Highly Effective Expression Of Two Antimicrobial Peptide Scygonadin And Hepcidin Fusion Gene Derived From Marine Animals In Algae And Arabidopsis Thaliana And Evaluation Of Its Antimicrobial Activities In Vitro And In Vivo

Antimicrobial peptides(AMPs),a kind of widely existed low molecular weight polypeptides possessing antimicrobial activity,consist an important part of innate immunity.AMPs possess advantages such as broad antimicrobial spectrum,freely soluble in water,no hazard residues in vivo,no drug resistance,etc.,making it a potential new pattern and eco-friendly substitutes for antibiotics.Because of the low level of expression of AMPs in vivo,it is difficult and costly to extract and purify AMPs.The effective method to produce a large quantity of AMPs for commercial application at present is using genetic engineering techniques.Biological expression system can be used as a "Bioreactor" for mass production of heterologous protein which has important economic value.Chlorella,a eukaryocyte that has short life cycle,is easy for large-scale culture at a low cost.Thus,transgenic Chlorella is one of the popular research focus as a bioreactor.In our previous study,we successfully constructed a fusion sequence of PC-hepc combined with scygonadin and obtained the recombinant product of this fusion peptide,which is now produced as an additive of feedstuffs to be used for fish.In this study,the AMPs Scy-hepc fusion gene was stably expressed in transgenic Chlorella sp.using genetic engineering technique.Furthermore,we investigated the antimicrobial activity of the AMPs Scy-hepc produced by the transgenic Chlorella and explored the potential application of the transgenic Chlorella sp.in aquaculture.This study provides us with the theoretical foundation and technical support for future industrialization of antimicrobial peptide Scy-hepc.Meanwhile,we obtained a new species of transgenic Arabidopsis thaliana with Scy-hepc gene.Further study of the expression and antimicrobial activity of Scy-hepc from transgenic A.thaliana provides us with the theoretical foundation and technical support for future application of antimicrobial peptide Scy-hepc in crop production.The main results are as followed:1.Construction,stable expression and antimicrobial activity of transgenic Chlorella with AMPs Scy-hepc fusion gene:A binary expression vector containing AMPs Scy-hepc fusion gene was constructed and transformed into Chlorella sp.using electroporation.Transformed Chlorella sp.were submitted for hygromycin B resistance screening,PCR identification,Western blot analysis and GFP fluorescence detection,then a strain of transgenic Chlorella sp.that stably expressing AMPs Scy-hepc was obtained.Antimicrobial activity assay showed that its expression product could inhibit the growth of Aeromonas hydrophila and Staphylococcus aureus.2.Transgenic Chlorella sp.with antimicrobial peptide Scy-hepc fusion gene could pro.vide immuneprotection:In this section,Sparus macrocephalus and Epinephelus lanceolatus(?)× Epinephelus fuscoguttatus(?)were chosen as experimental subjects.Each kind of fish were fed by transgenic and wild-type Chlorella sp.respectively and subsequently infected with A.hydrophila.Results showed that survival rate of both experimental groups(fed by transgenic Chlorella sp.)was higher than the control group(fed by wild-type Chlorella sp.).3.Expression of antimicrobial peptide Scy-hepc in Gracilarla tenuistipitata v.liui Zhang et Xia:By optimizing enzymolysis condition,protoplasts were successfully isolated from the explants of G.tenuistipitata v.liui Zhang et Xia.The Scy-hepc fusion gene was transfromed into the protoplasts using electroporation and transient expressed in the protoplasts.A specific target band was detected by Western blot analysis.The callus of the explants was induced by plant hormones.Agrobacterium-mediated method was applied to realized the expression of Scy-hepc fusion gene in callus.In transgenic callus,cell clusters with green fluorescence of GFP could be observed by fluorescence microscope.4.Construction,stable expression and antimicrobial activity of transgenic Arabidopsis thaliana with Scy-hepc gene:Using Agrobacterium-mediated method,the inflorescens of A.thaliana were infected with Agrobacterium carried Scy-hepc fusion gene.Then the transgenic seeds were obtained from the trangeinc A.thaliana.Using hygromycin B screening,PCR identification and Western blot analysis,the first generation(F1)of transgenic seeds were obtained.Using the same screening approaches,the second generation(F2)of the transgenic A.thaliana with Scy-hepc gene was acquired.The leaf protein extracts of F2 transgenic A.thaliana showed antimicrobial activity against S.aureus.The leaves of F2 transgenic A.thaliana had the ability to resist Fusarium graminearum infection...