Abstract:The spotted halibut (Verasper variegatus) is a rare and valuable marine fish species that inhabits the coast of northern China. Mature females are much larger than males because of their faster growth rate. It will create substantial economic benefits to establish an all-female breeding technique for V. variegatus. A better understanding of sex-related genes will contribute to the improvement of a single-sex breeding technique. In this study, we successfully isolated the piwil2 gene of V. variegatus, which was named Vvpiwil2. The total length was 3872 bp, including a 3192 bp open reading frame (ORF), encoding 1063 amino acids; the 5′UTR was 140 bp and the 3′UTR was 540 bp. Based on ExPASy, SMART, Signal4.1, and the NCBI Conservative Domain Database (CDD) biological analysis, the ORF encoded a putative protein, with a predicted molecular weight of 118.6 kDa and an isoelectric point of 9.02. No transmembrane structure or signal peptide site was detected. There were three domains: the ArgoL1, PAZ, and PIWI domains. Real-time fluorescence quantitative PCR technique was used to analyze the expression patterns of the Vvpiwil2 gene at different stages of embryo and larvae. The results showed that the Vvpiwil2 gene was abundantly expressed from early development to the high blastocyst stage, and then declined until the hatching stage. The developmental stage of the embryo from cleavage to the blastocyst stage was mainly guided by the cytoplasmic component. The mRNAs began to be transcribed and synthesized in a large amount at the early gastrula stage, then the transition from maternal to zygote occurred. Therefore, the results indicated that the Vvpiwil2 gene was a maternal gene. After hatching, the expression of the Vvpiwil2 gene at 68 days post hatching was significantly higher than during other stages, which demonstrated that the Vvpiwil2 gene was associated with gonadal differentiation. The expression level of the Vvpiwil2 gene in gonads was significantly higher than in other tissues, and the expression level in the ovary was significantly higher than in the testis, revealing that the Vvpiwil2 gene might play an important role in the maintenance of ovarian functions. The results of this study provide a potential sex determinant gene for V. variegatus and lay a solid theoretical foundation for the establishment of an all-female breeding technique.
