The Role Of DNA Methylation In The Regulation Of Diapause In Bombyx Mori

DNA methylation is an evolutionarily conserved epigenetic modification associated with insect embryonic development and diapause.The silkworm,Bombyx mori is not only a lepidopteran insect with egg diapause,but also a good model organism for studying the relationship between DNA methylation and embryonic development,including diapause.The methyltransferase system of B.mori is composed of Dnmt1 and Dnmt2,without Dnmt3.According to their differences in diapauses,silkworm strains are divided into univoltine,bivoltine and multivoltine strains.The diapause of bivoltine strains is regulated by environmental and maternal genetic factors.If the parental eggs are incubated at a high temperature of 25°C,their progeny eggs are diapause destinted;while the parental eggs are incubated at a low temperature of 15°C,their progeny are non-diapause destined.The regulation of diapause by this environmental signal is an epigenetic modification,but the mechanism is not fully understood.Therefore,in this study,silkworm strains of different voltinism and individuals of bivoltine strains from eggs incubated under different temperature and light conditions were used as materials,the transcription levels of the two Dnmt genes(Bm Dnmt1 and Bm Dnmt2)at different developmental stages and the DNA methylation level of the offspring silkworm eggs were analyzed.Furthermore,RNA interference(RNAi)was performed for the two Dnmt genes on the second day of pupae,which were obtained from the bivoltine strains Qiufeng under different incubation conditions,in order to see insight into the role of DNA methylation in the regulation of embryonic development and diapause in B.mori,and enrich the study of epigenetic regulation of life process.The main research points are as follows:1.Transcriptional characterization of Bm Dnmt in different voltinism strains of B.moriUnivoltine strains Lu1(Lu1)and Ankang 4(AK4),bivoltine strains Qiufeng(QF)and P50,multivoltine strains Nohai(NH)and Nistari were used as materials.Total RNAs were extracted in different tissues in larval stage,pupal ovary and silkworm eggs in early embryonic development stage of above strains,respectively.And the transcriptional characteristics of Bm Dnmt genes were analyzed by q RT-PCR method.The results showed that Bm Dnmt was expressed in egg stage,larval stage and pupal stage,and the expression levels in early embryonic development stage were higher than that in pupal stage and larval stage.In early embryonic development stage,the transcription level and trend of Bm Dnmt among different voltinism strains revealed different profiles.The transcription level of Bm Dnmt in Nistari was significantly higher than that in NH;in larval stage,the transcription level of Bm Dnmt in ovary or testis tissue of multivoltine strains was significantly higher than that in bivoltine strains,and the transcription level in Nistari was significantly higher than that in NH;In ovarian tissue,the transcript level of Bm Dnmt1 in NH strains was significantly higher than that in the bivoltine strains(QF and P50).Therefore,it is speculated that there is to some extent correlated between the voltinisms and DNA methylation,and diapause of offspring eggs and DNA methylation as well.2.Analysis on DNA methylation levels in early embryonic development stage of progeny eggs of different voltinism strains of B.moriThe transcription levels of Bm Dnmt are different in the early embryonic development stage of silkworm eggs of different voltinism strains,so whow about their DNA methylation levels? To answer this question,the progeny silkworm eggs of the above 6 strains were used as materials,and the DNA methylation levels was detected in the early embryonic development stage by immunofluorescence method.The results showed that during the period of 2-96 h post oviposition,difference the DNA methylation levels of progeny eggs were different among different voltinism strains of B.mori;and the DNA methylation level of Nistari was significantly higher than that of NH,which was consistent with the expression trend of Bm Dnmt in the progeny eggs of different voltinism strains above.These results suggest that DNA methylation is involved in the regulation of silkworm voltinism and diapause of offspring eggs.3.Transcriptional characteristics of Bm Dnmt and DNA methylation level in early embryonic development stage of progeny eggs with different destinies from bivoltine strainsThe above results show that DNA methylation is involved in the regulation of diapause in silkworm offspring,but the regulatory mechanism is still remained unclear.Here,the expression levels of Bm Dnmt in the non-diapause eggs(ND),the diapause destined eggs(DD)and the HCL acid-treated eggs(IA)of the bivoltine strains were analyzed.It showed that the expression level of Bm Dnmt in ND and IA was significantly higher than that in DD.Furthermore,immunofluorescence method was used for detecting the DNA methylation levels of eggs with different diapause fates.And the results revealed that their DNA methylation levels were of great differences,which was consistent with the expression trend of Bm Dnmt in silkworm eggs.It suggests that the incubation conditions during the parental embryonic stage and HCl acid treatment of offspring eggs affect the DNA methylation level,thereby participating in construction of diapause of the offspring eggs.4.Effects of knockdown of Bm Dnmt expression by RNAi in pupal stage on DNA methylation level and diapause-related gene expressionTo investigate how DNA methylation responds to changes in embryonic incubation conditions and thus participates in the regulation of diapause in silkworm offspring,the q RT-PCR method was used for analysis the expression of Bm Dnmt in QFHT group and QFLT group on day-2 to day-5 in pupal ovarian tissue.The results showed that the expression of Bm Dnmt in the two treatment groups showed a downward trend,and the overall expression level of the QFLT group was significantly higher than that of the QFHT group.Furthermore,RNAi technology was used to interfere the Bm Dnmt genes in day-2female pupae in QFLT and QFHT group,respectively.The results showed that the transcription levels of Bm Dnmt1 and Bm Dnmt2 were significantly decreased in both groups after Bm Dnmt interference,and the DNA methylation levels were also significantly decreased.After knockdown of Bm Dnmt1,FAR-1 and AKR were down-regulated;and DH and DHR-1 were not significantly affected in QFHT group,but in QFLT group,FAR-1 and DH were up-regulated,and AKR and DHR-1 were down-regulated.After knockdown of Bm Dnmt2,the expression results of FAR-1 and DH in the two treatment group were consistent with the results of knockdown of Bm Dnmt1,while the expression results of DHR-1 and AKR were inconsistent.It suggests that DNA methylation may involved in diapause regulation by regulating the expression of FAR-1,AKR,DH and DHR-1 in B.mori.In summary,DNA methylation modification is related to the diapause of silkworm progeny eggs.Different incubation conditions of bivoltine strain during the embryonic stage affect the level of DNA methylation modification,thereby further affecting the expression of diapause-related genes,resulting changes in diapause of silkworm offspring.The results laid a foundation for further explaining the diapause mechanism of silkworm from the epigenetic level of DNA methylation.