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Preliminary Study Of Transforming Of Helicobacter Pylori Antigens In Cherry Tomato

Posted on:2015-09-22Degree:MasterType:Thesis
Country:ChinaCandidate:W Q XieFull Text:PDF
GTID:2180330452951340Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
Helicobacter pylori is the major causal factor in gastrointestinal disorders such aschronic gastritis, peptic ulcer and gastric cancer. Through the2A sequences of FMDV,hpaA or ureB gene of Helicobacter pylori and LTB are assembled in one vector under thecontrol of fruit-specific promoter. The fusion gene is transformed into the genome ofcherry tomato (Lycopersivon esculentum Mill.) by the agrobacterium-mediatedplant transformation protocols. This is to lay the foundation for producing Helicobacterpylori oral vaccine by cherry tomato.CHE8-2391Z, CHE8-HpaA-eLTB and CHE8-UreB-eLTB fusion expression vectorhave been constructed, and the antigen expression strategy has been optimized. Tooptimize the cherry tomato regeneration system, effects of different hormonecombinations on callus induction, bud differentiation and roots induction of cherrytomato are studied. We also detect effects of kanamycin, cephalosporin and carbenicillinto the cotyledons regeneration to further optimize the transformation protocol. Thenumber of integrated copies following genetic transformation are estimated by real-timePCR (qPCR). RT-PCR are used to validate transcriptional expression of exogenous gene.The target protein are detected by total soluble protein extraction and the followingSDS-PAGE. To explain the low fruit-setting rate of transgenic plants, differences inpollen viability between non-transgenic plants, CHE8-2391Z transformed plants andCHE8-HpaA-eLTB transformed plants are explored by TTC staining. The mainresults are as follows:1. CHE8-2391Z, CHE8-HpaA-eLTB and CHE8-UreB-eLTB expression vectorshave been constructed.2. There are23,21and3transgenic plants which are transformed by CHE8-2391Z、CHE8-HpaA-eLTB and CHE8-UreB-eLTB vectors, respectively.3. The optimum medium to induce callus and regeneration buds is MS+6-BA2.5mg/L+IAA0.2mg/L, with the induction of callus rate up to100%and regeneration budsaveraged five. The optimum medium to induce roots is1/2MS+0.2mg/L IAA.4. The highest rate of callus induction is achieved when transformed cherry tomatoby Agrobacterium at OD600≈0.4. Kanamycin at50mg/L is the optimum concentration to select transformed cotyledons. The optimal concentration of cephalosporin andcarbenicillin to remove agrobacterium is both200mg/L.5.8,4and0transgenic plants which transformed by CHE8-2391Z, CHE8-HpaA-eLTB and CHE8-UreB-eLTB vectors are validated by PCR. The expression of targetgene at the transcriptional stage are detected in the fruits of CHE8-HpaA-eLTBtransgenic plants.6. qPCR show that transgene copy number in cherry tomatoes from0-20. Some ofthe transgenic plants which has been validated by PCR showed no target gene integration.Rearrangements occur in80%of the transformed plants.7. The pollen viability differs between transgenic and non-transgenic cherry tomato.The pollen viability of non-transgenic plants, CHE8-2391Z transformed plants andCHE8-HpaA-eLTB transformed plants are31%,4%and45.45%, respectively.This is the study that has both optimized the regeneration system and genetictransformation system of cherry tomato, which has laid the foundation for the furtherstudy of cherry tomatoes as transgenic material. The transgenic cherry tomato containinga single copy of hpaA could be used in the further study of the immunogenicity of cherrytomato. In our study, the number of integrated copies following genetic transformationare estimated by real-time PCR, and the possibility of apx genes as single copyendogenous reference gene for cherry tomato has been ruled out.
Keywords/Search Tags:Helicobacter pylori, cherry tomatoes, plant oral vaccine, hpaA, ureB, LTB
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