| Tobacco is the model plant for genetic engineering. Since the genetic engineering appeared in 1983, the transformation studies with tobacco have been at large. The reasons are as following: tobacco is easy to carry through tissue culture, regenerate and get the transformants; at the same time, it's regeneration period is short. In addition, the plant diseases and insect pests of tobacco are typical, so researchers often using gene transformation technique to study the resistant effect of different foreign gene. They select resistant plants to investigate the gene integration, transcribe, translation and the resistance to plant diseases and insect pests. These works lay a firm base for transformation of other plants.In this paper, I selected the leaf dish of Nicotiana benthamiana as the explants and introduced five antimicrobial peptide genes (MCEMA, SPTermicin, AFP, SPCEMA, ASP) into tobacco by Agrobacterium mediated transformation. I compared the resistant effect of different foreign genes in transgenic plants. The results are as following:1. Confirmation of selection pressureLeaves of tobacco were sliced into "leaf dishes (0.5cm2/tablef) and cultured on MSB1 (MSB0+ BA2.0mg/L+NAA0.1mg/L) medium containing different concentration Km (0,25,50, 75, 100mg/L). The results showed that the selection pressure was 100mg/L.2. Acquirement of transgenic tobacco of antimicrobial peptide genesSterile tobacco leaves sliced into leaf dishes (0.5cm2/tablet) were immerged into the Agrobacterium suspension of OD6oo 0.1-0.2 for 5min, and the leaf dishes were co-cultured on MSB1 medium covered with a sterile filter paper disc for 3d in darkness at 24 C.After co-culture was over, the explants were directly transferred to the selection medium MSB2 (MSB1+Cef500mg/L+Km100mg/L) for three weeks. The explants were selected twice. After green buds appeared, they were sliced and transferred to radication medium MSB3(MSB1+ Cef500mg/L+Kml50mg/L). Finally, green seedlings were selected for further assays.3. Molecular assays of transgenic plantExplants from the Kanamycin-resistant regenerated plants were stained for assaying GUS activity using a protocol derived from Jefferson (1987). Expression of the GUS gene confirmed that the T-DNA had been transferred and integrated into the regenerated plants. At the same time, the introduction of the target genes were confirmed by PCR analysis of the DNA samples isolated from the GUS positive regenerated plants.4. Resistance analysis(1) Inoculation with the living transformantsTransgenic plants of peptide genes were inoculated with Phytophthora nicatianae in greenhouse. The results showed that compared with control, the resistance of transgenic plants containing antimicrobial peptide genes was strengthened distinctively. Among five genes, the resistance of AFP and AFP-SPCEMA gene was more distinct. The incidence of a disease was 15.79% and 13.89% respectively.(2) Inhibition of fungi growth with the extracted proteinAfter extracting the whole proteins from the transformants, I tested their antifungal effect. We found the inhibitory effect was proportional to the concentration of added antimicrobial protein. The inhibitory circles produced by the production of AFP and ASP genes were the biggest. Their diameters were 1.6cm and 1.7cm respectively; MCEMA, SPTermicin and SPCEMA were 1.4cm, 1.3cm, 1.2cm respectively. The results showed that: the inhibitory effect is different with different genes, which is consistent with the inoculation experiment of living transformants.5. Assaying the stability of descendiblity in transgenic plantsAs for transgenic plants of every gene, we selected two plants randomly and germinated their seeds. After germinated, they were stained for assaying GUS activity. The x c2 aptness workout showed that the heritability of foreign gene accorded with the law of segregation.
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