| ETEC (Enterotoxigenic Escherichia coli) is the most common cause of diarrhea in human being and Enterotoxin and Colonization Factor Antigens (CFAs) play the main factor to cause diarrhea. ETEC can adhere to the epithelial by means of proteinaceous surface structures called colonization factors (CFs) to cause diarrhea. An effective ETEC oral vaccine can evoke responses of anti-colonization and anti-toxin. If ETEC vaccine contains Colonization Factor Antigen such as CFA/I and CS1-CS6, associating with Enterotoxin (LT), it can, theoretically, protect the user from diarrhea by percent 80 ETEC attacking. CSS is the one of the colonization factor antigens which is a protective antigen can cause immune reaction by the means taking orally. In this study, carrot was separately transformed with Agrobacterium tumefacience strain LBA4404 that contains CTB or CTB-CS3 fused gene in order to get oral diarrhea vaccine. This is a potent strategy to produce ETEC oral vaccine. In addition, we also focused on constructing a plant virus-based gene vector PVX to express LTB-ST fused gene by means of agroinfection. The possibility of producing vaccine was probed through plant virus-based gene vector expression system.The hormone condition and medium type of two cultivars "New Touxinhong" and "AC93" were established via somatic embryogenesis. The embryogenic callus of "New Touxinhong" was induced on the medium MSo supplemented with 0.1mg/L 2, 4-D, and embryo could produce only when the calli were transferred to MSo medium. The embryogenic calli of "AC93" were induced on the medium 850 supplemented with 0.2mg/L 2,4-D, and embryo could produce only when the calli were transferred to B50 medium. The embryo of these two varieties can grow into healthy plantlet after being transferred to medium with no hormone. The condition of somatic embryogenesis under different hormone conditions was discussed. The amount of embryo decreased but the amount of calli increased as the increase of KT concentration under the same 2,4-D level after embryogenesis calli were transferred to medium with no hormone. The suitable concentration of kanamycin for selecting transgenic explants in the course of carrot tissueculture was screened in order to strictly select transformant. The optimum concentration of kanamycin was different at different stage of culture, which was 50mg/L at calli induction stage,100mg/L at embryogenic calli selecting stage, and 50mg/L at plantlet root elongating stage, respectively. In all treatments of aseptic seedlings under 4℃ for different days, the treatment under 4℃ for 4 days produced the most amount (80%) of resistant embryogenic calli induced from hypocotyls. It has been obvious that the physiological state of explants has an effect on transformation.The optimum duration of co-cultivation of "New Touxinhong" hypocotyls together with Agrobacterium tumefacience PBinCTBS was determined among 1,2, 3, 5,7days, and which was 2-3 days when the hypocotyls was obvious surrounded by the Agrobacterium. The age of the explants was also studied to analyze the effect of the explants age on the genetic transformation. Hypocotyls were excised from 7, 11 and 30 days old aseptic seedling to transform under the same condition. It showed that the induction efficiency of the resistant calli were the same among all explants of different age, but the embryos on the calli from explants of 7 days appeared earlier than those from explants 11 and 30 days. In order to compare the resistant calli induction efficiency, petioles and hypocotyls were excised from 30days aseptic seedlings to transform with PBinCTBS under the same condition. The resistant calli induction efficiency of petioles was higher than those of hypocotyls. A more detailed analysis of the different types of explants showed that the number of PCR positive plants induced from petioles was higher than that from hypocotyls. This conclusion is corresponding with that of Pawlicki's.The result of PCR amplification, PCR-Southern, Southern blotting showed that CTB or CTB-CS3 was stabl...
|
PDF Full Text Request