牛筋草EPSPS酶联免疫试剂盒的研发及应用

doi:10.3864/j.issn.0578-1752.2022.24.006

Abstract

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

LI ZhiLing,LI XiangJu,CUI HaiLan,YU HaiYan,CHEN JingChao. Development and Application of ELISA Kit for Detection of EPSPS in Eleusine indica[J].Scientia Agricultura Sinica, 2022, 55(24): 4851-4862.

Figures/Tables 13

Table 1

Fig. 1

Fig. 2

Fig. 3

Table 2

Table 3

Table 4

Pairing effects of monoclonal and polyclonal antibodies"

抗体编号 Antibody number	效价 Titer	阴性对照 Negative control	重组蛋白 Recombinant protein	阳性样本 Positive sample	阴性样本 Negative sample
FL-374-01	1﹕100	0.1515	0.4524	0.1760	0.1417
FL-374-02	1﹕64K	0.1360	0.7681	0.7709	0.1614
FL-374-03	1﹕16K	0.1420	0.4806	0.3578	0.1321
FL-374-04	1﹕100	0.1322	0.5531	0.4250	0.1430
FL-374-05	1﹕256K	0.1309	0.3783	0.1352	0.1192
FL-374-06	1﹕256K	0.1221	0.4887	0.1345	0.1193
FL-374-07	1﹕1024K	0.1452	0.2869	0.1847	0.1290
FL-374-08	>1﹕1024K	0.1352	1.9016	1.5229	0.3065
FL-374-09	>1﹕1024K	0.1062	0.2559	0.2138	0.1429
FL-374-10	1﹕1024K	0.1102	0.2275	0.2050	0.1321
FL-374-11	>1﹕1024K	0.0870	0.4112	0.8804	0.1274
FL-374-12	>1﹕1024K	0.1037	0.2789	0.1690	0.1243
FL-374-13	>1﹕1024K	0.1015	0.2526	0.1591	0.1313
FL-374-14	1﹕1024K	0.0899	0.7826	0.5128	0.1306
FL-374-15	>1﹕1024K	0.0915	0.2666	0.1327	0.1160
FL-374-16	>1﹕1024K	0.0983	0.7068	0.6413	0.1355
FL-374-17	>1﹕1024K	0.1234	0.9342	1.3048	0.1977
FL-374-18	>1﹕1024K	0.1325	1.0463	3.4774	0.1905
FL-374-19	1﹕1024K	0.1102	0.2404	0.2307	0.1888
FL-374-20	>1﹕1024K	0.1519	1.6530	2.8649	0.6717
FL-374-21	1﹕256K	0.1215	0.8766	0.3307	0.1901
FL-374-22	1﹕1024K	0.1002	0.7049	0.8621	0.1706
FL-374-23	>1﹕1024K	0.1005	0.3511	0.9440	0.1478
FL-374-24	>1﹕1024K	0.1097	0.3207	0.2603	0.1486
FL-374-25	>1﹕1024K	0.1117	0.7694	0.2337	0.1512
FL-374-26	1﹕1024K	0.1062	1.3339	0.8813	0.5641
FL-374-27	>1﹕1024K	0.1311	1.4462	1.8647	0.5575
FL-374-28	1﹕1K	0.1269	0.9628	0.2446	0.1296
FL-374-29	>1﹕1024K	0.1291	0.9716	3.3775	0.4059
FL-374-30	1﹕1024K	0.1103	0.2361	0.3440	0.1240
FL-374-31	1﹕1024K	0.1077	0.3452	0.1224	0.1021
FL-374-32	1﹕1024K	0.0947	0.1878	0.1064	0.1033
FL-374-33	>1﹕256K	0.1017	0.1915	0.1317	0.1059
FL-374-34	1﹕1024K	0.1101	0.2415	0.1281	0.1101
FL-374-35	>1﹕1024K	0.0993	0.1928	0.1402	0.1118

Table 4

Table 5

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

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用途 Use	引物名称 Primer name	引物序列 Primer sequence (5′-3′)
克隆 Clone	EPSPS-F1	ACCAACCGCAGCCAAACCAACC
	EPSPS-R1	ACGTCGGCGCAGGCG
	EPSPS-F2	CGTGACCGCCGCTCG
	EPSPS-R2	TTAGTTCTTGACGAAAGTGCTG
表达量分析 Expression analysis	G2（目的基因Target gene）-F	GGTGGCAAGGTTAAGTTATCTGG
	G2-R	TCAACATAAGGGATGGAGATCAG
	ALS（内参Reference）-F	GCAATTTCCCCAGTGACGACC
	ALS-R	GCAAAAGCCTCTATCTTCCCTGT
相对拷贝数分析 Relative copy number analysis	ED-F	CTGATGGCTGCTCCTTTAGCTC
	ED-R	CCCAGCTATCAGAATGCTCTGC
	ALS（内参Reference）-F	GCAATTTCCCCAGTGACGACC
	ALS-R	GCAAAAGCCTCTATCTTCCCTGT

编号Number	100	1K	9K	81K	243K	阴性对照Negative control	效价Titer
R1711	1.789	1.764	1.586	0.692	0.360	0.150	1﹕243K
R1712	1.734	1.716	1.545	0.649	0.322	0.119	1﹕243K

动物编号 Animal number	100	1K	9K	81K	243K	阴性对照 Negative control	效价 Titer
M171069	2.154	1.566	1.054	0.535	0.331	0.104	1﹕81K
M171070	1.966	1.939	1.543	0.772	0.626	0.145	1﹕243K
M171071	1.939	1.944	1.744	1.028	0.759	0.125	1﹕243K
M171072	2.002	1.944	1.753	1.139	0.711	0.137	1﹕243K

名称Name		名称Name
包被酶标板 Coated enzyme label plate		样品提取液 Sample extracting solution
EPSPS标准品 EPSPS standard		终止液 Stop buffer
酶标工作液 Enzyme working solution		洗涤液 Cleaning solution
抗体工作液 Antibody working solution		显色剂 Chromogenic agent

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

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