| Dmrtl (double-sex and mab-3related transcription factor1) of Rana Chensinensis was cloned and its expression in different adult tissues was examined. Dmrtl location in testes was detected. Moreover, Expression of Dmrtl mRNA levels in testis of adult R. Chensinensis exposing to4-tert-octylphenol (OP,10-8,10-7,10-6and10-5mol/l) were also detected. Then, we focused on a reasonable speculation based on the results about the functions of Dmrtl. Dmrtl was cloned by reverse transcription PCR (RT-PCR) method and splicing technology. Multiple sequence alignment, physicochemical property, secondary/tertiary structure, phylogenetic, hydrophily and hydrophobicity, transmembrane, signal peptide and cell positioning analysis of Dmrtl were carried out. In addition, the tissue distribution of Dmrtl were analyzed with RT-PCR in testis, ovary, liver, brain and kidney. Dmrtl transcript was also detected at gonad-mesonephros complex (GMC) by PCR. The difference of Dmrtl level in male and female genome has also been simply estimated by PCR. Then, the expression level of Dmrtl mRNA in testis of R. Chensinensis, which exposed to10-8,10-7,10-6and10-5mol/l OP for10,20and30d were analyzed with means of quantitative real time-PCR (qRT-PCR). Moreover, Dmrtl location in testes were also tested by in suit hybridization. The mainly results and conclusions about this research are as follows:1. The full length of Dmrtl cDNA was1118bp, including a1005bp open reading frame (ORF) coding for334amino acids. Alignments of the Dmrtl amino acids sequence with other vertebrates showed that R. Chensinensis Dmrtl shared high similarity with the other species, especially Glandirana rugosa(97%) and Pelophylax nigromaculatus(95%), as well as the DM conserved domain, male-specific motif, and P/S rich region.2.2. Amino acid composition and physicochemical property analysis of frog Dmrtl shows that the molecular weight is36750.0Da, theoretical pI is8.45, Number of negatively charged (Asp+Glu) and positively charged (Lys+Asp+His) residues are28and39, the formula is C1582H2467N451O513S23, extinction coefficients is40340, estimated half-life is30h, instability index is61.89, aliphatic index is52.57, and grand average of hydropathicity is-0.656. Secondary structure analysis shows the protein contains α-helix, random coil and extended strand. Tertiary structure analysis shows the frog DM domain contains a zinc finger, the template used for build this model is 11pvA, the best alignment region ranges from24to76residues, the sequence identity is64%and Evalue is5.84e-12. Hydrophily and hydrophobicity, transmembrane, signal peptide and cell positioning analysis shows the protein belongs to hydrophilic protein, it has not formed transmembrane domain and signal peptide, frog Dmrt1probably belongs to DNA-binding protein (non-histone).3. Results of RT-PCR showed that Dmrt1was only expressed in testis and male kidney, the amount of Dmrt1in testis is a bit higher than in male kidney. Dmrt1gene amplified from male and female genomic DNA shows that Dmrt1gene copy number between male and female genomic DNA is almost the same. This indicates that Dmrt1gene has no sex dimorphism in the male and female frog genome.4. The Dmrt1transcript was detected at gonad-mesonephros complex (GMC) with surrounding muscle tissue during R. Chensinensis tadpole stage G30to G36, but it is no longer detected at stage G38to40, and no matter male or female, there is no Dmrt1transcript observed in GMCs during stage G42to G46. So Dmrt1plays an important role to the gonads differentiation of R. Chensinensis tadpole.5. In suit hybridization shows that Dmrt1mRNA was mainly located in Leydig cells, Sertoli cells and spematogenic cells in testis.6. Results of qRT-PCR showed that Dmrt1mRNA levels were significantly decreased at10d in all treatment groups compared to control (p<0.01) except10-8mol/L OP. Then slight up-regulation were observed in10-8mol/L OP group with comparison to control, but Dmrt1mRNA levels were decreased in10-7(0.01<p<0.05),10-6,10-5(0.01<p<0.05) mol/L OP treatment groups at20d, the expression level of Dmrt1mRNA were also decreased compared to control at30d, and the lowest expression of Dmrt1was observed at30d in10-6mol/L OP group (p<0.01), which group probably has the most strongest effects of estrogen. Down-regulation of Dmrt1expression was the combination of cumulative dose-effect and cumulative time-effect.In conclusion, Dmrt1has key role in gonads differentiation and functional maintainance. Dmrt1gene could also be used as a marker for identifying male frogs. And as a toxic environment pollutant, OP might plays a role of estrogen effects by surpressing Dmrt1mRNA expression level. |
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