Study On Safety Transgenic Tobacco Toward Breeding Varieties With High Resistace To Bacterial Wilt

Chinese tobacco production losses caused by pests and diseases reaches nearly 10 million every year. Bacterial wilt is an importance disease in tobacco, and so far, there is no specific drugs to cure it. As germplasm with high resistance genes often linked with the bad characters and the long period of breeding, progress for breeding high resistance varieties is slow by conventional method. With functional genomics and biotechnology devoping so swiftly, the breeding of transgenic tobacco will become an effective means of desease-resistant tobacco. Meanwhile, as transgene security become widespread concerns, finding a safe and effective means of transgenic breeding with biotechnology become popular. Based on available root-specific genes and its promoter sequences, and promoter specifically induced by bacteria, expression vectors harboring disease resistance gene driven by specific promoters were constructed and were transformed into tobacco for functional verification. Construction of laboratory self-deleted of antibiotics gene was verified. The results are showed as follows.1. According to tobacco root-specific genes NtR12 fragment, downstream fragment was got through 3' RACE primers, amplified 3' fragments of the NtR12 completed the full length of NtR12 gene. At the same time, the available NtR12 5 'upstream promoter has been analyzed by bioinformatics method, and series of expression vectors were constructed with various lengths of promoter sequences starting before ATG initiation codon, which are being used to identify the functional elements of the promoter.2.An antibiotics self-deleted vector, p35S-loxp-Gus, built in this laboratory before has been transformed into transgenic tobacco CuiBiyihao, mediated via Agrobacterium tumefaciens, and explants in bud differentiation stage were induced to remove antibiotics genes by dexamethasone and produce buds and grow into transgenic tobacco without antibiotics gene. Transgenic tobacco plants tested by GUS staining confirmed that under certain concentration of dexamethasone in the induction, the recombinant enzyme Cre-GRB can correctly identify the loxP sites and delete the expression units of both hygromycin marker gene and recombinant gene, and then recombine DNA so that 35S promoter can drive expression of GUS gene to produce a blue reaction. So the expression vector can be used to remove the hygromycin gene, producing unmarked transgenic tobacco plants.3. Ribosome inactivating protein gene of balsam pear was homologous cloned, and the function of the genes was analysed. The vector was constructed by binding root specific promoter with ribosome inactivating protein gene,which was transformed to tabacco CuiBiyihao. Then the transgenic tabacco was molecular detected and identified by inoculation with Rostonia solanacearum. The result showed that transgenic tabacco increase their resistance significantly to bacterial wilt,some plants showed immune rection to bacterial wilt. It was characterized that RIP expressed specifically in root and stem, not in leaves,, which provided basic explanation for safety of transgenics.4.Employing tobacco chitinase gene in associated with bacterium-induced promoter available in the lab, a plant expression vector Psc-PP1-Chi, was constructed and transformed into CuiBiyihao, mediated by Agrobacterium infection. More than 100 transgenic plants were received, and transgenic offspring were identified by PCR assay and nearly 100 positive transgenic plants were undergone R. solanacearum inoculation tests. And the results indicate that Ralstonia solanacearum infection could induce Chi gene expression that the resistance to bacterial wilt in transgenic plants were all seen to increase by different degrees, some showed high resistance.