Expression Of Mytilus Galloprovincialis Foot Protein Type 5(Mgfp-5) Gene In Chicory(Cichorium Intybus L.)

Mussel Adhesive Protein(MAPs),also known as Mytilus foot proteins(Mfps),is a class of sticky protein complex secreted by its byssus.MAPs often were used as biomedical adhesive and high quality medical adhesives due to its strong adhesion,biodegradability and excellent biocompatibility.Since the natural mussel extraction is a labor-intensive and inefficient process producing little purified protein.The genetic engineering technology has been used to study the properties and functions of recombinant proteins in the prokaryotic expression(E.coli)and eukaryotic expression(yeast,tobacco)system.However,only tyrosine modification can recombinant protein has adhesion in E.coli.The quantity and adhesion force of the recombinant protein were low.The quantity and adhesion force of the recombinant protein in eukaryotic expression system have not seen the relevant report.Compared with the in vitro modification of the recombinant protein,the expression of recombinant protein in plants can reduce the cost and toxicity due to the modification of the glycosylation and phosphorylation in vivo,and has the advantages of safety and large-scale production.The study of recombinant expression of Mytilus galloprovincialis Foot Protein(Mgfp-5)in tobacco has been studied.But it is still weak.Chicory(Cichorium intybus L.)is one of the commonly receptors for plant genetic engineering with the advantages of simple tissue culture,strong regenerative ability and high genetic transformation efficiency.Some genes have been successfully expressed in chicory such as ?-zein gene,Na+/H+ reverse transporter from wheat(NHX,NHE or NHA),animal oral vaccine,AFL2 gene,Mycobacterium tuberculosis tuberculosis antigen ESAT6.However,we have not seen the research of the expression of MAPs from chicory.Chicory was selected as the plant acceptor material.The plant expression vector p RI101-Mgfp with Mgfp-5 gene was introduced into chicory genome via Agrobacterium tumefaciens mediated method.Then,the resistance screening cultured.PCR,RT-PCR and Western blotting were used to identify the transgenic chicory plants.After purification,the adhesion of the recombinant protein Mgfp-5 was analyzed by atomic force microscopy(AFM)and Qaurtz Crystal Microbalance(QCM)ability.The main obtained results were as follows:1.An efficient protocol for the regeneration of chicory(Jiangjun)was established.By optimizing the combination of different phytohormones contained in medium.About 95.67±2.84% of callus and 79.18±5.58% of shoots were induced from leaf discs of chicory after being cultured on MS medium with 100 mg/L Vitamin C(Vc),1.5 mg/L 6-BA and 0.2 mg/L NAA.About 97.33±0.88% roots were induced after the regenerated shoots had been cultured on 1/2MS medium with 100 mg/L Vc and 0.1mg/L NAA.2.The optimum conditions for the Agrobacterium-medated transformation.The explants were pre-cultured for 3 d,infected by OD600=0.6 Agrobacterium for 10 min,co-cultured time for 3 d.A higher transformed plant could be obtained at a selective pressure of 50 mg/L of Kan concentration ultimately.3.Identification of transgenic plants.An expected fragment of about 260 bp can amplified in transgenic lines though PCR and RT-PCR.Out of 150 independent T0 plants,32.67 % plants were integrated Mgfp-5 gene with the expression rate of 73.47% at RNA level.4.Analysis of T1 transgenic plants.T0 seedlings were harvested and being cultured.Mgfp-5 gene of T1 transgenic plants were examined by PCR and RT-PCR.The heritability rate was 67.71% and the expression rate was 70.77%.About 44.44% of the transgenic plants followed the Mendelian genetics 3:1 by X2 test.5.Expression and purification of Mgfp-5 protein in transgenic plants.Western blotting identified that recombinant Mgfp-5 protein was expressed(58.70%)in T1 transgenic plants.The purified protein was purified(0.99 ?g/g,98.61%)by nickel column(Ni2+).6.The adhesion function analysis of recombinant Mgfp-5 protein.The roughness(Rz value)(848.11)and the adsorption(490.33)of recombinant Mgfp-5 protein cwere better than the modified protein by AFM and QCM analysis.