Abstract: The study aimed to explore the function of Apob gene in the lipid metabolism of chicken liver. The physical and chemical properties of chicken Apob protein were analyzed; RT-qPCR was used to detect the expression of Apob gene in the tissues of 4-week-old yellow broiler chickens, 3 replicates were set up in each group and 3 parallel experiments were carried out. Three pairs of sgRNA were designed according to the sequence of key domains of chicken Apob protein to construct Cas/gRNA vectors; T7 endonuclease I (T7EI) digestion method and TA cloning sequencing method were used to screen knockout active sites and calculate gene knockout efficiency in cells after the Cas/gRNA vector was transfected into DF-1 cells; The mRNA expression of Apob gene was detected in subcloned cells using RT-qPCR after gene knockout. The results showed that the relative molecular mass of chicken Apob was 523.356 ku and the average hydrophilicity was -0.300, which was a stable protein. And the Apob gene mainly expressed in chicken liver, kidney and small intestine tissues. T7EI digestion result showed that Cas/gRNA vectors could knock out the gene effectively. TA clone sequencing results showed that knockout efficiency of 3 active sites(Cas/gRNA6, Cas/gRNA7 and Cas/gRNA8) were 33.3%, 65% and 80%, respectively. At the same time, RT-qPCR results showed that the expression level of Apob genes in transfected Cas9/gRNA7, Cas9/gRNA6, Cas9/gRNA8 cells were down-regulated by about 99.96% (P<0.01), 85%(P<0.01),47%(P<0.05), respectively. In summary, we revealed the expression characteristics of Apob gene in chicken tissues and the physicochemical properties of the protein; Then we contructed Cas/gRNA knockout vectors, screened the optimal knock sites, and established Apob knockout subclonal cells successfully, which laid a foundation for exploring the function of Apob gene in chicken liver.

Key words: chicken, Apob gene, CRISPR/Cas9, lipid metabolism