Antifungal Effect Of Bombyx Mori Apolipophorin-Ⅲ On Beauveria Bassiana

Insect Apolipophorin-Ⅲ is a multifunctional protein,which not only has important physiological functions but also plays an important role in insect innate immunity.Our previous transcriptome and proteome studies indicated that the gene expression level of Bombyx mori Apolipophorin-Ⅲ(BmApoLp-Ⅲ)protein in silkworm larvae infected with Beauveria bassiana was significantly up-regulated.In this study,tissue-specific expression of BmApoLp-Ⅲ gene was analyzed by q RT-PCR detection,the ORF of BmApoLp-Ⅲ gene was cloned and the BmApoLp-Ⅲ protein was successfully expressed with prokaryotic expression system.Then the antifungal effect of the expressed BmApoLp-Ⅲ protein on B.bassiana was verified in vitro and in vivo.Furthermore,its influence on the onset and death of the larvae infected with B.bassiana was evaluated by overexpression and silencing of BmApoLp-Ⅲ gene,and at the same time,the expression changes of genes related with the immune signal pathways and the antimicrobial peptide gene BmCecropin A(BmCec A)in silkworm were detected at the transcript level.The main results are as follows:1.Tissue-specific expression patterns of BmApoLp-Ⅲ gene in silkworm larvae.The expression of BmApoLp-Ⅲ gene could be detected with q RT-PCR in the tissues including head,cuticle,hemolymph,midgut,silk gland,gonad,Malpighian tube and fat body in the silkworm larvae infected with B.bassiana,but with the highest expression in fat body,then in hemolymph,while the lowest in the silk gland.2.BmApoLp-Ⅲ protein was successfully expressed by prokaryotic expression system.The ORF of BmApoLp-Ⅲ was cloned from the fat body c DNA to construct the prokaryotic expression vector p ET-28a(+)-BmApoLp-Ⅲ.After transforming the expression vector to BL21 competent cells,the recombinant BmApoLp-Ⅲ protein was successfully expressed under the induction of IPTG.The expressed BmApoLp-Ⅲ protein was purified and the concentration was 6.4 μg/μL as determined using the BCA protein concentration determination kit.3.BmApoLp-Ⅲ has an antibacterial effect on B.bassiana.The fungistatic zone test showed that the recombinant BmApoLp-Ⅲ protein has a significant antifungal effect on B.bassiana.Injecting the purified BmApoLp-Ⅲ protein to the larvae inhibited the reproduction of B.bassiana and consequently delayed the onset and death of the infected larvae.Conversely,silencing BmApoLp-Ⅲ gene by RNAi resulted in an earlier morbidity and an earlier half-lethal time of the infected larvae.These further confirmed the antifungal activity of BmApoLp-Ⅲ protein on B.bassiana.4.BmApoLp-Ⅲ could regulate the expression of genes related with immune pathways in the silkworm.The fifth-instar larvae infected by B.bassiana were injected with BmApoLp-Ⅲprotein and the si RNA targeting BmApoLp-Ⅲ respectively.The expression changes of the key genes involved in the Toll,Imd,Jak/STAT signaling pathways,the genes of serine proteinase inhibitor and melanization and the antimicrobial peptide BmCec A gene were detected.The results indicated that injecting BmApoLp-Ⅲ protein up-regulated the expression of genes including BmβGRP4 and BmMyd88 in the Toll signal pathway,BmCTL5 and BmHOP in the Jak/STAT signaling pathway,serine proteinase inhibitor BmSerpin5,and antimicrobial peptide BmCec A,but down-regulated the expression of BmTak1 and BmRelish 2 in Imd signaling pathway and BmBAEE in melanization;while silencing BmApoLp-Ⅲ gene by RNAi down-regulated the expression of BmβGRP1 and BmSpaetzle,BmCTL5 and BmHOP,BmSerpin2 and BmSerpin5 of relevant pathways and BmCec A,but up-regulates the expression of BmPGRP-Lc,BmTak1 and BmRelish 2 in Imd pathway,serine protease inhibitor and melanin BmBAEE and BmPPO2.In summary,the above results indicate that the BmApoLp-Ⅲ could not only directly inhibit B.bassiana,but also participate in regulation of the expression of genes related with immune signal pathways,promote the expression of immune effect factors,and indirectly inhibit the reproduction of B.bassiana in the silkworm.The results of the study are helpful to understand the molecular defense mechanism of the silkworm against B.bassiana infection and may provide a useful reference for the development of effective measures for prevention and control of silkworm muscardine disease.