| Sex determination and differentiation and its molecular mechanisms are among the most basic biological issues.The sex of most vertebrates is determined by inheritance.However,in some amphibians,sex is controlled by multiple factors of the gene and environment.The Q.spinosa has both food and medicinal value,global warming,environmental pollution are increasingly serious,and excessive capture of Q.spinosa,resulting in a sharp reduction in the number of Q.spinosa,and is listed by the International Union for the Conservation of Nature(IUCN)as Vulnerable grade.In the Q.spinosa,there is a phenomenon of sexual dimorphism,male individuals are significantly larger than female individuals,and its united breeding has high economic value.Therefore,the study of the molecular mechanisms and gender control of gender determination and differentiation not only has theoretical significance,but also has great application prospects.The laboratory preliminary study found that Q.spinosa 'tadpoles development to 0-17 days to produce primitive gonads,0-44 days ovary began to differentiate,0-60 days ovarian differentiation completed,2 years after sexual maturity.The Dmartl,Sox9,Cyp17 and Cyp19 genes are important related genes in the sex determination of vertebrates.In this paper,first,different concentrations of methyltestosterone diet feeding experiments carried out hormone growth and development of Q.spinosa research;followed by differen concentrations of MT fed 0-17 days,0-44 days,0-60 days Real-time Quantitative PCR was used to detect the effect of MTT on the expression of Dmartl and Sox9 genes at concentrations of Omg/kg,30mg/kg,60mg/kg,80mg/kg,10Omg/kg and 200mg/kg.The expression level of CYP17 and CYP19 in two sexes was detected by qPCR.At the same time,the CYP17 and CYP19 gene were cloned and analyzed.The main findings are as follows:1.Different concentrations of methyltestosterone on the growth and development Q.spinosa 'tadpoles:Prepared by ethanol spray method,the concentration of MT was Ong/kg,10mg/kg,30mg/kg,60mg/kg,80mg/kg,100mg/kg and 200mg/kg of seven groups of feed,of which Omg/kg for the control group,the feeding method fed Q.spinosa 'tadpoles daily observation of the survival status of each group of tadpoles were recorded tadpole deaths,malformed individuals and metamorphosis.Every 20 days in the control group and the experimental group were taken 10 tadpoles body to measure length,weight,until the growth and development of tadpoles to 90 days.The results showed that:20d,30mg/kg,60mg/kg,80mg/kg MT can significantly promote the increase of body length of Q.Spinosa' tadpoles(P<0.01),and the high concentration(10Omg/kg and 200mg/kg)had no significant effect;On day 40,MT(60 mg/kg,80 mg/kg)promoted growth,and high concentrations(100 mg/kg,200 mg/kg)inhibited growth;at 60d and 80d,the lengths of tadpoles in 60mg/kg and 80mg/kg groups were significantly higher than those in the control group(P<0.01),but the inhibition of growth at high concentrations was reduced;for body weight,body weights of 30 mg/kg and 60 mg/kg in the experimental group at 60 days were significantly higher than those in the control group(P<0.05);after 80 days of growth,the body weight of the 80 mg/kg group was significantly higher than that of the control group(P<0.05).Low concentrations of MT did not cause teratogenicity and death(P>0.05);under medium and high concentrations,the Q.Spinosa' tadpoles deformity rate of showed a concentration increase.The deformity after the metamorphosis is mainly manifested as:the forelimbs are incomplete,the forelimbs are slender or the posterior incomplete,the jumping obstacles,the growth and development are severely inhibited,and the survival rate is lower.MT does not affect the metamorphosis of Q.Spinosa' tadpoles.2.The difference of tissue expression of Dmart1 and Sox9 in Q.spinosa and effect of different concentrations of methyltestosterone on it:qPCR was used to detect the expression differences of Dmart1 and Sox9 genes in 8 sexual tissues of sexual Q.spinosa.The results showed that the expression of Dmart1 gene was significantly higher in the testis of sexual maturity than that of other tissues,indicating that Dmartl gene might participate in male sex determination and testis differentiation.In addition to its high expression in the liver,the Sox9 gene is also expressed relatively higher in the intestine and testis,but its expression in the ovary is much lower than in the testis.Sox9 gene expression is not limited to a particular tissue and may be involved in the regulation of other life events in addition to sex determination and differentiation.The expression of Dmart1 gene was expressed 17 days after the tadpoles hatched.At 44 days,the Dmart1 gene was expressed with androgen treatment at high concentrations(100 mg/kg and 200 mg/kg).At 60 days,except at the low concentration of 30 mg/kg,Dmart1 gene expression was also significantly upregulated at medium concentrations.Throughout the tadpoles from primordial gonadal to ovarian differentiation throughout the completion of the control and low concentrations of MT,Dmart1 gene expression trend first high and then low,As the concentration of MT increases,this trend changes until the opposite trend occurs,the Dmart1 gene expression reached the maximum at the end of ovarian differentiation(60 days)at MT concentrations of 60 mg/kg,80 mg/kg,and 100 mg/kg,at the highest concentration(200 mg/kg),the peak of expression appeared at the beginning of ovarian differentiation.This upregulation may promote the reversal of the ovaries to the testis during gonadal differentiation.The Sox9 gene was also expressed at the beginning of the generation of the original gonad.This period was sensitive to androgens,and was induced by the low concentration of MT(p<0.05).However,the Sox9 gene and Dmartl gene are not synchronized with the hormone treatment dose,but show a synergistic effect.At the beginning of differentiation of gonads to completion of differentiation,different from the expression pattern of Dmartl gene,the expression level of Sox9 gene was significantly up-regulated at lower concentration of MT.3.Cloning and analysis of CYP17 and CYP19 Genes:through the extraction of total RNA from gonads,CYP17(331 bp)and CYP19(956bp)were cloned from the ovary of female Q.spinosa using reverse transcriptase cDNA as template.Homology analysis showed that CYP17 gene shares high homology with Pelophylax esculentus,R.dybowskii,R.sylvatica and N.parkeri,The CYP19 gene of Q.spinosa has high homology with R.pipiens,R.sylvatica,R.catesbeiana,R.rugosa and N.parkeri. |
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