Studies On The CDNA Sequences Of Dmrt1 And Sox9 And Construction Of A CDNA Library From The Testis In Rana Nigromaculata

The molecular mechanisms of sexual development and regulation are diversity between phyla, although sex is a highly conserved feature. In mammals, a male-specific gene located in the Y chromosome, named Sry (Sex-determining region of Y chromosome, Sry) is a sex-determination factor. In non-mammalian vertebrates there is no Sry gene. Both Dmrt1 (Double-sex and Mab-3 related transcription factor 1) and Sox9 (Sry related HMG-box 9) are conserved and would function as sex-dtermination in vertebrates. Dmrt1 and Sox9 are the prominent members of Dmrt and Sox gene families which take part in the development process of vertebrates respectively. These two genes encode transcriptional regulators, express in the gonads exclusively and may be required for testis differention. The dark-spotted frog, a widespread amphibian, is an ideal model for study the evolution and genetic mechanism of sexual development for it has highly specialized morphological and functional characteristics to adapt to both aquatic and terrestrial environments.Dmrt1 and Sox9 gene cDNA sequences of the dark-spotted frog(Rana nigromaculata)were obtained using RT-PCR(Reverse transcription), 3'RACE (Rapid amplification of cDNA ends) and 5'RACE. Full length Dmrt1 cDNA was 1807 base pairs long. The open reading fragment(ORF) contained 1005 nucleotides and encoded a 334-amino acid(aa) sequence. The 5'untranslated region(UTR) was 87bp and 3'UTR was 712bp. A database search for the predicted amino acid sequence of Dmrt1 revealed 93%, 77%, 76%, 76% identity with the homologous Dmrt1 of wrinkled frog(Rana rugosa), African clawed frog(Xenopus laevis), human(Homo sapiens), house mouse(Mus musculus), respectively. The Sox9 cDNA had 1448 base pairs and contained a 1446bp ORF which can encode 481 aa. Its predicted amino acid sequence showed 96%, 93%, 91%, 90%, 85%, 80%, 81%, 84% similarity with wrinkled frog, Chinese large toad(Bufo bufo gargarizans), western clawed frog(Xenopus tropicalis), African clawed frog(Alligator mississippiensis), American alligator, human, house mouse and chicken(Gallus gallus), respectively. These two genes expressed in dark-spotted frog testis and they were highly conservative in phylogeny.A full-length cDNA library from the testis of dark-spotted frogs was constructed with the SMART(switching mechanism at 5'end of RNA transcript) technique. The titers of optimal primary libraries and the amplified libraries were 2.0×106pfu/ml, 2.4×106pfu/ml and 0.48×109pfu/ml, 3.0×109pfu/ml, respectively. The percentages of recombinant clones of primary libraries and amplified libraries were all over 90%. The insert sizes were measured by PCR and the average length of the inserts was 1.0kb. A positive clone of 1170bp was sequenced and named based on sequence similarity with the known ubiquitin genes in Genbank. As RnUb was a full-length cDNA, with a complete coding sequence, it indicated that the library was qualified for screening full-length cDNAs. All those datas above showed that this library attained to the requirements of a standard cDNA library. This library provided a useful resource for the functional genomic researching of Rana nigromaculata.