The Level And Dynamic Changes Of DNA Methylation In Cephalopod

DNA methylation plays an important role in regulating gene expression,cell aging and trait differentiation.In order to investigate the differences of DNA methylation level and pattern in different genders and tissues of Sepia esculenta Hoyle and Octopus ochellatus Gray;To explore the changes of genomic DNA methylation level at three stages S.esculenta at the genomic level,and to verify the role of DNA methylation modification in the aging process.We applied Fluorescence-labeled Methylation sensitive Amplified Polymorphism(F-MSAP)and analyzed the methylation level and pattern of genomic DNA in the muscle,the heart,the pancreas and the gonad of female and male S.esculenta and O.ochellatus;and analyzed the methylation level and pattern of genomic DNA at three stages(Growing;Reproductive;Aging)in the brain,the optic lobe and the gonad.The main results of this research were as follows:1.Establishment and optimization of F-MSAP technology for detection methylation level of genomic DNA CephalopodsAccording to the reaction systems,the sequences of adaptors,pre-amplification primers and selective amplification primers used in F-MSAP technology for the detection of DNA methylation in invertebrates.We screened out 12 pairs of selective amplification primers with high specificity and amplification efficiency.These primers were applied to S.esculenta and O.ochellatus,and a simplified procedure for detecting DNA methylation data based on F-MSAP technology was developed.We established a set of F-MSAP detection systems that are stable,reproducible,and easy to use for both S.esculenta and O.ochellatus.For the study of cephalopods epigenetic modification(DNA methylation)provides a fast and efficient technical mean.2.The methylation level and pattern of genomic DNA the growing S.esculenta and O.ochellatusWe applied F-MSAP technology to analyzed the methylation level and pattern of genomic DNA in the muscle,the heart,the pancreas and the gonad of female and male S.esculen.ta and O.ochellatus.The result shows that the total methylation rate of S.esculenta and O.ochellatus is 23.97%?39.70%and 29.22%?42.46%,which was at a higher level in aquatic invertebrates.Comparing the total methylation levels of the same gender and tissue of S.esculenta and O.ochellatus,it was found that the tissues with significant differences all showed O.ochellatus total methylation levels higher than S.esculenta.We also found that the fully methylation level of female S.esculenta,female and male O.ochellatus was the largest contributor to the total methylation level and was consistent with the level of total methylation in the four tissues;In contrast,the hemi-methylation level was higher proportion of the total methylation level;It showed that DNA hemi-methylation and fully-methylation play different roles in regulatory and also have gender and species differences.This study can provide basic data for epigenetic regulation of the life processes such as the growth,differentiation,and death of S.esculenta and O.ochellatus.3.Methylation changes in genomics during aging of S.esculentaDNA methylation regulates the growth and development of organisms,while DNA m-ethylation levels and patterns change with aging.To analyze the relationship between agin-g and genomic DNA methylation of S.esculenta,F-MSAP technology was used to analyz-e the DNA methylation modifications of the brain,optic lobes,and gonadal tissue of the male and female S.esculenta at three development stages.The results showed:There was n-o significant difference in the total methylation level of three stages female S.esculenta brain and gonad.These showed that DNA methylation played an impo-rtant role in maintaining the stability of genomic DNA.The total methylation level of fem-ale and male S.esculenta optic lobes from the growth stage to the reproductive stage show ed a significant descending.This may be related to the function of the optic lobes as the ma in visual center.The optic lobes controlled producing different body model pigments to attr act the male S.esculenta of the opposite sex for mating.The total methylation levels of brain,optic lobes,and gonads ffrom the growth stage to the reproductive stage.The same organization showed a consistent trend in males and females;and from the reproductive stage to the aging stage,showed significant gender difference,which may be quickly died after mating with male S.esculenta,and female S.esculenta only died after spawning.In summary,DNA methylation played an important role in the growth,development,and tissue development of S.esculenta and O.ochellatus.It also played an important role i-n the growth,reproduction,and aging of S.esculenta.However,in different species,the le-vels of DNA methylation in different sexes,different developmental stages,and tissues w-ere not exactly the same.This showed that during the development of life,DNA methylatio-n modification was only one of many regulatory mechanisms.The research has obtained changes in the methylation level of genomic DNA in the life history of S.esculenta,and the purpose of preliminary exploration has been completed,which providing a theoretical basis for further research.