快速高效植物瞬时表达的实验室烟草无土栽培体系的构建

摘要

Objective To develop a hydroponic Nicctiana cultivation system for rapid and high-yield transient expression of recombinant proteins under laboratory conditions.Methods To establish the hydroponic cultivation system,several parameters were examined to define the optimal conditions for the expression of recombinant proteins in plants.We used the green fluorescent protein (GFP) and the geminiviral plant transient expression vector as the model protein/expression vector.We examined the impact of Nicotiana species,the density and time of Agrobacterium infiltration,and the post-infiltration growth period on the accumulation of GFP.The expression levels of GFP in Nicotiana leaves were then examined by Western blotting and ELISA.Results Our data indicated that a hydroponic Nicotiana cultivation system with a light intensity of 9000 LX/ layer,a light cycle of 16 h day/8 h night,a temperature regime of 28 ℃ day/21℃ night,and a relative humidity of 80% could support the optimal plant growth and protein expression.After agroinfiltration with pBYGFPDsRed,R/LBA4404,high levels of GFP expression were observed in both N.benthamiana and N.tobaccum (cv.Yuyan No.5) plants cultured with this hydroponic cultivation system.An optimal GFP expression was achieved in both Nicotiana species leaves 4 days after infiltration by Agrobacterium with an OD600 of 0.8.At a given time point,the average biomass of N.tobaccum (cv.Yuyan No.5) was significantly higher than that of N.benthamiana.The leaves from 6-week-old N.benthamiana plants and 5-week-old N.tobaccum (cv.Yuyan No.5) plants could be the optimal material for agroinfiltration.Conclusion We have established a hydroponic cultivation system that allows robust growth of N.benthamiana and N.tobaccum (cv.Yuyan No.5) plants and the optimal GFP expression in the artificial climate box.%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4d;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.