摘要
肌球蛋白(MyosinⅠ)是新型杀菌剂氰烯菌酯的靶标蛋白,为探究禾谷镰刀菌(Fusarium graminearum)中MyosinⅠ的三维结构及其与氰烯菌酯的互作机制,进一步阐释禾谷镰刀菌对氰烯菌酯潜在的抗性机理,本研究采用生物信息学方法对MyosinⅠ氨基酸序列进行分析,结果表明:该蛋白分子量大小为134.9ku,等电点为9.47,不具有信号肽,属非分泌型蛋白,无跨膜区,是亲水性蛋白,具有MYSc保守结构域。并采用结构生物学的方法,以禾谷镰刀菌(F.graminearum)cDNA为模板,根据MyosinⅠ重要功能域序列设计特异性引物进行PCR扩增,克隆了MyosinⅠ重要功能域基因并构建了三个功能域蛋白表达载体pHAT2-MyosinⅠ39-720,pETM20-MyosinⅠ39-720,pETM30-MyosinⅠ39-720,将重组表达载体转化到大肠杆菌BL21(BE3)中,并利用异丙基硫代半乳糖苷(IPTG)进行诱导表达,结果表明:主要功能域蛋白在大肠杆菌中表达,但主要以包涵体的形式存在。对该蛋白的生物信息学分析及表达纯化为其结构功能的分析及其与氰烯菌酯互作机制的研究奠定了基础。
The research was to investigate the structure of MyosinⅠand interaction between the MyosinⅠand JS399-19,further to explore the mechanism of resistance,for MyosinⅠis the target protein of a novel fungicide JS399-19.Some bioinformatics softwares were adopted to analyze the MyosinⅠsequence,and the results showed that MyosinⅠis hydrophilic non-secretory protein which molecular weight is 134.9 ku,pI is 9.47 and contains MYSc conserved domain but no signal peptide and transmembrane region.Special primers were designed based on the MYSc conserved domain sequence.The gene of Fg MyosinⅠwas obtained by PCR amplification with the template of F.graminearumcDNA,and three functional domain protein expression vectors pHAT2-MyosinⅠ39-720,pETM20-MyosinⅠ39-720,pETM30-MyosinⅠ39-720 were constructed respectively.Recombinant expression vectors were transformed into Escherichia coli BL21（BE3）and induced by IPTG.But the SDS-PAGE detection indicated that the functional domain protein of Fg MyosinⅠ was expressed in E.coli,but not solvable.It provides a basis for the study of MyosinⅠ structure and interaction mechanism between MyosinⅠ and JS399-19.
作者
迟梦宇
王健力
黄金光
HI Mengyu;WANG Jianli;HUANG Jinguang(College of Plant Health and Medicine/Key Lab of Integrated Crop Disease and Pest Management of Shandong province,Qingdao Agricultural Univeraity,Qingdao 266109,China;Yantai Entry-Exit Inspection and Quarantine of the People＇s Republic of China,Yantai 264000,China)
出处
《青岛农业大学学报（自然科学版）》
2018年第2期111-118,共8页
Journal of Qingdao Agricultural University(Natural Science)
基金
国家重点研发计划重点专项(2017YFD0201705)
国家自然基金(31471735)
青岛市现代农业产业技术体系(6622316106)
