中国农业科学 ›› 2022, Vol. 55 ›› Issue (24): 4851-4862.doi: 10.3864/j.issn.0578-1752.2022.24.006

• 植物保护 • 上一篇    下一篇

牛筋草EPSPS酶联免疫试剂盒的研发及应用

李志玲(),李香菊,崔海兰,于海燕,陈景超()   

  1. 中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京 100193
  • 收稿日期:2022-07-18 接受日期:2022-09-03 出版日期:2022-12-16 发布日期:2023-01-04
  • 通讯作者: 陈景超
  • 作者简介:李志玲,E-mail:82101215278@caas.cn
  • 基金资助:
    北京市自然科学基金(6222051)

Development and Application of ELISA Kit for Detection of EPSPS in Eleusine indica

LI ZhiLing(),LI XiangJu,CUI HaiLan,YU HaiYan,CHEN JingChao()   

  1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193
  • Received:2022-07-18 Accepted:2022-09-03 Online:2022-12-16 Published:2023-01-04
  • Contact: JingChao CHEN

摘要:

【目的】克隆牛筋草(Eleusine indica)中草甘膦的靶标基因EPSPS,获得牛筋草EPSPS重组蛋白并制备其单克隆及多克隆抗体,组装酶联免疫试剂盒,检测牛筋草植株不同组织EPSPS的表达水平及蛋白含量,为快速鉴定抗性牛筋草提供简便工具。【方法】利用PCR方法克隆EPSPS基因全长;构建pET30a-EPSPS阳性质粒并转化宿主菌BL21,经IPTG诱导表达后获得重组蛋白;利用该重组蛋白免疫小鼠与新西兰大白兔,制备单克隆及多克隆抗体,并利用Western blot检测特异性;利用间接ELISA方法进行免疫配对试验,筛选出最佳配对抗体,优选各试剂工作浓度并组装试剂盒;用研制的试剂盒对不同种群牛筋草进行EPSPS含量检测,并与qPCR结果分析比较。【结果】牛筋草EPSPS的开放阅读框含有1 620 bp的核苷酸,编码540个氨基酸,理论等电点为8.80,该蛋白无跨膜区域。进化树分析结果表明牛筋草EPSPS与水稻EPSPS进化关系最近。诱导蛋白表达的培养条件为1 mmol·L-1的IPTG,25℃,获得了纯度大于90%,浓度为3 mg·mL-1的重组蛋白12 mg;编号为R1711和R1712的家兔免疫后产生的多克隆抗体有较高的效价,编号为M171070、M171071、M171072的小鼠产生的单克隆抗体效价较高。进一步筛选出单抗FL-374-08能与多克隆抗体组成最佳配对抗体;酶联免疫试剂盒包被抗体的最优质量工作浓度为2 μg·mL-1,酶标抗体的最优工作浓度为10 μg·mL-1;试剂盒线性检测范围为5—80 μg·kg-1,检出限为5 μg·kg-1。抗性牛筋草植株叶片中EPSPS的表达量是敏感植株的52.9倍,而茎中EPSPS的表达量是敏感植株的63.0倍。在抗性牛筋草叶片中EPSPS相对拷贝数是敏感植株的53.5倍,而茎中EPSPS的相对拷贝数是敏感植株的78.6倍。Western blot结果表明单抗与牛筋草EPSPS有特异性免疫,并能准确区分不同敏感性植株及不同组织的蛋白含量差异。酶联免疫检测结果发现抗性牛筋草茎中的EPSPS浓度可以达到6.0 μg·L-1,而敏感植株叶片和茎中EPSPS蛋白的浓度分别为0.22和0.43 μg·L-1。【结论】获得的牛筋草EPSPS单克隆抗体特异性强、灵敏度高,研发的EPSPS酶联免疫试剂盒能够快速、准确检测牛筋草EPSPS蛋白含量并鉴定牛筋草由EPSPS过量表达导致的草甘膦抗药性。

关键词: 牛筋草, 草甘膦, 抗体, 抗药性, 快速检测, ELISA

Abstract:

【Objective】The objective of this study is to clone the target gene EPSPS of glyphosate in Eleusine indica, obtain the recombinant protein and antibody against EPSPS protein, then assemble the EPSPS ELSIA kit, finally, to detect the EPSPS expression and protein content of different plants and tissues of E. indica, and to provide a powerful tool for rapid identification of glyphosate resistant individuals. 【Method】The full-length of EPSPS was cloned by PCR method. The constructed pET30a-EPSPS positive plasmid was transformed into host bacteria BL21, which was induced by IPTG to express and purify the protein. Monoclonal and polyclonal antibodies were prepared by immunizing mice and New Zealand white rabbits with EPSPS recombinant protein as immunogen, and the specificity of the antibodies was detected by Western blot. Best matching antibody was screened by indirect ELISA. The working concentration of each reagent was optimized and the kit was assembled. The content of EPSPS in different E. indica populations was detected by the assembled kit and compared with the results of qPCR. 【Result】The open reading frame of EPSPS contains 1 620 bp nucleotide and encodes 540 amino acids, with a theoretical isoelectric point of 8.80. The protein has no transmembrane region. The evolutionary tree analysis showed that the evolutionary relationship between EPSPS of E. indica and EPSPS of rice was the closet. The protein could be expressed under a condition of 1 mmol·L-1 IPTG, 25℃. After purification, the purity of prokaryotic expression protein was more than 90%, the concentration was 3 mg·mL-1, and the protein content was 12 mg. Polyclonal antibodies produced from rabbits numbered R1711 and R1712 showed higher potency. Monoclonal antibodies produced from mice numbered M171070, M171071 and M171072 showed higher potency. A group of optimal matching antibodies (FL-374-08 monoclonal antibody and polyclonal antibody) were screened. The optimal concentration of coated antibody was 2 μg·mL-1. The optimal concentration of enzyme-labeled antibody was 10 μg·mL-1. The linear detection range of ELISA kit was 5-80 μg·kg-1, and the detection limit was 5 μg·kg-1. The expression of EPSPS in leaf of resistant individuals was 52.9 times higher than that in the susceptible individuals. Similarly, it was 63.0 times higher in stem. In the resistance individuals, the relative copy number of EPSPS in leaf was 53.5 times higher than that in the susceptible individuals, and it was 78.6 times higher in stem. Western blot results showed that monoclonal antibodies had specific immunity to EPSPS and could accurately distinguish the difference of protein content in different plants and tissues. The concentration of EPSPS in stem of resistant individuals was 6.0 μg·L-1, while, it was only 0.22-0.43 μg·L-1 in susceptible individuals, which detected by ELISA kit. 【Conclusion】The monoclonal antibody of EPSPS obtained in this study showed higher specificity, and sensitivity. The ELISA kit can be used to quickly and accurately identify glyphosate resistance in E. indica which caused by EPSPS overexpression.

Key words: Eleusine indica, glyphosate, antibody, resistance, rapid detection, ELISA