Cloning, Expression Analysis And Preliminary Studies On Functions Of Japanese Flounder (Paralichthys Olivaceus) Dazl Gene

Studies on germ cells have drawn more and more attention in recent years. Besides in mammals and other vertebrates, related studies in lower vertebrates, especially in teleosts, are also conducted and have achieved great progress. Dazl (deleted in azoospermia-like) is a member of DAZ family and an important RNA-binding protein. Existed studies show that, from invertebrates to vertebrates, dazl expression is confined to female and male germ cells, implying its critical functions in germ cell development. Dozens of studies have identified dazl in teleosts and analyzed its expression pattern. This study identifies two dazl variants generated from alternative splicing in teleost for the first time. Two dazl mRNAs in Japanese flounder(Paralichthys olivaceus) and their proteins are identified and analyzed, and the expression pattern is compared from both mRNA level and protein level. This study also predicts their functions by analyzing their target mRNAs.By gene cloning and a series of methods, the complete Japanese flounder dazl gene is identified to be 5413 bp in length. It contains nine exons and nine introns, with the last intron locating in the 3’UTR (untranslated region). Its exon 1 only contains the start codon ATG, which is a feature shared by most homologous genes. A pair of dazl alleles differ in their intron 6 sequences. Due to the alternative splicing of exon 8, Japanese flounder dazl gene can be transcripted into two different mRNAs, namely Podazl-Ⅰ and Podazl-Ⅱ. Podazl-Ⅱ has a 51 nt-deletion, which corresponds to the exon 8. Alignment of DAZL peptide sequences of various species shows that seven amino acid residues in the RRM (RNA recognition motif) differ between teleosts and non-teleosts. PODAZL-Ⅰ and PODAZL-Ⅱ share the highest sequence identity with Lates calcarifer DAZL, which is 80.6% and 80.4%, respectively. Prediction results show the transcription of Japanese flounder dazl may be regulated by several transcription factors, including GATA, CEBP and SOX.Results of RT-PCR and qRT-PCR prove the gonad-specific expression patterns of both Podazl-Ⅰ and Podazl-Ⅱ, and they both expressed in main embryo developmental stages. Among the expressed tissues and stages, Podazl-Ⅱ has a higher expression level than Podazl-1. Both the two mRNAs are expressed higher in ovary than in testis. They show relatively higher expression levels until the embryos develop to the middle gastrula stage. After that, the expression levels of the two mRNAs are very low, except in the pre-hatching stage, when the two mRNAs show instantaneously higher expression levels. Results of in situ hybridization, western blot and immunohistochemistry prove that both Podazl-Ⅰ and Podazl-Ⅱ are located in male and female germ cells, and that their proteins are also germ cell-specific. Two CpG islands are predicted to locate in the upstream of Japanese flounder dazl gene. But their methylation levels detected by bisulfite sequencing show no significant disparity between gonads and brain, implying the expression of Japanese flounder dazl may be regulated by some mechanisms other than methylation.RNA-binding protein immunoprecipitation (RIP) is conducted together with lllumina sequencing to find the target mRNAs of Japanese flounder DAZL proteins. Annotation and analysis results show altogether 3334 mRNA targets are directly or indirectly regulated by DAZL in Japanese flounder ovary. The most abundant targets are mRNAs encoding histones in the nucleosome, indicating the possible functions of DAZL in processing and modifying histone mRNAs. Some target mRNAs are related to growth, development and reproduction, such as oct4, gata6, vasa, dazl and dnd, implying DAZL may be involved in these important biological processes. Results of GO and KEGG pathway prediction indicate Japanese flounder DAZL may play roles in RNA processing and metabolism, translation regulation, cell cycle, protein metabolism and some other biological processes. Comparison of PODAZL-Ⅰ and PODAZL-Ⅱ target mRNAs show no apparent function difference of the two proteins. qRT-PCR further prove that both Podazl-Ⅰ and Podazl-Ⅱ may be the direct or indirect targets of their proteins.The present study identifies the two alternative splicing mRNA variants of Japanese flounder dazl and their corresponding proteins. The expression analysis and target mRNA study prove that Japanese flounder dazl can be used as a germ cell marker, which will be a powerful tool in related research. No apparent disparity is found between the expression patterns and possible functions of the two Japanese flounder dazl mRNA or proteins. So it may be reasonable to deduce that the generation of Podazl-11 does not bring too much new functions to this gene. The relatively time-saving transcription and translation of this shorter variant may help Podazl-Ⅰ to function more efficiently. These results will facilitate further reproduction and development studies of Japanese flounder and other teleosts. They may lay the foundation for the utilization and conservation of germplasm resources, also for the successful running of breeding projects.