The Role Of Bombyx Mori Storage Protein Genes Bm30K-19G1 And BmSP1 In Resisting The Infection Of Beauveria Bassiana

Silkworm(Bombyx mori)is not only an important economic insect,but also a significant model insect,playing an essential role in the scientific research of insect physiology,development,genetics and immune regulation.In the production of sericulture,the invasion of pathogenic microorganisms often causes great losses to the sericulture industry.Among them,Beauveria bassiana has become othe most serious pathogenic fungus to the silkworm because of its strong pathogenicity,easy transmission and wide host range.In our previous studies,some differentially expressed genes were identified in the B.bassiana infected silkworm larvae by digital gene expression profiling.It was found that the expression level of the storage protein(SP)gene of silkworm was significantly up-regulated after infection with B.bassiana.Therefore,it is considered that the storage proteins play anecessary physiological,biochemical role in the process of infection of B.bassiana and a certain antifungal effect.In this study,silkworm storage protein Bm30K-19G1 was successfully expressed to study its inhibitory effectson B.bassiana spores and hyphae in vitro and in vivo.At the same time,the silkworm storage protein BmSP1 was expressed by the baculovirus expression system to study its antifungal effect and inhibitory effect on haemolymph melanination and to identifiy its interacting proteins.The main results obtained are as follows: 1.Silkworm storage protein Bm30K-19G1 has a certain antifungal effects on Beauveria bassianaBombyx mori storage protein Bm30K-19G1 is a low molecular weight apolipoprotein that is abundant in the hemolymph of fifth instar larvae.In this study,the ORF sequence of Bm30K-19G1 was obtained by PCR amplification,which was 1311 bp in length and encodes a 436 amino acids peptide.Real-time quantitative PCR showed that Bm30K-19G1 was expressed in all the tested tissues of fifth instar larvae,with highest expression in fat body and the lowest expression in the midgut.Bm30K-19G1 protein was successfully expressed in the prokaryotic expression system using pET-28a(+)as vector and E.coli Arctic Express(DE3)as the expression bacterium strain.The expressed recombinant Bm30K-19G1 protein has an inhibitory effect on the conidial germination and hyphal growth of B.bassiana.Bm30K-19G1 also inhibited the growth and reproduction of B.bassiana in vivo,resulted in the postponed median lethal time of infected silkworms by 6.4 h and the postponed time for death of all infected larvae by 10 h.These results indicated that the silkworm storage protein 30K-19G1 is not only a storage protein but also an antifungal protein,which has a certain inhibitory effect on the growth and reproduction of B.bassiana.2.Expressional Analysis of Silkworm Storage Protein SP1 and Identification of its Interacting ProteinsThe expression profile of BmSP1 in silkworm larvae was detected by qRT-PCR.The results showed that the expression of BmSP1 in silkworm was gradually increased with the advance of silkworm age,and the expression level was the highest in fat body.The BmSP1 fusion protein was successfully expressed with silkworm baculovirus expression system to study its functions.Antifungal activity of the expressed BmSP1 fusion protein was not detected against B.bassiana in the fungistatic zone experiment.While,the overexpression of the recombinant protein BmSP1 in silkworm larvae showed strong ability of inhibiting melanization of hemolymph.In order to further elucidate the role of up-regulated expression of BmSP1 gene in the B.bassiana infected silkworm,co-immunoprecipitation technique was used to screen the proteins interacting with BmSP1 with EGFP as an antigenic determinant,and consequently the low molecular weight 30 kDa lipoprotein PBMHP-6 precursor was found to be a possible interacting protein.The interaction between BmSP1 and BmLP6 was confirmed with yeast two-hybrid assay.Therefore,it is speculated that although BmSP1 has no direct fungistatic effect on B.bassiana,it may play a certain role in the development and the innate immune regulation network of silkworm by interaction with BmLP6.The results of the study will help to further elucidate the function of BmSP1 and the regulatory network of innate immunity in the silkworm.