灰霉菌MyosinⅠ的体外表达纯化及抑制剂筛选  

In vitro expression,purification and inhibitor screening of MyosinⅠfrom Botrytis cinerea

作　　者：宋国红 刘松涛 李林蔚 刘飞[2] 张峰 SONG Guohong;LIU Songtao;LI Linwei;LIU Fei;ZHANG Feng(College of Plant Protection,Nanjing Agricultural University,Nanjing 210095,China;Institute of Botany,Jiangsu Province and Chinese Academy of Sciences,Nanjing 210014,China)

机构地区：[1]南京农业大学植物保护学院,江苏南京210095 [2]江苏省中国科学院植物研究所,江苏南京210014

出　　处：《南京农业大学学报》2023年第3期482-488,共7页Journal of Nanjing Agricultural University

基　　金：江苏省农业自主创新资金[CX(20)3129];江苏省六大人才高峰高层次人才项目(NY-035)

摘　　要：[目的]本文旨在体外表达纯化灰霉菌Ⅰ型肌球蛋白(MyosinⅠ)及筛选抑制剂。[方法]采用SF9昆虫细胞蛋白表达系统表达灰霉菌MyosinⅠ(BcMyosinⅠ)马达结构域,用亲和层析和凝胶过滤层析纯化方法纯化蛋白,用SDS-PAGE和Western blot检测蛋白表达情况。从实验室已有的化合物库中,用ATPase活性试剂盒测定化合物对BcMyosinⅠ的酶活性抑制率。采用菌丝生长速率法测定部分化合物对灰霉菌的菌丝生长抑制活性。以禾谷镰刀菌Ⅰ型肌球蛋白(FgMyosinⅠ)三维结构为模板,同源模建预测BcMyosinⅠ三维结构。用AutoDock-Vina软件将部分化合物与BcMyosinⅠ进行分子对接。[结果]成功在体外表达纯化BcMyosinⅠ。20μmol·L^(-1)条件下,化合物B1和B17对BcMyosinⅠ的酶活性抑制率分别为56.24%和65.39%,显著高于对照药剂氰烯菌酯对酶活性的抑制率(27.29%);B1和B17对灰霉菌的菌丝生长抑制率分别为32.90%和83.44%,显著高于对照药剂氰烯菌酯对菌丝生长的抑制率(1.06%)。对照药剂氰烯菌酯与BcMyosinⅠ的结合自由能为-29.26 kJ·mol^(-1),而化合物B1和B17与BcMyosinⅠ的结合自由能分别为-35.53和-36.78 kJ·mol^(-1),结合自由能低,说明化合物B1和B17对BcMyosinⅠ的抑制活性均高于氰烯菌酯。[结论]成功获得BcMyosinⅠ单体蛋白;筛选出B1和B17两个灰霉菌肌球蛋白抑制剂。[Objectives]The objective of this study was to obtain MyosinⅠfrom Botrytis cinerea and screen its inhibitors.[Methods]MyosinⅠmotor domain was expressed by SF9 insect cell protein expression system.The protein was purified by affinity chromatography and gel filtration chromatography,and the protein expression was detected by SDS-PAGE and Western blot.The inhibition rate of BcMyosinⅠwas determined by ATPase activity kit from the library of compounds in the laboratory.The mycelial growth inhibition activity of some compounds against B.cinerea was determined by mycelial growth rate method.The 3D structure of BcMyosinⅠwas predicted by homologous modeling using the 3D structure of FgMyosinⅠas a template.Some compounds were docked with BcMyosinⅠby AutoDock-Vina software.[Results]BcMyosinⅠwas successfully expressed and purified in vitro.At 20μmol·L^(-1),the inhibition rates of B1 and B17 on BcMyosinⅠactivity were 56.24%and 65.39%,respectively,which were significantly higher than that of control phenamacril(27.29%);the inhibition rates of B1 and B17 against the mycelial growth of B.cinerea were 32.90%and 83.44%,respectively,which were significantly higher than that of control phenamacril(1.06%).The binding energy of the control phenamacril to BcMyosinⅠwas-29.26 kJ·mol^(-1),while the binding energy of compound B1 and B17 to BcMyosinⅠwere-35.53 and-36.78 kJ·mol^(-1),respectively.The low binding free energy indicated that the inhibitory activities of compounds B1 and B17 on BcMyosinⅠwere higher than those of phenamacril.[Conclusions]BcMyosinⅠmonome protein was successfully obtained;B1 and B17 MyosinⅠinhibitors of B.cinerea were screened.

关 键 词：灰霉菌 MyosinⅠ 表达 纯化 抑制剂筛选 

分 类 号：S482.2[农业科学—农药学]