EGG CHORION ZP GENE MUTANTS AND TRANSGENIC LINES CONSTRUCT IN RARE MINNOW AND ZEBRAFISH

This study aims to identify the zona pellucida glycoproteins (Zps) enriched in the sticky egg chorion of the rare minnow (Gobiocypris rarus) by sequencing the egg chorion proteome, and further aims to construct G. rarus mutants and zebrafish (Danio rerio) transgenic lines of zp genes to investigate the mechanism of egg chorion formation during evolution. After collecting G. rarus embryos at 1hour post fertilization (hpf), the egg chorion was separated from embryos, and proteins were extracted for sequencing analysis. Protein sequences were assembled sequentially, and full sequences were BLASTed against cyprinid proteins. Genes encoding these egg chorion proteins were selected as candidate genes, and based on BLAST results, we focused on zp2l2 and zp4l. We designed CRISPR/Cas9 target site in exon 1 and exon 6 of zp2l2 and zp4l to knockout genes and construct G. rarus mutants. The upstream promoter (2874 base pair, bp) of zebrafish zp3.2 gene and the coding sequence (CDS) of G. rarus zp2l2 (1137 bp) and zp4l (3009 bp) were amplified separately. The promoter of zp3.2 was firstly ligated into the vector pT2AL-ef1a-eGPF vector (containing Tol2 transposon elements) through In-fusion cloning to construct the plasmid pT2AL-zp3.2-eGPF. And then, the amplified CDS of zp2l2 and zp4l were cloned into pT2AL-zp3.2-eGPF plasmid to construct the transgenic plasmids pT2AL-zp3.2-zp2l2-eGPF and pT2AL-zp3.2-zp4l-eGPF, which were used for embryo injection. Transposase (TPase) mRNA was synthesized by in vitro transcription, mixed with constructed transgenic plasmids, and injected into zebrafish embryos to generate transgenic strains. By injecting gRNA/Cas9 protein complexes and applying a traditional three-generation screening protocol, zp2l2 and zp4l were knocked out, and stably inherited homozygous mutants were obtained in G. rarus. Additionally, using the Tol2 transposon system, we successfully created stable and inheritable transgenic zebrafish lines, Tg(zp3.2:zp2l2-eGPF) and Tg(zp3.2:zp4l-eGPF). The expression of enhanced green fluorescent protein (eGFP) was first detected at 15 days post-fertilization (dpf) in both transgenic zebrafish ovary and liver. In general, the construction of egg chorion genes zp2l2 and zp4l knocked out G. rarus mutants, as well as transgenic zebrafish lines, provide workable experimental ideas and technical routes for the mechanism research of the egg chorion formation in cyprids.