萤火素酶互补实验检测蛋白互作

doi:10.11983/CBB19229

摘要/Abstract

摘要： 蛋白质-蛋白质相互作用在真核生物的各项生命活动中发挥重要作用。与其它蛋白质互作研究技术相比, 借助于烟草(Nicotiana benthamiana)瞬时表达系统的萤火素酶互补实验(LCA)具有简单、灵敏、可靠、高效和低背景等优点, 并可轻松扩展为大规模蛋白质互作的筛选和验证研究。该文介绍了萤火素酶互补实验的具体操作过程, 通过2种数据收集方法来定性并定量分析生物发光或发光强度, 从而检测植物目标蛋白之间的相互作用。

关键词: 萤火素酶互补实验, 蛋白质相互作用, 烟草瞬时表达

Abstract: Protein-Protein interactions play important roles in various eukaryotic biological processes. Compared to other techniques measuring protein-protein interactions in plants, the Luciferase Complementation Assay (LCA), based on Agrobacterium-mediated transient expression in Nicotiana benthamiana, is a simple, sensitive, reliable, highly quantitative and low background method that can be easily scaled up for high-throughput interactome studies. Here, we describe a protocol that includes two alternative data collection methods to qualitative and quantitative analyse luminescence or luminous intensity to detect protein-protein interactions in plant cells.

Key words: luciferase complementation assay, protein-protein interactions, tobacco transient expression

赵燕,周俭民. 萤火素酶互补实验检测蛋白互作. 植物学报, 2020, 55(1): 69-75.

Yan Zhao,Jianmin Zhou. Luciferase Complementation Assay for Detecting Protein Interactions. Chinese Bulletin of Botany, 2020, 55(1): 69-75.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB19229

https://www.chinbullbotany.com/CN/Y2020/V55/I1/69

图/表 3

图1 表达载体示意图 (A) pCAMBIA1300-NLuc (pNL); (B) pCAMBIA1300-CLuc (pCL); (C) pCAMBIA1300-HA-NLuc (pHNL); (D) pCAMBIA1300-CCLuc (pCCL)。构建的表达载体中均包含pCAMBIA1300骨架, NLuc位于目标基因C末端, CLuc位于目标基因的N末端或C末端; 序列中显示的是单酶切位点。MCS: 多克隆酶切位点; L: 连接序列; RBS: 转录终止子; ATG: 翻译起始密码子; TGA: 翻译终止密码子; HA: 标签蛋白

Figure 1 Plasmid maps (A) pCAMBIA1300-NLuc (pNL); (B) pCAMBIA1300-CLuc (pCL); (C) pCAMBIA1300-HA-NLuc (pHNL); (D) pCAMBIA1300-CCLuc (pCCL). All the vectors have a framework of pCAMBIA1300 and allow expression of the target genes with a C-terminal NLuc fusion and N-terminal or C-terminal CLuc fusion. Unique sites are shown in sequence. MCS: Multiple cloning sites; L: Linker sequence; RBS: Transcription terminator; ATG: Translation initiation codon; TGA: Translation stop codon; HA: Tag protein

图2 萤火素酶互补实验示意图 (A) 选取4周龄完全伸展的烟草叶片进行农杆菌注射, 24-48小时后取样观察(Bar=2 cm); (B) 截取完整的农杆菌注射后的烟草叶片, 叶背向上平放于培养皿中(Bar=1 cm); (C) 利用植物活体分子影像系统检测烟草叶片的发光情况并拍照; (D) 选取农杆菌注射后的烟草叶片, 置于培养皿中用打孔器打孔(Bar=1 cm); (E) 将直径为0.6 cm的烟草圆片置于装有200 μL无菌水的96孔酶标板中; (F) 利用luminometer检测烟草叶片的荧光强度。图中圆圈示农杆菌注射部位。

Figure 2 Schematic diagram of the Luciferase Complementation Assay (A) Select fully expanded young leaves of Nicotiana benthamiana (four-week old) for Agrobacterium injection, observe after 24-48 h (Bar=2 cm); (B) Cut the infiltrated tobacco leaves and put them into the petri dish (Bar=1 cm); (C) Use CCD imaging system to detect the luminescence of the complete tobacco leaves and captured the pictures; (D) Select infiltrated leaves for punch (Bar=1 cm); (E) Punch tobacco leaf discs of 0.6 cm in diameter from infiltrated leaf areas by a cork borer and place leaf discs in a 96-well plate containing 200 μL sterile water in each well; (F) Use luminometer to scan the fluorescence intensity of the tobacco leaves. The circles show the injection sites of Agrobacterium.

图3 蛋白质OsXLG2-OsBIK1互作实例将分别含有CLuc-OsXLG2和OsBIK1-NLuc质粒的农杆菌液1:1体积比混合后注射4周龄烟草叶片, 24-48小时后进行检测, 以CLuc-OsPPI和OsBIK1-NLuc作为负对照(未发表)。(A) 利用植物活体成像系统采集烟草叶片的发光图像(Bar=2 cm); (B) 使用luminometer检测萤火素酶活性, 误差表示平均值的标准偏差(n=8); (C) Western blot检测CLuc和NLuc融合蛋白的表达, 融合蛋白分别用anti-luciferase和anti-CLuc抗体来检测。

Figure 3 An example of dynamic OsXLG2-OsBIK1 interactions Four-week old Nicotiana benthamiana leaves co-infiltrated with Agrobacterium expressing CLuc-OsXLG2 and OsBIK1-NLuc. CLuc-OsPPI and OsBIK1-NLuc are used as negative control (unpublished). (A) The luminescence images were captured using a CCD imaging system (Bar=2 cm); (B) The luciferase activity measurement using a luminometer, the error bars represent the standard deviation from the mean (n=8); (C) Western blot analysis of the expressed proteins of CLuc- and NLuc- fusions, the fusions are detected using anti-luciferase and anti-CLuc antibodies, respectively.

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