Transcriptome Analysis Of Yellow Catfish And Functional Study Of P15^PAF In Zebrafish Embryonic Development

Sex dimorphic growth pattern has significant theory and application implications in fish.A Y and X specific allele marker-assisted sex control technique has been developed for mass production of all-male population in yellow catfish?Pelteobagrus fulvidraco?,but the genetic information for yellow catfish breeding has remained unclear.Here,we attempted to provide the first insight into a comprehensive transcriptome covering multiple tissues from XX females,XY males,and YY super-males of yellow catfish.A total of 1,202,933 high quality reads?about 540 Mbp?were obtained and assembled into 28,297 contigs and141,951 singletons.BLASTX searches against the NCBI non-redundant protein database?nr?led a total of 52,564 unique sequences including 18,748 contigs and 33,816 singletons to match 25,669 known or predicted unique proteins.All of them with annotated function were categorized by gene ontology?GO?analysis,and 712 were assigned to reproduction and reproductive process.Some potential genes relevant to reproductive system were further identified by Kyoto Encyclopedia of Genes and Genomes?KEGG?analysis;and at least 21 sex determination and differentiation-related genes,such as Dmrt1,Sox9a/b,Cyp19 b,WT1,and AMH were predicted.Additionally,a total of 82,794 simple sequence repeats?SSRs?,26,450 single nucleotide polymorphisms?SNPs?,and 4,145 insertions and deletions?INDELs?were revealed from the transcriptome data.Therefore,the current transcriptome resources highlight further studies on sex-control breeding in yellow catfish and will benefit future studies on reproduction and sex determination in teleost fish.In the analysis of the transcriptome data,we found that the expression of p15^PAF,a proliferating cell nuclear antigen?PCNA?binding factor involved in tumorigenesis and cell proliferation,is significant higher in the ovary of XX female yellow catfish than in the testis of XY male yellow catfish.However,P15^PAFfunction in vertebrate development is yet unknown.To investigate the roles of p15^PAFin zebrafish development,we generated p15^PAFAF mutant zebrafish using CRISPR/Cas9 technology.Maternal loss of zebrafish p15^PAF(p15^PAF-M)led to severe defects in early embryonic development including axis formation that was indicated by reduced or dislocated expression of a number of marker genes for early embryonic dorsoventral patterning.Strikingly,the normal degradation of maternal factors and activation of zygotic genes during maternal-to-zygotic transition?MZT?were severely disturbed,such as the down-regulated expression of several components of the Wnt/?-catenin signaling pathway.The dorsal-ventral patterning defects in p15^PAF-M embryos were rescued by ectopic expression of?-catenin.The TOPFlash luciferase reporter assay that was used to analyze TCF/LEF activity of Wnt/?-catenin signaling showed that p15^PAFand?-catenin cooperate to regulate the downstream genes.Co-immunoprecipitation assays confirmed that zebrafish P15^PAFcould bind to?-catenin.In summary,our results reveal that zebrafish p15^PAFinteracts with?-catenin and regulates its activity and plays important roles in early embryonic development including MZT and axis formation.