Study On The Antibacterial Activity Of Silkworm Defensinb And Its Sequence Diversity And Differential Expression Among Varieties

Antimicrobial peptides?AMPs?are the major effector molecules in the humoral immunity.AMPs play an important role in immune defense of inscects.Silkworm is not only an important economic insect,but also a model of Lepidopitera insects.Silkworm AMPs are divided into seven families based on the characteristics of their structures and functions.The antimicrobial functions of six families,Cecropin,Attacin,Lebocin,Moricin,Enbocin and Gloverin have been reported.However,the activities of DefensinA and DefensinB,two members of Defensin family,are still undetermined.In this paper,Bombyx DefensinB was expressed using baculovirus expression system,and its antimicrobial activity was investigated.We amplified the DefensinB gene from genome DNA,analyzed and compared the sequences from PCR products of DefensinB gene from different varieties of silkworm.Next,we analyzed expression profile of DefensinB induced by bacterial and fungal infections among different varieties of silkworm by quantitative real-time PCR,and tried to explain the differences between varieties of mulberry silkworms.The main results are as follows:1.The recombinant plasmid pFastBac1-DefensinB was constructed and subsequently transformed into E.coli AcDH10Bac.After identified and isolated from positive clones,the recombinant bacmid was transfected into Sf9 cells,and the recombinant viruses were obtained.Then,DefensinB was produced in Sf9 cells and culture medium by infection with recombinant viruses.The recombinant protein DefensinB was successfully purified using Ni-IDA affinity chromatography column.2.Antimicrobial activity of recombinant protein DefensinB was investigated by agar diffusion and growth curve method.The results showed that DefensinB was inactive against Staphylococcus aureus?G⁺?,Escherichia coli?G^-?and three strains of fungi,Neurospora crassa,Rhizoctonia solani and Fusarium oxysporum.3.PCR results showed that the gene size of DefensinB from different varieties of silkworm were different.And sequencing results showed that the size and sequence of single intron in DefensinB were different between varieties of silkworm.Only one amino acid residue was different in CDS region for mature peptide.4.The expression profiles of DefensinB induced by S.aureus,E.coli K₁₂D₃₁ and Beauveria bassiana were analyzed by quantitative real-time PCR.The results showed that both basic expression level and induced expression profile of DefensinB were varied from different varieties of silkworm.Our results indicated that B.mori DefensinB could response to the invasion of different pathogenic microorganisms,and both basic expression level and induced expression profile were varied from different varieties of silkworm.Moreover,the size and sequence of single intron in DefensinB and CDS of DefensinB were also varied from different varieties of silkworm.DefensinB in vitro expressed and purified using baculovirus expression system didn't show activities against all tested bacteria and fungi,and its activity need to be further studied.All of the above results provided the experimental basis to further research on DefensinB.