Abstract:

Elongation of very long chain fatty acids protein 3(ELOVL3),mainly involved in the synthesis of very long chain fatty acids(VLCFA),plays an important role in fatty acid metabolism.The objectives of this study were to clone the protein-coding sequence of ELOVL3 gene from Jiulong yak,to construct the eukaryotic expression vector of ELOVL3 gene,and to predict and analyze its biological functions.The CDS region of the ELOVL3 gene was amplified by PCR and inserted into the pcDNA3.1 vector by homologous recombination to construct a recombinant plasmid.The recombinant plasmid was validated by restriction digest and PCR,and the biological functions of its protein coding sequence were analyzed by online prediction software in combination with sequencing results.In addition,qPCR and Western Blot were performed for the expression of ELOVL3 gene at mRNA level and protein level.The results showed that the protein-coding sequence of Jiulong yak ELOVL3 gene was 813 bp in length,encoding a total of 270 amino acids.Enzymatic cleavage,PCR and sequencing identification confirmed the successful construction of the pcDNA3.1-ELOVL3 eukaryotic expression vector.Bioinformatics analysis showed that the protein encoded by the ELOVL3 gene was a hydrophobic protein,with 6 transmembrane structural domains and 27 phosphorylation sites.The phylogenetic tree showed that the closest relation to the Jiulong yak was the common cow,and the furthest was the original chicken.In addition,the ELOVL3 gene was most highly expressed in the lung tissue of the Jiulong yak.

Key words: Jiulong yak, ELOVL3 gene, Gene cloning, Eukaryotic expression vector construction