Expression Pattern And Function Analysis Of Bucky Ball Gene In Medaka

In most animals,the fate of germ cells is determined and maintained by the maternally inherited germ plasm.The germ plasm is structurally and functionally related to the female germ cell specific organelle-Balbiani body(Bb).Bb is a nuclear protein complex that contains RNA and its binding proteins,such as buc,dazl,and mitf,which control oogenesis and germ cell development by participating in RNA metabolism.In zebrafish,the bucky ball(buc)gene is a key regulator of germplasm formation and plays a vital role in regulating and developing germ plasm.Previous studies have shown that Buc protein can recruit RNA-binding proteins.The RNA of buc recruited is translated into proteins and initiates a positive feedback regulation mechanism.The buc gene induce the increase of primordial germ cells(PGCs)with Bb.The buc plays an indispensable role in the development of oocytes.Overexpression of buc m RNA can lead to an increase in the number of PGCs and the production of ectopic PGCs in zebrafish.PGCs are progenitor cells of germ cells,which can produce eggs and sperm.PGCs is a basis for reproduction and reproduction.Studies on zebrafish and other fish indicate that the number of PGCs affects sex determination and gender differentiation.The formation,proliferation,migration,and differentiation of PGCs are determined by several maternal germplasm,including dazl,dnd,nanos,piwi and vasa.Buc protein is a highly conserved germ plasm component and plays an important role in the development of PGCs.Therefore,the study of the buc provides a basis for exploring the development of maternal germplasm and PGCs.It is a great significance to the theory of reproductive development,protection of germplasm resources,and breeding applications.The medaka is a model organism for cell biology and developmental biology research.It is a representative small fresh-water fish that often used for its advantages of fast reproduction,transparent embryos,and small and complete genome sequences.At present,our laboratory has obtained a variety of germ cell-specific transgenic lines such as vasa-GFP and nanos-GFP,and gene knockout technology has also been successfully applied in barley,which has laid a foundation for reproductive development and the functional impact of related genes.In addition,the laboratory has long been committed to exploring and verifying gonad genes on germline development and gender regulation.The genes piwi and vasa can affect the migration of PGCs.The loss of dazl and dnd gene function leads to the loss of PGCs.Oct4 and nanog genes affect the development of embryonic stem cells.Based on this,the role of buc gene in the development of sex-generating gonads was studied using barley.In this study,the full-length c DNA sequence of the buc gene was obtained by RACEs(Rapid-Amplification of c DNA Ends).The expression pattern of the gene in embryos and gonads was analyzed by RT-PCR,chemical in situ hybridization and fluorescent two-color in situ hybridization.The buc 3’UTR fusion red fluorescent protein(RFP)of buccal carp was constructed into rfp-buc 3’UTR expression vector,and the function and conservation of buc 3’UTR-labeled PGCs were investigated using RNA site-directed expression technology.The effect of Buc gene on the PGCs specialization was explored by anti-sense RNA(ASbuc m RNA)knockdown of the medaka fish and overexpression of the Olbuc m RNA of the buc gene of medaka fish.In the present study,a buc homolog(Olbuc)was identified in medaka(Oryzias latipes),and the roles of Olbuc on medaka PGCs was further elucidated.The full length of Olbuc was 2148 bp,which contains a 1724 bp CDS(Coding sequence),a 167 bp 5’ UTR(Untranslated region)and a 257 bp 3’ UTR.By RT-PCR,the Olbuc RNA expression was maternally provided during embryogenesis and was restricted in ovary of adult tissues.By in situ hybridization,Olbuc RNA was abundant in meiotic stage oocyte,but gradually decreases during the oogenesis proceed.Surprisingly,Olbuc was not colocalized with dazl,the marker gene of Bb.Interestingly,Olbuc 3’ UTR could induce specific and stabilize GFP reporter expression in PGCs.Furthermore,overexpression of Olbuc m RNA in medaka embryos can increase PGCs number and generate ectopic PGCs.In summary,our results show that Olbuc performs conserved function in PGCs development that is independent of Bb in medaka.