| Orange-spotted grouper(Epinephelus coioides)is a kind of valuable marine fish species in Southeast Asia and China,which is fast-growing,adaptable,delicious and nutritious.It is suitable for aquaculture in subtropical area and tropical area,like in southern Guangdong and Hainan.However,it is difficult to obtain parent fish,especially male parent fish,because of its special sexual development pattern(hermaphroditism and late maturity).This problem seriously affects the breeding and production of grouper and shows the urgent need for the reproductive research.Most previous reproduction studies are about endocrine regulation and sex-determining genes while nowadays,epigenetic regulation of physiological activities,such as histone modification regulation,has attracted much attention.Histone modification changes the status of chromosomes,thus affecting the transcription of genes and ultimately affecting the genetic traits of organisms.In this research we talked about the role histone modification plays in the development of orange-spotted grouper gonads from two different perspectives: histone modification enzymes and histone modification sites.The results of this article demonstrated that histone modification plays active roles in the early gonadal development and sexual reversal of orange-spotted grouper,through the researches about histone modification enzymes and genomic histone modification sites.Research contents were divided into three parts:(1)The basic molecular genetics research of histone modification enzymes.In this part,we identified the histone-modifying enzymes in the gonads of orange-spotted grouper,using the results of genome sequencing and transcriptome sequencing as well as the known histone-modifying enzymes sequence in other species.Then we speculated the correlation between histone-modifying enzymes and gonadal development of grouper.Next,we analyzed the histone modifying enzymes of Jmj family through phylogenetic tree,qPCR and in situ hybridization.We found that histone modifying enzymes like EZH1 may play a role in the gonadal development of juvenile orange-spotted grouper,while histone modifying enzymes like Jmjd3b(Kdm6bb)may play a role in the sexual reversal process.These results provide the basis for further study.(2)The feeding experiment of UNC1999,the inhibition of EZH1,one of the histone modification enzymes in orange-spotted groupers.The feeding experiment was performed on orange-spotted groupers(70 days after hatching),and the experiment last for 6 months.Results of western blot showed that the activity of EZH1 in the gonads of juvenile groupers was inhibited and the level of H3K27me3 was decreased after UNC1999 feeding.As shown in results of hematoxylin-eosin staining,the number of oogonia and oocytes increased after UNC1999 feeding.Results of qPCR revealed that the expression of foxl2 and other ovarian development-related genes were up-regulated after UNC1999 feeding.In conclusion,in this part we demonstrated that histone modifying enzyme EZH1 inhibits the gonadal development(toward mature ovary)of juvenile orange-spotted grouper.(3)The ChIP-seq(Chromatin-immunoprecipitation-seq)experiment.In this part,we performed the chromatin immunoprecipitation and got the chromosomal DNA fragments samples of orange-spotted grouper gonads(including ovary,bisexual-phase gonad and testis).Then high-throughput sequencing analysis was performed using these chromosomal DNA fragments.By the means of genome mapping,enrichment region detection and visualization analysis,it was proved that histone modification indeed plays a role in sexual reversal of orange-spotted grouper,especially h3k27 ac modification which enhances the transcriptional activity of amh,dmrt1 and other male-related genes and then promots the sex reversal. |
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