Evolutionary and Molecular Characterization of liver enriched gene 1

Liver enriched gene 1 (Leg1) is a newly identified gene with few functional information. To know the function of Leg1 in mammals, a phylogenetic study was first conducted and indicated that Leg1 is an ancient gene, existing from bacteria to mammals. Analysis of pig genome shows the presence of three copies of pig Leg1s (pLeg1), which could be grouped to Leg1a, Leg1b, and Leg1c clades, respectively. Purifying force acts upon the evolution of Leg1 genes, making them highly likely be functional constraint.Additionally, expression analysis of pLeg1 in combination with the available expression data of human and mouse Leg1s suggests that the eutherian Leg1s might preserve similar function in contrary to those of zebrafish and platypus. Therefore, pLeg1a might provide essential information regarding eutherian Leg1a. In our work, pLeg1a and its coding protein pLEG1a display high similarities to its human and mouse counterparts in terms of gene organization, expression pattern, glycosylation, structures. Moreover, preliminary functional study using RNA-seq suggested that pLeg1a involves in the lipid homeostasis. In conclusion, our study has provided some basic information regarding the evolution, and function of Leg1 genes, which could be applied to further validation of Leg1. HEK293T cells were cultured in DMEM (Hyclone) with 10% fetal bovine serum (Hyclone) in a 6-well plate until reaching 80% confluency.Then,the cells were transfected with 3 μg pCAG-pleg1a-3×FLAG vector (pleg1a) or 3 μg empty pCAG-3×FLAG vector (NC) using Lipofectamine 3000 (Thermofisher). After 48 hours, cells are harvested (n=4), and total RNA was isolated. Then mRNA was seperated with oligo(dT)25 and was used to prepare sequencing libraries.