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目的构建适用于快速高效植物瞬时表达系统的实验室烟草无土栽培体系。方法优化烟草的无土栽培条件,基于Geminivirus新型植物高效瞬时表达系统,观察烟草品种、侵染时机、侵染工程菌浓度、侵染后叶片采收时间对无土栽培烟草表达绿色荧光蛋白(GFP)的影响,以Western blot和ELISA分析GFP表达情况。结果烟草无土培育的最佳条件为:光照强度9000LX/层,光照时间16 h/d,日/夜温度28/21℃,相对湿度80%。侵染工程菌的最佳D600为0.8;叶片采收最佳时间为侵染后4 d;本明烟和豫烟5号最佳侵染时机分别为第6周和第5周;工程菌pBYGFPDsRed.R/LBA4404侵染的本明烟和豫烟5号均能高效表达GFP;豫烟5号的平均生物量高于本明烟。结论基于人工气候箱成功构建了实验室烟草无土栽培体系,并可实现GFP在本明烟和豫烟5号的快速高效表达。
Objective To construct a laboratory tobacco soilless culture system suitable for rapid and efficient plant transient expression system. Methods The soilless culture conditions of tobacco were optimized. Based on the new highly efficient transient expression system of Geminivirus, the effects of tobacco varieties, the timing of infection, the concentration of infecting engineering bacteria and the harvest time of leaves on the expression of green fluorescent protein (GFP) ), Western blot and ELISA analysis of GFP expression. Results The optimum conditions of tobacco soilless culture were light intensity 9000LX / layer, light time 16 h / d, day / night temperature 28/21 ℃ and relative humidity 80%. The optimal D600 for infecting engineering bacteria was 0.8; the best time for leaf harvesting was 4 days after infection; the best time of infection for Benmingyan and Yuyan 5 was the 6th week and 5th week respectively. The engineered bacteria pBYGFPDsRed The R / LBA4404 infected tobacco and Yuyan 5 could express GFP efficiently. The average biomass of Yuyan 5 was higher than that of the present invention. Conclusion The laboratory tobacco soilless cultivation system was successfully constructed based on the artificial climate chamber, and the rapid and efficient expression of GFP in Benmingyan and Yuyan 5 was achieved.