The Differences Of Inhibiting BmNPV Proliferation In Silkworm And The Expression Differences Analysis Of BmTc25 In Response To BmNPV

The silkworm（Bombyx mori L.）,as an important economic insect,is one of the important economic sources in developing countries.Meanwhile,silkworm is also an important lepidopteran model insect,but silkworms are often endangered by different pathogenic microorganisms during the feeding process.Among the pathogens,Bombyx mori Nucleopolyhedrovirus（BmNPV）is the most serious pathogen and causes huge economic losses to the silk industry.The purpose of this thesis is to analyze the differences in BmNPV replication and proliferation and tissue differences in silkworms of different resistant strains,to study the resistance differences to BmNPV between two different resistant strains,and to clarify the physiological basis of the resistance strain;In this thesis,the candidate differentially expressed protein BmTc25 obtained from the proteome of silkworm digestive juice was identified and the expression pattern in response to BmNPVwas also analyzed.It provides a reference for further analysis of the resistance mechanism of silkworm against BmNPV.The main results are as follows:1.Analysis of virus proliferation after oral administration of BmNPV occlusion-derived virus（ODV）.The expression patterns of BmNPV virus early gene ie-1,gp41,envelope protein gene gp64,late gene vp39 as well as the the very late virus gene polh were analyzed in the midguts of different resistant strains P50 and A35 after BmNPV infection to study the replication and propagation of the virus.The results showed that the rate of virus propagation in resistant silkworm strain A35 was significantly lower than that of the susceptible strains.And the analysis of the virus copy number in the fat body has also obtained similar results to the midgut tissue.2.Analysis of virus proliferation after budded virus（BV）inoculation in the blood cavity.The expression patterns of BmNPV virus early gene ie-1,gp41,envelope protein gene gp64 and late gene vp39were analyzed in the midguts of different resistant strains P50 and A35 after BmNPV infection to study the replication and propagation of the virus.The results showed that the rate of virus propagation in resistant silkworm strain A35 was significantly lower than that of the susceptible strains.And the analysis of the virus copy number in the fat body has also obtained similar results to the midgut tissue.3.Comparative analysis of the virus proliferation in different resistant strains after ODV and BV infection.In order to further study the physiological basis of resistant strains resistant strains to BmNPV,we compared the expression pattern of the gene ie-1 in the midgut and fat body tissues in different resistant strains after ODV and BV infection,respectively.The results showed that the resistant strain A35 shows strong resistance to both ODV and BV infection,and the resistance to ODV significantly higher than BV.This result proved that the resistance of the resistant strain of silkworm A35 is largely derived from the midgut tissue.4.Identification and expression analysis of the candidate differentially expressed protein BmTc25 The candidate differentially expressed protein BmTc25 obtained from the silkworm digestive protein proteome was analyzed after the BmNPV infection,and the expression level of the resistant strain was higher than that of the sensitive strain,and by RT-q PCR Technical analysis of the expression of this gene in midgut tissue,the results are consistent with the proteome data;bioinformatics analysis of the protein found that BmTc25 protein has a trypsin domain,belonging to the serine protease family,evolutionary evolution and tobacco moth The close relationship of trypsin.The spatiotemporal expression profile of the BmTc25 showed that the gene was highly expressed in the midgut,and the expression level in the middle midgut was significantly higher than that of the anterior and posterior parts;The expression of this gene gradually increased,and its expression level during molting of 4^thinstar was significantly lower than the before molting of 4^thinstar and 1^stday of 5^thinstar.5.Prokaryotic expression of BmTc25,preparation of polyclonal antibody and analysis of expression of BmNPV in response to recombinant BmTc25 was successfully obtained by prokaryotic expression,and polyclonal antibody was prepared.Using RT-q PCR technology and Western blot method to analyze the expression of BmTc25 in susceptible strain P50 and resistant strain A35 after BmNPV infection,the results showed that compared with the control group,the sensitive strain and resistant strain were After BmNPV infection,BmTc25 protein was up-regulated in midgut digestive juice and midgut tissues,and the expression level of BmTc25 in resistant strain A35 was significantly higher than that in susceptible strain P50.