechanism of PKA.

关 键 词：玉米大斑病菌 蛋白激酶A(PKA) 原核表达 GST pull-down 互作蛋白 

分 类 号：S453.131.4[农业科学—植物保护]