Integrating Analyse Of MRNAs And MiRNAs In The Gonad Of Smphysodon Aequifasciatus

Discus fish(Symphysodon aequifasciatus)belonging to perciformes,the family Cichlidae(Teleostei)is naturally distributed in the east and middle Amazon River basin of South America.It endowed as ‘the king of tropical fish',has been widely cultured for its bright colors and pretty disc-shaped body.It is very popular in aquaculture industry in several Asian countries and it occupys important position on the freshwater aquarium trade.At present,the reproduction of discus fish still carried out by natural pairing and natural spawning.Which greatly restricted the yield of seed breeding and limited the farming of discus fish.Adult male exhibit a larger ultimate size than female,males are more economically valuable.However,due to the lack of genome and transcriptome data resources,there are few studies on the gene selection breeding of discus fish.Therefore,in order to understand the transcriptome of the gonad of this species,it was found that genes expressed differently in the male and female glands are very important for the further study of gene selection breeding of the discus fish.In this study,the advanced next-generation high-throughput sequencing technology was used to perform transcriptome sequencing using the mature testes and ovary tissues of discus fish.After de novo assembly and functional gene annotation of high quality sequence,we identified differentially expressed genes mRNA and miRNA between the testis and ovaries.Screening candidate genes associated with important life processes such as gametogenesis and the produce of steroid hormones about gonad function,performing the integration analysis of the miRNA and mRNA,the results could contribute to further research regarding underlying miRNA and mRNA pairs of gametogenesis and steroidogenesis in discus fish.The results are as the followings.A.High-throughput mRNA sequencing between testes and ovary in discus fishWe utilized the next generation high-throughput sequencing technology.A total of six cDNA libraries with three testes and three ovaries,were constructed and sequenced using Illumina sequencing.Two differentially expressed genes were compared The differentially expressed genes between the two organs were compared.50,082 nonredundant genes(unigenes)and 65,496 transcripts were obtained.Genes differentially expressed in the two tissues were compared,the results of differences in expression shown that 29,752 differentially expressed unigenes were detected,including 18,570 upregulated unigenes and 11,182 down-regulated unigenes.Results of functional annotation showed that 28,989(57.88%)unigenes were annotated in the Nr database,20,176(40.29%)unigenes were assigned to the GO database,21,311(42.55%)unigenes were assigned to the KOG database,and 15,702(31.35%)unigenes were assigned to the KEGG database.We used bioinformatics analysis methods,the candidate genes associated with important gonadal functions such as gametogenesis and steroidogenesis were screened.To validate the sequencing data,8 mRNAs were selected randomly,and specific primers were designed and used to quantify mRNA for qRT-PCR validation.Quantitative PCR validation results for 8 random mRNAs were similar with RNA-Seq results.It showed the reliability and accuracy of high-throughput sequencing.The gametogenesis of discus fish and the molecular regulation mechanism of steroid synthesis remain to be further studied.The detailed molecular regulation mechanism of gametogenesis and steroidogenesis of the discus fish is still to explore.B.High-throughput miRNA sequencing between testes and ovary in discus fishIn this study,miRNA sequenced in the testes and ovaries by Illumina Hiseq 4000,and detected the differentially expressed miRNA.There are 47 miRNA were differentially expressed in the testis and ovary,including 31 up-regulated miRNA and 16 downregulated miRNA.We perform the quantitative PCR technology to randomly select 8 miRNA from the 47 differentially expressed miRNA,and verified them experimentally.The differentially expressed miRNA in the gonad indicated that these miRNA play an important biological role in the testis and ovary.In order to reveal the molecular regulation mechanism of miRNA in the gametogenesis and steroidogenesis,it provides a basic theoretical basis.C.Integrated analyses of miRNA and mRNA between testes and ovary in discus fishTo predict the genes targeted by differentially expressed miRNA,two computational target prediction algorithms(TargetScan 50 and miRanda 3.3a)were used to identify the miRNA binding sites.Finally,the data predicted by both algorithms combined,and the overlaps were calculated.By predicting miRNA target genes in testis and ovary,the results indicated that the miRNA target genes were very rich,and found that each miRNA corresponds to a number of target genes,a mRNA was regulated by the number of miRNA at the same time.In order to understand miRNA and mRNA regulation of potential relationships of the gametogenesis and steroidogenesis,we carried out bioinformatics methods,screening to its functional genes related to 41 pairs,miRNA with mRNAs network relationship were established.