植物蛋白质SUMO化修饰体外高效检测系统

doi:10.11983/CBB22080

摘要/Abstract

摘要：

蛋白质SUMO化修饰是一种调控蛋白命运的关键修饰方式, 广泛参与植物生长发育及逆境胁迫响应。SUMO化修饰过程主要由激活酶(E1)-结合酶(E2)-连接酶(E3)组成的级联酶促反应催化, 其关键酶组分将SUMO分子缀合至底物蛋白的赖氨酸残基, 形成共价异肽键以完成SUMO化修饰过程。该文报道了1种植物蛋白质SUMO化修饰体外高效检测系统, 通过在大肠杆菌(Escherichia coli)中构建拟南芥(Arabidopsis thaliana) SUMO化修饰的关键通路实现对底物蛋白的SUMO化修饰, 结果可通过免疫印迹进行检测。该系统可以简化植物蛋白质SUMO化修饰的检测流程, 为植物细胞SUMO化修饰的功能研究提供了有力工具。

关键词: SUMO化修饰, 体外, 检测系统, 植物

Abstract:

Protein SUMOylation is a key modification for regulating the fate of proteins and it is widely involved in plant development and stress responses. The SUMO molecules are conjugated to the lysine residues of substrate proteins via isopeptide bonds by enzyme reaction. SUMOylation is mediated by an enzyme cascade composed of a SUMO activating enzyme complex (E1), a SUMO-conjugating enzyme (E2) and usually a SUMO ligase (E3). Here, we report an efficient in vitro detection system for SUMOylation of plant proteins. We established a system for SUMOylation detection of plant proteins by reconstructing the Arabidopsis SUMOylation enzyme cascade in Escherichia coli. Using this system, SUMOylation of several substrates were detected via immunoblotting. Therefore, this system simplifies the SUMOylation detection of plant protein substrates and provides a powerful tool for functional analysis of SUMOylation in plant cells.

Key words: SUMOylation, in vitro, detection system, plant

黄俊文, 冯琦伊, 郑凯勇, 黄俊杰, 王林博, 赖瑞强, 赖建彬, 阳成伟. 植物蛋白质SUMO化修饰体外高效检测系统. 植物学报, 2022, 57(4): 490-499.

Huang Junwen, Feng Qiyi, Zheng Kaiyong, Huang Junjie, Wang Linbo, Lai Jianbin Lai Ruiqiang, Yang Chengwei. An Effective in Vitro SUMOylation Detection System for Plant Proteins. Chinese Bulletin of Botany, 2022, 57(4): 490-499.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22080

https://www.chinbullbotany.com/CN/Y2022/V57/I4/490

图/表 3

图1 体外SUMO化修饰检测流程 (A) 体外SUMO化修饰检测过程(输入: 将SUMO修饰关键组分基因和候选底物蛋白基因导入大肠杆菌表达; 激活: E1结合以激活SUMO分子参与修饰; 结合: E2将E1激活后的SUMO分子连接至底物完成修饰); (B) 体外SUMO化修饰检测实验流程(共转化: 将SUMO化修饰关键组分载体导入大肠杆菌, 通过卡那霉素和氯霉素平板筛选阳性克隆; 制备感受态细胞: 将获得的阳性克隆制备成感受态细胞; 共转化: 将构建好的底物蛋白表达载体导入感受态细胞, 通过卡那霉素、氯霉素和链霉素平板筛选阳性克隆; 诱导: 通过IPTG诱导蛋白表达; 凝胶电泳: 将获得的菌体样品裂解变性后通过SDS-PAGE凝胶电泳将不同分子量的蛋白分离, 将蛋白从凝胶转移至PVDF膜上; 抗体孵育: 将封闭后的PVDF膜与抗体进行孵育; 显影: 观察并记录实验结果)

Figure 1 The process of in vitro SUMOylation analysis (A) The process diagram of in vitro SUMOylation detection (Input: the key component genes of SUMO modification and the candidate substrate protein gene were introduced into Escherichia coli for expression; Activating: E1 activates SUMO molecules to participate in the modification; Conjugating: E2 attaches the activated SUMO molecules to the substrate); (B) The experimental procedure of in vitro SUMOylation detection (Co-transformation: The plasmids for expressing key SUMOylation components were introduced into E. coli, and the positive colonies were screened on LB agar plates containing kanamycin and chloramphenicol; Preparation of competent cells: The obtained positive colonies were prepared into competent cells; Co-transformation: The constructed plasmid for substrate protein expression was introduced into the competent cells mentioned above, and positive colonies were screened on LB agar plates by kanamycin, chloramphenicol and streptomycin; Induction: Protein expression was induced by IPTG; Gel electrophoresis: After lysed and denatured, samples were separated by SDS-PAGE. Then the proteins were transferred from gel to PVDF membrane; Antibody incubation: The blocked PVDF membrane was incubated with antibody; Immunoblotting: Observation and recording of the experimental results)

图2 E3连接酶介导的体外SUMO化修饰检测 (A) E3连接酶介导的体外SUMO化修饰检测过程(输入: 将SUMO化修饰关键组分基因和候选底物蛋白基因导入大肠杆菌表达; 激活: E1结合以激活SUMO分子参与修饰; 结合: E2将E1激活后的SUMO分子连接至底物完成修饰, 在E3存在的条件下, E3促进了SUMO分子从E2到底物蛋白的连接); (B) E3连接酶介导的体外SUMO化修饰检测实验流程(共转化: SUMO关键组分载体导入大肠杆菌, 通过卡那霉素和氯霉素平板筛选阳性克隆; 制备感受态细胞: 将获得的阳性克隆制备成感受态细胞; 共转化: 将构建好的底物蛋白表达载体(带有独立的E3表达框)导入感受态细胞, 通过卡那霉素、氯霉素和链霉素平板筛选阳性克隆; 诱导: 通过IPTG诱导蛋白表达; 免疫印迹: 通过SDS-PAGE凝胶电泳将不同分子量的蛋白分离, 将蛋白从凝胶转移至PVDF膜上, 将封闭后的PVDF膜与抗体进行孵育, 通过显影观察并记录实验结果)

Figure 2 The process of in vitro SUMOylation analysis mediated by an E3 ligase (A) The process diagram of the E3 ligase-mediated in vitro SUMOylation detection (Input: The key component genes of SUMO modification and the candidate substrate protein gene were introduced into Escherichia coli for expression; Activating: E1 activates SUMO molecules to participate in the modification; Conjugating: E2 attaches the activated SUMO molecules to the substrate. In the presence of E3, E3 facilitates the attachment of SUMO molecules from E2 to the substrate protein); (B) The experimental procedure of the E3 ligase-mediated in vitro SUMOylation detection (Co-transformation: The plasmids for expression of key SUMOylation components were introduced into E. coli, and the positive colonies were screened on LB agar plates containing kanamycin and chloramphenicol; Preparation of competent cells: The obtained positive colonies were transformed into competent cells; Co-transformation: The constructed plasmid for substrate protein expression (with independent E3 expression cassette) was introduced into the competent cells mentioned above, and the positive colonies were screened by LB agar plates containing kanamycin, chloramphenicol and streptomycin; Induction: Protein expression was induced by IPTG; Immunoblotting: After lysed and denatured, samples were separated by SDS-PAGE gel electrophoresis, then the proteins were transferred from gel to PVDF membrane, the blocked PVDF membrane was incubated with antibody, the result was observed and recorded by immunoblotting).

图3 检测实例 (A) AtDREB2A蛋白SUMO化修饰检测; (B) 温度影响AtACR4蛋白的SUMO化修饰水平; (C) E3连接酶AtSIZ1促进AtPHR1蛋白的SUMO化修饰; (D) E3连接酶AtSIZ1促进AtMYB30蛋白的SUMO化修饰

Figure 3 Experimental cases (A) SUMOylation test of AtDREB2A; (B) Temperature affects SUMOylation of AtACR4; (C) The E3 ligase AtSIZ1 facilitates SUMOylation of AtPHR1; (D) The E3 ligase AtSIZ1 facilitates SUMOylation of AtMYB30

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