防龋用转基因番茄外源蛋白表达稳定性研究

The Study of Expression Stability of Exogenous Gene in Transgenic Anti-Caries Tomatoes

摘要: 目的：追踪检测转基因防龋可食番茄中外源蛋白的表达稳定性。方法：田间种植含有编码变异链球菌表面蛋白粘附功能A区(Cell surface protein antigen A, PAcA)、表面蛋白免疫活性P区(Cell surface protein antigen P, PAcP)的外源基因pacA-ctxB、pacP-ctxB的转基因番茄种子，获得连续的四代番茄植株F0，F1，F2，F3。在植株的开花坐果期，每七天提取样本，通过生物分子技术检测外源蛋白的表达。结果：1) 四代转基因番茄中含嵌合基因pacA-ctxB的植株中有31株表达外源蛋白；含pacP-ctxB的植株中有28株表达。2) 果实中外源蛋白PAcA/CTB占总蛋白的含量是：F0代0.248%，F1代0.088%，F2代0.103%，F3代0.078%；PAcP/CTB的含量是：F0代0.308%，F1代0.300%，F2代0.278%，F3代0.176%。3) 在开花坐果期的第6~8周，果实中外源蛋白PAcA/CTB含量分别为0.236%，0.276%，0.257%；PAcP/CTB为0.568%，0.600%，0.554%。结论：防龋疫苗中外源蛋白能够较稳定地表达，表达含量较高；PAcP/CTB的表达阳性率及含量均较PAcA/CTB高；外源蛋白在果实中的表达较叶片高；外源蛋白在坐果期的6~8周的表达最高；同株的不同果实同期表达含量及比重有差异。

Abstract: Objective: To trace the expression stability of exogenous protein PAcA/CTB, PAcP/CTB in the filial generations of the edible anti-caries tomato vaccine. Methods: Four generations of transgenic to-matoes carrying fusion gene pacA-ctxB or pacP-ctxB were planted in the field. Soluble protein of leaves and fruits was extracted every 7 days during fruit-bearing period. Western Blot, BCA and ELISA are used to analyze the content changes of exogenous protein. Results: 1) Exogenous protein is confirmed to be expressed in four generations. 2) The percentage of PAcA/CTB is 0.248% in the F0, 0.088% in the F1, 0.103% in the F2 and 0.078% in the F3. The percentage of PAcP/CTB is 0.308% in the F0, 0.300% in the F1, 0.278% in the F2 and 0.176% in the F3. 3) The percentage of PAcA/CTB during 6th - 8th week was 0.236%, 0.276% and 0.257%; that of PAcP/CTB was 0.568%, 0.600% and 0.554%. Conclusions: Exogenous protein PAcA/CTB and PAcP/CTB were successfully expressed in four filial generations. The proportion of PAcA/CTB was lower than PAcP/CTB. The proportion of PAcA/CTB and PAcP/CTB reached the peak in 6th - 8th week. Expression of exogenous protein in different fruits of one plant at the same time is of significant difference.