大豆24 kDa油体蛋白基因与aFGF融合转化红花的研究  

Study on human aFGF fusion gene transformation with soybean 24 kDa oleosin and expression in safflower

作　　者：盖玉红[1] 王旺[2] 金潇[2] 王嘉琪[2] 李宏博[2] 王刚[2] 杨晶[3] 魏健[2,3] 

机构地区：[1]吉林农业大学农学院,吉林长春130118 [2]长春师范大学生命科学学院,吉林长春130032 [3]吉林农业大学生物反应器与药物开发教育部工程研究中心,吉林长春130118

出　　处：《中国中药杂志》2013年第12期1898-1904,共7页China Journal of Chinese Materia Medica

基　　金：国家高技术研究发展计划(863)项目(SQ2010AA1000691008);教育部科技创新工程重大项目培育基金项目(70S018);吉林农业大学2012年校内基金项目

摘　　要：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 investigate the expression of acidic fibroblast growth factor （aFGF） in transgenic safflower and lay the foundation for the use of the plant bioreactor large-scale production aFGF. Method： The haFGF gene was transformed into plant preference of the aFGF sequence as a basis for design of primers, plant preferences aFGF gene sequences was amplified by PCR. The vegetable body expression vector was constructed by using digested connection method and then transferred to Agrobacterium tumefa- ciens EHA105 by the freeze-thaw method. It transferred to safflowers by agrobacterium-mediated transformation method, and identified by PCR,southem blot and RT-PCR. Result： The full-length aFGF gene sequences were amplified through PCR and constructed into plant expression vector with soybean oleosin and promoter, and transformed into safflower. Three independently transformed safflower plant units with point insertion were successfully obtained, which showed the same size of aFGF expression at the transcriptional level. Conclusion： The plant oil body expression vectors were successfully constructed, and the optimal condition for genetic transformation was selected. The transgenic safflower plants were obtained.

关 键 词：AFGF 油体蛋白 红花 

分 类 号：R346[医药卫生—基础医学]