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Human Epidermal Growth Factor Gene Cloning And Genetic Transformation In Plant Mediated By Agribacterium Tumefaciens

Posted on:2003-10-31Degree:DoctorType:Dissertation
Country:ChinaCandidate:T S ChenFull Text:PDF
GTID:1100360065456303Subject:Crop Cultivation and Farming System
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Banana(Musa spp.) is one of the most important fruits in the south of China and there are many problems in banana industry at present such as the quality of fruits, the serious diseases and insect pests etc. Some useful foreign genes could be transfered into banana by genetic engineering to improve banana varieties and enhance the competition ability of banana production in market. Banana could be served as bio-reactor to produce some medical proteins which has broad perspectives.In the present study, the human epidermal growth factor(hEGF) was cloned by polymerase chain reaction(PCR), and both plant and Escherichia coli expression vectors were constructed, and bio-activities of the protein expression by Escherichia coli were studied. Meanwhile, the regeneration system of both banana and aloe using the thin cell layer was optimized, and banana transformed with the gene hEGF for the first time, and the gene was expressed in transgenic banana buds. At the same time, the factors for both banana and aloe genetic transformation mediated by Agrobacterium tumefaciens were optimized. The results were as follows:1. The human epidermal growth factor was cloned by PCR.2. Two expression plasmids were constructed for transformation. In the plant plasmid plSOlhEGF, the CaMV35S promoter and nos terminator fragments were syncretized with reporter gene and targeted gene. The selectable maker gene terminated by polyA fragment for transformation of plants was hygromycin phosphotransferase gene which allowed selection with hygromycin, and the selectable marker gene for plasmids was neomycin phosphotrasferase gene, which allowed selection with kanamycin.3. The fusion protein GST-hEGF expressed in Escherichia coli in the experiments with mice showed the recombinant protein had good immunogenecity and the purifid protein hEGF could stimulate the growth of Hela cells efficiently.4. The regeneration system of banana using the thin cell layers with the materials from lowest generation was optimized.5. The transformation of banana thin cell layers via particle bombardment was tested but no transgenic bud was obtained.6. An efficient banana transformation system mediated by Agrobacterium tumefaciens was established. Among three Agrobacterium tumefaciens strains that is, EHA105, LBA4404 and AGL1, the banana thin cell layers were the most sensitive to EHA105, and various factors that affect the genetic transformation of banana via Agrobacterium-mediated such as acetosyringone(AS), sucrose, the pH value of resuspension solution, condition of co-culture, strains of Agrobacterium tumefacients, time of co-culture and the preparation of Agrobacterium tumefacients liquid using for transformation were optimized.7. For the first time, banana was transformed with the gene hEGF mediated only by Agrobacterium tumefacients. The presence and integration of foreign DNA in the transgenic banana buds were confirmed by polymerase chain reaction(PCR), PCR-Southern blotting and genomic dot-blotting analysis.8. The regeneration system of aloe also was optimized by using the thin cell layers with the materials from plantlets and many factors that influence the bud differentiation of aloe thin cell layers were optimized.9. Aloe was transformed with the gene hEGF mediated by Agrobacterium tumefacients and obvious GUS gene transient expression were observed, and a genetic transformation system of aloe was preliminarily set up.
Keywords/Search Tags:human epidermal growth factor(hEGF), banana, aloe, genetic transformation, thin cell layer
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