本研究研究了AMP脱氨酶1(AMPD1)在靖远鸡中的功能及其表达载体的生物学活性。通过RT-PCR从鸡胸肌组织中克隆和测序AMPD1,并使用聚类分析。DNASTAR,和在线生物信息学软件,以及矢量构造,qPCR,西方印迹,酶消化,和测序。编码序列为2162bp,编码683个氨基酸并产生约78.95kDa的蛋白质。经过验证,发现过表达质粒pEGFP-AMPD1,Cas9/sgRNA2和Cas9/sgRNA3在鸡肌肉细胞和个体鸡中具有生物学活性,和两个sgRNA(sgRNA2,sgRNA3)被鉴定为可以编辑AMPD1。qPCR和Western印迹结果表明,pEGFP-AMPD1质粒显着增加了AMPD1的mRNA和蛋白表达。T7EI消化显示编辑效率约为35%,37%,AMPD1的sgRNA2、sgRNA3和sgRNA2+sgRNA3在鸡肌细胞中的表达为33%。相比之下,TA克隆测序显示编辑效率约为36.7%,86.7%,和26.7%,鸡个体的编辑效率约为71%,45%,76.7%,分别。这些结果为进一步研究AMPD1基因的功能提供了理论基础和支持。
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.