Identification And Application Of Vasa Gene In Mylopharyngodon Piceus

Black carp(Mylopharyngodon piceus)is one of the most commercially important aquaculture species in China.Of the four major Chinese carps,the black carp has a larger body and long maturation period.During recent years,there has been a trend of the germplasm resources degradation,the diseases increasing and breeding efficiency decreasing.Visualization,isolation and preservation of black carp germplasm resources provide an ideal system for its protection and reference for the preservation of large freshwater fishes’ germplasm,even to reduce the reproductive cycles of black carp by transplant and surrogate broodstock production.Isolation and identification of molecular markers is the first step in black carp germplasm preservation.As a molecular marker of germ cells especially primordial germ cells(PGCs),vasa was widely used in analysis the origin,migration and differentiation of PGCs,and also in gametogenesis study.Here,we identified the Vasa homologue in black carp and analyzed its expression in tissues and embryos,the result indicated it mainly expressed in germ cells.Then we observed the origin,migration and differentiation of PGCs by using in situ hybridization.By microinjection of the m RNA containing fluorescence protein gene and 3’ untranslated region(3’UTR)and transgenic plasmid containing vasa 5’ transcriptional regulatory sequence,3’UTR and fluorescence gene in zebrafish and medaka embryos,we found they can label the PGCs.In addition,we established 2 black carp somatic cell lines which expressed vasa and stem cell related gene nanog and oct4.In regenerated medaka tail fin,the expression of vasa m RNA was also detected.Therefore,vasa RNA can be present in fish adult stem cells besides germ cells.The main results are as follows:1)A 2972-bp black carp vasa full-length c DNA was cloned using RACE.The nucleic acid and protein structure of Vasa and its similarity and homology with other species were analyzed.The results showed that black carp vasa was 81%,74%,58%,46%,51%,51%,and 37% identical to the Vasa homologues from the grass carp,zebrafish,medaka,frog,human,mouse,and fly,respectively.The phylogenetic tree revealed black carp Vasa was clustered together with VASA sub-family and showed closer association with Cypriniformes Vasa homologues.Sequence comparison with zebrafish genomic DNA demonstrated that black carp has 25 exons.2)sq PCR,q RT-PCR and western blot analysis revealed black carp vasa expression was restricted to ovary in adult organs.The dynamic patterns of tempo-spatial expression and subcellular distribution during gametogenesis of vasa were examined by in situ hybridization and fluorescent immunohistochemistry.vasa transcripts were restricted to oocyte and follicular and enriched in immature oocytes(stages I,II and III oocytes)and barely in mature oocytes(stages IV and V oocytes).q RT-PCR and western blot analysis revealed black carp vasa RNA continuously expressed in embryogenesis.Whole mount in situ hybridization showed vasa transcripts aggregated in the ends of the cleavage plane.During the embryogenesis,the vasa transcripts have scattered to cytoplasm,and distributed at the marginal region as four clusters.At the gastrula stage,vasa positive cells were migrating towards the dorsal side and moved to the putative gonad area.The dynamic expression patterns of black carp vasa were very similar with the migration process of PGCs in other fish,which indicated this gene is Vasa homologue and could act as the first molecular markers in black carp.Both of the enlarging in situ hybridization in hatching stage and subcellular localization results showed that Vasa was mainly expressed in cytoplasm.Moreover,the vasa transcripts were highly detected in unfertilized eggs,evidently supported that vasa transcripts were maternally inherited.vasa m RNA and protein all expressed in unfertilized eggs.In addition,the in situ hybridization showed,vasa was also located in ends of the cleavage plane in unfertilized eggs as same as fertilized eggs.3)As a germ cell specific marker,Mp＿vasa 3’UTR and red fluorescent reporter gene were constructed into a plasmid named as p C-Cherry-Mpvs3 U.The Cherry-Mpvasa3’UTR m RNA was microinjected into embryos from zebrafish and medaka at 1-cell stage,and delineated the PGCs in both zebrafish and medaka promptly.We have isolated the 3157 bp 5’ transcriptional regulatory sequence of black carp vasa gene by Tail-PCR strategy and constructed a transgenic plasmid called p Mpvs P-Cherry-3U,which could also label the PGCs in zebrafish by microinjection.Besides,we have acquired the Mpvasapromoter-Pac Red-3UTR transgenic chimera medaka by microinjection of a transposon system termed p T2/BH Mpvs P-Pac Red-3UTR transgenic plasmid composed of transcriptional regulatory sequence and dual marker genes of both fluorescence and resistance.4)The bladder and kidney cell lines developed from black carp were established and the cryopreservation and viability,optimal growth condition,chromosomal analysis,species identification,transfection efficiency and cell susceptibility to SVCV were analyzed.It’s indicated that the optimal growth temperature,basic cell culture medium and addition of fetal bovine serum of MPB and MPK were 28-32℃,DMEM and 10%.The 16 s r RNA sequence analysis revealed both of the cell lines were from black carp and the modes of chromosomal number were 48.Viral infectivity assays demonstrated significant CPEs could be observed in MPK while no CPEs in MPB,which reflected MPK was a sensitive cell line to SVCV.The liposome and electronic transfection efficiency of MPB were up to 21% and 34% while a lower efficiency at 10% and 18% in MPK,respectively.The MPB and MPK expressed vasa and stem cell related gene nanog and oct4,which indicated MPB and MPK might be black carp bladder and kidney adult stem cell lines.In regenerated medaka tail fin,the expression of vasa m RNA was detected.The expression of vasa in MPB,MPK and regenerated tail fin suggested that vasa may has a role in fish adult stem cells besides germ cells.There were 3 kinds of vasa variants in MPB,and 2 kinds of vasa variants in MPK.It remains to be further investigated whether those kinds of variants play a role in black carp cell line and related to its expression in adult stem cells.In conclusion,we obtained the first germ cell marker gene vasa in black carp,and successfully labeled the PGCs in zebrafish and medaka as a germ cell marker.We also obtained two vasa expressed cell lines from adult organs.