南方医科大学学报2012，Vol.32Issue(6)：772-777,6.DOI:10.3969/j.issn.1673-4254.2012.06.004
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
A hydroponic cultivation system for rapid high-yield transient protein expressioin in Nicotiana plants under laboratory conditions
摘要
Abstract
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.关键词
烟草/瞬时表达/无土栽培/绿色荧光蛋白Key words
Nicotiana plants/transient expression/hydroponic cultivation/green fluorescent protein分类
生物科学引用本文复制引用
莫倩珍,麦荣嘉,杨志晓,陈敏芳,杨铁钊,赖华芳,杨培梁,陈强,周晓红..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[J].南方医科大学学报,2012,32(6):772-777,6.基金项目
国家自然科学基金(30872204,30400367,81071378) （30872204,30400367,81071378）
国家留学基金(留金发[2010]009号) （留金发[2010]009号）
南方医科大学公共卫生与热带医学学院院长基金(GW201109) （GW201109）
