Study On The Relationship Between The Expression Level Of M~6A Modification Tool Enzyme In Silkworm And Its Resistance To BmNPV

Previous studies have shown that N6-methyladeosine(m~6A)modification plays an important role in the process of viral infection and replication.The silkworm(Bombyx mori)nuclear polyhedrosis virus disease is caused by Bombyx mori nuclear polyhedrosis virus(BmNPV)infection.There are two infection approaches of BmNPV,oral infection and traumatic infection.During oral infection,BmNPV first infects the midgut tissue of larvae.,Then is there any correlation between the m~6A modification level of total RNA in the silkworm and the resistance of silkworm strains to BmNPV?To answer this question,we used Bombyx mori cultured BmN cell line,larvae of common B.mori strain Qiufeng and BmNPV-resistant strain 898WNB as materials to study the expression level of m~6A modification tool enzyme genes after BmNPV infection,the effect on replication and proliferation of BmNPV,and the difference of m~6A modification level between resistant strainand commonstrain,respectively.The main results are as follows:1.Changes of gene expression levels of m~6A modification tool enzyme genes in BmN cells before and after BmNPV infectionBmN cells was infected with BmNPV,and Quantitative Real-time PCR(q RT-PCR)method was used to quantified changes in the transcription levels of m~6A modification tool enzyme genes METTL3,METTL14,Fl(2)d and YTHDC1 in BmN cells before and after infection.The results showed that at 24 hours after infection of BmNPV,the METTL3transcription level of BmN cells were increased significantly.2.Effect of RNA interference of METTL3 gene on BmNPV proliferation in BmN cellsRNA interference(RNAi)was performed on METTL3 in BmN cells by transfection of ds RNA of METTL3,and BmN cells were infected with BmNPV Subsequently.Changes in transcription levels of BmNPV nucleocapsid protein gene VP39 and capsule fusion protein gene GP64 were detected by q RT-PCR.The results showed that after RNAi of METTL3gene,the expression levels of VP39 and GP64 were significantly reduced,indicating that the reduction of METTL3 transcription level,namely the m~6A modification level of RNA,could inhibit the replication of intracellular BmNPV to a certain extent.3.Characterization of expression of m~6A modification tool enzyme genes in the midgut of Bombyx mori larvaeSilkworm strain Qiufeng was selected as the material,and q RT-PCR was used to quantified the transcription characteristics of m~6A modified tool enzyme genes METTL3,METTL14,Fl(2)d and YTHDC1 in the midgut tissues of silkworms in 72 hours after first feeding of 5th instar larvae.The changes of METTL3 protein at different times were detected by Western-bloting.The results showed that the transcription level and protein level of METTL3 in the midgut tissue of Qiufeng larvae were consistent.4.Corelation between the expression level of m~6A modification tool enzyme genes in the larval midgut of Bombyx mori and its resistance against BmNPVBmNPV resistant strain 898WNB was selected as the material and the common strain Qiufeng was servedas the control,and the 5th instar larvae were feed with BmNPV polyhedron(1×108 PIBS/m L)at the first feeding.Total RNAs of the midgut tissue were extracted and the expression levels of m~6A modification tool enzyme genes METTL3,METTL14,Fl(2)d and YTHDC1 before and after BmNPV infection were detectedby q RT-PCR.The results showed that after infection of BmNPV,in 898WNB larvae the METTL3 transcription level in the midgut tissue decreased,while in Qiufeng larvae the METTL3 transcription level in the midgut tissue of increased,indicating that there is a correlation between the METTL3 transcription level in thelarvalmidgut of B.mori and its resistance against BmNPV,i.e B.mori strains with strange restance against BmNPV has lower m~6A level.The above results are beneficial to elucidate the mechanism of resistant B.mori strains against BmNPV,and will proved a reference of breeding of B.mori.