Abstract: HIGH EXPRESSION OF OSMOTICALLLY RESPONSIVE GENES 1（HOS1） is an integration factor of several signaling pathways in plants, it plays important roles in processes such as growth and development, stress response, photomorphogenesis and flowering time regulation etc., but its function in soybean has not been revealed. By using reverse genetics, we cloned two HOS1 homologous genes in soybean, GmHOS1a and GmHOS1b, and analyzed their protein structures, expression patterns and functions by bioinformatics analysis. Through phylogenetic analysis, we found that GmHOS1a and GmHOS1b are highly conserved to Arabidopsis and rice HOS1 proteins, both of which have N-terminal ring finger domain and conserved motif ELYS-like. This suggests that GmHOS1a and GmHOS1b may have similar functions to Arabidopsis HOS1. However, subcellular localization showed that GmHOS1a and GmHOS1b proteins were only localized in the nucleus, which was different from that of Arabidopsis HOS1 protein. Therefore, we speculated that the action mode of GmHOS1a and GmHOS1b may be different from that of Arabidopsis. Further analysis showed that the expression patterns of GmHOS1a and GmHOS1b were similar, the highest expression of GmHOS1a and GmHOS1b exited in trifoliate leaves, followed by flowers and single leaves. The expression pattern of GmHOS1a and GmHOS1b were rhythmically. The expression of GmHOS1a increased after exposure to light and reached a peak before darkness. These results suggest that GmHOS1a and GmHOS1b in soybean may be involved in the regulation of circadian rhythm. To further explore the function of GmHOS1a and GmHOS1b, we constructed nine knockout vectors of GmHOS1a and GmHOS1b using CRISPR/Cas9 system, and screened out six efficient vectors through hair root experiments, which will be used in the future soybean genetic transformation. This study provides a theory basis for exploring the function of GmHOS1a and GmHOS1b, and provides vector materials for further genetic analysis.