The Excavating Of MiRNA Related TSD And Study Of Promoter Methylation Of Regulatory Candidate Cyp19a1 Gene In Reeves' Turtle (Mauremys Reevesii)

Temperature-dependent sex determination(TSD)is a type of environmental sex determination in which the sex of the embryos depends on the ambient temperature remaining during embryonic incubation,however,the molecular mechanisms governing this process remain unknown.The primary research of TSD focused on the description of sex-determine system in different species,and the role of the sex hormone,and excavating genes that regulated TSD.Recent studies suggest that DNA methylation modification and non-coding RNAs may be an important regulatory mechanism of the TSD model.Furthermore,as the product of cyp19a1 gene,aromatase,is a steroidogenic enzyme that irreversibly converts androgens into estrogens,and therefore cyp19a1 gene is considered to be important gene that regulate TSD in reptiles and an potential target gene for gender-related miRNA.Through hatching the eggs of Reeves' Turtle(Mauremys reevesii)at different temperatures(26?,29?and 32?),we would confirm the sex determination model of the Chinese pond turtle and identify the difference expression of miRNAs under different incubation temperatures.Because cyp19a1 gene had been regarded as candidate gene,we would analyze the effect of DNA methylation modification of cyp19a1 gene promoter on turtle's sex determination.Specific results is as follows: 1.The influence of incubation temperatures on sex determination in tutleWe hatched the eggs of Reeves' Turtle(Mauremys reevesii)at 26?,29? and 32?,and the gender of infant turtle were identified.Results showed the sex ratio(female/male)was 3.3%,36.6% and 100% for the 26?,29? and 32? group,respectively.Suggesting that Chinese pond turtle is the species of temperature-dependent sex determination,belong to TSD Ia,meaning high temperature product females,and low temperature product males.It also provides a theoretical basis for subsequent experiment research.2.The identification of difference expression of miRNA in the turtle gonad under different incubation temperaturesAt thermo sensitive period,we obtained AKG and through Next Generation Sequencing,identified the difference expression of miRNAs during TSP in the turtle gonad under different incubation temperatures.The 1013 known miRNAs and 730 novel miRNAs had been determined.Among them,a total of 18 miRNAs showed discrepant expression under different incubation temperature.Through qRT-PCR,seven miRNAs expression pattern were confirmed in accordance with the sequencing results.Based on the KEGG analysis of the predicted miRNA,the results showed that the endocytosis and axon pathway might be an important pathway that participated in the process of the turtle's TSD.These findings demonstrated that the miRNAs might participate in sex determination of the turtle.3.The study of methylation pattern of the turtle's cyp19a1 gene promoter in different incubation temperaturesThrough TA cloning and DNA sequencing,we successfully obtained 5'flanking sequences of the turtle's cyp19a1 gene,and predicted the putative transcriptional start site(TSS)and transcription factor binding sites(TFBSs)on this region by bioinformatics,therefore preliminarily determined the core promoter of cyp19a1 gene.And the result of dual-luciferase reporter gene assay showed this sequence possess the function of promoter.The methylation level of turtle cyp19a1 promoter was examined in different incubation temperature by bisulfite sequencing.Results showed that the overall methylation percentage of three CpG sites demonstrated significant differences between different incubation temperatures.The percentage of methylation was significantly lower in female-producing temperature(32 ?)compared to the male-producing temperature(26 ?)(P<0.01)and medium temperature(29?)(P<0.05).Meanwhile,the results of real-time quantitative PCR and ELISA showed cyp19a1 mRNA expression and estradiol levels in the female-producing temperature(32?)was significantly higher than in male-producing temperature(26?)and medium temperature(29?)(P <0.01).Taken together,the data obtained from all assays demonstrated that during the TSP,female-producing temperature(32?)could cut down the level of DNA methylation at the promoter region,and low level of methylation was responsible for high expression of cyp19a1 gene and oestrogen synthesis in Reeves' Turtle(Mauremys reevesii).Thus,this confirmed the response of the methylation modification in cyp19a1 gene promoter to the incubation temperature,might be an essential regulatory mechanism of the turtle's TSD.