The Construction Of A Marker-free Co-transformation Vector With A Melon A-ACO1 Gene And Its Co-transformation To Tobacoo

Co-transformation is one of the important ways for producing transgenic plant with marker-free gene, improving the safty of transgenic plants , accumulating the commercialization process of genetically modified products,and the key is to construction the co-transformation expression vector and obtain the co-transformation plants firstly. In this experiment, the co-transformation vectors containing melon anti-ACC-oxidase were built and then directed into the leaves of tobacco which gave rise to co-transformation plants.1.The gene of nptⅡwas exsected from the vector named pCB-aACO1 by restriction endonuclease Xhol. And then the residual big segment linked with itself to form a new vector pCD-aACO1, the T-DNA area harboring target gene a-ACO1 and reported gene GUS, without gene nptⅡ.2.The gene of GUS was exsected from the vector named pCAMBIA2301 by restriction endonucleases, And then the residual big segment linked with itself to form a second vector pCAMBIA2302, the T-DNA area harboring only gene nptⅡ.3. The dish-like leaves of tobacco were infected with the mixed inocula([pCD-aACO1]∶[pCAMBIA2302]=1:1,2:1 and 3:1),and the result showed that different outcomes responded to different mixed inocula:,gave rise to the differentiation rate of 52.5%,48.3% and 25.8%, the co-transformation rate of 17.5%,35% and 33.3%,respectively. Obviously, it was the best one that the volume ratio of pCD-aACO1∶V pCAMBIA2302 = 2∶1.4.In this experiment,the highest co-transformation rate is 52.5%,while the transformation rate only with one strain is 85%,so the co-transformation rate is lower than the transformation rate only with one strain so far.