Functional prediction of AMP deaminase 1 in Jingyuan chicken and evaluation of the biological activities of its expression vectors.

ABSTRACT

This study investigated the function of AMP deaminase 1 (AMPD1) in Jingyuan chicken and the biological activity of its expression vector. AMPD1 was cloned and sequenced from chicken breast muscle tissue by RT-PCR and further analyzed using Cluster, DNASTAR, and online bioinformatics software, as well as vector construction, qPCR, Western blotting, enzymatic digestion, and sequencing. The coding sequence was 2162 bp, encoding 683 amino acids and producing a protein of approximately 78.95 kDa. After verification, the overexpression plasmids pEGFP-AMPD1, Cas9/sgRNA2, and Cas9/sgRNA3 were found to have biological activity in chicken muscle cells and individual chickens, and two sgRNAs (sgRNA2, sgRNA3) were identified that could edit AMPD1. The qPCR and Western blotting result showed that the pEGFP-AMPD1 plasmid significantly increased both mRNA and protein expression of AMPD1. T7EI digestion showed editing efficiencies of approximately 35 %, 37 %, and 33 % for sgRNA2, sgRNA3, and sgRNA2 + sgRNA3 of AMPD1 in chicken muscle cells. In comparison, TA cloning sequencing showed editing efficiencies of approximately 36.7 %, 86.7 %, and 26.7 % and editing efficiencies in chicken individuals of approximately 71 %, 45 %, and 76.7 %, respectively. These results provide a theoretical basis and support for further investigation into the function of the AMPD1 gene.