Studies On Promoting Efficiency Of Tobacco Protoplast By Agrobacterium Tumefaciens Transient Expression Protein

Study showed the processing and transfer of T-DNA are mediated by products encoded by the virulence (vir) region of the Ti plasmid in agrobacterium. Vir region contain three key genes—VirD₁,VirD₂ and VirE₂. VirD₁ protein is a topoismerase,which makes DNA have a change from superholix to relaxtant condition.VirD2 protein can cut relaxtant T-DNA, which results in a nick at the border of it.Then the single T-DNA are released. VirE₂ protein is a single-stranded-DNA-binding protein,which makes T-DNA from a slim nuclitid-protein complex.The complex can protect T-DNA from endocytic/extrocytic nuclinase degeneration.The T-complex can penetrate cell wall and memberane of agrobacterium and plant,enter nucleus and integrate it into plant genome. Both VirD₂ and VirE₂ contain short peptide sequences that act as nuclear localization signals (NLSs).This study aim to use three key virulence proteins--VirD₁,VirD₂ and VirE₂ genes of agrobacterium tumefaciens in the course of transformation to assist Green Fluorescence Protein (GFP) transforming into tobacco protoplast. The purpose is to combine the Pollen-tube path way give a base to explore a high effective, no-host-limit and rebirth-system-limit.It will be much convenience to our scientific research and technology of the gene's transfer to plant.According to the agrobacterium tumefaciens Ti plasmid gene sequences published by U.Washington we designed a pair of primers, Which include BamH I and Xho I endonuclease sites, amplified the VirDi Gene Situating in the Virus region of agrobacterium tumefaciens Ti Plasmid with the method of PCR, joined VirDi and GFP respectively to the express vector PET30a, and gained prokaryotic expression protein.The transient expression vector--pUC-pBin was built, the gene VirD₁, VirD₂, VirE₂ and GFP was joined in it respectively. The stable cultivate system of tobacco protoplast established. The frequency of VirD₂,VirE₂ events was about 7-13% of the total number of transformants than VirDi mediated³-10%.The transformation efficiency of VirD₁,VirD₂ and VirE₂ mediated was about 60%-66% of the total number of transformants.This result was more than the transformation efficiency of GFP without VirD₁,VirD₂ and VirE₂ gene--0.3%.