| Our study was to make transgenic tomato to produce correctly cholera toxin B subunit (CT-B). Our hope was to use this transgenic tomato as an edible vaccine to stimulate mucosal immune system producing protective antibody. Institutions and industries in most advanced countries actively study transgenic plants as bioreactor to replace the traditional microbie or yeast fermentation system in order to produce high- value pharmaceutical proteins. Using plant seeds to produce foreign proteins could be used as food addition or oral vaccines. Our purpose was to make microbial antigen gene into tomato as an edible vaccine to protect infectual diarrhea. Tomato is a very important fruit in the world, rich in nutrition ,easy to eat raw. We designed to transfer CT-B gene into tomato (L. Esculentum, jingfeng 1) to express CT-B oligerma. We set up effective tomato system through a great deal experiment and got lransgenic tomato plant PCR, Southen blot, ELISA and Western blot proved that Cl-B protein was expressed in tomato. CT-B gene was amplified by polymerase chain reaction using high-fidility DNA polymerase, and then subcloning into 2 middle vectors (pRCTB and pRCTBK) and 4 binary plant vectors (pGA-CTB, pGA-CTBK, pBI-CTB and pBI-CTBK) were constructed. The 3?end of CT-B nucleotide sequence in pBI-CTBK pGA-CTB were added micmsomal retentional signal sequence (SEKDEL). The 4 binary plant vectors , which contained CT-B under control of CaMV 35S promoter and NOS terminator, were introduced into Agrobacterium tumefaciens strain LBA 4404 via electroporation and used to transfer tomato plant. We made an effective tomato plant transform system through a serial experiment. 5 Th~t: ~LM4i B ~ hi order to transfer CT-B gene into tomato plant, we have developed a new and highly efficient tomato regeneration and transformation system by testing different explants and different plant regulators. We found that: I. For the culture of tomato抯 cotyledon, zeatin and 6-BA alone, had~ significant effect on the regeneration of cotyledon. Zeatin was better than 6-BA, which could enhance the prevelance of cotyledon and also increase the number of tomato buds; 2)The combination of cytokine and atudn such as IAA, 2,4-D, NAA had inhibitory effects; 3)213梒ut cotyledon was the best recipient for Agrobacterium-mediated transformation, although the regeneration system of this explant was not better than others including petiol-cotyledon, multicutting cotyledon and so on.; 4) Our protocol of plant transformation system was follow From ten to thirteen days post germination cotyledons cocultivated for 48 hoi.u~s ~ith an overnight gIv~n culture of Agzubaterium Tumefaciens on medium of Murashige and Skoog (MS) adding 1.0 mg/L ZT and 02 mg/L IAA; 5) Cotyledon post-culturation ~4th A. Tumefaciens caning pBI-CTB and pBI-CTBK were directly placed onto the selective medium containing 100 rng/L kanamycin, 500 mg/L cartenicilhin 6) Alter six weeks, kanamycin-resistant shoot regeneration were removed from callus, and lmnsfened to a rooting medium. In plant genetic transformation experiment, we fourrd:l) There was close relationship between transfomiation fi~quency and tomato varieties; 2) The size of plasmid had an effect on tomato transformation frequency. 13 kanamycin-resistant plants were checked for the presence of transgene in leaf tissue using PCR and Southern blot analysis. 10 plants...
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