Characterization of gdnfa gene expression and identification of gdnfa^+/-F₁ genotypes in zebrafish

【Objective】The purpose of the study was to clarify the expression characteristics and evolutionary conser-vation of gdnfa gene in zebrafish,to establish the heterozygous mutant of gdnfa^+/-F₁ generation using CRISPR/Cas9 gene editing technology,to lay a foundation for the subsequent generation of gdnfa^-/-F₂ homozygous mutant,and to provide a theoretical basis for exploring the role of gdnfa gene in gonad differentiation and development of zebrafish.【Method】By downloading the GDNF protein amino acid sequences of different species and analyzing the conserved structural domain,the evolutionary conservation of this gene in different species was explored.Real-time fluorescence quantitative PCR was used to detect the expression of gdnfa gene in different tissues of 6-month-old zebrafish,and gdnfa F₀ generation mutant individuals were obtained by CRISPR/Cas9 gene editing technology,and then gdnfa^+/-F₁ generation mutant individuals were obtained by backcrossing with wild type,and their genotypes and mutation types were analyzed.Finally,paraf-fin sections of tissues were used to analyze the differences between 3-month-old gdnfa^+/-F₁ generation individuals and con-temporaneous wild-type gonad tissues.【Result】The molecular weight of zebrafish GDNFA protein was 26.89 kD,consisting of 235 amino acid residues,including 13 hydrophobic amino acids and 51 acidic amino acids.The theoretical isoelec-tric point (p I) was 8.455,and there was a TGF-β structural domain in the protein.The GDNF protein sequence of zebrafish was evolutionally-conserved,and had the same TGF-β structural domain as the GDNFA protein of Xenopus laevis,mouse and human,and it was hypothesized that the GDNFA protein sequence was evolutionally-conserved in fish,human and amphibian.The gdnfa gene was expressed in gonads (testis and ovary),kidney,liver,gill and muscle tissues of 6-month-old zebrafish,and the relative expression of gdnfa gene in testis was significantly higher than that in other tissues(P<0.01).The 48 zebrafish gdnfa^+/-F₁ generation heterozygous mutants produced three types of mutations,which were in-sertation of two bases(△+2 bp) near the target,missing two bases(△-2 bp) and missing twelve bases(△-12 bp).The sex ratio of 48 gdnfa^+/-F₁ generation individuals was male∶female=14∶34,showing an obvious female sex bias.Com-pared with wild-type zebrafish GDNFA protein,the tertiary structure of GDNFA protein in Zebrafish gdnfa^+/-F₁ generation individuals had significant changes.Paraffin section of gonad showed that the spermatogonial cell clusters of type A were reduced in some gdnfa^+/-F₁ generation individuals(△-2 bp),and the spermatogonial cell clusters of type B were more sparsely arranged.Some individuals(△-12 bp) accumulated more cortical follicular oocytes in their ovaries.【Conclu-sion】The gdnfa gene in zebrafish has evolved in parallel with other bony fish,and gdnfa gene is highly conserved in the evolution process of bony fish.Due to the role of gdnfa gene in the development of zebrafish testis,the sex ratio of gdnfa^+/-F₁ generation individuals is biased toward females,and it is hypothesized that it is involved in the male sex differentiation of zebrafish.The partial function loss of gdnfa^+/-F₁ generation heterozygote inhibits the production of spermatogo-nial cell and the transition phase from cortical follicular stage oocytes to primary growth stage oocytes.The gdnfa^+/-F₁ indi-viduals obtained are used to generate gdnfa^-/-F₂ homozygous individuals.