Identification Of Resistance Of Bombyx Mori To Nuclear Polyhedrosis Virus

Bombyx mori is a typical representative of Lepidopteran insects and one of the important model insects.It has abundant variety resources,breeding lines and mutants,and is an excellent material for molecular biology research.At the same time,silkworm is a silk-secreting insect with high economic value and occupies a position in agricultural production of China.However,the annual production of sericulture is affected by a variety of diseases,among which the silkworm pyosis caused by the nuclear polyhedrosis virus BmNPV has caused great losses to the sericulture.Therefore,it is extremely important to study the resistance to BmNPV.A highly resistant strain NB was found in 344 silkworm varieties,and its resistance is 1000 times higher than that of susceptible strain 306.This is the first highly resistant silkworm variety resource identified in the world,but it has not been systematically investigated.In this study,we systematically identified the antiviral properties of NB from molecular biology,pathology,transcription level and protein level.The main results are as follows:?1?Antiviral identification of different silkworm strainsThe survival rate,mortality rate,growth and color change of hemolymph of resistant strain NB,susceptible strain 306 and near isogenic strain BC₈ were counted and compared by adding 5?L BmNPV with a concentration of 1x10⁸/mL or puncturing 2?L 1x10⁸/mL BmNPV solution,respectively.The results showed that the survival rate of resistant strain NB was 100%;all susceptible strain 306 died,and the survival rate was 0%.The survival rate of near-isogenic strain BC₈ was also 100%,indicating that NB and near-isogenic strain BC₈ were highly resistant to BmNPV.?2?Pathological identification of resistant strain NB and susceptible strain 306EGFP-BmNPV was fed or punctured into the fifth instar silkworms as described above.At 24 h,48 h,72 h,96 h,and 120 h after the addition of EGFP-BmNPV,the silkworm body,midgut,silk gland,haemolymph,fat body and sperm-ovary of resistant strain NB and susceptible strain 306 were collected respectively to observe the fluorescence through stereomicroscope with fluorescence,fluorescence microscope and typhoon scanner.The proliferation of virus particles and the formation of polyhedron in hemolymph were observed under microscope,and the changes of blood cells were observed under phase contrast microscope.The results showed that there was no fluorescence in all tissues and at time points of resistant strain NB and no polyhedron formation in blood cells,while fluorescence appeared in the silkworm body and the tissue of susceptible strain 306 at different time periods.Meanwhile,with the passage of time,the fluorescence in susceptible strain 306 became stronger and stronger.At 96h,a large number of polyhedrons could be observed in blood cells?without normal cell morphology?.According to the above results,it can be inferred that the virus did not proliferate in NB,but rapidly proliferated in 306 and resulted in the death of 306.?3?Resistance identification from transcriptional level to BmNPV in resistant strain NB and susceptible strain 306After the same treatment as described above,the midgut,silk gland and haemolymph of the fifth instar silkworm were collected at 0 h,24 h,48 h,72 h,and 96 h.The specific sequences of the early gene ie-1 and the late gene vp39 of BmNPV were used as primers,and the relative expression levels of the viral genes in different strains were analyzed by semi-quantitative PCR and real-time PCR?internal parameter:?-tubulin?.The results showed that the early and late genes of BmNPV in NB almost were not replicated,but proliferated in 306,thereby confirming the resistance of NB to BmNPV at the transcriptional level.?4?Resistance identification from protein level to BmNPV in resistant strain NB and susceptible strain 306The expression of viral proteins in different tissues and at different time periods was studied at the protein level by Western blot.The results showed that the resistant strain NB did not express the viral proteins IE1 and VP39 in the midgut,silk gland and hemolymph tissues,while the susceptible strain 306 expressed the viral proteins IE1and VP39 in different tissues and at different time periods,which confirmed the resistance of NB to BmNPV at the protein level.To sum up,the results showed that silkworm NB was highly resistant to BmNPV.At the same time,it was found that the infection mode of susceptible strain 306 was different from resistant strain NB,and the time of death,the site of invasion and the route of invasion were also different.