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目的:旨在建立一种在红花油体中表达EGF的转基因植物的方法。方法:通过PCR技术把大豆油体基因(DDoil)与EGF构建成融合基因,克隆至植物表达载体pCAMBIA1390R中,构建成植物表达载体p1390Do-EGF,然后转化进农杆菌LBA4404中用于侵染红花外植体,通过甘露糖筛选培养基培养可获得红花转化苗。通过PCR、实时荧光相对定量RT-PCR、SDS-PAGE和Western blot分析目的基因的表达情况,通过MTT法检测EGF的促细胞增殖活性。结果:PCR结果显示,红花叶片中能检测到EGF基因;实时荧光相对定量RT-PCR结果显示,在红花种子中EGF能成功实现转录;SDS-PAGE和Western blot检测证明,在转基因红花种子中能有效表达出EGF,并具有其原有的免疫原性,MTT法实验结果表明EGF具有促进balb/c 3T3细胞增殖的生物活性。结论:大豆油体和EGF融合基因已经成功转化进红花细胞的基因组中,并实现了EGF外源蛋白在红花种子油体中的表达,为EGF蛋白的产业化生产探讨了一种新的生产途径。
OBJECTIVE: To establish a method of expressing EGF transgenic plants in Safflower oil. Methods: The soybean oil body gene (DDoil) and EGF were constructed into a fusion gene by PCR and cloned into the plant expression vector pCAMBIA1390R to construct the plant expression vector p1390Do-EGF, which was then transformed into Agrobacterium LBA4404 for infection with safflower The explants, safflower transformed seedlings can be obtained by mannose selection culture medium. The expression of target gene was analyzed by PCR, real-time fluorescence quantitative RT-PCR, SDS-PAGE and Western blot. The cell proliferation activity of EGF was detected by MTT assay. Results: The results of PCR showed that the EGF gene was detected in leaves of safflower. The results of real-time fluorescence quantitative RT-PCR showed that EGF could be successfully transcribed in safflower seeds. The results of SDS-PAGE and Western blot showed that in transgenic safflower EGF can be effectively expressed in seeds and has its original immunogenicity. The results of MTT assay showed that EGF has the biological activity of promoting the proliferation of balb / c 3T3 cells. CONCLUSION: The soybean oil body and EGF fusion gene have been successfully transformed into the genome of safflower cells, and the expression of the foreign protein of EGF in oil safflower seed oil has been achieved, and a new type of EGF protein industrialization has been explored Production path.