| Artificially induced gynogenesis is a highly effective sex control technology,combined with the sex reversal make the product of all-female group possible. In thisstudy, we do a preliminary research of gynogenesis in goldfish(Carassius auratus), inorder to handling the key technology in the gynogenesis process. Then, the offspringwere dealt with bathing in methyltestosterone to make sex reversal. At the same time,we took a analysis at the expression of Cyp19a, Dmrt2a and Dmrt2b gene in thegoldfish, and cloned the full length DNA of Cyp19a, and the full length cDNA ofDmrt2a, providing a theoretical basis for the regulation of goldfish differentiation at themolecular level.The thesis includes the following three parts:1.The sperm were irradiated by UV with Hank’s solution, the dose of UV was30MJ/cm2, the time was3min, take300eggs of red pearl goldfish to artificialinsemination, then the DNA was duplicated with cold shock in0℃cold water for45min to retain the second polar body. In the gynogenetic haploid control group, weirradiated the sperm with UV, but without cold shock.Goldfish embryonic and larval development of gynogenetic diploid was observed,it is no different from normal diploid goldfish, but, in the gynogenetic haploid controlgroup, the phenomenon of "haploid syndrome" was validated. Karyotype analysis showthe chromosome of gynogenetic diploid is2n=100. At last,49larvae were hatched out.2.Cytochrome P450aromatase (Cyp19) is the terminal enzyme in the steroidogenic pathway that converts androgens (e.g., testosterone) into estrogens (e.g., estradiol),which is a key reaction in the sex differentiation in vertebrates.In this study, a cytochrome P450aromatase (Cyp19a) gene from Carassius auratuswas identified by genome-walking PCR and designated the encoded protein as Cyp19a.The Cyp19a gene extends6,905bp, and contains nine exons and eight introns. The openreading frame (ORF) of the Cyp19a transcript has1,557bp which encodes518aminoacids.Cyp19a was mainly expressed in the ovary and at a very low level in the brain,while it was barely expressed in other tissues. During fiffrent development stage,Cyp19a was only expressed at a very low level after hatching, untill25day afterhatching it began to increase obviously.Immunohistochemical analysis revealed that Cyp19a is distributed in the ovary ofadult fish and gonad of some48-dah-larvae.We have isolated the promoter regions of the C. auratus Cyp19a genes via genomewalking technique to search for regulatory sequences that bind to transcription factors.Sequences like estrogen receptor (ER) recognition site, steroidogenic factor1(SF-1)and SOX-5were found in the5-fanking regions of Cyp19a gene. EGFP checkingsystem demonstrated it possesses promoter function in vitro experiment.3.Dmrt2, dsx and mab-3related transcription factor2are members of a gene family ofputative transcription factors which contain a common zinc finger-like DNA bindingmotif, DM domain. The full-length cDNA of Dmrt2a gene was obtained from totalRNA isolated from the liver of goldfish by the technology of RACE (RapidAmplification of cDNA Ends). The complete cDNA of goldfish Dmrt2a is1755bp andits ORF (opening reading frame) includes1500bp which codes499amino acid residues.Dmrt2a in goldfish shares high homology with Dmrt2a genes from other fishes viaphylogenesis analysis. The sequence homologies between deduced goldfish Dmrt2apeptide and that from zebrafish, takifugu, medaka and tilapia are85%、61%、58%and58%, respectively. SYBR Green I Quantitative Real-time PCR analysis was used toassess the expression of Dmrt2a and Dmrt2b at mRNA level. The Dmrt2a and Dmrt2bgene were expressed from early embryo stage to juveniles of15days post hatching(dph). Dmrt2a was expressed highly in embryos, reached a peak at24hours postfertilization (hpf), and decreased at36hpf; Dmrt2b was expressed lowly in embryos and increased obviously after hatching. In adult goldfish, Dmrt2a and Dmrt2b genewere expressed highly in ovary and testis, had a certain expression in intestin, kidney,liver, heart and brain. |
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