Expression analysis of zpax4 gene in northern pike（Esox lucius） and establishment of its knockout model

【Objective】 The objective of this study was to explore the expression of zpax4 gene in the gonadal growth and development of Esox lucius in the ZP gene family, and to establish a knockout model of zpax4 gene in Esox lucius by CRISPR/Cas9 technology, which could provide reference for the subsequent exploration of its biological function. 【Method】 The full-length cDNA sequence of zpax4 gene was cloned by RACE, and the physicochemical properties, structural characteristics and phylogenetic analysis of zpax4 protein were carried out by bioinformatics software. Real-time fluorescence quantitative PCR was used to analyze the relative expression levels of zpax4 gene in 8 tissues at 126, 180 and 320 d of Esox lucius. Appropriate sgRNAs were designed and screened, and a CRISPR/Cas9 knockout model of zpax4 gene was established by microinjection of fertilized eggs, and tissue sections were performed to observe the obtained F₀ generation knockout chimeras. 【Result】 The total cDNA length of zpax4 gene was 2771 bp, of which 162 bp was in the 5’ noncoding region（5’-UTR） and 150 bp in the 3’ non-coding region（3’-UTR）. zpax4 gene encoded 811 amino acid residues, the molecular formula of the protein was C₄₁₁₇H₆₃₆₄N₁₀₉₂O₁₂₀₉S₃₉, the theoretical molecular weight was 91.752 kD, the theoretical isoelectric point（pI） was 5.43, and the encoded protein had a signal peptide sequence at the N-terminus, a ZP domain near the C-terminus, and no transmembrane domain, and its secondary structure was composed of α-helix（14.3%）, random coil（54.5%） and extended chain（31.2%）. Multiple sequence alignment and phylogenetic tree analysis showed that the amino acid sequence similarity of zpax4 protein in Esox lucius and other fish species was 54.85%, and the position of functional domain was relatively conserved. The results of real-time fluorescence quantitative PCR showed that the zpax4 gene was basically not expressed in the intestine, muscle, gills, head kidney, kidney, liver and sperm nest, but was extremely significantly expressed in the ovaries at the 3 stages（P＜0.01）, and the relative expression level in the ovaries increased first and then decreased at the three stages of 126, 180 and 320 d, and the relative expression level was the highest at 180 d. In addition, there was also a certain amount of expression in the brain tissue of 320 d male fish. CRISPR/Cas9 technology was used to screen suitable sgRNAs, and the zpax4 gene knockout model of Esox lucius was successfully established, and a variety of mutation types were obtained, and the missing base numbers were 1, 4, 7 and 8, all of which were not multiples of 3. The whole fish section analysis of the F₀ generation chimera fry showed no obvious phenotypic abnormalities in gonads and other parts. 【Conclusion】 CRISPR/Cas9 technology is used to knock out the zpax4 gene of Esox lucius, and the knockout model has been successfully constructed, the zpax4 gene knockout chimera of Esox lucius is obtained, and the better sgRNA target sequences are screened. The zpax4 gene may play an important role in the early and middle stages of ovarian development in Esox lucius.