利用转基因番茄表达人成纤维细胞生长因子21(FGF21)

Expression of fibroblast growth factor 21 in transgenic tomato plants

【目的】在番茄中表达人成纤维细胞生长因子-21(Fibroblast growth factor-21,FGF21),为利用植物反应器规模化生产FGF21奠定基础。【方法】以甘露糖-6-磷酸异构酶基因(pmi)作为转基因植物的选择标记,将人源fgf21基因克隆至表达载体pCAMBIA1390RⅡ(简写为p1390RⅡ)上,构建重组表达质粒p1390 RⅡMFGF21。采用冻融法将质粒p1390RⅡMFGF21转入根瘤农杆菌EHA105,叶盘法转化番茄(Lycopersicon esculentum)"中蔬6号",采用PCR检测、Southern杂交筛选转基因番茄阳性植株,并对FGF21蛋白在转基因番茄叶片中的表达进行了Western blot分析。【结果】成功构建了带pmi安全选择标记基因的植物双元表达载体p1390RⅡMFGF21,在番茄中初步建立了以pmi为选择标记基因的遗传转化体系,共获得了26株转基因番茄植株,其中5株为阳性克隆,转化率为19.2%。采用PCR扩增和Southern blot分析进行检测,结果表明,重组fgf21基因已整合到转基因番茄基因组中。Western blot分析检测结果显示,FGF21蛋白在转基因番茄叶片中有一定水平的表达,并具有良好的抗原性。【结论】获得了以pmi为选择标记成功表达FGF21蛋白的转基因番茄遗传体系。

【Objective】 The study investigated the expression of human fibroblast growth factor 21（FGF21） in transgenic tomato plants.【Method】 Phosphomannose isomerase gene（pmi） was used as a selection marker for transgenic plants;the plant expression vector p1390RⅡMFGF21 was obtained by cloning fgf21 gene into the pCAMBIA1390RⅡ（p1390RⅡ） vector.After p1390RⅡMFGF21 was transferred into Agrobacterium tumefaciens strain EHA105,then fgf21 gene was introduced into cultivated tomato（Lycopersicon esculentum） 6 via Agrobacterium-mediated leaf disc transformation.The positive transgenic tomato plants were determind by PCR,Southern blotting,and Western blotting analysis.【Result】 The plant binary expression vector p1390RⅡMFGF21 that carries pmi selection marker was successfully constructed,and five positive tomato plants which correctly accumulated FGF21 protein were obtained from twenty-six transgenic plants,the transformation efficiency value being 19.2%.Subsequent PCR and Southern blotting analyses confirmed that fgf21 gene was integrated into the genome of tomato plants.Western blotting analysis showed transgenic tomato plants had positive expression of FGF21 protein and good antigenicity.【Conclusion】 Human fgf21 was successfully expressed in transgenic tomato plants using pmi as a selectable marker.