Preparation of polyclonal antibodies against the EIL3 protein and its expression in soybean seedlings

Ethylene is closely related to the floral organ development and yield of soybeans, and ethylene insensitive 3 gene (EIN3) and its homologous gene EIN3-like 3 (EIL3) may be key node genes involved in soybean yield regulation; however, there is still a lack of sufficient evidence. The aim of this study was to prepare EIL3 antibodies, verify their effectiveness and preliminarily explore the biological function of EIL3 in the development of soybean seedlings. EIL3 was expressed in a prokaryotic strain, followed by the preparation of polyclonal antibodies and immunohistochemical detection of EIL3 in soybean seedlings. The results showed that the EIL3 protein (Glyma.15G031800) coding region (486-590 amino acids) was cloned into Escherichia coli Rosetta strain, and the EIL3 protein was expressed in the form of inclusion bodies. Two Japanese male large-ear white rabbits were immunized with purified EIL3 protein, and two EIL3 polyclonal antisera were obtained. When they were diluted 81 000 times, the absorbance values were much greater than 0.4, indicating that they had high-quality serum titers. The lower detection limit for the antigen was both 1 ng. The expression of the EIL3 protein was strongly induced by 1-aminocyclopropane-1-carboxylic acid (ACC), and eil3 mutant reduced the accumulation and growth inhibition of EIL3, which improved the plant height, hypocotyl elongation, and lateral root development of the seedlings. This study provides an important means for exploring the biological function of soybean EIL3 at the protein level.