Full Length CDNA Cloning Of Aj-vasa And Its Developmental Expression Pattern In The Germline Of Apostichopus Japonicus

Ontogenesis of germ cells is an important field in developmental biology. Up to now, vasa expression is exclusively restricted to the germ line cells in most animals, so vasa gene provides the most reliable molecular marker for studies on the germ cell development and reproductive regulation. Sea cucumber, Apostichopus japonicus is one of the most commercially important Echinodermata in China. While, the germline origin and the gonad genesis of A. japonicus haven't been reported till now. In this thesis, full-length cDNA of Aj-vasa from A. japonicus was first cloned using a homologous cloning strategy and rapid amplification of cDNA end (RACE) technology, and then the spatio-temporal expression of Aj-vasa transcripts was analysed by RT-PCR and in situ hybrization. Furthermore, Aj-vasa mRNA was used as a molecular marker to establish the origin model of sea cucumber primordial germ cells (PGCs). Finally, early genesis of A. japonicus gonad was observed by histological technology.Aj-vasa cDNA was 2167 bp in length, comprising an open reading frame (ORF) of 1593 bp encoding 530 amino acids, a 5' untranslated region (UTR) of 449 bp and a 3' UTR of 125 bp. The putative amino acid sequence of Aj-VASA shared all the nine conserved motifs characteristic and GG repeats of the DEAD-box protein family. An ARKF motif, appeared in VASA and PL 10 proteins, but not in other DEAD-box helicases, was also found in the Aj-VASA protein. Homology and phylogenetic analysis showed that Aj-VASA appeared more closely related to the other members of VASA sub-familie, which indicates that this gene is highly conserved through evolution. The three-dimensional structure of Aj-VASA suggested that this protein should possess essential functions of DEAD-box proteins such as RNA binding, ATPase and helicase activities.Semi-quantitative RT-PCR showed that Aj-vasa was specifically expressed in the gonads of adult sea cucumbers and in situ hybridization analysis revealed that Aj-vasa mRNA located abundantly in the cytoplasm of spermatogonia, spermatocytes and oocytes, which suggested that Aj-vasa could be involved in gametogenesis of A. japonicus. Aj-vasa mRNA was detected by RT-PCR from fertilized eggs to auriculariaes. The expression level increased rapidly after fertilization, and reached the peak at blastulas, then gradually decreased from gastrulaes to auricularias. Whole-mount in situ hybridization showed that Aj-vasa mRNAs were detected in each cell in early developmental stage from unfertilized egg to blastula. Cells containing Aj-vasa mRNAs were observed in endoblast and mesoblast during gastrulation. At early auricularia, Aj-vasa mRNAs were detected in the cells of alimentary tract and hydroentorocoel. These positive cells subsequently appeared at hydroeoele and somatocoel and located symmetrically at hyaline spheres at both sides of the body during doliolaria and pentactula stage. Then the signals appeared at the forside of the body and finally centralized at the dorsal mesentery at juveniles. These cells were the primodial germ cells(PGCs).The gonad genesis was invesgated by observing serial sections of 1mm to 3 cm juveniles. The results suggested that several PGCs were embedded in the dorsal mesentery in 1-3mm juveniles, these PGCs and the dorsal mesentery composed the gonad rudiment. When A. japonicus developed to 5mm juvenile, a cavum occurred at the gonad rudiment and an embranchment appeared at 3cm juvenile. At this time, its configuration is very near to the adult's.