Construction Of Plant Expression Vector With Multiple Denes And Transformation Analysis

Selectable marker gene plays a pivotal role in the transformation of genetically modified crops（GMC）. But in order to ensure the safety of GMC and dispel people’s concerns about security, it isnecessary that selectable marker gene must be removed after selection. Nurturing marker-free transgenicplants has been becoming one of the hotspots. In addition, Stacked traits plays an important role inGMC. Therefore, in this study, a double T-DNA plant expression vector is constructed with severaldifferent expression cassettes of target genes. Then it is transformed to the Nicotiana tabacum NC89byAgrobacterium tumefaciens. We analyse the transformation of the vectors and the random integration ofthe two T-DNAs. All of this can lay the foundation of stacked traits and it is also play a platform formarker-free. The main results are as follows:1） We construct a double T-DNA plant expression vector with pCAMbia2300. The two T-DNAsare arranged with left-right-left-right borders. Each of T-DNAs has different Multiple CloningSites (MSC). We name it DT2300.2） We successively and separately add the expression cassettes of selectable marker gene （nptII）and target genes(bt、hptII、cp4、bar、gus、gfp）into DT2300’s two T-DNAs with isocaudarner,andobtain several different expression cassettes of target genes vectors: DT1、DT2、DT3、DT4、DT5、DT6、DT7. For avoiding the gene silencing, The target genes nptII、bt、gfp are under theCaMV35s promoter; The genes cp4、gus are under the nos promoter; The bar、hptII genes areunder the cab5promoter. The vectors are transformed into the Nicotiana tabacum NC89byAgrobacterium tumefaciens. We obtain genetically modified tobaccos with a series of vectorswhich includes7strains with DT1,15strains with DT2,10strains with DT3,7strains withDT6,27strains with DT7. From the polymerase chain reaction(PCR)test of DT7, we find thatDT7’s foreign genes have been integrated into genome of tobacco.3） We can see from the consequence of PCR test of DT7,6out of27strains only contain theT-DNA with the marker gene, the others contain the two T-DNAs. So the co-transformationratio is21/27=77.8%.4） In the21strains which contain the two T-DNAs,15strains which contain the two T-DNAs arerandomly integrated into the tobacco genome. What is more, we obtained9strains with thesingle copy number. In the21strains which contain the two T-DNAs, we find that there areonly12strains which contain all of the traget genes (seven genes), and9out of21strains haslost with different sizes of fragment by the integration. Most of the lost are adjacent to the leftborder. From the PCR test of12strains which contain seven genes,6strains transform into tobacco genome with the two T-DNAs locked(that is to say,the two T-DNA can be recognizedas one T-DNA). The other6strains are randomly in tegrated.