Construction Of The Gene Encoding Plastid-Targeted Protein Pt-mVirD2 And The Tobacco Plastid Transformation Vector Containing A T-DNA Structure

It has a number of advantages:higher transgene expression level;site-specific introduction of foreign genes which leads to the absence of gene silencing and position effect;the ability to express polycistronic messages from a single promoter;uniparental inheritance that prevents pollen transmission of foreign DNA;provision of more favorable environments for certain biochemical reactions and for accumulating large amounts of gene products.It has become a useful tool in many fields,the superiority of chloroplast transformation are favored by us.Now the vast majority of of transgenic plastids are almost using particle bombardment,a small number of experiments are also using PEG-mediated protoplast method.However,these methods exist lots of disadvantages,such as low efficiency of transformation,long time of homogenization, pollutinon of plastid genes and so on.This study was ready for cloning VirD2 gene and AtCor15a from the Agrobacterium and Arabidopsis respectively,constituting a plastid-targeted fusion protein coding genes pt-mVirD2;At the same time the tobacco plastid transformation vector including T-DNA structure was constructed, it lay the solide foundation for directional chloroplast transformation,the current problems of plastid transformation research.The main experimental results are as follows:1 Construct of plant gene expression vector containing pt-mVirD2-GUS and pt-mVirD2-GFP dual-geneNuclear localization signal loss mVirD2 gene and plastid targeting signal sequence pt were amplified from Agrobacterium tumefaciens C58 genome,pMD-AtCor15a plasmid respectively, successfully constructed the plant gene dual expression vector pVCT2155 and pVCT2210 containing pt-mVirD2-GUS chimeric gene and pt-mVirD2-GFP chimeric gene.2.Obtaining transgenic plants after transforming Pt-mVirD2-GUS and pt-mVirD2-GFP gene into the tobacco respectively. Leaf discs by Agrobacterium-mediated method in the 50mg/1 hygromycin selection pressure screening,to be hygromycin-positive plants,transform pt-mVirD2-GUS into tobacco.Transgenic plants are identfied through the PCR and the GUS staining.Transgenic plants of PCR-positive and hygromycin-resistant are GUS staining,with 70%ethanol,there is no expected blue spot to be appeared.Gus is replaced with GFP,then transform pt-mVirD2-GFP into tobacco suceully,I can watch green through microscope.3.Construction of tobacco plastid transformation vector containing T-DNA-typeThe tobacco plastid transformation vector pVCT2164 are constructed with a T-DNA left and right borders.It contains DNA fragment trnI and trnA for homologous recombination GFP reporter gene and the aadA resistance gene spectacular ADM controlled by Prrn promoter and terminator Tpbs,4 control;nptâ…¢kanamycin resistance gene T-DNA outside and E.coli replicon ColE1.