The Antimicrobial Peptide Genes Of Hermetia Illucens Enhance Resistance Of Transgenic Silkworm To Bacterial Diseases

The silkworm,Bombyx mori,is a lepidopteran model insect with great economic value in sericulture.Each year the sericulture industry faces great economic loss due to the silkworm diseases caused by entomopathogenic infection.Developing resistance strain with better economical property always takes an important part in sericulture researches.However,the long breeding time and lackage of natural resistance genes are the disadvantages of traditional silkworm breeding.The Black Soldier Fly(BSF),Hermetia illucens,often exposed to high dosage of entomopathogens duing the convertion of biowaste,exhibits high resistance to those pathogens.It is a promising way to dig the immune resources of BSF and combines it with modern molecular technology for silkworm breeding.In present study,we explored the function of three antimicrobial peptide(AMP)genes cloned from BSF immune pathway regulation in vivo and the functional study of those proteins in vitro,then the silkworm that can endogenously express three AMPs were constructed by using silkworm transgenic technique to improve the entomopathogen resistance.The main results were as follows:1.The function research of black soldier fly AMP genes during pathogen infectionHidefensin-1,Hi CG13551 and Hidiptericin-1 gene was respectively cloned at first,then we proved that the expression of Hidefensin-1 would increase upon B.bombyseptieus infection whereas Hidiptericin-1 would increase upon S.marcescens infection.The expression level of Hi CG13551 was not significantly changed upon both pathogen infection.The RNA interference(RNAi)was done on three AMP genes respectively,the IMD pathway related genes and Hidiptericin-1 were singnificantly downregulated either Hi CG13551 or Hidiptericin-1 was knockdown while Hidefensin-1 knockdown would upregulate the expression level of IMD pathway and Hidiptericin-1 gene.Toll pathway would be downregulated when Hidefensin-1 and Hi CG13551 gene were silenced.Among those three AMP gene downregulations,only the resistance of Hi CG13551 RNAi black soldier fly was significantly decreased upon two pathogen infection.2.BSF AMP genes in vitro expression,function identification and antibacterial mechanism initial exploration.The Hidefensin-1,Hi CG13551 and Hidiptericin-1 sequences were aligned with the database and all three AMP genes got conserved domain that closely related to antimicrobial function.Then the prokaryotic expression and purification were established on these three AMPs and bioassay was tested on E.coli TG1,S.aureus,S.pneumoniae,B.bombyseptieus and S.marcescens.All three proteins got satisfying inhibition effect on E.coli but exhibit no significant inhibition on B.bombyseptieus.Hidefensin-1 and Hi CG13551 could inhibit the growth of S.pneumoniae and S.aureus,respectively.The Hidiptericin-1 showed significant inhibition on S.pneumoniae and S.aureus and Beauveria bassiana,proved that it got potential to be broad spectrum bactericide.Through the morphological obsertvation,the working mechanism of Hidiptericin-1 was disruption of cell memberane of E.coli and S.aureus.3.Construction of Hermetia illucens AMP gene endogenous expression silkwormThe transgenic vector which express AMP cassette driven by IE1 promotor was constructed at first,then the AMP transcription was successfully detected after transfection of the vector.Then after newly oviposited preblastoderm embryos were prepared and microinjected with DNA solution containing transgenic vector and the helper plasmids,the transgenic silkworm line was successful obtained.Three AMP protein could be expressed and detected independently.The fertility and development time were not affected by transgenic oversxpression of AMP genes,furthermore,the cocoon weight and pupae weight were increased compared to Nistari silkworm.4.The entomopathogen resistance evaluation of transgenic silkworm lineThe transgenic silkworm line showed higher resistance on Gram-positive S.aureus,B.bombyseptieus and Gram-negative S.marcescens pathogens compared to Nistari silkworm.Among them,the B.bombyseptieus showed resistance to three AMPs in vitro,this phenomenon indicated that there exists synergistic effect between AMPs in vivo.In conclusion,the present study confirmed three Hermetia illucens AMPs exhibit antibacterial activity in vitro and it is the first time to express heterogeneous AMPs in transgenic silkworm.The transgenic silkworm line was constructed to express the three Hermetia illucens AMPs constitutively and proved that the heterologous expression of BSF AMPs could protect the silkworm from bacterial pathogen infection.Our findings present new approaches for utilization of AMP resources from BSF and shed light on a new strategy in silkworm disease resistant breeding.