Research On Transgenic Tobacco Of WIN1Gene By Agrobacterium And The Culture Of Transgenic Tobacco’s Explan

In recent years, the Plant gene engineering technology develop rapidly and remarkable results were achieved in many areas of the transgenic plants research. The transgenic technology made wild type plants resist drought, cold, disease and so on and these transgenic plants were widely used. The methods of Plant genetic engineering include Agrobacterium-mediated system, Plant virus system and Direct induction of exogenous DNA. Agrobacterium-mediated transformation system includs Ti plasmid and Ri plasmid transformation system. Ti plasmid transformation system includes Cointegrative vector systems and Binary vector systems. Three methods (Inoculation method, Cocultivation method, Leaf-disk transformation) were used in the Agrobacterium-mediated system has:Plant virus vector include the ssRNA plant virus vector, ssDNA plant virus vector and the dsDNA plant virus vector. The direct induction of exogenous DNA usually has:PEG-mediated, Electroporation, Liposome-mediated, Pollen-tube Pathway Method and Particle bombardment. At the same time, the detection methods of transgenic plants were widely used and detects in the whole level, intranscription level and inexpression level. PCR, Southern blot and Chromosome in situ hybridization were used to detect in the whole level; In the transcription level RT-PCR, Northern blot was usually used; Inexpression level many methods were used:Immunohistochemical staining, Western blot, the method of Green Fluorescence Protein, ELIS A.The WIN1 genes are related with the process of gene expression in waxy metabolic. Total RNA was extracted from the wild type arabidopsis buds, amplificat them into SHN1-WIN1, ligated to the expression vector PBI121. The recombinant vector was named PBI121-SHN1/WIN1, and transformed into tobacco k326 by using the agrobacterium-mediated technique.Four different cultures were used in this study. TI is the minimal medium:MS+ 1mg/L 6-BA+0.1 mg/L NAA; T2 is the medium for filtration sterilization:MS+ 1mg/L 6-BA+0.1 mg/L NAA+500mg/L Carb; the medium for screen tobacco is T3:MS+100mg/L Kan;the medium for tobacco subculture is T4:MS+80mg/L Kan+300mg/L Carb; the medium for rooting culture of tobacco is T5:MS+0.1 mg/L Naa+300mg/L Carb+80mg/L Kan.In this study, WIN1 gene was inserted into tobacco k326 by the Plant Genetic Engineering to change the total gene sequences of tobaccok326. PCR and RT-PCR were used to detect WIN1 gene, the results indicate that WIN1 genes was successfully inserted into tobacco k326 and transgenic tobacco k326 is eatablished.In this reserch, WIN1 genes was inserted into tobacco k326 by the Plant Genetic Engineeringand expected results were achieved:cutin membrane become thick and it’s composition changed the drought-resistance, cold-tolerance and disease resistance of tobacco K326 were increased.the ability of resistance of tobacco was improved. The experiment is not only meaningful to reveal the function of plant cutin membrane structure, but also has great important application value in plant gene engineering technology and cultivate quality variety of plants and agricultural production.