| Destruxins are serial analogues of cyclodepsipeptidic compounds isolated from the entomopathogenic fungus,Metarhizium anisopliae.Destruxin A(DA)is the most common anologue of the destruxins,which has insecticidal activities against host insect’s innate immunity system,but the molecular mechanism is still not clear.In our previous research,the silkworm’s Bm12 cells were treated with DA and the total proteins were extracted,after they were lysed by protease K,a specific protein strip was separated by gelelectrophoresis.Consequently,by means of MS(mass spectrum),several HSPs(heat shock proteins)were identifiedin the strip,which implied that interactions were likely in DA and HSPs.Therefore,in this study,we intend toconstruct the prokaryotic expression system of five HSPs(Bm HSP70,Bm HSP75,Bm HSP90,Bm HSCP and Bm HSP97)to obtain these HSPs by N ickel column affinity chromatography.Then,the affinity between DA and HSPs will be measured by Forte Bio Octet QK of Bio-Layer Interferometry system to verify the interactions of HSPs with DA.Finally,the expression levels of HSP genesin Bombyx mori Bm12 cells treated with DA were analyzedby SYBR Green quantitative real-time PCR(q PCR).The results of this study will help to elucidate the molecular mechanisms of DA.The main results indicated as follows:(1)Construction of prokaryotic expression system of Bombyx mori HSPsFirstly,total RN A was extracted from Bm12 cells.Next,the methods reverse transcription-polymerase chain reaction and PCR amplification were used to clone the encoding sequence of HSPs.Then,products of PCR were respectively cloned into p EASY-T vector,and a positive clone was selected by using the methods of blue-white selection and PCR amplification.O n the basis of double-enzyme cleavage method,the HSP fragment were linked into p ET-30 a plasmids and the competence cells were transformed.After that,the positive clones were pickedby kanamycin and blue-white selection,and sequenced with p ET-30 a vector identification primer T7/T7 ter.The results of gene sequencing showed that HSPs gene recombinants were constructed successfully.(2)Protein purification with Nickel affinity chromatographyThe expression of target protein was induced with IPTG.Proteins of E.coli BL 21(DE s3)were extracted with ultrasonication,andwere74.9k Da,88.6k Da and 71.7k Da in SDS-PAGE as we expected.After N i-NTA affinity chromatograph,we obtain the target protein.Moreover,the expression level of recombinant recognized by a commercial Micro UV spectrophotometer was about 30% of total cell protein.Further more,western blot analyses showed the recombinant proteins had a good immunore activity with the reference positive serum.(3)Detection of affinity between destruxin A and heat shock proteinsThe interaction between destruxin A and heat shock proteins was analysised by Forte Bio Octet QK systembase on Bio-layer interferometry,which was commissioned by the Nanjing Detex Biotechnology Company.The result showed that there was no molecular interactions between destruxin A and heat shock proteins.We conjecture that the differential protein after DA treatment may bethe stress response of silkworm cells which lead to high expression of heat shock protein,and was not hydrolyzed byhydrolases.(4)Effects of DA on Bombyx mori HSP genes expressionsAfter the treatment with DA,the total RN A was extracted from the cells of Bm12,which was divided into experimental group and control group.Then,the c DNA obtained by Reverse transcription-polymerase chain reaction(RT-PCR)was used for template of q PCR.The results showed that the Bm HSP75,Bm HSP90,Bm HSCP,Bm HSP97 and Bm HSP70 genes were significantly up-regulatedin the DA treated group,when compared with the control group.The results of this study provided further confirmation of the stress response of silkworm cells treated with DA,which lead to high expression of heat shock protein.In conclusion,DA can induce the five HSPs expression in Bm12 cells,but DA has no molecule interaction between these HSPs. |
