| Objective: To obtain the gene sequence of 14-3-3?(MD14-3-3?)gene from the cDNA library of Musca domestica.Then MD14-3-3? gene was cloned,expressed,the molecular characterization and antibacterial activity and antibacterial mechanism of the recombinant protein of the gene was analyzed.MD14-3-3? gene expression patterns were also investigated in different tissues of 3 instar larvae and different developmental stages of Musca domestica.the expression profiles of MD14-3-3? gene was discussed after induction by Escherichia coli.In order to provide experimental basis for further study of its function.Methods: 1.Sequence analysis: The basic physical,chemical properties,signal peptide,secondary structure of MD14-3-3? gene was analyzed by the bioinformatics-related software and the protein function was predicted.2.cDNA cloning and prokaryotic expression: primers were designed according to the cDNA sequence of MD14-3-3?,the recombinant plasmid pET-28a(+)-MD14-3-3? was constructed and the recombinant plasmid was transformed into E.coli BL21(DE3),the protein was induced with IPTG.The solubility of the expressed product was showed by SDS-PAGE,and the recombinant protein was purified by Ni2+ affinity chromatography.3.Western blot and mass spectrometry: New Zealand white rabbits were immunized by the purified protein to obtain polyclonal antibody.The recombinant protein was identified by Western blot.The SDS-PAGE of the purified protein was excised and analyzed by mass spectrometry.4.Primary study on antibacterial activity and antimicrobial function of recombinant MD14-3-3? protein: The antimicrobial activity of recombinant protein was detected by micro-liquid dilution method,Escherichia coli and Staphylococcus aureus as the indicator bacteria.The effect of MD14-3-3? on membrane permeability of Escherichia coli was tested by bacterial permeability test.5.Spatio-temporal developmental expression patterns of MD14-3-3?:1)The insect body of different developmental stages from Musca domestica life history(eggs,age,pupae,adult)andthe different tissues of third instar larva(body wall,fat body,malpighian tubules,midgut,salivary gland and trachea)were collected.Total RNA was extracted for all the tissue samples and was reversed to cDNA.As RPS18 gene was a reference gene,the expression of MD14-3-3? gene were detected by qPCR technique in differential developmental stages.Each experimental group included 3 biological samples,each sample was repeated 3 times.2)The change of expression profiles of MD14-3-3?gene induced by Escherichia coli: In the contrast of PBS group,the second larvae of Musca domestica was injected quantitatively by Escherichia coli,and then the samples were detected to observed MD14-3-3? gene expression levels in different time points(3 h,6 h,12 h,18 h,24 h,36 h,48 h)by qPCR.Results: 1.MD14-3-3?gene is 771 bp,encodes 257 amino acids,its theoretical molecular weight 29.35 kD;tisoelectric point of the protein is 4.87,which is a hydrophilic acidic protein,contains a variety of enzyme binding sites,has 14-3-3 family domain and active site,locates in the nucleus,Its secondary structure is mainly composed of alpha helix and irregular curl.2.MD14-3-3? expression vector was constructed,recombinant protein was expressed by IPTG inducing.The purified MD14-3-3? recombinant protein was obtained by Ni2+affinity chromatography.3.The results of Western blot showed that the size of clear bands were consistent with the predicted bands.Mass spectrometry showed that the recombinant protein sequence was consistent with the MD14-3-3? sequence.4.MD14-3-3? showed significant antibacterial activity against Escherichia coli and Staphylococcus aureus.The MIC value was 0.16 mg/m L against Escherichia coli,the MIC value was 0.25 mg/m L against Staphylococcus aureus.The results of bacterial endometrial permeability showed that the A430 nm value increased from 0.1 to 0.7 with the prolongation of the time.5.Spatial and temporal expression patterns of MD14-3-3? gene in Musca domestica indicated: 1)the expression of MD14-3-3? gene in each developmental stage of the Musca domestica was different.As the egg was a reference,The expression of MD14-3-3? gene was the highest in adult,the expression level character is as follows: adult> second instar larvae > pupae> first instar larvae> third instar larvae>egg.The expression of MD14-3-3? gene in adult raised 141.773 times(P<0.05)as against egg.The second instar larva increased79.7967 times(P<0.05).In the third instar larvae,the expression of the gene was the highest in the body wall,followed by trachea(P<0.05)and salivary gland(P<0.01).2)The expression of MD14-3-3? gene was induced by Escherichia coli: By comparing with PBS group,MD14-3-3? genes were up-regulated in 3 h,24 h after Escherichia coli,expression of 3 h were significantly up-regulated by 14.704 times(P<0.05);the expression of 24 h were up-regulated by 2.503 times.MD14-3-3? gene expression trends was low levers in 36 h,48 h(P<0.01).Conclusion: 1.In this study,the14-3-3? gene of Musca domestica were analyzed in the molecular characteristics and cloned,expressed,the recombinant protein of MD14-3-3? was obtained and identified;The results showed that the recombinant protein had antibacterial effects on Staphylococcus aureus and Escherichia coli,the antibacterial activity of Escherichia coli was better and could change the permeability of its intima.2.MD14-3-3? gene was expressed in different growth stages of Musca domestica and different tissues of third instar larvae.After induction by Escherichia coli,the expression of this gene was significantly up-regulated at 3 h,suggesting that the gene was involved in immune defense process of Musca domestica. |
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