| Nile tilapia(Oreochromis niloticus) is a fish, which sex determination is influenced by a combination of genotype and temperature. Sex determination in Nile tilapia can be affected by temperature changes when the gonad is undifferentiated. But it is not clear that how temperature override the role of genetic factors and switch the mechanism to affect the sex determination and differentiation of Nile tilapia. Aromatase, which encodes by cyp19a1 a gene, is a key enzyme controls the process that androgen turn into estrogen. Aromatase plays a important role in the period that Nile tilapia sex differentiation, it can decide which sex the Nile tilapia will be. In recent years, Epigenetics is a hot research issue. DNA methylation and histone methylation can both affect gene expression, even decide the direction of fish sex differentiation. But how epigenetics guide Nile tilapia sex differentiation and affect gene expression is unknown. And what exactly is the high temperature role in this process, this is a worthwhile question. In this study, Nile tilapia fry which in TSP were induced in high temperature,the temperature is 36℃, then feed up to 4-months and get the samples, then extracted DNA were sequenced by Me DIP-seq to explore the genome-wide methylation levels in Nile tilapia(control female, control male, induced female, induced male). Cyp19a1 a plays an important role in sex differention process. So Bisulfite sequencing to detect the methylaiton level of cyp19a1 a promoter in different ages and temperature induced Nile tilapia. The expression of these genes were systematically analyzed by q RT-PCR techniques,the histone methylation level detected by Ch IP. The main results are as follows:Using Me DIP-seq detect genome-wide methylaiton level in induced groups and control groups of Nile tilapia. We have four groups: control female, control male, induced female,induced male.We obtained 128055474, 50323361, 69696162, 75132883 raw data from fourgroups respectively, in total to 16.15 Gb raw reads, which contains 105101871, 47468675,55024475, 67759606 clean data, in total 13.75 Gb accounting for 82.08%, 92.33%, 78.95%,90.19% respectively. There were 105101871, 47468675, 55024475, 67759606 reads could be mapped to the Nile tilapia reference genome, and there were 49917355(47.49%),22480291(47.36%), 26099799(47.43%), 32506902(47,79%) reads could be uniquely mapped to the Nile tilapia genome. There were many different methylation genes in four groups:CF/CM group has 344 hypermethylation genes and 661 hypomethylation genes, IF/IM group has 331 hypermethylation genes and 215 hypomethylation genes, IF/CF groups has 248 hypermethylation genes and 126 hypomethylation genes, IM/CM groups has 568 hypermethylation genes and 680 hypomethylation genes. We screened out four genes from these genes and detect the expression of m RNA through q RT-PCR techniques, and the results are in accordance with methylation level. It suggested that high temperature will affect the degree of genome-wide methylation level in Nile tilapia.Bisulfite sequencing to detect cyp19a1 a promoter methylation level under different temperature of Nile tilapia. It is shown that cyp19a1 a promoter methylation level is increasing after high temperature induction in female and male(P<0.05). At the same time, the expression of m RNA is declining(P<0.05). The methylation level of cyp19a1 a could be increased in male and female Nile tilapia exposed to high temperature.Ch IP technology detects cyp19a1 a histone H3K9 methylaiton level under different temperature of Nile tilapia. It is shown that cyp19a1 a histone H3K9 methylation level is increasing after high temperature induction in male(P<0.05). It is indicated that high temperature can affect histone methylation level and affect the transcription of cyp19a1 a,significantly decrease in male Nile tilapia.Bisulfite sequencing to detect cyp19a1 a promoter methylation level in different ages of Nile tilapia. It is shown that the cyp19a1 a promoter methylation level is gradually decline in female, and on the contrary in male(P<0.05). At the same time, the expression of m RNA in female is increasing, and on the contrary in male(P<0.05). This suggests that the demand foraromatase, which encode by cyp19a1 a gene, gradually increased in female with age, while declined in male.In conclusion, the results of the present study showed that genome-wide methylation level can be affected and the methylation level of cyp19a1 a promoter could be incresed,expression of m RNA could be decresed in Nile tilapia exposed to high temperature. It preliminarily proved that epigenetic mechanisms may mediate the process of masculinization of Nile tilapia. The results of the present study provide an important foundation for further exploring the molecular mechanism of high temperature effects on Nile tilapia masculinization. |
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