| In the field of the mechanism of sex determination and differentiation, it is evident that the studies on amphibians are lagged behind other non-mammalian vertebrates. According to research data at present, genetic factors may play an important role in sexual development in most amphibians. The widespread Chinese toad, Bufo bufo gargarizans, is a kind of common experimental organisms. To date, its genes related sex determination and differentiation have not been obtained. The Dmrt gene family and the Sox gene family participate in the development process of vertebrates. In this paper, we studied the Sox and Dmrt genes of Chinese toad, the results were as follows:1. The Dmrt gene family involved in sexual development encodes putative transcription factors including a DNA-binding homology motif, the DM domain. In present paper, seven distinct Dmrt genes were cloned and sequenced from Chinese toad using highly degenerate primers. A database search for the cloned sequences revealed the following percentage identity with the homologous Dmrt genes of the human: BgDmrt1=97%, BgDmrt2=97%, three isoforms of BgDmrt3 (BgDmrt3a=93%, BgDmrt3b=95%, BgDmrt3c=100%) and two isoforms of BgDmrt5 (BgDmrt5=97%, BgDmrt5=91%). Based on similarities of amino acid sequences in the DM domain, phylogenetic tree of vertebrate and invertebrate Dmrt genes was reconstructed. These Dmrt genes were grouped into seven distinct subfamilies.2. Sox proteins, all of which harbors a HMG box, form a large class of transcriptional regulators implicated in the control of a variety of developmental processes. One member of this family, Sox9, is a transcription factor that plays critical roles in developmental processes including sex determination and chondrogenesis. In this study, we isolated the Chinese toad Sox9 coding sequence in adult testis tissue by RT-PCR. The Chinese toad Sox9 encoded a 481-amino acid (aa) protein containing a HMG-box from aa 103 to 181 in the N-terminus region and a TA domain from aa 373 to 481 in the C-terminus region. The deduced amino acid sequence of toad Sox9 showed 92.0% similarity to that of Rana rugosa and 81.0% similarity to that of Homo sapiens. Phylogenetic analysis further supported that our isolated gene was a true homolog of Sox9. The detection of Sox9 mRNA in the testis suggested that this gene might be involved in the gonad development of Chinese toad.3. Constructing and screening cDNA library is a reliable method to obtain new genes. A full-length cDNA library from the testis of Chinese toad was constructed with the SMART (switching mechanism at 5'end of RNA transcript) technique. Total RNA was extracted from the testis and reversely transcripted into full-length cDNA using PowerscriptTM reverse transcriptase. First-strand cDNA was amplified by long-distance PCR. After Sfi I digestion and Chroma spin-400 fractionation, cDNAs (>500bp) were ligated toλTriplEx2 vector and were packaged with Gigapack? III Gold Packaging Extract. The optimal primary library contained 2.21×10~6 pfu/ml clones and the amplified library had a titer of 2.94×10~9 pfu/ml, in which over 90% clones were recombinant and the average sizes of inserted cDNAs was about 1.0 kb. These results showed that the testis cDNA library can be used for screening genes. A full-length gene with 5'and 3'untranslated regions was isolated from the cDNA library. Sequence analysis showed that this 561bp cDNA encoded a 128-aa ubiquitin/L40e extension protein.
|
PDF Full Text Request