烟草基因瞬时表达体系的建立与优化研究
Establishment and Optimization of Agrobacterium-Mediated Transient Gene Expression System in Tabacco

作者: 文莉薇 , 朱鸿亮 :中国农业大学,食品科学与营养工程系,北京;

关键词: 烟草叶片瞬时表达Tobacco Leaf Transient Expression System

摘要:
本研究以GUS基因和RIN基因作为报告基因,利用农杆菌(Agrobacterium tumefaciens) GV3101对烟草叶片注射侵染,探究了农杆菌侵染浓度对本氏烟草(Nicotiana benthamiana)叶片瞬时表达效果的影响,建立并优化了烟草中的瞬时表达体系。结果表明,β-葡萄糖苷酶(GUS β-glucuronidase)基因与RIN基因在OD600 = 1.0的侵染浓度时表达效果最佳。农杆菌介导的烟草叶片瞬时表达方法简单高效,结果准确可靠,从种子播种到收获蛋白只需25天左右。该体系的建立与优化为基因表达和蛋白功能性研究等方面提供了一定支持。

Abstract: To establish and optimize a transient expression system in Nicotiana benthamiana, the method was developed with the β-glucuronidase (GUS) and Ripening Inhibitor (RIN) as marker genes. Using the agrobacterium-mediated transformation method, GV3101 was used for the effects of different bacteria concentration on the efficiency of protein transient expression in Nicotiana benthamiana. Observed by the results, a higher transient expression level of GUS & RIN gene could be obtained as OD600 value of A. tumefaciens for intiltration 1.0. The entire process only took 25 days from sowing seed to protein analysis. Therefore, this method is simple and rapid. It has a potential application in dissecting gene expression and function in Brassica napus.

文章引用: 文莉薇 , 朱鸿亮 (2015) 烟草基因瞬时表达体系的建立与优化研究。 植物学研究, 4, 25-31. doi: 10.12677/BR.2015.42004

参考文献

[1] Goodin, M.M., Zaitlin, D., Naidu, R.A. and Lommel, S.A. (2008) Nicotiana benthamiana: Its history and future as a model for plant-pathogen interactions. Molecular Plant-Microbe Interactions, 21, 1015-1026.

[2] Bombarely, A., Rosli, H.G., Vrebalov, J., Moffett, P., Mueller, L.A. and Martin, G.B. (2012) A draft genome sequence of Nicotiana benthamiana to enhance molecular plant-microbe biology research. Molecular Plant-Microbe Interactions, 25, 1523-1530.

[3] 谭小力, 诸葛锐军, 李冠英, 卢长明, 王政, 张志燕 (2013) 农杆菌介导的油菜子叶瞬时表达. 生物学杂志, 6, 93-96.

[4] 吴英杰, 姜波, 张岩, 李彦邦, 贺琳, 王玉成 (2010) 农杆菌介导的烟草瞬时表达试验条件优化. 东北林业大学学报, 9, 110-112.

[5] 赵文婷, 魏建和, 刘晓东, 高志晖 (2013) 植物瞬时表达技术的主要方法与应用进展. 生物技术通讯, 2, 294-300.

[6] 王长松, 刘友生, 陈燕平 (2005) 蛋白质体外表达与进化技术. 生命的化学, 4, 333-336.

[7] Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. (1987) GUS fusions: Be-ta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. The EMBO Journal, 6, 3901-3907.

[8] Martel, C., Vrebalov, J., Tafelmeyer, P. and Giovannoni, J.J. (2011) The tomato MADS-box tran-scription factor RIPENING INHIBITOR interacts with promoters involved in numerous ripening processes in a COLORLESS NONRIPENING-dependent manner. Plant Physiology, 157, 1568-1579.

[9] 侯文秀, 李景富, 刘剑峰, 康立功, 张贺, 许向阳 (2010) 番茄成熟突变体rin基因的AFLP分子标记. 东北农业大学学报, 02, 20-24.

[10] 王辉, 李文丽, 王富, 石丽 (2008) 番茄成熟突变体rin的生长特性和果实耐贮性的初步研究. 青岛农业大学学报(自然科学版), 1, 21-23.

[11] Zheng, L., Liu, G.F., Meng, X.N., Li, Y. and Wang, Y. (2012) A versatile agrobacterium-mediated transient gene expression system for herbaceous plants and trees. Biochemical Genetics, 50, 761-769.

[12] 刘敏, 滕文静, 董清华, 晁慧娟, 李凤岚, 邱德有 (2009) 草莓果实外源基因瞬时表达系统的建立. 果树学报, 1, 60-65.

[13] 李静, 陈敏, 刘现伟, 沈法富, 王鹏 (2006) 莴苣高效瞬时表达体系的建立. 园艺学报, 2, 405-407.

[14] 马亮, 梅晓宏, 许文涛, 罗云波, 周忻, 唐小革, 黄昆仑 (2010) 玉米In-5 启动子功能区域在洋葱瞬时表达系统中的分析. 农业生物技术学报, 6, 1073-1078.

[15] 徐冉, 汤雪燕, 缪旻珉, 曹碚生 (2010) 黄瓜疏松愈伤组织瞬时表达系统的建立. 北方园艺, 13, 127-128.

[16] Grefen, C., Donald, N., Hashimoto, K., Kudla, J., Schumacher, K. and Blatt, M.R. (2010) A ubiquitin-10 promoter-based vector set for fluorescent protein tagging faci-litates temporal stability and native protein distribution in transient and stable expression studies. The Plant Journal, 64, 355-365.

[17] Sparkes, I.A., Runions, J., Kearns, A., et al. (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nature protocols, 1, 2019-2025.

[18] 邱礽, 陶刚, 李奇科, 邱又彬, 刘作易 (2009) 农杆菌渗入法介导的基因瞬时表达技术及应用. 分子植物育种, 5, 1032-1039.

分享
Top