Several Germ Line Marker Genes Of Spinibarbus Caldwelli: Molecular Cloning, Identification And Expression In Germ Cell Development

Germ cell marker genes are a class of instructions, tracking physiological activities related to germ cell gene, so it could be widely used as a molecular marker for the study of gonadogenesis, gametogenesis, the origin, migration and differentiation of the original germ cells (primordial germ cells, PGCs).And it could be also used as a useful tool for the separation of germ cells in vitro and transplantation, for the procreation protection of the endangered species by means of the closely related species.In this study, we cloned successfully the germ cell marker genes of Sc-vasa, Sc-nanos and Sc-scp3(related germ cell meiosis I gene) in the Spinibarbus caldwelli by homology cloning strategy: At last, we obtained full-length open reading frame(1962 bp) of Sc-vasa gene, encoding 654 amino acids.The Spinibarbus caldwelli Vasa showed 90%,89%,86%,79%,79%,77%,77%and 74% similarty to Carassiusauratus gibelio, Carassius auratus, Ctenopharyngodon idellus, Oncorhynchus mykiss, Thunnus orientalis, Danio rerio, Oreochromis niloticus and Monopterus albus Vasa protein respectively; we gained full-length open reading frame (456 bp) of Sc-nanos gene,encoding 125 amino acids. The Spinibarbus caldwelli Nanos showed 81%,63%,52%,50% and 44% similarty to Cyprinus carpio, Danio rerio, Strongylocentrotus purpuratus, Bombyx mori and Ailuropoda melanoleuca Nanos protein respectively; we also gained part-length open reading frame (477 bp) of Sc-scp3 gene, encoding 159 amino acids.The Spinibarbus caldwelli Scp3 showed 93%,84%,72%,69%,66% and 65% similarty to Danio rerio, Oncorhynchus mykiss, Xenopus laevis, Bos Taurus, Homo sapiens and Mus musculus Scp3 protein respectively.Semi-quantitative RT-PCR showed that Sc-vasa, Sc-nanos and Sc-scp3 were specifically expressed in the gonadal tissue of adult Spinibarbus caldwelli. In situ hybridization analysis revealed:During spermatogenesis, Sc-vasa mRNA, Sc-nanos mRNA and Sc-scp3 mRNA transcription results were very similar. They were transcripted both in spermatogonia and primary spermatocytes , but none was found in the latter stages. During oogenesis, Sc-vasa mRNA, Sc-nanos mRNA and Sc-scp3 mRNA were found in all time phases of oocytes. They were transcripted strongly in oogonia and early oocytes, but the positive signals gradually weakened in the latter phases of oocytes and finally these signals gathered round the cell edge.