| The Chinese sea perch is a fish of aquaculture importance and an excellent model for adaptation biology,as it grows fast and can be cultured in a wide range of temperature and salinity.Chinese sea perch is one of the most flavored species,which shows special advantages in a mild taste.Given the limited wild resources,Chinese sea perch has become an important farmed fish in C hina.Studies on this valuable aquatic fish have focused on genetic marker development,genetic map construction,and characterization of functional genes involved in reproduction,stress,growth,and immunity.As a eurythermal and euryhaline species,Sea perch is widely cultivated in China and a potential valued marine fish model.It can be cultured in fresh water after desalting.However,adult fish cannot complete the entire reproduction process without sea water,which is a major obstacle in the culture of sea perch inland.In this study,we use Chinese sea perch as the experimental material,brain and gonadal of sea perch use for trascriptase sequencing.A number of important reproductive related genes with significant differences in expression were analyzed,and the expression and function of cyp19 a,a major reproductive gene,were further studied.The main results are as follows:1.Analysis of transcriptome sequencing data of brain and gonadA total of 78,256,909 clean reads were generated from the adult brain,ovary and testis by using the Illumina Hi Seq2000 platform and assembled into 274,909 contigs.A total of 31,683 unigenes were annotated based on sequence similarity,and 20,702 unigenes were found to exhibit 8237 gene ontology terms and 3888 pathways.Transcripts of 26,623 unigenes were present in all of the organs,whereas those of 748,349,and 319 unigenes were unique to the brain,ovary or testis,respectively.In addition,pair-wise comparisons revealed that 671/367,496/315,and 1668/580 unigenes were up/down regulated.Homology search led to the identification of reproduction-associated genes of the brain-gonad axis,including those involved in sex differentiation and maintenance.The data provided an integrated and comprehensive unigene resource for Sea perch,which could be used for further research on HPG axis gene function,reproduction regulation,and sex-biased gene expression.2.HPG axis-related gene identification and expression analysisAnalysis by transcriptome data,All of those genes(56 in the Gn RH signaling pathway(KEGG number: map04912),31 in the ovarian steroidogenesis(KEGG number: map04912))were identified in this study.such as Gn RH receptor,LH,FSH,LH receptor,FSH receptor,androgen receptor(AR),estrogen receptor(ER),GSDF,CYP19 a etc.Six of those genes were selected to verify their expression level among different development stages and tissues.The cyp19 a was mainly expressed in the gonad and brain,but minor expression was observed in other organs.However,another hormone synthesis enzyme,3?-HSD,was widely expressed in seven tissues,AR and ER,were both expressed in all the selected tissues.GSDF was highly expressed in the testis than in the ovary.PIWI specifically expressed in the testis and ovary.3.Detection of genomic DN A methylation status in the brain and gonad tissues of Chinese sea perch by MSAPIn the brain,testis and ovary,eight pairs of selective amplification primers produced a total of 596,580 and 796 clear bands within 100 bp to 1000 bp,in which each individual of eight pairs of primer combinations averaged 149,145 and 199 amplified bands,brain,testis and ovary of Chinese sea perch genomic DNA methylation levels were 34.23%,38.62% and 47.74%,respectively.4.Detection of cyp19 a gene promoter methylation status in brain and gonad tissues of Chinese sea perch by BSPPCR amplify and clone the promoter sequence of cyp19 a by Genomic Walking methods.The genomic DNA of the three tissues were treated with bisulfite,respectively.The BSP primers were designed and the three promoter sequences were amplified.The methylation of Cp G sites was detected by cloning and sequencing.The results showed that the methylation degree of cyp19 a gene promoter in testis and brain was significantly higher than that in ovary,about twice as much.Promoter methylation level and cyp19 a expression in three tissues were negatively correlated.5.Transcription function analysis of cyp19 a promoter in vitroThe spcyp19 a promoter activation was determined by construction of PGL3-basic-cyp19 a luciferase reporter vector.Deletion of region of-140 to-46 significantly decreased the basal activities of cyp19 a promoter(decrease to~58.90%),so the longer promoter showed higher transcriptional activity.suggesting this region,including several transcription binding sites of SF-1,SOX,PPARE,CRE,and TATA box,is important for the regulation of cyp19 a promoter activities,Cp G methylation can inhibit the transcriptional activity of the promoter. |
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