| Due to bony fishes have primitive nature of evolution and the complexity of living conditions,they have a very complicate of sex determination and differentiation.In most bony fishes,there are both genetic sex determination(GSD)and environmental sex determination(ESD).Apparent regulation plays an important role in ESD.It can adapt to environmental changes by regulating gene expression.It is considered to be a bridge between environment and genotype.Previous studies have shown that long non-coding RNAs are important Apparent regulatory factors can play a very important role in apparent regulation through chromatin remodeling and histone modification.However,there is still a lack of research in this area,especially the role in the sex determination process of bony fishes is rarely reported.Chinese tongue sole is an important marine aquaculture fish in my country.There is a huge difference in growth between males and females.The growth rate of males and the body length and weight of adults are significantly lower than that of females.Previous studies have shown that the sex chromosome type of Chinese tongue sole is the ZZ/ZW type of the female hetero ligand,and in the early sex differentiation of the semi-smooth tongue sole,the hereditary female will be sexually reversed to form a pseudo-male with a male phenotype,and the sex reversal is in the proportion in the natural population is approximately 14%.Under the influence of high temperature,the sex reversal rate of pseudo males is as high as 60%.In addition,the pseudo-males of Chinese tongue sole are heritable,that is,about 90% of the offspring of pseudo-males will also be sexually reversed to pseudo-males.Therefore,Chinese tongue sole can be an ideal object to study the sex determination differentiation and sex reversal mechanism of bony fishes.This article mainly focuses on the study of the regulation mechanism of non-coding RNA on the sex-related genes of the semismooth tongue sole,and explores the mechanism of long non-coding RNA in the sex determination and differentiation of the Chinese tongue sole,in order to solve the scientific problems existing in the process of sex reversal of the semi-smooth tongue sole.The main research contents are as follows:In order to identify the non-coding RNAs in the gonads of Chinese tongue sole before and after sex differentiation,this study first used the genetic female(GF),genetic male(GM),female(F),male(M),and male fish before and after sex differentiation.A total of 15 sex gland samples of pseudo male(PM)were used as the research object,and the lncRNA library constructed by the Chinese tongue sole sex glands was sequenced using the second-generation high-throughput sequencing technology.The results showed that the lncRNA libraries of 15 sex glands got 224.4 G Valid Data in total,and each library got 14.96 G Valid Data on average.A total of 13,386 lncRNAs were identified through whole transcriptome sequencing and analysis.These lncRNAs are evenly distributed in the sense strand and the negative sense strand of the chromosome,and most of them are antisense lncRNAs.Compared with m RNA,the length of lncRNA,the number of exons,the length and expression of ORF are significantly lower than the level of m RNA.We further used DESeq to compare and analyze the differentially expressed non-coding RNAs between the two samples before and after sex differentiation of the semi-smooth tongue sole.41 lncRNAs and 18 m RNAs were only detected in the gonads of male fish after sex differentiation.33 lncRNAs and 21 m RNAs were only detected in females after sex differentiation.Twenty-six lncRNAs and 12 m RNAs were only detected in pseudo males,which revealed the difference and specificity of the expression of lncRNA and m RNA in the testis and ovary of the semi-smooth tongue sole.We further performed KEGG and GO enrichment analysis on differential m RNAs.Among them,male fish were mainly enriched in pathways related to male development,and in female fish,they were enriched in pathways related to oogenesis and steroid hormone synthesis.The difference analysis of lncRNA shows that in order to further confirm the regulatory relationship between m RNA and lncRNA.We performed co-expression analysis of m RNA and lncRNA and predicted that 8,760 lncRNA and 5820 m RNA have regulatory effects.Among them,93 lncRNAs are predicted to have a targeted relationship with 53 sex-related genes,and play a role in sex determination and differentiation of semi-smooth tongue sole,laying the foundation for further research functions.According to the prediction results,we identified lncRNA TCONS＿00624903 and got its c DNA full length of 532 bp.Through comparison and analysis,it is concluded that the lncRNA is located in the reverse complementary region of the fourth exon and the third intron of dmrt2,and it is named DMRT2-AS(dmrt2 antisense).Predictions by Inta RNA and RNAup indicate that there are interaction sites between DMRT2-AS and dmrt2.RT-qPCR experiments show that DMRT2-AS is highly expressed in the testis but not in the ovary,and the expression trend in the testis increases with the differentiation of the gonads.Further in vitro overexpression experiments show that DMRT2-AS can promote the expression of dmrt2,reveal the function of DMRT2-AS,and provide a basis for studying the involvement of lncRNA in sex differentiation of Chinese tongue sole. |
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