Effects Of RNAi On The Proliferation Of BmNPV By Ie1, Gp64 And Ie0 Genes

Bombyx mori is a kind of Lepidoptera insect with important economic value.There are many kinds of pathogens causing silkworm disease.Bombyx mori polyhedrosis virus?BmNPV?is one of the most serious diseases.The traditional method of disinfection using chemical agents to prevent silkworm disease has great limitations,cultivating resistant varieties has become an effective way to prevent silkworm disease.In recent years,many researchers have made a lot of research on the cultivation of silkworm resistant varieties,but the conventional methods of cultivating resistant varieties have shortcomings such as long cycle and low efficiency.RNAi is a common phenomenon in organisms and has been widely used in insect control.RN Ai-binding transgenic technology is expected to improve the resistance of Bombyx mori to BmNPV.The study showed that ie1,gp64 and ie0 genes play an important role in the proliferation of BmNPV.In this study,the 1-545 bp of the ie1 gene,the 1-507 bp of the gp64 gene and the 1-525 bp region of the ie0 gene were ligated in reverse tandem to construct transfer vectors pF-A3-die1-IM?pF-A3-dgp64-IM?pU-A3-die0-IG and pF-A3-die1-dgp64-IM,which are capable of stable transcription of ds RNA in cells,Construction of recombinant strain rSw106-Bm-pF-A3-IM?rSw106-Bm-pF-A3-die1-IM?rSw106-Bm-pF-A3-dgp64-IM?rSw106-Bm-pF-dA3-die1-dgp64-IM-pU-A3-die0-IG?rSw106-Bm-pF-dA3-die1-dgp64-IM?rSw106-Bm-pF-A3-die1-IM-pU-A3-die0-IG?rSw106-Bm-pU-A3-die0-IG and rSw106-Bm-pF-A3-dgp64-IM-pU-A3-die0-IG by using Invasin-mediated DAP auxotrophic E.coli BmMultiBac/?asd Sw106/PGB2?Inv strain.BmN cells were infected with recombinant strains and recombinant baculoviruses BmBV-pF-A3-IM?BmBV-pF-A3-die1-IM?BmBV-pF-A3-dgp64-IM?BmBV-pU-A3-die0-IG?BmBV-pF-A3-die1-dgp64-IM?BmBV-pF-A3-die1-IM-pU-A3-die0-IG?BmBV-pF-A3-dgp64-IM-pU-A3-die0-IG and BmBV-pF-A3-die1-dgp64-IM-pU-A3-die0-IG were obtained by using baculovirus multi-gene expression system.The recombinant baculovirus titer was determined by TCID50 method,The recombinant virus was d iluted 10¹,10² and10³ times and injected 5^{t h} instar 1 day silkworm larvae,24 h observed once and recorded the results.The results showed that the results showed that the time of onset and death of the larvae of the experimental group were delayed by 24 h after injection of the recombinant virus at the concentration of 10¹ dilution.The mortality of silkworm larvae after injection of BmBV-pF-A3-dgp64-IM was 10%lower than the positive control group.The time of onset and death of the larvae of the experimental group were delayed by24 h after injection of the recombinant virus at the concentration of 10² dilution.The morbidity of silkworm larvae after injection of BmBV-pF-A3-die1-IM-pU-A3-die0-IG was10%lower than the positive control group,the mortality of silkworm larvae after injection of BmBV-Sw106-pF-A3-dgp64-IM-pU-A3-die0-IG?BmBV-Sw106-pF-A3-die1-IM?and BmBV-Sw106-pF-A3-die1-IM-pU-A3-die0-IG was 10%lower than the positive control group,the mortality of silkworm larvae after injection of BmBV-pF-A3-dgp64-IM was 20%lower than the positive control group.After injection of 10³ dilution concentrations of recombinant virus BmBV-Sw106-pF-A3-die1-IM-pU-A3-die0-IG?BmBV-Sw106-pF-A3-die1-dgp64-IM?BmBV-Sw106-pF-A3-dgp64-IM-pU-A3-die0-IG?BmBV-Sw106-pF-A3-dgp64-IM and BmBV-Sw106-pF-A3-die1-dgp64-IM-pU-A3-die0-IG,silkworm larvae began to develop symptoms on the 5th day,the morbidity reached 70%?66.67%?60%and 50%respectively on the 10th day,the morbidity were reduced by 10%,20%,22.22%,30%and 50%respectively compared with the positive control group.After injection of 10³ dilution concentrations of recombinant virus BmBV-Sw106-pF-A3-die1-dgp64-IM-pU-A3-die0-IG?BmBV-Sw106-pF-A3-dgp64-IM-pU-A3-die0-IG?BmBV-Sw106-pF-A3-die1-dgp64-IM and BmBV-Sw106-pF-A3-dgp64-IM,silkworm larvae began to die on the 8th day,the mortality reached 90%,80%,77.78%,70%and 50%respectively on the 10th day,the mortality were reduced by 20%?22.23%?30%and 40%respectively compared with the positive control group.In summary,the use of RNAi technology to interfere ie1,gp64 and ie0 genes on BmNPV proliferation replication has played a significant inhibitory effect,which provided a theoretical basis for the cultivation of transgenic silkworm with BmNPV resis tance combined with transgenic technology.