The Expression And Function Analysis Of The DNA Methylase From Siniperca Chuatsi

Siniperca chuatsi(mandarin fish)is a carnivorous freshwater fish of the genus Perch and an important economic fish in Chinese aquaculture.As an important part of epigenetics,DNA methylation participates in the regulation of genes,and one of its main components is DNA methylase.As a type of gene family,DNA methylases(dnmts)have been mainly concentrated in model organisms such as humans,mice,fruit flies and zebrafish in many previous studies,and the research direction is single and one-sided.Therefore,based on the perspective of molecular biology,this experiment analyzed in detail the expression and distribution of dnmts in the early development process of mandarin fish and different tissues,discussed its role in fish gonadal development and embryonic development,and studied the different Changes in the expression pattern of Sc-dnmts stimulated by environmental conditions(nutrients and salinity).Firstly,17 different tissues(skin,gills,opercular gill,muscle,liver,mesonephro,head-kidney,myocardium,brain,spleen,gallbladder,esophagus,stomach,pyloric caecum,intestine,testis and ovary)of mandarin fish were treated and 9 developmental stages(2-cell stage,1K-cell stage,blastocyst stage,gastrulation stage,pigment stage,tail bud stage,before exiting the membrane,1 day exiting the membrane,5 days exiting the membrane)for high-throughput sequencing,and Using the sequencing results,4 mandarin fish DNA methylases Unigene were isolated.In this study,we cloned and characterized Sc-dnmts,namely dnmt1,dnmt2,dnmt3 a and dnmt3 b,and carried out molecular characterization analysis.There are differences in the general structure of Sc-dnmts,which are divided into N-terminal regulatory domains and C-terminal catalytic domains.Multi-species amino acid sequence alignment and spatial structure showed that the N-terminus of Sc-dnmts is not conserved and there are multiple domains.The C-terminus contains only one domain of Dcm and is highly conserved.The difference in structure is related to the different functions of dnmts.The phylogenetic tree results suggest that compared with DNNT1 and dnmt2,the biological evolutionary course of dnmt3 family genes is not conservative and there are differences.Although Sc-dnmts are expressed in different tissues and embryonic developmental stages,the expression profile still has differences in tissues and developmental stages.In the gonads of mandarin fish,Sc-dnmt1 is highly expressed in the ovary,Sc-dnmt2 can be highly expressed in the testis and ovary,and Sc-dnmt3 a and dnmt3 b are highly expressed in the testis.At the same time,the results of immunohistochemistry also proved that there is dnmt2 protein in the seminiferous tubules of mandarin fish testis,dnmt2 and dnmt3 a in spermatocytes;dnmt2 is highly expressed in the primary oocytes of phase II or III mandarin fish ovaries,Sc-dnmt3 a showed a weak positive signal in primary oocytes.Therefore,this study shows that Sc-dnmt2 and Sc-dnmt3 a may play an important role in the development of mandarin fish testis and ovaries.In different stages of embryonic development,the expression of Sc-dnmts has obvious temporal and spatial expression differences.Sc-dnmt1 and Sc-dnmt2 were highly expressed at the 2-cell stage immediately after fertilization,and then declined;Sc-dnmt3 a was expressed from the gastrulation stage;Sc-dnmt3 b was expressed very low at the first fertilization stage,but expressed at the blastocyst stage rise.The expression levels of the four Sc-dnmts all showed ups and downs during embryonic development,and combined with the bony fish protein interaction network and the analysis of the TPM value of the different developmental stages of the mandarin fish,it was found that there are some common proteins and Sc-dnmts co-expressed in the different developmental stages of the mandarin fish Or synergistic effects may occur,including EZH2,QTRT1,MAT2 B and H3,indicating that dnmts can play an important role in the development of biological embryos,including the differentiation and growth of specific organs during fish development.Under the stimulation of different living environment conditions(nutrient composition,salinity and drugs),the expression pattern of Sc-dnmts changed.⑴ After the artificial feed domestication,the expression level of Sc-dnmts in the liver,stomach and intestines has changed.For example,the expression level of Sc-dnmt1 in the liver after domestication increased significantly;the expression of Sc-dnmt2 only increased significantly in the liver after domestication,but decreased in the intestine;the expression of Sc-dnmt3 a in the liver and stomach after domestication The expression level increased;the expression level of Sc-dnmt3 b in the stomach increased significantly after acclimation.(2)After 10 ppt salinity stimulation,the expression profile of Sc-dnmts in the gills and intestines also changed.In the gills,the content of Sc-dnmt2 and 3a increased significantly;in the intestine,the expression levels of Sc-dnmt1,3a,and 3b increased significantly.⑶ In a short period of time after LPS and Poly I:C injection,almost all of the Sc-dnmts expression ability in blood and skin declines.It implies that Sc-dnmts may be related to the digestion and ingestion,energy metabolism,osmotic pressure regulation and inflammation of the mandarin fish.Especially Sc-dnmt3 a showed significant sensitivity to different stimuli.This study provides basic information for future research on the application of DNA methylases in more areas,and also increases the abundance of DNA methylases in teleost.