The Construction Of Fmdv Plant Expression Vectors And Transformation Into Soybean

Much attention has been focused on the plant-derived oral vaccine production. Compared with the traditional vaccines, the plant-derived oral vaccines are safe, stable, efficient and low-costed. Plant as the delivery vehicle for vaccine can express antigen in edible tissue such as seeds and tubers. The plant-derived vaccine can induce protective mucosal immune response when the transgenic plants are provided as food or feed. With the advances of plant biotechnology, plant bioreactor may become an available approach and will be an alternative means for vaccine production in the future. Foot and Mouth Disease(FMD) is a highly contagious viral disease, leading to widespread outbreaks in the world. So far there is still none medicine to cure this disease, and the commercial yeast-derived vaccines are widely used. But the high produce cost, cold requirement during store and carry, and requiring specialties when inoculating restrict the utilization of the vaccine. Transgenic plant as plant-derived vaccine is prospect in the production of FMD vaccine. The antigen of FMDV has been transformed into tobacco, potato, and alfalfa et al. but there is no report in soybean yet. Primers of FMDV-P1(FP1) gene (including FVP1, the procedure same as FP1 gene) were designed according to the nucleotide sequence with the endonuclease recognition site BamHI and SaLI. The FP1 gene was amplified from pGEM-FP1 vector by polymerase chain reaction (PCR) and inserted into the pUCM-T vector constructed to pUCM-FP1. The DNA sequence analysis results showed that the amplified target fragment was correct completely. The pUCM-FP1 vector and plant expression vector pBin438 were digested with corresponding restriction endonuclease and constructed to pBin-FP1. The recombinant plant expression vector was transferred into E.coll JM109. After identification by restriction enzymes and PCR, the correct clone was transferred into Agrobacterium tumefaciens GV3101 by electrotransformation. we constructed two plant expression vectors successfully, named pBin-FP1 and pBin-FVP1. GV3101 which contained the pBin-FP1 and pBin-FVP1 plant expression vectors was used to infect soybean.The FMDV gene was introduced into soybean with Agrobacterium-cotyledonary node transformation. cotyledonary node explants were prepared from 5-7 day old seedings, infected and co-cultivated with GV3101. Adventitious buds emerged after 10 days on shoot induction medium. the buds were transferred onto elongation medium and selected with 50μg/ml Kanamycine. The shoots were cut off and transferred onto rooting medium when the shoots grew to 3-4 cm high. We abtained 51 kanamycine resistant plants. 27 of them had already flowered and seeded normally, 15 of which were PCR positive. PCR analysis proved that foreign gene was transferred into soybean genome. Candidate: Zhu HuiyingSpeciality: BotanySupervisor: Hu Baozhong Han Tianfu...