The Removal Of Marker Genes In PCAMBIA-E8-APB-DOCK8 Fusion Gene Expression Plasmid And The Experimental Study Of Transient Expression Of Tomato

Dental caries is a bacteritic infectious disease, with a chronic progressive destruction of hard tissue.With high morbidity rates and widely distributed. Streptococcus mutans(S.mutans) has been implicated as a causative organism of dental caries. Surface protein antigen(PAc) and glucosyltransferases(GTFs) are indicated as two of important virulence factors of S.mutans. The research suggests that, the antibody for the two kinds of virulence factor can effectively inhibit its function. immunization is an effective control measures of dental caries When not damage to tooth structure.This research is the prophase’s continue and thorough.Based on lasmid p CAMBIA-E8-APB-DOCK8(p E8AD3) was constructed,and the removal of Km and GUS marker genes. The exprements is to transient expression system. Establishment of transient expression system of tomato Cotyledon. In order to make experimental basis for understanding the structure of target protein and its biological function.Objective:This research is the prophase’s continue and thorough.Based on plasmid p E8AD3 was constructed,and the removal of Km and GUS marker genes. It will increase the food and environmental safety of transgenic plants if theselectable mark er genes were removed from the transgenic plants afterwards.Establishment of transient expression system of tomato Cotyledon. Analysis of the expression of exogenous gene.Methods: 1.With the DNA recombinant techniques,Based on plasmid p E8AD3 was constructed,and the removal of Km and GUS marker genes. 2.Cultured on MS medium to obtain tomato sterile cuttings.The main countermeasure arefilter variety.The plasmid was then transferred into Agrobacterium strain EHA105.The transformation efficiency can be obviously raised by using the optimal transient expression,consisted of the material of tomato leaf,concentration of Agrobacterium, injection of Agrobacterium,Seedling age of tomato Cotyledon and time of co-cultivati. With the DOCK8 reporter gene of the Agrobacterium as the injected form of the transient transformation for detection,and detected their expression in plant cells with transient expression. 3.The establishment of antibacterial antibiotics and concentration in the regeneration and transformation of tomato.Through the cultivation of Tomato Cotyledon Explant and Bacteriostatic Experiment of Agrobacterium infection observed Carbenicillin, Cefotaxime, Timentin three kinds of antibiotics in different inhibitory concentration the inhibition effect and explant morphology changes.And Preliminary screening of transgenic tomato fruits was carried out by PCR detection method.Results: 1.Successful removal of marker genes Km and GUS in plant expression vector p E8AD3. 2.Establishment of transient expression system of tomato Cotyledon.Tomato varieties suitable for this experiment were selected. Tomato varieties suitable for this experiment is the European red(Apple type).The best material of tomato leaf is tomato Cotyledon. The best concentration of Agrobacterium is OD600: 0.2-0.7.The best injection of Agrobacterium is 50 μL.The best Seedling age of tomato Cotyledon is 7-10 d.And the best co-culture time is 24-48 h. 3.Timentin as an effective antibiotic inhibiting Agrobacterium has obvious antibacterial effect, little influence on plant.The concentration of antibiotics in regenerated tomato is 400 mg/L.And Preliminary screening of transgenic tomato fruits was carried out by PCR detection method.Conclusion: 1.Fusion gene expression plasmid p E8AD3 was obtained without Km and GUS selective marker genes can be used for plant genetic transformation.There are E8,APB,DOCK8 inthis vector. 2.Establishment of transient expression system of tomato Cotyledon. Advantages are simple operation, no need for expensive reagents and instruments.In order to make experimental basis for understanding the structure of target protein and its biological function. 3.The concentration of antibiotics in regenerated tomato is 400 mg/L.The primary advantage is Timentin as an effective antibiotic inhibiting Agrobacterium has obvious antibacterial effect, little influence on plant. 4.Obtained transgenic(p E8AD3) positive tomato fruit containing GUS reporter gene, and successfully used for the preservation and breeding of seeds.