Screening And Identification Of Non-coding RNA Related To Sex Differentiation Of Takifugu Rubripes

Takifugu rubripes,commonly known as puffer fishes.There is no obvious difference in growth between males and females,but the ovaries of females are highly poisonous and cannot be eaten.Testis is non-toxic and delicious,but expensive.Parthenogenetic breeding,especially all-male breeding,can improve its economic benefits.The research of the molecular mechanism of its sex determination and differentiation can lay a theoretical foundation for all-male breeding.The T.rubripes is a dioecious fish with an XX/XY sex determination system,in which a SNP locus of the amhr2 gene is linked to sex.Therefore,the T.rubripes is also a model fish for studying the mechanism of fish sex differentiation.Therefore,it is of great significance to systematically carry out the research on the sex determination and differentiation mechanism of T.rubripes,whether it is for changing the breeding mode in the future aquaculture industry of T.rubripes,or for interpreting the mechanism of sex determination and differentiation of them.Non-coding RNA（nc RNA）is a class of RNA molecules that are transcribed from genes but do not encode proteins,mainly including mi RNA,si RNA,pi RNA,t RNA,r RNA,lnc RNA and circ RNA.Among them,there are many studies on mi RNA,lnc RNA and circ RNA.Previous studies have found that non-coding RNAs are differentially expressed in male and female gonads during the sex differentiation process of fish.Therefore,non-coding RNAs may regulate the process of sex differentiation in fish.In this experiment,the T.rubripes was used as the research object,and the whole transcriptome library of male and female undifferentiated gonads was constructed,and the non-coding RNAs with sex differential expression were screened.The aromatase inhibitor（AI,letrozole）and 17β-estradiol（E₂）was used to treat juvenile T.rubripes,and the expression changes of the screened candidate mi RNA-novel＿167 and its target gene dmrt1 were detected.At the same time,the targeting relationship between the candidate mi RNA-novel＿167 and the target gene dmrt1 was confirmed by in vivo experiments by injecting mi RNA agomir and mi RNA antagomir.The main findings are as follows:1.High-throughput sequencing was performed on the whole transcriptome of the undifferentiated gonads of juvenile T.rubripes 40 days after hatching,and the sequencing data was compared with the genome.Eight differences were found in the male and female undifferentiated gonads of juvenile T.rubripes.The expressed mi RNAs,of which 5 mature mi RNAs are highly expressed in female gonads and 3mature mi RNAs are highly expressed in male gonads,of which mi RNA-novel＿167targets the dmrt1 gene and fru-mi R-15b targets the foxl2 gene.A total of 130 lnc RNAs showed sex differential expression,of which 51 lnc RNA were highly expressed in male gonads and 79 lnc RNAs were highly expressed in female gonads,of which lnc RNA＿000338 targeted the gsdf gene.A total of 60 circ RNAs transcripts were differentially expressed in the gonads of redfin pufferfish,among which 34 circ RNAs were highly expressed in male gonads and 26 circ RNAs were highly expressed in female gonads.We selected some of them for q PCR detection,and the results were all consistent with the screening results,indicating that the sequencing results were reliable.2.We verified the targeting relationship between mi RNA-novel＿167 and dmrt1using dual luciferase experiments.The juvenile T.rubripes were treated with AI and E₂at 25 day after hatching,and the treatment period was 55 day.The gonads of the treated T.rubripes were observed by tissue section,and candidate mi RNAs and relative expression regularity of mi RNA-novel＿167 and its predicted target gene dmrt1 were determined by q PCR.Histological results showed that in the E₂-treated experimental group,the XY gonads had ovarian lumen and no gonads were found to be normal testis,and all gonad phenotypes were all feminized.In the AI-treated experimental group,no individuals with normal ovaries were found,and all gonads were masculinized.The results of q PCR showed that compared with the control group,The expression level of mi RNA-novel＿167 in XY gonads after E₂ treatment was significantly higher than that in XY gonads in control group,and the expression level in XX gonads after AI treatment was significantly lower than that in XX gonads in control group.Compared with the control group,The expression of dmrt1 was significantly decreased after E₂ treatment（P<0.05）,and the expression of dmrt1 was significantly increased after AI treatment（P<0.05）.3.In order to verify the function of mi RNA-novel＿167,the juvenile T.rubripes were injected intraperitoneally with mi RNA-novel＿167 agomir（at a concentration of66μg/g fish weight）and mi RNA-novel＿167 antagomir（at a concentration of 66μg/g fish weight）.The relative expression of dmrt1 and its mi RNA-novel＿167 in male and female gonads after injection was detected by q PCR.The results showed that the expression of mi RNA-novel＿167 in the experimental group injected with agomir was significantly higher than that in the control group（P<0.05）,and the expression of mi RNA-novel＿167 in the experimental group injected with antagomir was significantly lower than that in the control group（P<0.05）,and the expression of dmrt1 was significantly lower than that of the control group（P<0.05）.Therefore,the targeting relationship between mi RNA-novel＿167 and dmrt1 was further demonstrated by in vivo experiments.In order to further study the function of dmrt1,we carried out RNA interference experiment on dmrt1 gene,and designed three pairs of primers（dmrt152,dmrt352 and dmrt430,66μg body weight）to knock down dmrt1.The results showed that the relative expression of dmrt1 after injection of dmrt152,dmrt352 and dmrt430was significantly lower than that of the control group.We selected one of the groups（dmrt152）to detect the q PCR of mi RNA-novel＿167,and the results showed that there was no significant difference in mi RNA-novel＿167 compared with the control group.mi RNA-novel＿167 may not be the only factor regulating the expression of dmrt1 gene,it may regulate the expression of dmrt1 together with other genes,which lays a foundation for the follow-up study of the function of dmrt1.