Molecular Mechanism Underlying The Regulation Of MRNA M~6A Methylation On The Diapause Of Bivoltine Bombyx Mori

The function of diapause of the silkworm,Bombyx mori is to survive under poor living environment andseanson.The diapause characteristics of B.mori is induced by environmental signals,and its formation and development are closely related to epigenetic inheritance.Epigenetics is multi-dimensional and coregulated.But how this combined effects response to environmental signals induced by diapause and how epigenetic regulation induced by environmental signals precisely regulates diapause in insects remains unknown.m~6A methylation is one of the most common internal modifications of m RNA,but its regulation on diapause is rarely reported.Based on fluorescence immunoassay technique,we analyzed the apparent transcriptomic sequencing(Me RIP-seq)data of bivoltine silkworm Qiufeng in the 3d ovarian tissues of the pupa stage developed from eggs incubated at a normal temperature(QFHT,diapause egg producer)compared to those from eggs incubated at a low temperature(QFLT,nondi-apause egg producer)to explore the potential effect of m~6A methylation modification on diapause of the silkworm.Firstly,we validited Me RIP-seq data,enriched the differentially modified peaks and screened out some pathway genes and N6-methyladenosine(m~6A)methylases related to diapause as candidate.Secondly,RNAi technology was used to further screen the potential regulatory effects of candidate genes on diapause characteristics of the silkworm eggs.Thirdly,RT-q PCR was used to analyze the transcriptional characteristics of genes with potential role in diapause regulation in AK,QF,SH and Nistari;And Me RIP-q PCR was used to analyze the methylation level of m~6A gene in disinfected QFHT and QFLT.Fourthly,m~6A modification sites were determined by m~6A-RT-QPCR and site-recognition proteins were determined by RNA immunoprecipitation.Then actinomycin D treatment and double luciferase reporter system was used to determine the regulatory effect of reading protein on target genes.Finally,the effect of overexpression on diapause traits in QFLT strains was analyzed,and the effect of m~6A modification on gene expression and its association with diapause in silkworm were analyzed.The main results are as follows:1.YTHDF3 is involved in the diapause process of bivoltine B.mori strains by regulating the expression of Cyp307a1 and Cyp18a1 in the ecdysone synthesis pathwayReading protein YTHDF3(YTH N6-Methyladenosine RNA Binding Protein 3)knockout and overexpression experiments were carried out in bivoltine B.mori strains.We observed that the knockdown of B.mori YTHDF3 resulted in delayed embryo development,while the overexpression of YTHDF3 resulted in the transformation of nondiapause-destined eggs into a mixture of diapause and nondiapause eggs,which confirmed that YTHDF3 was involved in the regulation of diapause of bivoltine silkworm.Combined with Me RIP-seq sequencing,further studies showed that YTHDF3,as a reading protein,can recognize the m~6A site of Cyp307a1 and Cyp18a1 genes in the ecdysone synthesis pathway(ESP),The increase of Cyp307a1 can promote the expression of 20-Hydroxyecdysone(20E),while Cyp18a1 is a downstream gene of 20E and affects the expression of 20,26-dihydroxyecdyone gene.Both of these genes are important genes in ESP and important hormones affecting diapause traits.Our results show that m~6A modification mediates YTHDF3 to affect the expression levels of its target genes,Cyp307a1 and Cyp18a1,in the ESP to regulate diapause in bivoltine B.mori.2.m~6A-dependent mevalonate kinase in juvenile hormone synthesis pathway regulates the diapause process of bivoltine silkwormThe modification rate of m~6A methylation of mevalerate kinase(MK)was significantly different in the pathway of juvenile hormone synthesis.Expression profile analysis showed that there were significant differences in the expression of MK transcripts in the ovary and head of the QFHT and QFLT pupa at 1-6 d and in the eggs at 2-72 h;the m~6A methylated MK in ovary and eggs of QFLT was significantly higher than that of QFHT.Further studies showed that the MK knocked down in the QFLT pupal stage caused the non-diapause eggs to become diapause eggs.In addition,the reading protein YTHDF3 recognizes m~6A-modified MK and facilitates translation.3.RNA N6-methyladenosine of DHAPAT and PAP involves in regulation of diapause of Bombyx mori via the lipid metabolism pathwayThe analysis of methylation sequencing data showed that the m~6A methylation levels of dihydroxyacetone phosphate acyltransferase(DHAPAT)and phosphatidyl phosphatase(PAP)in lipid metabolism pathway were significantly different in QFHT and QFLT pupal 3 d ovarian tissues.We knocked down DHAPAT in the pupal stage of the QFLT group,resulting in the non-diapause destined eggs becoming diapausing eggs.In the PAP knockdown group,the colour of the non-diapause destined eggs changed from light yellow to pink 3 days after oviposition,but they hatched as normal.Further analysis of transcriptome expression profiles showed that there were significant differences between DHAPAT and PAP in ovarian and adipose tissue at 1-6 d and eggs at 2-72 h.The m~6A methylated transcripts in ovaries and eggs of QFLT were significantly higher than those of QFHT.In addition,the methylated reading protein YTHDF3 recognizes m~6A-modified DHAPAT and PAP,increasing m RNA stability and promoting translation.These results indicate that m~6A-mediated methylation can change the expression of diapause related genes,and then regulate the synthesis of related pathways to affect the diapause traits of silkworm.In conclusion,m~6A methylation modification can regulate diapause of disinfected silkworm in response to environmental induced signals.The results of this study partially explained the molecular mechanism of diapause changes induced by environmental signals,and provided a reference for the study of the relationship between epigenetic inheritance and diapause,as well as a new target for pest control in agriculture and forestry.