Research On The Relevance Of M6~A Modification Of RNA And Diapause In Bombyx Mori

N6-methyladenosine(m~6A)is a hot spot in epigenetic modification of nucleic acids.m~6A modification in RNA can regulate a series of downstream events including RNA splicing,nuclear transport,RNA degradation and translation,and plays an important role in the growth,development,diseases and stress response of organisms.Silkworm is an important economic insect and lepidopteran model organism with clear genetic background,which is a good material for epigenetics research.The occurrence and termination of diapause in bivoltine silkworm offspring are regulated by external signals,which is probably related to epigenetic regulation.This research explores the connection of m~6A mofification and diapause of silkworn based on epigenetics.The main research contents are as follows:Bioinformatics methods were used to analyze the related information of m~6A methylating tool enzyme genes in silkworm.The transcription characteristics and expression level of m~6A methylated tool enzyme in univoltine,bivoltine and multivoltine silkworm were analyzed by q PCR and Western blot techniques.The changes of m~6A abundance in related tissues of silkworm with different voltinism and diapause fate were detected.The relationship between external factors causing diapause fate changes in silkworm and m~6A methylation modification was analyzed.Me RIP-seq was used to sequence the ovaries of different diapause fate bivoltine silkworm on the 3rd day of pupal stage to reveal the correlation between m~6A modification and silkworm diapause.The main results of this research are as follows:1.There are five m~6A methylation tool enzyme homologous genes in Silkworm.The m~6A methylation tool enzyme sequences in some reported species were searched from NCBI,and five m~6A methylating tool enzyme homologous genes were found from silkworm genome database by homologous alignment,namely,METTL3,METTL14,Fl(2)d,YTHDC1,YTHDDF3.Phylogenetic analysis showed that the five genes are highly conserved in different species.2.The transcription level of m~6A methylation tool enzyme is different in silkworms with various diapause-fate.qRT-PCR method was employed to analyze the transcription characteristics of m~6A methylating tool enzyme in ovary,head and egg from 5 groups silkworm with different diapause fate of univoltine(AK4),bivoltine(QFHT,incubated at 25?and QFLT,incubated at 17?)and multivoltine(NH and Nistari)from pupal stage of to eggs at different developmental phase.Especially for silkworm with the same voltinism but different diapauses fate,the gene transcription patterns are significantly different.The results indicated that the voltinism and diapause fate in silkworms may be telated to m~6A modifiacation.3.The expression of methyltransferase METTL3 protein in different diapause silkworm has specificity.The protein expression of METTL3 in ovaries of different diapause silkworm pupae at different developmental stages was analyzed by western-blot method.The experimental results further confirmed the existence of METTL3 in silkworm,and found that there are divergence in the protein expression of METTL3 for silkworm with different diapause.the change of protein level was consistent with the results of transcriptional characteristics analysis.4.The dissimilarity of m~6A abundance was existed in multivoltine with diapause and non-diapause fate.The changes of m~6A abundance in the ovaries and eggs of NH(multivoltine with diapauses fate)and Nistari(multivoltine with nondiapause fate)pupae were detected by fluorescence immunoassay.The results showed that the abundance of m~6A in different diapause fate silkworms had significant changes in pupal and egg in different developmental stages.The abundance of m~6A in the ovary and eggs of multivoltine non-diapause silkworms was generally higher than that of multivoltine diapause silkworms.The changes in abundance were in good agreement with the expression changes of METTL3.5.Modification system of silkworm m~6A can be influenced by external factorsThe bivoltine silkworm eggs produced at 25?for 18 h were treated by acid soaking treatment method and various concertration of diapause hormone treated on Bm N cell to analyze the influence of environmental factors related to diapause on the modification of m~6A from the organism and cell levels respectively.The results showed that environmental factors significantly changed the expression of m~6A methylated tool enzyme gene and m~6A abundance in silkworm.6.RNAi and Trim-Away can knockdown METTL3 significantly.RNA interference(RNAi)and Trim-away methods respectively,combined with microinjection technology to inject ds RNA into silkworm eggs,or inject exogenous antibody and ubiquitin protease system were used to target silencing the expression of METTL3.The results showed that RNAi can significantly inhibit the expression of METTL3 at transcription level,and Trim-away can rapidly reduce the expression of METTL3 at protein level.7.The epitranscriptome of ovary of bivoltine silkworm in 3rd days pupae with different diapause fate was significantly different.MeRIP-seq epigenetic transcriptome sequencing and data analysis were carried out on the ovaries in 3rd day of pupae from QFHT and QFLT.The results revealed that there were significant differences in the epitranscriptome between the two groups of silkworm,which were not only reflected in the number,type and function of modified genes,but also in the modification frequency of gene loci.The above results show that the diapause change of silkworm is closely related to the epimodification of m~6A,and environmental factors may further affect the expression of diapause related genes through m~6A modification system,resulting in the change of diapause.This study has laid a foundation for further elucidating the diapause mechanism of silkworm from the epigenetic level of m~6A.