| The DM (Doublesex and Mab-3) domain was identified by the homologous analysis between the sex determining gene doublesex(dsx) in Drosophila melanogaster and Mab-3 in Caenorhabditis.elegans. DM domain-containing genes act as transcription regulators. Then they were named DMRT (Doublesex and Mab-3 related transcription factor). DMRT1 has been suggested to play significant roles in sex determination and differentiation in the testis of vertebrates.To isolate the DMRT1 gene, we amplified the DMRT1 DM-domain by PCR, using the forward and backward primers designed on the basis of the conserved region amino acid sequence of the DMRT1 genes registered in Genbank. Then the full-length cDNA of CcDMRT1 were cloned, using 5’-and 3’-rapid amplification of cDNA ends(RACE). The transcript contains a 750 base pair (bp) open reading frame (ORF) encoding a 249 amino acid sequence. The relative molecular mass of the Yellow River carp CcDMRTl is 27.2202 KDa, isoelectric point (pI) of 6.93, It is with 22 kinds of nitrogen amino acids in amino acid sequence, which negatively charged amino acid (Asp+Glu) 21, positively charged amino acids (Arg and Lys) 21. It is amino acid composition for 84 non-polar hydrophobic amino acids,118 polar amino acid,21 acidic amino acids,26 basic amino acid. In the Yellow River carp CcDMRT1 protein secondary structure analysis of alpha helix is occupied 20.9%, beta Angle is occupied 6.83%, random coil accounted for 53.82%, extending chain accounted for 17.27%. Homology comparison data showed:the Yellow River carp with the homology is very high between bony fish, the Yellow River carp CcDMRT1 and zebrafish DMRT1 is the similarity, the highest was 85.0%. The similarity of Lowest DMRT1 is 38.3%. These data suggested that the Yellow River carp CcDMRT1 genes close with carp shape on the evolutionary purpose genetic relationship.Several technologies, such as Semi-quantitive PCR and fluorescent quantitative RT-PCR, were used to analyse the expression pattern of CcDMRT1. The result revealed that the expression of CcDMRT1 was testis-specific in adults, with very week expression in ovary. This pattern is in line with that of other species’s CcDMRT1. Contrary to expectations, all the CcDMRT1 transcripts were detectable in all embryos tested. However, take the result by and large, each isoform’s expression trend was risie at first and then decline. By in situ hybridization, CcDMRT1 gene positioning analysis in the maturation of the Yellow River carp showed that, in males, CcDMRT1 around only born in seminiferous tubule cells expressed in the cytoplasm; in females, only in the primary oocyte and growth period of oocyte expression in the cytoplasm. Taken together, these results provided the basic data for elucidating CcDMRT1 roles for sex-determination and gonadal differentiation in vertebrates.The laboratory was respectively in the programd gain control (PGC), March (age period of gonadal differentiation period, T1) and mature (TA) gonad mRNA pool as a template, constructed the three gonadal of differentiation period RNA-seq transcriptome library. Through comprehensive analysis of the DMRT gene family, we found that four members CcDMRT1a2, CcDMRT1b, CcDMRT2, CcDMRT3 are in all three periods, but the frequency of appearance and expression level of CcDMRTla2 gene was different, not only in the expression quantity but also reproduce are far higher than the others. Our further study illustrate show that it plays an important role in the decision of the species in the male gonad development process. While the others expression level in the gonadal undifferentiated stage is very low, even for 0 (such as CcDMRT2). More importantly, the choice of shear gene CcDMRT1a2 and CcDMRT1 was also found in the database, the existence of the phenomenon of selective shear illustrate that the different transcripts involved in the collaborative the Yellow River carp early embryonic development and the regulation of sex determination. Through analysis of quantity expression, we found that DMRT family genes were up-regulated in gonadal differentiation period overall, which shoes that they played their role in the family members involved in sex differentiation and spermary function maintaining. Our depth study of the family genes which can lay a foundation for the molecular mechanism of male gender development and differentiation of the Yellow River carp and other fish.In conclusion, my study established that the Yellow River carp CcDMRT1 closes with car p shape on the evolutionary purpose genetic relationship; the CcDMRT1 has obvious correlation wi th the male gonads differentiation and development; the CcDMRTla2, CcDMRT1b, CcDMRT2, CcD MRT3 play an important role in the decision of the male gender; the alternative splicing has been occured; the DMRT family genes members play their role in the formation and function maintaina nce of testis. |
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