农杆菌介导的棉花子叶瞬时表达系统的建立

The Establishment of Agrobacterium-mediated Transient Expression System in Cotton Cotyledons

植物瞬时表达系统在研究功能基因的亚细胞定位、蛋白质互作、启动子活性等方面具有重要作用。选取棉花(Gossypium hirsutum)子叶为材料,以β-葡糖醛酸酶基因(GUS)、增强型绿色荧光蛋白基因(eGFP)为报告基因,对棉花子叶生长时间、农杆菌浓度、注射量、共培养时间等因素进行了分析,建立了农杆菌介导的棉花子叶瞬时表达系统。结果表明,对萌发6天的棉花子叶注射50μLOD600为0.5的含目的基因的农杆菌LBA4404或GV3101,共培养2–3天后,外源基因表达效率最高。利用此方法从棉花萌发到观测结果,仅需8–13天,且操作简单易行,可快速检测棉花来源的启动子活性、亚细胞定位、蛋白质互作等。该方法在棉花基因功能研究方面有很好的应用价值。

The plant transient expression system plays an important role in analysis of subcellular localization, interaction and promoter activity of protein. Here, we established a transient expression system in cotton cotyledons based on Agrobacterium-mediated transformation with GUS and eGFP as reporter genes. We investigated the optimal conditions for the system, including cotyledon growth time, Agrobacterium concentration, injection volume and co-culture time. The highest efficiency of exogenous gene expression was 50 μL Agrobacterium LBA4404 or GV3101 with OD600=0.5 and co-culture for 2–3 days in cotton cotyledons germinated for 6 days. The transient expression system is convenient and only needs 8 to 13 days to detect exogenous protein expression from germination to results. The system may have a good application for analysis of gene function in cotton.