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目的:利用红花表达酸性成纤维细胞生长因子(acidic fibroblast growth factor,aFGF),为利用植物生物反应器规模化生产aFGF奠定基础。方法:将人源aFGF基因改造为植物偏好性的aFGF基因序列,通过PCR搭桥技术克隆植物偏好的aFGF基因,利用酶切连接方法构建植物油体表达载体,通过冻融法转到根瘤农杆菌EHA105,利用农杆菌介导的方法转化到塔城红花中,对转化红花进行PCR,Southern杂交,RT-PCR分子检测。结果:扩增出植物偏好的aFGF基因的全长序列,并与大豆油体蛋白基因Doil基因融合,成功地插入到改造好的含有大豆油体蛋白启动子表达载体中,探索了进行红花遗传转化的最佳条件,成功获得单位点插入的3个独立转化红花植株,在转录水平都有大小一致的aFGF的表达。结论:该实验成功构建了含aFGF植物油体表达载体,并筛选出最佳的遗传转化条件,A为0.6,侵染时间为10 min,共培养3 d,获得了aFGF转录水平表达的转基因红花植株,这将红花油体蛋白本身的皮肤保护作用,与FGFs的创伤修复作用累加,快速开发出外用创伤药物奠定基础。
OBJECTIVE: To express acidic fibroblast growth factor (aFGF) by safflower and lay foundation for the large-scale production of aFGF using plant bioreactor. Methods: The human aFGF gene was transformed into a plant-preferred aFGF gene sequence. The plant-preferred aFGF gene was cloned by PCR-based bridging technique. The plant oil expression vector was constructed by restriction enzyme digestion and transferred to Agrobacterium tumefaciens EHA105 by freeze- The Agrobacterium tumefaciens-mediated transformation into Tara sativa was carried out, and the transformed safflower was detected by PCR, Southern hybridization and RT-PCR. Results: The full-length sequence of plant aFGF gene was amplified and fused with the Doil gene of soybean oil body protein. The fusion gene was successfully inserted into the engineered expression vector containing soybean oil body protein promoter. Transformed successfully, three independently transformed plants of saffron were successfully obtained, and the same size of aFGF was expressed at the transcriptional level. CONCLUSION: This experiment successfully constructed the aFGF plant oil-bearing expression vector and screened the best genetic transformation conditions, A was 0.6, the infection time was 10 min, co-cultured for 3 d, obtained aFGF transcript level expression of transgenic safflower Plant, which will safflower oil body protein itself skin protection, and FGFs trauma repair accumulated, rapid development of topical trauma drugs to lay the foundation.