Effects of Different Regulatory Elements and Their Combinations on Transient Expressions of Exogenous Proteinsin Nicotiana benthamiana

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

Abstract

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

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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