Research On Transgenic Tobacco Expressing Pokeweed Antiviral Protein Gene(PAP)

Tobacco is the model plant for genetic engineering. Since the genetic engineering appeared in 1983, the transformation studies with tobacco have been at large. The reasons are as following:tobacco is easy to carry through tissue culture, regenerate and get the transformants; at the same time, it's regeneration period is short. In addition, the plant diseases and insect pests of tobacco are typical, so researchers often using gene transformation technique to study the resistant effect of different foreign gene. They select resistant plants to investigate the gene integration, transcribe,translation and the resistance to plant diseases and insect pests. These works lay a firm base for transformation of other plants.Pokeweed antiviral protein gene of Phytolacca americana L.was one of antiviral protein gene. In this paper, I selected the leaf dish of Nicotinan tabacium as the explants and introduced PAP gene into tobacco by Agrobacterium mediated transformation.It was cloned and inserted into binary vector under CaMV35S promoter to construct the plant constitutive expression vector pBinARpap. The leave discs of tobacco (Nicotinan tabacium L. cv. K326) were transformed with pap gene via Agrobacterium-mediated transformation. Shoots were regenerated on MS medium supplemented with 30mg/L Kan, 0.1mg/L IAA and 0.5mg/L BA and rooted on MS medium without hormone. PCR-Southern analysis proved foreign pap gene intergration in one Kanamycin resistance plantlet. RT-PCR indicated the pap gene of transgenic plantlet had expressed at the transcriptional level. Disease challenge test of the detached leaves of transgenic plantlet by inoculation of Tobacco mosaic virus showed that the resistance was dramatically enhanced compared with non-transgenic plants. The result implicated the potential application of pap gene in plant disease-resistance engineering.