生物技术通报 ›› 2022, Vol. 38 ›› Issue (10): 54-65.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1384
收稿日期:
2021-11-03
出版日期:
2022-10-26
发布日期:
2022-11-11
作者简介:
陈臣,男,博士,副教授,研究方向:食品生物技术、食品风味;E-mail:基金资助:
CHEN Chen(), HUANG Zhi-yang, YU Hai-yan, YUAN Hai-bin, TIAN Huai-xiang()
Received:
2021-11-03
Published:
2022-10-26
Online:
2022-11-11
摘要:
原核生物基因表达调控主要发生在转录水平,研究原核生物的转录调控有利于了解其基因表达调节机制。近年来,随着分子生物学及相关技术的突破,转录调控研究技术也不断发展,因此主要综述了原核生物转录调控的技术方法及其新进展,包括凝胶电泳迁移率实验、DNase I足迹技术、染色质免疫共沉淀技术、微量热泳动技术、等温滴定量热法及细菌单杂交系统,以期系统地了解这些方法的优缺点和适用性,帮助研究者更好的利用原核生物转录调控为人类造福。
陈臣, 黄芝阳, 于海燕, 袁海彬, 田怀香. 原核生物转录调控研究技术及进展[J]. 生物技术通报, 2022, 38(10): 54-65.
CHEN Chen, HUANG Zhi-yang, YU Hai-yan, YUAN Hai-bin, TIAN Huai-xiang. Research Technology and Progress in Transcriptional Regulation in Prokaryotes[J]. Biotechnology Bulletin, 2022, 38(10): 54-65.
研究技术 Method | 英文名称/缩写 Name/Abbreviation | 优缺点 Advantages/Disadvantages | 适用性 Applicability | 参考文献 Reference | |
---|---|---|---|---|---|
体外方法 | 凝胶电泳迁移率实验 | EMSA | 方法简单、灵敏度高,放射性标记探针安全性低成本高,电泳运行条件下蛋白质-DNA复合体不稳定 | 适用于验证转录因子与假定DNA结合位点直接相互作用及结合位点突变对结合作用的影响 | [ |
等温滴定量热法 | ITC | 蛋白质无需固定化或修饰,样品消耗量少,可区分结合常数相近的配体相互作用及比较结构与结合作用的关系,对温度适应范围广但难以解释复杂系统中的相互作用 | 适用于成分简单的超高/超低亲和力相互作用系统及复杂的相互作用,可获得丰富的热力学信息 | [ | |
DNase I footprinting技术 | DNase I footprinting | 分辨率高、可区分同一DNA片段多个不连续结合位点,但需要较多蛋白质才能产生清晰足迹,易产生超敏位点受到切割 | 适用于未纯化蛋白样品的检测,判断同一片段是否存在多个结合位点获得结合序列及比较各自亲和力 | [ | |
微量热泳动技术 | MST | 对相互作用的分子大小或质量无选择性,具有较好的适用性,对结合亲和力准确测定,可检测低至pM级别的结合亲和力,可在溶液环境中进行,无需固定分子避免结合假阳性 | 适用于结合亲和力弱,样品量小,样品所处环境复杂的情况 | [ | |
体内方法 | 染色质-免疫共沉淀技术 | ChIP | 接近细胞内真实情况,可研究转录因子对启动子结合的动态过程,但实验重复性不佳,获得良好实验结果对经验依赖性较高,对实验环境的要求严格 | 适用于确定转录因子修饰位置及低丰度转录因子结合分析 | [ |
细菌单杂交 | B1H | 细菌转化效率高构建文库质粒容量更大,无需复杂的仪器,转录因子需要能在大肠杆菌中表达,可能存在假阳性和假阴性的情况,结果需要进一步验证 | 适用于未纯化蛋白样品的检测,缺少相应仪器,用分子生物学手段进行筛选,转录因子要求能在大肠杆菌中表达,可发现新的转录因子 | [ |
表1 原核生物转录调控研究技术总结
Table 1 Summary of research techniques on transcriptional regulation in prokaryotes
研究技术 Method | 英文名称/缩写 Name/Abbreviation | 优缺点 Advantages/Disadvantages | 适用性 Applicability | 参考文献 Reference | |
---|---|---|---|---|---|
体外方法 | 凝胶电泳迁移率实验 | EMSA | 方法简单、灵敏度高,放射性标记探针安全性低成本高,电泳运行条件下蛋白质-DNA复合体不稳定 | 适用于验证转录因子与假定DNA结合位点直接相互作用及结合位点突变对结合作用的影响 | [ |
等温滴定量热法 | ITC | 蛋白质无需固定化或修饰,样品消耗量少,可区分结合常数相近的配体相互作用及比较结构与结合作用的关系,对温度适应范围广但难以解释复杂系统中的相互作用 | 适用于成分简单的超高/超低亲和力相互作用系统及复杂的相互作用,可获得丰富的热力学信息 | [ | |
DNase I footprinting技术 | DNase I footprinting | 分辨率高、可区分同一DNA片段多个不连续结合位点,但需要较多蛋白质才能产生清晰足迹,易产生超敏位点受到切割 | 适用于未纯化蛋白样品的检测,判断同一片段是否存在多个结合位点获得结合序列及比较各自亲和力 | [ | |
微量热泳动技术 | MST | 对相互作用的分子大小或质量无选择性,具有较好的适用性,对结合亲和力准确测定,可检测低至pM级别的结合亲和力,可在溶液环境中进行,无需固定分子避免结合假阳性 | 适用于结合亲和力弱,样品量小,样品所处环境复杂的情况 | [ | |
体内方法 | 染色质-免疫共沉淀技术 | ChIP | 接近细胞内真实情况,可研究转录因子对启动子结合的动态过程,但实验重复性不佳,获得良好实验结果对经验依赖性较高,对实验环境的要求严格 | 适用于确定转录因子修饰位置及低丰度转录因子结合分析 | [ |
细菌单杂交 | B1H | 细菌转化效率高构建文库质粒容量更大,无需复杂的仪器,转录因子需要能在大肠杆菌中表达,可能存在假阳性和假阴性的情况,结果需要进一步验证 | 适用于未纯化蛋白样品的检测,缺少相应仪器,用分子生物学手段进行筛选,转录因子要求能在大肠杆菌中表达,可发现新的转录因子 | [ |
图1 EMSA实验的基本原理 星号表示标记的探针,TFs表示转录因子,TFBS表示转录因子结合位点
Fig. 1 Basic principle of EMSA experiment Asterisks indicate labeled probes,TFs indicate transcription factors,and TFBS indicate transcription factor binding sites
图2 DNase I footprinting技术基本原理 对DNA片段中的一条链进行标记,加入适当浓度脱氧核糖核酸酶I(DNaseI),与目的蛋白结合的DNA基序不被DNase I水解,经过对比未结合目的蛋白DNA片段的放射自显影图谱可得到中断的DNA梯度条带
Fig. 2 Basic principle of DNase I footprint technology Adding appropriate concentration of deoxyribonuclease I(DNase I)to label strand in the DNA fragment,the DNA motif bound to the target protein will not be hydrolyzed by DNase I. After comparing the autoradiograph of the DNA fragment not bound to the target protein,interrupted DNA gradient bands can be obtained
图3 ChIP实验的基本原理 在体内通过交联剂将目的蛋白与DNA交联,再将交联得到的染色质复合物片段化处理后通过目的蛋白特异性抗体沉淀富集目的蛋白结合的DNA片段,对复合物解交联、纯化并分析DNA产物
Fig. 3 Basic principle of ChIP experiment Cross-link the target protein with DNA in vivo through a cross-linking agent,and then fragment the DNA and protein complex. After that,precipitate with the specific antibody of the target protein to enrich the DNA fragments bound to the target protein. Reversal of crosslink and DNA extraction,finally analysis of purified DNA
图4 MST实验的基本原理 A:荧光探测器;B:MST光学元件示意图;C:MST实验信号曲线;D:MST结合实验
Fig. 4 Basic principle of MST experiment A:Fluorescence detector. B:MST optical element schematic diagram. C:MST experiment signal curve. D:MST combination experiment
图5 ITC实验的基本原理 已知浓度的反应物滴定到样品中引起样品池中组分之间反应吸热或放热,温度补偿系统使样品池与参比池之间维持恒定的温差,通过记录数据模拟整合加热的等温线之后,可以得到反应焓变、结合亲和力和结合化学计量等信息
Fig. 5 Basic principle of ITC experiment The titration of reactants of known concentration into the sample causes the reaction between the components in the sample cell to be endothermic or exothermic. The temperature compensation system maintains a constant temperature difference between the sample cell and the reference cell,and the data are recorded to simulate the integrated heating isotherm. After that,information about the reaction enthalpy change,binding affinity and binding stoichiometry can be obtained.
图6 细菌单杂交系统基本原理 A:转录因子识别结合位点启动报告基因表达;B:分别构建α-TF表达载体和结合位点文库;C:消除自激活现象纯化结合位点文库;D:筛选并分析结合位点
Fig. 6 Basic principle of bacterial one hybrid system A:Transcription factor recognition binding sites activate the expressions of the reporter genes. B:Construct α-TF expression vector and binding site library respectively. C:Eliminate self-activation to purify binding site library. D:Selection and analysis of binding sites
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