Study On The Anti-BmNPV Mechanism And Key Enzymes Involved In Biosynthesis Pathway Of Moracin N In Mulberry Leaves Under UV-B Induction

Silkworm,Bombyx mori L.,is a Lepidoptera,bombycidae insect.Bombyx mori nucleopolyhedrovirus(BmNPV)is one of the most acute infectious diseases in silkworm,which has caused great economic loss in sericulture.Currently,the specific mechanism for BmNPV-resistance is unknown.Silkworms feed on mulberry leaves,which are also important medicinal plants.Previous studies have shown that the content of secondary metabolites in mulberry leaves was altered following UV-B irradiation.Specifically,moracin N level was increased.This is the first study which shows that UV-B treated mulberry leaves have antiviral effects against BmNPV,and moracin N maybe the the active ingredient.Label-free proteomics analysis provided insights towards understanding of the anti-BmNPV mechanism of UV-B treated mulberry leaves and moracin N.The discovery of new approaches to control BmNPV infection in silkworm promotes the development of a treatment for Bombyx mori.Meanwhile,the key enzymes involved in biosynthesis pathways of moracin N are discussed in this paper.The results show that a new oxidative cyclization enzyme in UV-B treated mulberry leaves can catalysis oxyresveratrol for moracin M,which is the important precursor of moracin N.It provides the basis for this biosynthesis of secondary metabolites respond to ultraviolet-b radiation in mulberry leaves.The main contents and results of this paper are as follows:(1)Study on the antr-BmNPV mechanism of moracin N in mulberry leaves under UV-B inductionBmNPV is one of the most acute viruses.Silkworms infected with BmNPV,often outflow pus,containing a large number of polygonal body virus,which is known as septic disease.The disease has caused great economic loss.In anti-virus experiments,we show that silkworms fed with UV-B treated mulberry leaves and moracin N are less likely to die following BmNPV infection.To uncover the mechanism of enhanced BmNPV resistance,silkworm midguts from UV-B treated mulberry leaves(BUM)and moracin N(BNM)groups were analyzed by proteomic technique.The results showed that 413 proteins and 102 proteins from mid guts isolated from the BUM and BNM group were identified as differentially expressed proteins respectively.The results of bioinformatics analysis of the differentially expressed proteins show that UV-B treated mulberry leaves and moracin N can resist BmNPV by some ways.Some changed ribosomal proteins can control the total protein synthesis.Up-regulated V-ATPase and down-regulated ATP synthase can affect the intercellular environment,stopping virus uncoating or entrancing into cell nucleus.Cytochrome c oxidase,Ca2+related to protein and programmed cell death protein were up-regulated,which can promote apoptosis and inhibit viral replication.Lipase-1 and serine protease precursor were up-regulated,which can improve the body immunity against BmNPV.Rab protein and histone play special roles in the process of anti-BmNPV The anti-BmNPV process of silkworms reared on UV-B treated mulberry leaves and moracin N provid insight into the mechanism of the defense response towards BmNPV,which also provide a new treatment for BmNPV.(2)Study on key enzymes involved in biosynthesis pathway of moracin N in mulberry leaves under UV-B inductionMorus alba L.is Moraceae morus,deciduous trees or shrubsis,is also an impoant medicinal plant It has a variety of pharmacological effects,and is the main source of food for silkworms.Moracin is one important secondary metabolite of mulberry leaf,which has anti-tumor,anti-inflammatory,antiviral activity,etc.The content of secondary metabolites in mulberry leaves,particularly for moracin N,was increased after UV-B irradiation At present,the chemical synthesis of morusin N has been reported,but there is no detailed report about its biosynthesis pathway.According to previous experimental results,the important precursor of moracin N was moracin M.This is the first study to explore a new oxidative cyclization enzyme,and oxyresveratrol is the important substrate of the enzyme,which can be catalysised for moracin M.The enzyme reactions show that the enzyme activity of mulberry leaves under UV-B induction or darkness was increased.To optimize the extraction condition of oxidative cyclization enzyme,the single factor experiments and orthogonal experiments were designed.The results show that the condition with the specific activity of enzyme highest are as follows:pH of extracting so lution is 7.5,the ratio of solid to liquid(g/mL)is 1:10,the extract concentration is 100 mM,the concentration of Na2-EDTA is 10 mM,the extracting time is 2 h,the reaction temperature is 30℃.This laid a foundation for the separation and purification of oxidative cyclization enzyme which from mulberry leaf.