南方农业学报2025，Vol.56Issue(6)：1715-1728,14.DOI:10.3969/j.issn.2095-1191.2025.06.003
白斑狗鱼zpax4基因表达分析及其敲除模型的建立
Expression analysis of zpax4 gene in northern pike(Esox lucius)and establishment of its knockout model
摘要
Abstract
[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,struc-tural 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 ob-tained 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 expres-sion level in the ovaries increased first and then decreased at the three stages of 126,180 and 320 d,and the relative ex-pression 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.关键词
白斑狗鱼/zpax4基因/表达特征/基因敲除/CRISPR/Cas9Key words
Esox lucius/zpax4 gene/expression characteristics/gene knockout/CRISPR/Cas9分类
农业科技引用本文复制引用
刘璎慧,蒋丽,雷静,刘祎,王顺哲,杨倩,孙浩然,范静,张俊杰..白斑狗鱼zpax4基因表达分析及其敲除模型的建立[J].南方农业学报,2025,56(6):1715-1728,14.基金项目
新疆自然科学基金项目(2022D01A66) （2022D01A66）
新疆农业大学大学生创新项目(S202210758035) Xinjiang Natural Science Foundation(2022D01A66) （S202210758035）
Xinjiang Agricultural University Student Innovation Project(S202210758035) （S202210758035）
