摘要
农田杂草抗性不断加剧,除草剂靶标蛋白积累水平检测对于解析杂草对除草剂的抗性机制非常重要。为检测除草剂靶标酶乙酰乳酸合酶(acetolactate synthase, ALS),丰富对该酶的研究手段,本研究将多花黑麦草Lolium multiflorum Lamk.的ALS基因片段插入到原核表达载体pGEX-2TK上,转化导入大肠杆菌BL21感受态细胞中,诱导表达并纯化获得重组融合蛋白GST-LmALS,通过免疫小鼠获得抗ALS多克隆抗体。采用间接ELISA法测定多克隆抗体的效价,结果显示,该抗体的效价可达到1∶256 000。采用Western blot方法进一步对ALS抗体的特异性进行测试,结果表明,制备的抗ALS多克隆抗体可以检测到多花黑麦草ALS,并且还可以用于其他多种杂草的ALS检测;该抗体的成功制备将为后续除草剂靶标酶ALS的生化特性研究奠定良好的基础。
In order to achieve serological detection of the target enzyme ALS of weeds and enrich the detection methods of this enzyme,the ALS gene of Lolium multiflorum was ligated into the prokaryotic expression vector pGEX-2TK,the recombinant expression plasmid pGEX-2TK-Lm ALS was transformed into Escherichia coli BL21 cells,and the recombinant fusion protein GST-Lm ALS was induced and purified.After immunizing mice with purified fusion protein as immunogen for four weeks,anti-ALS polyclonal antibody was obtained.The titer of the antibody was determined by indirect ELISA,and the results showed that the antibody had a good titer,which could reach 1∶256000.Western blot analysis demonstrated that the prepared anti-ALS polyclonal antibody can specifically detect the ALS of L.multiflorum,and can also detect the ALS of various weeds.Preparation of polyclonal antibodies will lay a solid foundation for the subsequent study on the biochemical characteristics of herbicidal target enzyme ALS.
作者
刘宇
高海涛
董立尧
冯致科
LIU Yu;GAO Haitao;DONG Liyao;FENG Zhike(College of Plant Protection,Nanjing Agricultural University,Nanjing 210095,China)
出处
《植物保护》
CAS
CSCD
北大核心
2024年第6期254-261,共8页
Plant Protection
基金
国家重点研发计划(2023YFD1400501)。
