不同调控元件及组合对烟草外源蛋白瞬时表达的效果分析

doi:10.13560/j.cnki.biotech.bull.1985.2019-1226

摘要/Abstract

摘要： 植物瞬时表达是一种高效快速获得外源基因表达的方法,该系统主要应用于蛋白互作研究、调控元件功能分析、蛋白亚细胞定位和蛋白制剂合成等方面。为了进一步提高外源基因在瞬时表达体系中稳定表达效果,本研究对瞬时表达载体的多种作用元件及转化条件进行优化。首先,我们在目前最常用的双元表达载体pCambia1300骨架上,分别添加可增强特异性转录和稳定蛋白表达的新的调控元件,构建pREU-EF、pREUR-EF和pREUR-p24-EF重组表达载体,并且通过定性和定量方法分析不同元件组合、不同菌液浓度对报告基因eGFP表达的影响。激光共聚焦显微镜观察结果证明3个重组载体可在烟草叶片的细胞膜、细胞质和细胞核中快速表达报告基因;定量PCR分析表明3个重组载体中报告基因在转录表达水平上分别比原pCambia1301-eGFP载体提高了4倍、20倍和28倍;蛋白水平分析表明烟草叶片转化48 h后,pREUR-EF外源蛋白表达量明显高于pREU-EF;并且当菌液注射液浓度OD₆₀₀= 0.4左右时,外源蛋白的表达效率最高。此外,我们还进一步把改造后的瞬时表达重组载体运用到双荧光素酶（Dual-Luciferase）报告系统中,实验证明改造后的瞬时表达重组载体可以更加快捷和有效地用于转录调控分析。因此,烟草瞬时表达载体中作用元件增加和优化组合,可以有效地提高外源蛋白的表达。

关键词: 植物瞬时表达, 调控元件, 烟草, 转录调控, 双荧光素酶

Abstract: The plant transient expression is an efficient way to have foreign gene expressed,and this system can be used for analyzing the protein interactions,identifying the localizations of proteins,investigating the functions of regulatory elements,producing proteins of interest,etc. To further improve the expressions of foreign genes in transient expression system,this study focused on the optimization of various functional elements and transformation conditions of the transient expression vectors. Firstly,the commonly used binary vector pCambia1300 was used as backbone for the insertions of functional elements that can increase the specific transcription and the expressions of stable proteins,and thenrecombinant vectors pREU-EF,pREUR-EF,and pREUR-p24-EF were constructed. Next,the qualitative and quantitative methods were applied to evaluate the effects of different element combinations and different agrobacterial suspension concentrations on the expression of reporter gene Enhanced Green Fluorescent Protein（eGFP）. The results of laser scanning confocal microscope showed that the eGFP in the recombinant vectors expressed rapidly in the cell membrane,cytoplasm,and nucleus of tobacco leaves. The quantitative PCR results indicated that the expression ofthe eGFPin thepREU-EF,pREUR-EF and pREUR-p24-EF increased by 4 times,20 times,and 28 times,respectively,while compared with the pCambia1301-eGFPvector. Moreover,the results from protein level analysis suggested that compared with pREU-EF,pREUR-EF and pREUR-p24-EF could obtain more proteins 48 hours post agroinfiltration. Furthermore,the expression of the foreign protein reached the highest when the concentration of agrobacterial suspension OD₆₀₀ was about 0.4. In addition,the transcription regulation analysis in dual-luciferase reporting system implied that the process was more rapidly and efficiently while using the modified recombinant transient expression vectors. Therefore,this study demonstrates that the combinations of regulatory elements in transient expression vectors can effectively increase the expressions of foreign genes at the transcription and translation levels.

Key words: plant transient expression, regulatory elements, Nicotiana benthamiana, transcriptional regulation, dual luciferase

刘文浩, 王瑞丰, 刘琬琳, 许杰. 不同调控元件及组合对烟草外源蛋白瞬时表达的效果分析[J]. 生物技术通报, 2020, 36(7): 62-71.

LIU Wen-hao, WANG Rui-feng, LIU Wan-lin, XU Jie. Effects of Different Regulatory Elements and Their Combinations on Transient Expressions of Exogenous Proteinsin Nicotiana benthamiana[J]. Biotechnology Bulletin, 2020, 36(7): 62-71.

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