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Among eukaryotic transcription factors, the basic leucine zipper (bZIP) transcription factor is the most widely distributed and the most conserved protein. Alfalfa (Medicago sativa) is the largest forage crop in the world. However, few studies have evaluated the bZIP family in Alfalfa. In this study, bZIP genes in Alfalfa were identified, with 57 MsbZIPs being distributed into 11 sub-families in the phylogenetic tree. Motif and gene structure analysis revealed that the eleven subfamilies had similar motifs and gene structures, 15 cis-acting elements in MsbZIPs, including eight elements in hormones and four in plant stress resistance; 57 MsbZIPs were localized on 8 chromosomes, among which chr3 and chr4 had more collinearities, and MsbZIPs had more collinearity with the soybean. Tissue specific expression revealed that MsbZIPs were highly expressed in leaves and roots. These findings formed a foundation for further studies on functional characteristics, evolution and biological functions of bZIP transcription factors in alfalfa.

Background: Metformin (MET), a worldwide used drug for treating type 2 diabetes but not metabolized by humans, has been found with the largest amount in the aquatic environment. Two MET chlorination byproducts, including Y and C, were transformed into drinking water during chlorination. However, the potential toxicity of the byproducts in hepatotoxicity and reproduction toxicity remains unclear. Methods: The TOPKAT database predicted the toxicological properties of metformin disinfection by-products. The targets of metformin disinfection by-products were mainly obtained from the PharmMapper database, and then the targets of hepatotoxicity and reproductive toxicity were screened from GeneCards. The overlapping targets of toxic component targets and the hepatotoxicity or reproduction toxicity targets were regarded as the key targets. Then, the STRING database analyzed the key target to construct a protein-protein interaction network (PPI) and GO, and KEGG analysis was performed by the DAVID platform. Meanwhile, the PPI network and compound-target network were constructed by Cytoscape 3.9.1. Finally, Discovery Studio 2019 software was used for molecular docking verification of the two toxic compounds and the core genes. Results: Y and C exhibited hepatotoxicity, carcinogenicity, and mutagenicity evaluated by TOPKAT. There were 22 potential targets relating to compound Y and hepatotoxicity and reproduction toxicity and 14 potential targets relating to compound C and hepatotoxicity and reproduction toxicity. PPI network analysis showed that SRC, MAPK14, F2, PTPN1, IL2, MMP3, HRAS, and RARA might be the key targets; the KEGG analysis indicated that compounds Y and C caused hepatotoxicity through Hepatitis B, Pathways in cancer, Chemical carcinogenesis-reactive oxygen species, Epstein-Barr virus infection; compound Y and C caused reproduction toxicity through GnRH signaling pathway, Endocrine resistance, Prostate cancer, Progesterone-mediated oocyte maturation. Molecular docking results showed that 2 compounds could fit in the binding pocket of the 7 hub genes. Conclusion: This study preliminarily revealed the potential toxicity and possible toxicity mechanism of metformin disinfection by-products and provided a new idea for follow-up research.