| Caries is a bacterial infectious disease, serious harm to human health. Therefore, to explore the effective measures to control caries is an urgent task of today's caries research. Immunization against dental caries is an effective way of controlling caries. Preparation of transgenic plants to produce vaccines with the advantages such as simple, low cost, oral administration has been directly caused widespread concern of domestic and overseas.Objectives:To detect the expression of exogenous recombinant protein PAcA/CTB, PAcP/CTB in transgenic tomatoes, and to analyze the content changes of exogenous recombinant protein in the transgenic tomato, fruits and leaves, to determine the best harvest period of fruits, discuss the changes in the mechanisms of exogenous recombinant protein, and obtain a stable and effective expression of exogenous recombinant protein in individual transgenic tomato plants, so as to make a foundation for produce edible anticaries vaccine and commercialized cultivation of the transgenic tomatoes by using the transgenic technology in the next step.Methods:Extracting soluble protein from transgenic fruit, using Western Blot to detect the expression of exogenous recombinant protein PAcA/CTB, PAcP/CTB in transgenic tomatoes fruit, using ELISA to calculate the exogenous recombinant protein PAcA/CTB, PAcP/CTB of the total soluble protein in fruit and leaves and analyse the change of the content.(1) Extracting soluble protein from transgenic fruit, using Western Blot to detect the expression of exogenous recombinant protein in transgenic tomatoes, making a statistics of the total positive incidence and the positive incidence of exogenous recombinant protein PAcA/CTB, PAcP/CTB in transgenic tomatoes with generation F0, F1, F2, F3.(2) Transgenic tomato feeding and field planting;(3) Sampling, Every 7 days collecting young leaves and fruits samples in the Plant flowering fruit-bearing period;(4) Using BCA to detect the content of the soluble protein in the fruit and young leaves of transgenic tomato;(5) Using ELISA to detect the concentration of exogenous recombinant protein expression in the leaves and fruits of transgenic tomatoes and to calculate the content percentages of the exogenous recombinant protein through the content of the total soluble protein;(6) Observing the percentages of the content of the exogenous recombinant protein changes and to studying the following:A. Expression differences of four generations exogenous recombinant protein PAcA/CTB and PAcP/CTB in transgenic tomatoes at the same time.B. Expression changes of the exogenous recombinant protein PAcA/CTB and PAcP/CTB in the leaves and fruits of the transgenic tomatoes followed as the content changes in the growth process of the transgenic tomatoes.C. The content of exogenous recombinant protein expressed in one transgenic tomato plant in the fruit period.Results:1. Target protein strip is separated by the SDS-PAGE electrophoresis; Molecular size for 5.5 x 104KDa confirmed by Exposure, coincided with the theoretical molecular weight of exogenous recombinant protein PAcA/CTB, PAcP/CTB.2. Detecting the total protein of plant by Western Blot,31 plant of transgenic tomato contains exogenous protein PAcA/CTB (9 trees in the F0,9 trees in the F1,7 trees in the F2,6 trees in the F3); The positive rate of transgenic tomato exogenous recombinant protein PAcA/CTB was 70.45%.28 plants express the exogenous protein PAcP/CTB (9 trees in the F0,8 trees in the F1,4 trees in the F2,7 trees in the F3), and the positive rate of the transgenic tomato exogenous recombinant protein PAcP/CTB was 63.6%.3. Detecting the total protein content by the BCA and extracted the soluble protein of fruit, detecting the content of exogenous recombinant protein by the way of ELISA, and calculating exogenous recombinant protein content percentage of the total soluble protein. we found that the pencentage of exogenous recombinant protein PAcA/CTB is 0.248% in the F0 in the same period,0.088% in the F1, 0.103% in the F2,0.078% in the F3; and the pencentage of exogenous recombinant protein PAcP/CTB is 0.308% in the F0,0.300% in the F1,0.278% in the F2, 0.176% in the F3.4. In the growth of the transgenic tomato plant, we found that the content of the recombinant protein was the most between 6-8 weeks. The percentage of exogenous recombinant protein PAcA/CTB was 0.236% in the sixth week, 0.276% in the seventh week,0.257% in the eighth week; the percentage of exogenous recombinant protein PAcP/CTB was 0.568% in the sixth week,0.600% in the seventh week,0.554% in the eighth week.5. The recombinant protein content was expressed in one transgenic tomato plant. The percentage of exogenous recombinant protein in plant A15 was 0.627%, 0.467%, and 0.463%; The percentage of exogenous recombinant protein in plant A17 was 0.379%,0.496%,0.233%; The percentage of exogenous recombinant protein in plant P15 was 0.580%,0.481%,0.758%; The percentage of exogenous recombinant protein in plant P19 was 0.498%,0.692%,0.597%.Conclusion:1. Exogenous recombinant protein PAcA/CTB and PAcP/CTB in every generation of transgenic plants was successfully expressed in the fruits. Positive incidence of exogenous recombinant protein PAcA/CTB and PAcP/CTB are tending to degressive tendency. The positive incidence of Exogenous recombinant protein PAcP/CTB was significantly reduced in every generation of transgenic plants compared with exogenous recombinant protein PAcA/CTB of F1 generation that express the greater degree of reduction of the positive incidence than PAcA/CTB.2. Observing the expression of exogenous recombinant protein in transgenic tomato at the same time, we found that the content of exogenous recombinant protein are tending to a degressive tendency followed as hereditary. And the content of the exogenous recombinant protein PAcA/CTB was at a lower level than the content of the exogenous recombinant protein PAcP/CTB in F0, F1, F3 and F3 generations of plant.3. The proportion of the exogenous recombinant protein PAcA/CTB in plant was at a lower level than the proportion of the exogenous recombinant protein PAcP/CTB in the whole process of growth in plant. And the proportion of the recombinant protein PAcA/CTB, PAcP/CTB was reached the highest level almost at the same period of 6-8 weeks in transgenic tomato.4. Expression of exogenous recombinant protein PAcA/CTB and PAcP/CTB in one transgenic tomato plant with different mature fruits at the same time has significant difference. |
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