The Expression Patterns And Antifungal Activities Of Enbocin 1 Proposed For Silkworm(Bombyx Mori) Against Beauveria Bassiana Infection

Beauveria bassiana is an important pathogenic fungus of silkworm, Bombyx mori and the white muscardine disease of silkworm caused by its infection always lead to huge losses to the sericulture industry. At present, the pathogenicity mechanism of fungi, including B. bassiana, to silkworm is still unclear. Researches on the function of silkworm antimicrobial peptide enbocin1 against B. bassiana infection would be helpful to better understand the molecular mechanism of silkworm immune response to the fungus and the molecular pathogenicity of B. bassiana to the silkworm.Newly exuviated fifth instar larvae of silkworm P50 were infected with the conidia of B. bassiana. The hemolymph, fat body, Malpighian tube, integument of randomly selected silkworm larvae in both the experimental group and control group were collected at 10 different time points after the infection, and the temporal and spatial expression patterns of enbocin1 in these tissues were analyzed with real-time PCR. The result showed that the expression of the enbocin1 gene was up-regulated obviously mainly in the fat body and kept in a higher level up to 30 hours post infection, only little up-regulated in Malpighian tube and integument and almost undetectable in hemolymph, implying that the enbocin1 may play an important role in silkworm response against B. bassiana infection.Recombinant expression vector PET-30a(+)-enbocin1 was successfully constructed and transformed into Escherichia coli BL21(DE3) for expression of enbocin1 protein. High purity recombinant protein enbocin1 was obtained by Ni-NTA affinity chromatography. SDS-PAGE electrophoresis detection demonstrated that the protein band was about 10.8kD just as expected. The concentration of recombinant protein was 10mg/mL. Its high activity in inhibiting the growth of B. bassiana was verified with fungistatic zone experiment. Furthermore, polyclonal antibodies against the recombinant enbocin1 was prepared and successfully used for western blot detection of the expressed enbocin1.These results provided new experimental evidences for better understanding of the molecular mechanism of silkworm defense against B.bassiana infection and theoretical reference to develop new measures for the prevention and control of white muscardine disease of the silkworm. At the same time, the results are also helpful for further study on the function of enbocin gene.