生长因子FGF10转基因本氏烟草植株的获得及其表达分析

成纤维细胞生长因子10(FGF10)具有非常重要的科研和医疗价值,但其目前有限产量难以满足市场需要,而植物生物反应器能为解决这一问题提供切实可行的途径。本研究构建了由组成型强启动子CaMV 35S驱动且人工修饰的FGF10基因植物表达载体,通过农杆菌介导法将其导入本氏烟草基因组中,利用筛选标记基因Bar进行草铵膦抗性筛选,获得413个抗性株。经PCR检测鉴定,其中398株为转基因阳性,阳性率为96.4%。通过RT-PCR和ELISA检测分析,最终筛选得到5株高表达转基因后代,其目的蛋白表达量占可溶性总蛋白的0.1%以上,表达量最高的达到0.24%,意味着利用植物生物反应器表达平台生产FGF10蛋白的可行性。这些结果表明,通过该高通量遗传转化平台可以获得具有市场应用潜力的烟草转基因种质资源,并为后续FGF10蛋白相关产品的开发提供了支撑。

An Approach to Obtain Transgenic Plants of Growth Factor FGF10 and Its Expression Analysis in Nicotiana benthamiana

Fibroblast growth factor 10(FGF10)is of very important value in scientific research and medical field,but its limited yield is hard to meet the demand in the market,and plant bioreactors would provide the practical channel to solve this problem.In this study,a plant expression vector harboring the artificially modified FGF10 gene driven by the constitutively strong promoter CaMV 35 S was constructed and introduced into the N.benthamiana genome by Agrobacterium-mediated transformation.A total of 413 glufosinate-resistant plants were obtained after selection by using the marker gene Bar,and 398 of them were positive transgenic plants confirmed by PCR analysis,and therefore the positive rate is 96.4%.Five high-expression transgenic lines with more than 0.1%of the target protein expression among all of the positive transgenic plants were obtained by RT-PCR and ELISA analyses.The highest expression level of target protein can reach up to 0.24%of total soluble protein in the mature leaves of the transgenic lines,indicating the feasibility of production for FGF10 protein by using plant bioreactor expression platform.These results would provide the transgenic tobacco germplasm resources with market potential application by this high-throughput genetic transformation platform technology and offer the support for the development of FGF10-related products subsequently.