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