EMSA Experiments with Fluorescently Labeled Probes to Analyze the Binding Sites of Opaque2 Protein with the ZmGRAS11 Promoter

doi:10.19586/j.2095-2341.2022.0084

Current Biotechnology ›› 2022, Vol. 12 ›› Issue (6): 880-887.DOI: 10.19586/j.2095-2341.2022.0084

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EMSA Experiments with Fluorescently Labeled Probes to Analyze the Binding Sites of Opaque2 Protein with the ZmGRAS11 Promoter

Jiameng ZHU¹^,²(), Haiyang JIANG¹, Rumei CHEN², Xiaojin ZHOU²()

^1.School of Life Science，Anhui Agricultural University，Hefei 230000
^2.Institute of Biotechnology，Chinese Academy of Agricultural Sciences，Beijing 100081

Received:2022-05-24 Accepted:2022-06-30 Online:2022-11-25 Published:2022-11-30
Contact: Xiaojin ZHOU

通过荧光标记的凝胶阻滞技术分析Opaque2蛋白与ZmGRAS11启动子的结合位点

朱佳梦¹^,²(), 江海洋¹, 陈茹梅², 周晓今²()

^1.安徽农业大学生命科学学院，合肥 230000
^2.中国农业科学院生物技术研究所，北京 100081

通讯作者: 周晓今
作者简介:朱佳梦 E-mail: zhujiameng2021@163.com；
基金资助:
国家重点研发计划项目(2021YFF1000304)

Abstract

Abstract:

The electrophoretic mobility shift assay （EMSA） is an experimental technique for studying the binding between proteins and nucleic acids. Since the establishment of the EMSA technology， radioactive isotope and biotin labeling are the main techniques for tracing nucleic acid probes， but these two traditional labeling techniques have some disadvantages， such as poor stability of radioactive probe and complex detection steps of biotin. Although fluorescently labeled probes have been used in EMSA recently， the systematic workflow has not been reported. This study optimized and systematically summarized the technical procedure of fluorescently labeled EMSA. The 6-carboxy-fluorescence （FAM） labeled ZmGRAS11 promoter probe was applied to detect its binding to Opaque2， and the appropriate protein to probe ratio was 8∶1. Several mutated GCN4 motif sequences were applied to determine the binding site between Opaque2 and ZmGRAS11 promoter， and the results showed that the core motif， TGAC， may play an essential role in the recognition by Opaque2. This study provided data surport for further exploring the mechanism of the Opaque2-ZmGRAS11 transcriptional regulatory module in maize kernel development.

Key words: electrophoretic mobility shift assay, fluorescence-labeled probes, ZmGRAS11 promoter, opaque2 protein

摘要：

凝胶阻滞实验（electrophoretic mobility shift assay，EMSA）是研究蛋白质与核酸结合的一种关键实验技术。EMSA技术兴起以来，使用放射性同位素、生物素标记核酸探针的手段已经非常成熟，但这两种传统的标记技术分别具有放射性探针稳定性差和生物素检测步骤复杂等缺点。近年来，尽管荧光标记探针逐渐被应用于EMSA中，但是对于利用荧光标记探针的EMSA仍缺乏系统的报道。对荧光标记的EMSA技术流程进行了优化和系统总结；利用6-羧基荧光素（6-carboxy-fluoroscine，FAM）标记ZmGRAS11启动子探针，通过EMSA检测其与Opaque2蛋白的结合，明确了蛋白和探针的适宜比例为8∶1。对GCN4 motif序列碱基进行突变并利用EMSA分析Opaque2与ZmGRAS11启动子之间的结合位点，结果表明GCN4 motif的“TGAC”核心基序在ZmGRAS11启动子与Opaque2蛋白的结合中可能起到了关键作用。研究结果为进一步探究Opaque2-ZmGRAS11转录调控模块在玉米籽粒发育中的作用机理提供了数据支撑。

关键词: 凝胶阻滞实验, 荧光标记探针, ZmGRAS11启动子, Opaque2蛋白

CLC Number:

Q943

Jiameng ZHU, Haiyang JIANG, Rumei CHEN, Xiaojin ZHOU. EMSA Experiments with Fluorescently Labeled Probes to Analyze the Binding Sites of Opaque2 Protein with the ZmGRAS11 Promoter[J]. Current Biotechnology, 2022, 12(6): 880-887.

朱佳梦, 江海洋, 陈茹梅, 周晓今. 通过荧光标记的凝胶阻滞技术分析Opaque2蛋白与ZmGRAS11启动子的结合位点[J]. 生物技术进展, 2022, 12(6): 880-887.

Figures/Tables 10

References 30

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试剂名称	配方	pH
1 mol·L^-1 IPTG	2.38301 g IPTG,10 mL 超纯水	—
裂解缓冲液	0.02 mol·L^-1 咪唑，0.02 mol·L^-1 Tris，0.5 mol·L^-1 氯化钠，10% 甘油，0.5% TritonX-100	8.0
洗杂缓冲液A	0.02 mol·L^-1咪唑，0.02 mol·L^-1 Tris，0.5 mol·L^-1 氯化钠，10% 甘油，0.5% TritonX-100	8.0
洗杂缓冲液B	0.04 mol·L^-1咪唑，0.02 mol·L^-1 Tris，0.5 mol·L^-1 氯化钠，10%甘油，0.5% TritonX-100	8.0
洗杂缓冲液C	0.08 mol·L^-1 咪唑，0.02 mol·L^-1 Tris，0.5 mol·L^-1 氯化钠，10% 甘油，0.5% TritonX-100	8.0
洗脱缓冲液	0.25 mol·L^-1咪唑，0.02 mol·L^-1 Tris，0.5 mol·L^-1 氯化钠，10% 甘油	8.0
1×置换缓冲液	0.05 mol·L^-1 Tris，0.25 mol·L^-1 氯化钠，0.005 mol·L^-1 DTT	7.5
5×TBE缓冲液	0.45 mol·L^-1 Tris，0.45 mol·L^-1 硼酸，0.01 mol·L^-1 EDTA	8.3

试剂名称	配方	pH
1 mol·L^-1 IPTG	2.38301 g IPTG,10 mL 超纯水	—
裂解缓冲液	0.02 mol·L^-1 咪唑，0.02 mol·L^-1 Tris，0.5 mol·L^-1 氯化钠，10% 甘油，0.5% TritonX-100	8.0
洗杂缓冲液A	0.02 mol·L^-1咪唑，0.02 mol·L^-1 Tris，0.5 mol·L^-1 氯化钠，10% 甘油，0.5% TritonX-100	8.0
洗杂缓冲液B	0.04 mol·L^-1咪唑，0.02 mol·L^-1 Tris，0.5 mol·L^-1 氯化钠，10%甘油，0.5% TritonX-100	8.0
洗杂缓冲液C	0.08 mol·L^-1 咪唑，0.02 mol·L^-1 Tris，0.5 mol·L^-1 氯化钠，10% 甘油，0.5% TritonX-100	8.0
洗脱缓冲液	0.25 mol·L^-1咪唑，0.02 mol·L^-1 Tris，0.5 mol·L^-1 氯化钠，10% 甘油	8.0
1×置换缓冲液	0.05 mol·L^-1 Tris，0.25 mol·L^-1 氯化钠，0.005 mol·L^-1 DTT	7.5
5×TBE缓冲液	0.45 mol·L^-1 Tris，0.45 mol·L^-1 硼酸，0.01 mol·L^-1 EDTA	8.3

试管号	菌液加入量/mL	IPTG加入量/μL	IPTG诱导浓度/(mmol·L^-1)
a	6	0	0
b	6	1.2	0.2
c	6	2.4	0.4
d	6	3.6	0.6
e	6	4.8	0.8
f	6	6.0	1.0

试管号	菌液加入量/mL	IPTG加入量/μL	IPTG诱导浓度/(mmol·L^-1)
a	6	0	0
b	6	1.2	0.2
c	6	2.4	0.4
d	6	3.6	0.6
e	6	4.8	0.8
f	6	6.0	1.0

探针名称	序列（5'→3'）	标记
FAM-F	5'-GGCACTAGTCATGCTAGCTTGTGACTCATCCAACAAACAATAGTTATCC-3'	5'-FAM
FAM-R	5'-GGATAACTATTGTTTGTTGGATGAGTCACAAGCTAGCATGACTAGTGCC-3'	5'-FAM
WT-F	5'-GGCACTAGTCATGCTAGCTTGTGACTCATCCAACAAACAATAGTTATCC-3'	—
WT-F	5'-GGATAACTATTGTTTGTTGGATGAGTCACAAGCTAGCATGACTAGTGCC-3'	—
M1-F	5'-GGCACTAGTCATGCTAGCTTGTCCAACAAACAATAGTTATCC-3'	—
M1-R	5'-GGATAACTATTGTTTGTTGGACAAGCTAGCATGACTAGTGCC-3'	—
M2-F	5'-GGCACTAGTCATGCTAGCTTGTTTTTTTTCCAACAAACAATAGTTATCC-3'	—
M2-R	5'-GGATAACTATTGTTTGTTGGAAAAAAAACAAGCTAGCATGACTAGTGCC-3'	—
M3-F	5'-GGCACTAGTCATGCTAGCTTGGGGGGGGTCCAACAAACAATAGTTATCC-3'	—
M3-R	5'-GGATAACTATTGTTTGTTGGACCCCCCCCAAGCTAGCATGACTAGTGCC-3'	—
M4-F	5'-GGCACTAGTCATGCTAGCTTGCAGCTCATCCAACAAACAATAGTTATCC-3'	—
M4-R	5'-GGATAACTATTGTTTGTTGGATGAGCTGCAAGCTAGCATGACTAGTGCC-3'	—
M5-F	5'-GGCACTAGTCATGCTAGCTTGTGGTCCATCCAACAAACAATAGTTATCC-3'	—
M5-R	5'-GGATAACTATTGTTTGTTGGATGGACCACAAGCTAGCATGACTAGTGCC-3'	—
M6-F	5'-GGCACTAGTCATGCTAGCTTGTGACCTGTCCAACAAACAATAGTTATCC-3'	—
M6-R	5'-GGATAACTATTGTTTGTTGGACAGGTCACAAGCTAGCATGACTAGTGC-3'	—

EMSA Experiments with Fluorescently Labeled Probes to Analyze the Binding Sites of Opaque2 Protein with the ZmGRAS11 Promoter

通过荧光标记的凝胶阻滞技术分析Opaque2蛋白与ZmGRAS11启动子的结合位点

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Figures/Tables 10

References 30

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各个组分加入量/（μL）	泳道
各个组分加入量/（μL）	1	2	3	4	5	6	7	8
超纯水	14.0	13.5	13.0	12.0	10.0	8.0	6.0	4.0
5×结合缓冲液	4	4	4	4	4	4	4	4
O2蛋白	0	0.5	1.0	2.0	4.0	6.0	8.0	10.0

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