褐飞虱类酵母共生菌菌体蛋白质提取方法的比较  

Extraction methods for mycoprotein, a yeast-like symbiote isolated from brown planthopper, Nilaparvata lugens, in rice

作　　者：张海强[1,2] 陈建明[2] 张珏锋[2] 

机构地区：[1]杭州师范大学生命与环境科学学院,浙江杭州310036 [2]浙江省植物有害生物防控重点实验室省部共建国家重点实验室培育基地、浙江省农业科学院植物保护与微生物研究所,浙江杭州310021

出　　处：《浙江农林大学学报》2015年第2期173-180,共8页Journal of Zhejiang A&F University

基　　金：国家自然科学基金资助项目(30771411,31272040);浙江省自然科学基金资助项目(LY13C140008)

摘　　要：为了筛选出适合褐飞虱Nilaparvata lugens类酵母共生菌蛋白质提取的细胞破碎方法,选择超声波破碎法、反复冻融法和液氮研磨法等3种细胞破碎方法提取共生菌吡虫啉敏感菌株和抗性菌株蛋白质。结果表明:菌体显微形态观察发现,使用超声波破碎法和液氮研磨法处理类酵母共生菌的细胞破碎效果均优于反复冻融法,处理后菌体分布均匀,破碎彻底;72 h是提取菌体蛋白质的最佳培养时间。定量分析表明:用超声波破碎法提取蛋白质质量浓度最高,液氮研磨法次之,反复冻融法提取蛋白质质量浓度的效果最差,各方法所提蛋白质质量浓度差异显著(P<0.05)。培养72 h后,超声波破碎法、液氮研磨法和反复冻融法提取的蛋白质质量浓度分别为2.82 g·L-1,2.62g·L-1和2.12 g·L-1(敏感菌株)及2.64 g·L-1,2.53 g·L-1和2.05 g·L-1(抗性菌株)。十二烷基磺酸钠-聚丙烯酰胺凝胶(SDS-PAGE)电泳分析发现:超声波破碎法获得的蛋白质不仅得率高,且条带清晰,丰度好。液氮研磨法虽得率高,但蛋白质部分降解,条带不清晰;而反复冻融法获得的蛋白质得率最低。超声波破碎法更适合于褐飞虱类酵母共生菌的蛋白质提取,该研究为后续蛋白质的双向电泳实验和分离差异蛋白质提供技术支撑。To select the most suitable cell breaking method for protein extraction of the yeast-like symbiote （YI_S） isolated from brown planthopper, Nilaparvata lugens, three cell breaking methods： ultrasonication, re- peated freeze-thaw, and liquid nitrogen grinding, were used to extract the mycoprotein of susceptible- and re- sistant-imidacloprid strains of YLS. Analysis was conducted by Duncan＇s new multiple range test （DMRT） and sodium dodecyl sulfate--polyacrylamide gel electrophoresis （SDS-PAGE）. Results of observation using cell mi- croscopic morphology showed that the breaking effect of the YLS cells treated with ultrasonication and liquid nitrogen grinding were greater than repeated freeze-thaw. After treatment, cells were uniformly distributed and thoroughly broken. Optimum culture time for mycoprotein extraction was 72 h. Protein concentration for the three methods was significantly different fd=8, P〈0.05） with uhrasouication highest, then liquid nitrogen grinding, and lastly repeated freeze-thaw. After being cultured for 72 h, protein concentrations for the suscepti- ble strain were ultrasonication, 2.82 g·L-1; liquid nitrogen grinding, 2.62 g·L-l; and repeated freeze-thaw, 2.12 g· L-a; protein concentrations for the resistant strain were ultrasonication, 2.64 g· L-1; liquid nitrogen grinding, 2.53 g· L-l; and repeated freeze-thaw, 2.05 g· L-1. Electrophoresis showed that proteins extracted by uhrasonica- tion had clear bands, greater abundance, and more protein; by liquid nitrogen grinding had partial protein degradation and unclear bands; and by repeated freeze-thaw had the least protein. Thus, the uhrasonication method was best for mycoprotein extraction of YLS from N. lugens, and in the future this will provide technical support for two dimensional eleetrophoresis protein experiments and separation of differential proteins. [ Ch, 4 fig. 1 tab. 22 ref. ]

关 键 词：植物保护学 褐飞虱 类酵母共生菌 菌体蛋白质 提取方法 

分 类 号：S476[农业科学—农业昆虫与害虫防治]