Study Of DEAD Box RNA Helicase Family In Two Species Of Flatfish(Pleuronectiformes)

1 Molecular cloning and functional analysis of tongue sole vasa geneBased on the transcriptome of tongue sole, three vasa transcripts are isolated, namely vas-l, vas-m, and vas-s. Quantitative real-time PCR (qPCR) and in situ hybridization (ISH) results show that tongue sole vasa transcripts are specifically expressed in gonads. Embryonic development expression profiles reveal the onset of zygotic transcription of vasa mRNAs and the maternal deposit of the three transcripts. The genetic ZW female juvenile fish are discriminated from genetic ZZ males by a pair of female specific primers. Only the expression of vas-s can be observed in both sexes during early gonadal differentiation. Before PGCs started mitosis, there is sexually dimorphic expression of vas-s with the ovary showing higher levels and downward trend. Due to the germline specific expression of tongue sole vasa gene, its promoter can be used to construct vectors that specifically expression marker gene in primordial germ cells (PGCs). In this study, the 3’flanking region of vasa gene is inserted into the multiple cloning sites (MCS) of pEGFP-C1 vector by double digestion. Then, the Pvas-EGFP-3’flank vector is constructed by replacing the original CMV promoter with the promoter of vasa gene. The results demonstrate the benefits of vasa as a germline specific marker for PGCs during embryonic development and gonadal differentiation. This study will help us label PGCs in tongue sole and trace the formation and migration of PGCs to give insights into the formation and maturity of gonads in tongue sole. Thus, we will develop more efficient reproductive control methods and promote the development of selective breedings. This study lays the groundwork for further application of tongue sole PGCs in fish bioengineering, such as cryopreservation of the genetic resources and transplantation for surrogate broodstock production.2 Evolution analyses of Japanese flounder Ddx3 genesIn this study, two homologous Ddx3 genes from the genome of teleost are initially identified. Synteny and phylogenetic analysis of Ddx3 genes in vertebrates are performed to compare the evolutionary relationship of teleost Ddx3 genes with human Ddx3X/Y. Human Ddx3X originates from the translocation of the ancient Ddx3 ortholog on the autosome,while human Ddx3Y derives from the allele of Ddx3X. On the contrary, the teleost Ddx3a and Ddx3b genes originate from the teleost-specific genome duplication (TGD). Synteny analysis of Ddx3a also finds the alterations of upstream genes in teleosts. And a chromosomal inversion, an interchromosomal translocation, and an intrachromosomal translocation are found related to Ddx3a in teleost evolutionary history.Comparisons of genomic structure reveal the conserved function between Ddx3a and Ddx3b in teleosts. Molecular evolutionary analysis finds that teleost Ddx3b gene is under strong purifying selection in evolution and 10 sites in Ddx3a gene are under positive selection. Through qPCR of flounder Ddx3a and Ddx3b confirmed that mutation of these positively selected sites may affect the RNA helicase and ATPase activities of Ddx3a, resulting in the functional divergences between Ddx3a and Ddx3b. All the results indicate the sub-functionalization of teleost duplicated Ddx3 genes.3 Molecular cloning and evolutionary analysis of Japanese flounder p68 genesBased on the genome and transcriptome of Japanese flounder, two p68 genes are isolated. Genomic structure analysis finds that the flounder p68 genes encode three similar types of transcripts. The conventional isoforms comprise of 13 exons, such as p68a-1 and p68b-1. The other two isoforms result from alternative splicing. For p68a-s and p68b-s, they are attributed to the alternative splicing of intron 11. Inclusion of this intron introduces a stop codon at the 5’terminal of the intron. For p68b-m, the intron 11 is spliced while several nucleotides at the 5’terminal of intron 12 are retained with the remaining part spliced. In addition, inclusion of this part results in the reading frame shift and terminates the coding in advance at the 5’terminal of exonl3. Amino acid sequences analysis reveal that the deduced amino acid sequences of p68b-m share significant similarities with the same highly conserved motifs characteristics of the DEAD box protein family, while those of p68a-s and p68b-s lack the last two motifs. Then, qPCR results indicate that flounder p68 genes showed differential tissue specific expression pattern, especially in the ovary. In addition, ISH finds that p68b-1 and p68b-m are specially expressed in the oocytes and p68b-s is only expressed in somatic cells in ovary. The results indicate the p68b-s may perform a regulatory role in gene expression. Two homologous p68 genes are identified from many teleost genomes. Synteny and phylogenetic analysis of p68 genes in vertebrates are performed to confirm that the two p68 genes in teleost are originated from the TGD as well.