CDNA Cloning And Expression Analysis Of Two DEAD-box Family Genes, Fc-vasa And Fc-PL10a, From Fenneropenaeus Chinensis

DEAD-box family proteins are a putative ATP-dependent RNA helicase, which form a widely dispersed family of proteins ranging from viruses to mammals, and are associated with nearly all RNA processes. Among the DEAD-box proteins, VASA and PL10 are required for germ cell formation. Up to present, vasa expression is exclusively restricted to the germ cell lineage in most animal species and provides the most reliable molecular marker for studies on the germ cell development and reproductive regulation. Chinese shrimp (Fenneropenaeus chinensis) is one of the most commercially important shrimp in China. The extensive investigation on reproduction and development of Chinese shrimp would be very important. In this thesis, we first cloned the full-length cDNA of two DEAD-box genes, Fc-vasa and Fc-PL10a from F. chinensis using a homologous cloning strategy, followed by analysis of characterization and expression.Fc-vasa was obtained with 2893 bp comprising an open reading frame (ORF) of 2139 bp encoding 712 amino acids. The complete cDNA sequence of Fc-PL10a with 2398 bp contained a 1983 bp ORF encoded 660 amino acids. The predicted amino acid sequences of both Fc-VASA and Fc-PL10A shared all the nine conserved motifs characteristic of the DEAD-box protein family and contained four arginine-glycine-glycine (RGG) motifs in common. An ARKF motif, found in VASA and PL10 proteins, but not in other DEAD-box helicases , was also found in the two proteins. Three tandemly arranged zinc-finger CCHC motifs in the N-terminal region of Fc-VASA were conserved among the VASA-related proteins but not present in PL10-related proteins. Homology and phylogenetic analysis showed that Fc-VASA and Fc-PL10 appeared more closely related to other members of their sub-families, respectively, which indicates that these genes are highly conserved through evolution. The three-dimensional structures of Fc-VASA and Fc-PL10A suggest that the two proteins should have essential functions of DEAD-box proteins such as RNA binding, ATPase and helicase activities.Semi-quantitative RT-PCR showed that Fc-vasa was specifically expressed in the gonads of adult shrimps and decreased as germ cell maturation. In situ hybridization analysis revealed that a high level of Fc-vasa RNA was found in the cytoplasm of spermatogonia and oocytes. The data suggested that Fc-vasa could be involved in gametogenesis of F. chinensis. Fc-vasa mRNAs were detected by RT-PCR in early developmental stages from 2-cells embry to intramembranous nauplius, and not in any stages after nauplius. Based on above results, it is postulated that Fc-vasa is required for germ cell determination and formation of F. chinensis as a maternal component. Moreover, expression of Fc-PL10a was widely detected in gonads and other adult tissues of F. chinensis by RT-PCR, which suggests that Fc-PL10a gene may have wider functions.