| BmNPV(Bombyx mori nucleopolyhedrovirus)is very harmful to silkworm,causing huge economic losses to sericulture industry.At present,the infection of BmNPV is unpreventable and difficult to control,mainly because the molecular mechanism of BmNPV infection is still unclear.Therefore,it is very important to elucidate the infection mechanism of BmNPV and the antiviral mechanism of silkworm for sericulture science and production.Current researches on antiviral mechanisms mainly focus on gene and protein levels,and a series of important advances have been made.However,the mechanism of BmNPV infection and the antiviral mechanism of Bombyx mori are still unclear.Mammalian-based studies have demonstrated that viruses alter host metabolic pathways and rely on these changes to achieve optimal viral replication.Determining which key metabolites,as well as key enzymes involved in metabolic pathways,that are significantly altered during viral invasion is crucial to unravel how the virus uses the cellular metabolic environment to replicate and proliferate.Currently,there are few reports on how BmNPV regulates and changes the metabolic pathways of silkworm,therefore,this work aims to unveil how BmNPV regulates the host metabolic pathways,including those of lipids and other small molecules,and the mechanisms behind the regulation.Key lipids and small molecule metabolite markers involved in BmNPV infection and host antiviral mechanism will be screened and analyzed,and the key enzymes associated with the metabolic pathways of these markers will be investigated,which will lay a foundation for elucidating the mechanism of silkworm resistance to BmNPV infection.The main findings are as follows:1.The lipid profiles of the BmNPV susceptible strain 306 and resistant strain NB were analyzed by liquid chromatography mass spectrometry.A total of 905 lipids have been identified in silkworm,most of them belong to glycerolipids,fatty acids,sphingolipids and sterol lipids.The contents of sterols,phosphatidylinositols,phosphatidylserines and spingolipids in silkworm were significantly lower than those in typical mammals.Lipid profiles were compared between the BmNPV-susceptible and resistant strains,and it was found that the contents of lipids such as phosphosphingolipids,diacylglycerols,ceramides and quinones were significantly increased in the susceptible strain 306.Fatty acyl chain length and degree of saturation were analyzed and compared between the two silkworms and after virus inoculation,and no specific trend associated with virus treatment was observed.2.The lipidomes of the two silkworms and their corresponding BmNPV treatment groups were compared at the individual lipid level,and 288 lipids were significantly different between the susceptible strain 306 and resistant strain NB,while only 36 and 77 significant lipids were found between the silkworms and the corresponding virus treated groups,respectively.This indicates that the lipid profiles between the two silkworm strains are much more different than those caused by the virus treatment.After the susceptible strain 306 was infected by BmNPV,the whole diacylglycerol levels decreased,while the whole triacylglycerol levels increased,indicating that BmNPV infection altered the glycerolipid biosynthesis pathway.After the resistant strain NB was treated by BmNPV,the sphingolipids including glucosylceramide(Glc-Cer)and phosphatidylethanolamine ceramide(PE-Cer)were reduced,indicating that glycosphingolipids and sphingomyelin play a certain role in the host’s immune response against the virus.Real-time fluorescent quantitative PCR was used to analyze the expression of key enzyme genes in the susceptible strain 306 and resistant strain NB lipid synthesis pathway before and after virus treatment,and it was found that the three genes 1-acyl-sn-glycerol-3-phosphate acyltransferase 5(AGPAT5),sphingomyelin synthase 1(SGMS1)and ceramide synthetase 3(CERS3),which were involved in the lipid metabolic pathway had significantly different transcription levels between the susceptible and resistant strains of the silkworms.Subsequently,RNA interference technology was used to knock down the expression of key enzyme genes CERS3 and AGPAT5.The CERS3 gene reduced expression was conducive to the infection and proliferation of BmNPV.3.In order to further analyze the mechanism of BmNPV regulating host metabolic pathway,we analyzed the levels of small metabolites in the silkworm hemolymph before and at different time points after virus inoculation.The results showed that the abundance of most amino acids in susceptible strain 306,such as tyrosine,phenylalanine,tryptophan and leucine,decreased significantly with the increase of infection time,but in resistant strain NB,the abundance of most amino acids increased or remained basically unchanged.In addition,trehalose levels of virustreated 306 and NB groups were all higher than control group,but with the increase of infection time,trehalose levels showed a downward trend,and were always higher in the virus-treated NB group than those in the 306 virus-treated group.Real-time fluorescence quantitative PCR was used to analyze the expression levels of key enzymes in the pathways involved in these metabolic markers for small metabolic markers with significant changing trends.The results showed that the expression levels of genes related to the metabolic pathways of tyrosine,glutamine and trehalose increased hundreds of times after 120 h of virus innoculation in the susceptible strain 306.These results suggest that the virus can significantly alter the metabolic pathways in the susceptible strain306 to facilitate viral replication.However,resistant strain NB can rely on metabolic pathways such as glycolysis and TCA cycle to produce enough energy to resist virus infection,so as to maintain its metabolic balance and normal life activities. |
PDF Full Text Request