The Role Of DNA Methylation In The Developmental Regulation Of Gonads,Embryos And Larvae Of The Swimming Crab Portunus Trituberculatus

Swimming crab is one of the most important marine aquaculture crabs in the coastal areas of China and occupies an important position in the marine fishing target in China.At present,the scale of artificial breeding of the Portunus trituberculatus is gradually expanding,but there are problems such as gonadal dysplasia and small individuals in the Portunus trituberculatus during the large-scale breeding process.The quality of high-quality Swimming crab cannot meet people’s needs.In recent years,more and more attention has been paid to the research on the regulatory mechanism of reproduction and development of Portunus trituberculatus.The gonads are important organs for reproduction and development,and bear the heavy responsibility of breeding offspring.Many regulatory factors are involved in the regulation of gametogenesis.As an important component of epigenetics,methylation modification plays an important role in the growth and development of eukaryotes.This experiment will study the mechanism of the development of gonads,embryos and larvae of Portunus trituberculatus from the perspective of DNA methylation,and provide references for the study of reproduction and development of Portunus trituberculatus.Below are key research findings: 1.Detection and analysis of methylation levels of ovaries,embryos and larvae ofPortunus trituberculatusIn order to understand the role of DNA methylation in the development of Swimming crab,we used the Methylflash to detect the ovaries of Portunus trituberculatus.Embryonic and larval development at different stages of DNA methylation.The results showed that the overall methylation level was about 0.005%-0.07% at different stages of ovarian development,and DNA methylation was present at different stages of ovarian development.Among them,the overall methylation level of the VI stage ovary was the highest,followed by the V stage methylation level,which was significantly higher than other stages of ovarian development(P<0.05).During embryonic development,the overall methylation level of embryos was between 0.008% and 0.08%.The level of DNA methylation first decreased and then increased.The level of DNA methylation was highest during fertilized eggs and naupliar stages.The lowest methylation of the blastocyst.At the development stage of larvae,the level of DNA methylation first decreased and then increased.The methylation level was highest in stage I larvae(P<0.05).The results show that DNA methylation plays an important role in the development of gonads,embryos and larvae.2.Cloning of Dnmt2,Dnmt3 b,UHRF1,MBD2,MBD4 Genes Related to Methylationof Portunus trituberculatus and Their Expression in Different Stages of Embryonic,Larval and Gonadal DevelopmentTo investigate the role of DNA methylation-related genes Dnmt2,Dnmt3 b,UHRF1,MBD2 and MBD4 in the development of gonads,embryos and larvae of Portunus trituberculatus.In this experiment,the full-length c DNAs of the above five genes were cloned using RACE cloning gene technology,and sequence analysis,homology analysis and phylogenetic tree analysis were performed using Gene Tool,MEGA and other softwares.The results showed that the above 5 genes were relatively evolutionarily conserved and shared 55%-60% homology with other species.Among them,the Dnmt2 amino acid sequence of Portunus trituberculatus is closely related to the Dnmt2 evolution in Macrobrachium rosenbergii.The evolutionary relationship between Dnmt3 b and UHRF1 amino acid sequences is closest to that of A.cerana and A.persicae.The amino acid sequences of MBD2 and MBD4 are closely related to the evolution of M.mori.The results of expression analysis in each tissue of Portunus trituberculatus revealed that the five genes were expressed in hepatopancreas,gonads,thymus,iliac crest,brain ganglion,intestinal tract,and muscle.The expression of Dnmt2,Dnmt3 b and MBD4 in the ovary was significantly higher than that in other tissues(P<0.05).The analysis of the expression patterns of Dnmt2,Dnmt3 b,UHRF1,MBD2,and MBD4 ovarian tissues at different stages of the DNA methylation-related genes of Portunus trituberculatus showed that the expression of each gene was significantly different at different stages of ovarian development.The expressions of Dnmt2,UHRF1,and MBD4 genes in the different stages of ovarian development were basically the same,all increased first and then decreased.The expression levels of Dnmt2 and MBD2 genes showed a gradually increasing trend,and the expression level in V phase peaked.Phase VI has the lowest expression level.The analysis of the expression patterns of these five genes at different stages of the spermary tissue of Portunus trituberculatus found that the expression level of Dnmt2 showed a gradual upward trend in testis stage I-IV,and the expression level peaked in stage IV and was significantly higher than that in other periods(P <0.05).UHRF1 gene expression in different stages of the testis found that UHRF1 expression showed a decreasing trend,in the phase I expression was significantly higher than other periods(P <0.05).The expression trends of MBD2 and MBD4 were basically the same in the testis at different stages of development,with the peak expression in phase II and gradually decreased afterwards.Using Real-time PCR,we detected the relative expression levels of the three methylation-related genes Dnmt2,Dnmt3 b and UHRF1 in Portunus trituberculatus during embryo and larval development.In the early embryonic period,Dnmt2 was not expressed in the fertilized egg and multicell stage,and it was expressed from the blastocyst stage,and the expression level gradually increased.The expression level was the highest during the period of no hatchling.The expression of Dnmt2 increased during the period of large ocular larvae compared with the fourth stage of zoea,but the expression level of Dnmt2 decreased significantly at the fifth day of Large-eyed larvae.The expression of Dnmt3 b in the embryonic stage was similar to that of Dnmt2.The expression of Dnmt3 b was not expressed in the fertilized egg and multicell stage.The expression was highest in the blastocyst stage and then decreased gradually.The expression level of Dnmt3 b was basically the same in all stages of the juvenile larvae.The ocular larval period has a higher expression level.At the early stage of embryonic development,the UHRF1 gene showed a tendency of first decrease and then increase,and the expression in the fertilized egg stage was significantly higher than that in other periods(P<0.05).During larval stage,the expression of UHRF1 first increased and then decreased.On the third day of zoea II and large ocular larvae,the expression of UHRF1 was highest and significantly higher than other tissues in the same period(P<0.05).MBD2 and MBD4 showed a tendency to increase first and then decrease during embryonic development.In combination with the function of MBDs,it was speculated that the ability of MBD2 to inhibit the expression of related genes in the early stage of embryonic development was enhanced.The expression levels of MBD2 and MBD4 in the zoea stage were higher than those in the stage of macrolarvae,indicating that these two genes regulate the growth and development of the zoea stage.