The Changes Of Peritrophic Membrane Proteins Of Silkworm Larvae, Bombyx Mori After BmNPV Infection

Peritrophic matrix （PM） is a kind of acellular and transparent of permeable net-membrane structure which is secreted by the midgut cells of the invertebrates to adapt their physiological activity in the long evolutionary process and throughouts the midgut.PM is protein polysaccharide matrix which is mainly comprised of chitin, protein and carbohydrate.It exists between the food and the intestinal epithelial cells, playing an important role in the digestion and absorption of nutrients and protecting midgut epithelial cells from abrasive food particles. In addition, PM is also the first effective physical barrier to protect midgut epithelial cells from pathogenic microorganisms and toxins intaked with food. Bombyx mori nuclear polyhedrosis disease is one of the major diseases of the silk industry caused by BmNPV（Bombyx mori nuclear polyhedrin osis virus, BmNPV）.The disease caused huge economic losses of sericulture. Although there are more reports about BmNPV infecting silkworm cell,the systems analysis for BmNPV as a representative virus interacting with Bombyx mori in the individual level has not been elucidated, especially the study of viruses interacting with PM is still quite limited. Particularly PM proteins of Bombyx mori interacting with BmNPV have little knowledge. Based on this we research on the change of protein components of PM silkworm larval midgut after BmNPV infection, which can provide experimental data for the study that Bombyx mori react with BmNPV infection and complement theoretical knowledge for the study of the structure and function insect peritrophic membrane, but also provide a theoretical basis to identify the new potential and biological control targets of pest and develop more effective biological insecticide agents.In this paper, SDS-PAGE,biochemical assays,bioinformatics and quantitative PCR technology was used to analyze types and quantity of PM proteins and sugar,.The change of PM proteins after baculovirus infection was detected and analyzed.In order to get the exact information of host PM proteins in response to viral infection at different times,we used two-dimensional electrophoresis to identify and separate PM proteins of Bombyx mori PM proteins. We hope to find the detail informations of the host PM proteins in response to virus infection at different times.The main contents and results are as follows:1. Biological test results shows that;4th silkworm larvae were injected10μl three concentrations of BV particles （1×105,1×106and1×107）, we statistics consecutive mortality in four days, the average mortality rate of Bombyx mori was3.33%,6.67%,35.56%at the third days, the average mortality rate of Bombyx mori was100%at fourth days. It shows mortality relating wtih the infection dose of virus and the lethal dose is much less than oral infections.the lethal time is longer than oral infections two days in advance.it further confirmed that midgut tissue and peritrophic membrane play an important role in the process of virus infection.2. The protein and total sugar content of Bombyx mori PM proteins:SDS-PAGE result shows that total PM proteins are at least19in SDS-PAGE. The proteins are located in the11-245KDa.Most are medium and low molecular-weight proteins which are below100KDa.Content of total PM proteins are about41.04%,alkali-soluble proteins are23.46%,acidic proteins are17.61%.The sugar content of PM is only0.156%. Showing Peritrophic membrane protein content, type and molecular weight range are similar to other lepidopteran insects, but the sugar content is much lower than other insects, the significance of which is currently unknown.3. Detecting changes of PM proteins of Bombyx mori larvae after BmNPV infection.3.1SDS-PAGE suggests that6h,8h,12h and48h after feeding with BmNPV there have no significant difference in PM proteins.After4h,We found the content of proteins are decreased in the molecular weight about30KDa and175KDa. After infection24h and72h,the quantity of proteins at the58KDa are significantly decreased, showing PM proteins of Bombyx mori were significantly changed after BmNPV infection, and the change time of PM proteins consistent with the time that the change of PM surface structure after virus infection.3.2The strips of decline of the protein are cutted for mass spectrometry,the results is as follows:The quantity of seven PM proteins are decline after4h BmNPV infection,these are Aminopeptidase （BM₀8059）,serine protease （BM₁4428, BM12791, BM10024, BM14429）, chitin-binding protein2（BM01491） and the unknown sequence （XP004925459.1|）; The quantity of etight PM proteins are decline after24h BmNPV infection including the unknown protein （XP004923455.1）,3-1,3-glucan recognition protein precursor4（NP₀01159614.1）, chymotrypsin Bl-like BI （XP004928997.1）, transmembrane serine protease9（XP004925969.1）, alkaline phosphatase （BAG41975.1）,alpha-amylase4N-like （XP004933429.1）, serine protease3-like （XP004928982.1）, mucin-5AC-like （XP₀04931813.1）.The quantity often PM proteins are decline after72h BmNPV infection including insect midgut lipase6（BM₀2669）, chitin deacetylase （BMJ3756, BM13757）, serine protease （BM₁2777, BM08280, BM BM₀8818）, carboxypeptidase （BM₀4797, BM04801）.Protease inhibitors （BM01983）, chitin-binding protein （BM01504）.4. Changes of gene expression of Peritrophic membrane proteins after BmNPV infection.4.1The analysis about genes of the PM proteins after BmNPV infection by microarray. Tissue microarray analysis shows:Except that the gene expression levels of carboxypeptidase （BM.04797and BM.04801） was lower in the midgut, other genes were expressed at high levels in the midgut. Microarray analysis of different developmental stages displays that only the gene of serine protease （BM-08280） have high expression levels at the nest, the gene of other proteins are expressed during the5th instar of Bombyx mori feeding a lot of food. Suggests that degraded proteins that obtained in this experiment is mainly express in midgut and participate in intestinal digestion and resist microorganisms.4.2Quantitative PCR detect the changes of gene expression levels between the infected BmNPV group and control group:4h after BmNPV infection, the gene expression level of serine proteases （BM.14428） was significantly upregulated, while the gene expression level of serine protease （BM-12791） and aminopeptidase （BM-08059） were significantly downregulated.the expression level of trypsin （BM-10024） was no significant difference.72h after BmNPV infection,The gene expression levels of deacetylase （BM.13757） and serine protease （BM.12777） were no significant change.while mRNA expression levels of chitin deacetylase （BM.13756）, chitin-binding protein（CBP）（BM.01504） and metalcarboxypeptidaseA （BM.04797） were significantly Downregulated. We speculate that after infected4hours,downregulation of serine protease gene （Bm-12791） and aminopeptidase gene （Bm-08059） may be associated with virus infection; after infected72hours, downregulation of chitin-binding protein gene （Bm-01504）, chitin deacetylase gene （Bm-13756） and metal carboxypeptidase gene （Bm-04797） may be have a function to reduce resistance of host, promoting the effect of viral infection, and after infected72hours, the expression levels of serine protease gene （Bm-14428） was regulated which may inhibit the virus to infect host.The experimental show that the changes of the host PM proteins after virus infection at different times.The results lay the foundation to explane the defense order and mechanism of silkworm PM proteins in response to virus infection, and will be helpful to finally found the key antiviral factors in the host. Meanwhile, the results show that different member among the serine protease superfamily and deacetylase family may has diverse function after the host was infected by virus.