The Construction, Genetic Transformation Of The Insect-resistant Plant Expression Vectors And The Expression Analysis

On the base of the plant expression vectors constructed by Wang Y P, the plant expression vectors harboring Bt genes were constructed and transformed into tobacco using leaf-disk method. The Bt genes expression levels were tested. Selecting 741 poplar and pB29 clone as the experiment materials, the leaf regeneration systems were established and optimized the Agrobacterium tumefaciens mediated transformation system. Mediated by Agrobacterium tumefaciens, Bt cry3A (Bt3) gene was integrated into pB29 genome which have harbored Bt cry1A(c) (Bt1) gene already and transgenic 741 poplars with two Bt genes were obtained. Some transgenic plants were tested on DNA and protein levels. The main results were as follows:On the base of the plant expression vectors pCAMBIA1305-Bt1-Bt3, pCAMBIA1305-Bt1 and pCAMBIA1305-Bt3, using DNA recombination technology, the plant expression vector pCAMBIA3301-Bt1 fused Bt1 gene and the plant expression vector pCAMBIA3301-Bt3 fused Bt3 gene were separaterly constructed. Each gene was promoted with CaMV35S promoter, containing PAT gene as a selective marker. The plant expression vectors were transformed into EHA105.Hygromycin (Hyg) critical concentration was 50 mg.L-l and Cefotaxime (CTX) was 200 mg.L-l for the tobacco transformation. Mediated by Agrobacterium tumefaciens, using the plant expression vectors pCAMBIA1305-Bt1-Bt3, pCAMBIA1305-Bt1 and pCAMBIA1305-Bt3 and the plant expression vectors pCAMBIA3301-Bt1 and pCAMBIA3301-Bt3, the Bt genes were transformed into tobacco using leaf-disk method and the Hyg and PPT resistant plants have been regenerated. PCR detections indicated that Bt1 and Bt3 genes were integrated into the tobacco genome. ELISA analysis showed that in most of the transgenic tobacco plants the expression levels of Bt proteins were higher than the comparison. In five transformed Bt1-Bt3 genes tobacco plants, both Bt1 and Bt3 proteins expressed in three plants. The expression levels of Bt1 and Bt3 proteins were higher than that in the wild-type tobacco plant. The highest could be up to 0.030 1% and 0.293 8% of the leaf total soluble proteins, respectively. The leaf regeneration system of the 741 poplar was established and the Agrobacterium tumefaciens mediated transformation system was optimized. The shoot induction medium was MS+ 6-BA 1.0 mg.L-l + NAA 0.1 mg.L-l and the root induction medium was 1/2 MS+NAA 0.1 mg.L-l. Hygromycin (Hyg) critical concentration was 10 mg.L-l and Cefotaxime (CTX) 400 mg.L-l was a suitable concentration to control the propagation of Agrobacteria. Several crucial factors influencing the transformation efficiency were studied. It was found that OD600=0.4, infection of leaf explants for 8~10 min with Agrobacteria and cocultivation for 2 days after infection would befavorable for the transformation.Mediated by Agrobacterium tumefaciens, Bt3 gene was integrated into pB29 genome, which had already integrated Bt1 gene already, then transgenic 741 poplars with two Bt genes were obtained. Through several transformations and selections, 9 clones of transgenic 741 poplar with two Bt genes were obtained and the clone numbers were pC1~pC9.The results of PCR detection using special primers confirmed that Bt1 gene stably consisted in pB29 clones and Bt3 gene were integrated into the pB29 genome. ELISA analysis indicated that in the two clones pC1and pC2, both Bt1 and Bt3 proteins expressed. The expression levels of Bt1 protein were 0.009 4% and 0.011 0%, respectively. The expression levels of Bt3 proteins were 0.217 0% and 0.149 4%, higher than that of pB29 and that of the transformed single Bt3 gene 741 poplar clone CC71, of which the expression level of Bt3 protein was 0.033 7%.