Primary Study On Agroinoculation Virus Assay Transgenic Plant Vaccine

An almost complete sequence of the RNA genome of tobacco necrosis virus (TNV) strain A has been determined. Tobacco necrosis virus A is a positive-sense ssRNA virus, it contain six open reading frames. SphI was constructed between ORFâ…£and ORFâ…¤(coat protein)of TNV-A gene by overlap extension of PCR.TheintermediateplasmidspBSac-1,pMD18-TNV-1,pBSac-2, pMD1 8- TNV-S,pUCm-GFP,UCm-LTB have been successfully constructed. The pE-TNV-GFP,pE-TNV-LTB,and pE-TNV of virus vectors were constructed with the mutated TNV-A gene inserted into SalI and SmaI of pE1802. Then these three virus vectors were transformed into Agrobacterium EHA105 directly.The agrobacteria harboring the modified vectors were simply pressure-infiltrated into leaves. Two weeks later,there are visible disease dots in inoculability parts.The RT-PCR results for the GFP,LTB and TNV transcripts provided evidence for partial expression of the virus based vector with the foreign sequence.By using the leaf-discco-cultivated method,gene was transferred into tobacco cell respectively.Transformed shoots were selected on solidified medium containing Kanamycin. The expression of these genes in tobacco plants was confirmed by PCR, RT-PCR analyses.By method of agrobacterium mediated,K88-ST gene was transferred into alfalfa cells. Transformed shoots were selected on solidified medium containing Kanamycin. Protein was shown to be expressed in alfalfa plants by Western Dot.