Abstract: In the study, using Portulaca oleracea L. as materials, the ω-6 fatty acid desaturase PoFAD6 gene from Portulaca oleracea L. was obtained by homologous cloning, and bioinformatics analysis, prokaryotic expression and expression under low temperature stress were carried out. The results showed that the CDS of PoFAD6 gene was 1 380 bp in length, encoding 459 amino acids. The amino acid sequence had the conserved domain of the Membrane-FADS-like superfamily. PoFAD6 protein had no signal peptide at the N-terminus and had four transmembrane structures, which was an unstable hydrophilic protein. Subcellular prediction was located on the chloroplast. The secondary structure mainly included α-helix and random coil. The results of amino acid homology comparison and phylogenetic tree analysis showed that PoFAD6 had the highest amino acid sequence identity with FAD6 of Chenopodium quinoa, which was 77.54%, and the genetic relationship was the closest.The PoFAD6 fusion protein was successfully expressed in Escherichia coli BL21 strain, with a molecular weight of about 53 kD. The results of RT-qPCR showed that the expression pattern of PoFAD6 gene increased first and then decreased during 0-24 hours of low temperature treatment at 5 ℃, indicating that PoFAD6 gene could respond to low temperature stress.