基于水稻原生质体的染色质免疫共沉淀技术优化及应用

doi:10.13560/j.cnki.biotech.bull.1985.2021-1592

摘要/Abstract

摘要：

植物转录因子是植物体内调节基因表达的重要蛋白,参与多种生物学功能的调控。研究植物转录因子调控靶基因的常用方法为染色质免疫共沉淀法（chromatin immunoprecipitation,ChIP）,但抗体特异性、遗传材料构建的耗时性等因素却大大限制了ChIP技术的应用范围和效果。本文通过对水稻原生质体瞬时转化、甲醛固定、免疫沉淀核酸的超声波破碎等条件的优化,建立了基于水稻原生质体的染色质免疫共沉淀技术体系（chromatin immunoprecipitation system based on rice protoplasts,ChIP-RP）;并通过本技术体系验证了水稻转录因子OsNF-YA4蛋白对靶基因序列的富集作用。本技术体系将减少制备特异性抗体或构建稳定遗传材料的局限,有利于水稻转录因子直接调控靶基因的快速筛选和验证,推动水稻转录因子调控功能的分子机制解析。

关键词: 水稻原生质体, 染色质免疫共沉淀技术, 转录因子, OsNF-YA4, ChIP-RP

Abstract:

Transcription factors（TFs）are important regulatory proteins that control gene expression and regulate various biological functions in plant. Chromatin immunoprecipitation（ChIP）is a very widely used technique to investigate the direct target genes of TFs. However,ChIP is always limited by antibody specificity or time-consuming construction of genetic materials. In this paper,the chromatin immunoprecipitation system based on rice protoplasts（ChIP-RP）was established by optimizing the transient transformation of rice protoplasts,formaldehyde fixation and ultrasonic fragmentation of immunoprecipitated nucleic acid. Moreover,the enrichment of target genes by OsNF-YA4 protein,a transcription factor of rice,were verified via ChIP-RP method. This technical system will greatly reduce the limitations of antibody specificity or constructing stable genetic materials,facilitate rapid screening and verification of direct target genes of rice transcription factors,and promote the deciphering of the molecular mechanism of rice transcription factor regulatory functions.

Key words: rice protoplast, chromatin immunoprecipitation, transcription factor, OsNF-YA4, ChIP-RP

石佳, 朱秀梅, 薛梦雨, 余超, 魏一鸣, 杨凤环, 陈华民. 基于水稻原生质体的染色质免疫共沉淀技术优化及应用[J]. 生物技术通报, 2022, 38(7): 62-69.

SHI Jia, ZHU Xiu-mei, XUE Meng-yu, YU Chao, WEI Yi-ming, YANG Feng-huan, CHEN Hua-min. Optimization and Application of the Chromatin Immunoprecipitation Based on Rice Protoplast[J]. Biotechnology Bulletin, 2022, 38(7): 62-69.

图/表 7

表1 NRTs基因启动子区域调控元件的预测

Table 1 Prediction of regulatory elements in the promoter region of NRTs genes

家族Family	基因Gene	ID	CCAAT 数目及位置CCAAT number and position
OsNRT1家族（低亲和力）OsNRT1 family（low affinity）	OsNRT1.1	Os03g13274	2（-1404,-1055）
OsNRT2家族（高亲和力）OsNRT2 family（high affinity）	OsNRT2.1	Os02g02170	1（-175）
	OsNRT2.2	Os02g02190	2（-276,-53）
	OsNAR2.1	Os02g38230	2（-631,-460）

表2 RT-qPCR验证所需的引物序列

Table 2 Required primer sequences for RT-qPCR validation

基因名称 Gene name	引物名称 Primer name	引物序列 Primer sequence（5'-3'）	片段大小及内容 Segment size and content
OsNRT1.1	OsNRT1.1 Q-F2	TGCTACGGTCTCATCTTCTCT	199 bp 包含NRT1.1基因启动子序列中的第二个CCAAT-box 199 bp contains the second CCAAT-box in the promoter of NRT1.1
OsNRT1.1	OsNRT1.1 Q-R2	CAAGTAATCCATCTAACGCTACGA
OsNRT2.1	OsNRT2.1 Q-F1	ACGAATCTTAAGGCAAAT	194 bp包含NRT2.1基因启动子序列中的一个CCAAT-box 199 bp contains a CCAAT-box in the promoter of NRT2.1
OsNRT2.1	OsNRT2.1 Q-R1	CTTCTTGGAGATGGAATC
OsNRT2.2	OsNRT2.2 Q-F2	CACGAGGCAGATATTACAACTTGA	149 bp 包含NRT2.2基因启动子序列中的第二个CCAAT-box 149 bp contains the second CCAAT-box in the promoter of NRT2.2
OsNRT2.2	OsNRT2.2 Q-R2	CGGTGACGATGATCTTGGC
OsNAR2.1	OsNAR2.1 Q-F2	TTCCTCCATTAAGAACGCCTTC	119 bp 包含NAR2.1基因启动子序列中的第二个CCAAT-box 119 bp contains the second CCAAT-box in the promoter of NAR2.1
OsNAR2.1	OsNAR2.1 Q-R2	TTGAGTGCCTCGGTTGTTG
Negative	OsNAR2.1-NF	GATGGCTGTCCTGCTCTTG	183 bp 为NAR2.1基因启动子中不含CCAAT-box的序列 183 bp NAR2.1 gene promoter without CCAAT-box
Negative	OsNAR2.1-NR	ATCATTTCGCTCCTCCAAACTATT

图1 pRTV-NF-YA4-3Myc载体示意图

Fig.1 Schematic diagram of pRTV-NF-YA4-3Myc vector

图2 原生质体转化后OsNF-YA4蛋白表达水平的检测表达载体转化水稻原生质体后,孵育不同时间,提取蛋白,用anti-Myc抗体检测融合蛋白的表达量 1-4：分别为12 h,18 h,24 h,36 h时NF-YA4蛋白表达水平;考马斯亮蓝染色显示各个泳道蛋白上样量

Fig.2 Expressions detection of OsNF-YA4 protein after protoplast transformed After rice protoplast transformed with expression vector and the incubation was performed at different times,the transient expressed proteins were extracted,and the expression of fused protein was detected via anti-Myc antibody 1-4：NF-YA4 protein expressions at 12,18,24 and 36 h. Coomassie brilliant blue staining showed the loaded protein in each lane

表3 不同甲醛终浓度对原生质体细胞活性的影响

Table 3 Effects of different final concentrations of formal-dehyde on protoplasmic cell activity

甲醛终浓度 Final concentration of formaldehyde/%	细胞活性百分比 Percentage of cell activity/%
0	100+0.01
0.1	93+2.79
0.3	80+4.27
0.7	72+3.98
1.0	48+2.72

图3 OsNF-YA4蛋白ChIPs富集DNA的超声破碎时间优化（A）和效果检测（B） A：超声破碎时长的效果比较（M：DL2000 marker;1-5：超声时长分别为5 min,10 min,15 min,20 min,30 min）;B：ChIPs富集DNA的破碎效果（1：超声时长为15 min时,input样品的破碎效果）

Fig.3 Optimization of ultrasonic fragmentation time on OsNF-YA4 ChIPs-enriched DNA（A）and validation of the fragmentation（B） A：Effects of ultrasonic crushing time on fragment length（M：DL2000 marker;1-5：ultrasonic duration was 5,10,15,20 and 30 min respectivey）B：The fragmentation of DNA enrichment by ChIPs（1：the fragmentation of input when ultrasonic treated 15 min）

图4 染色质免疫共沉淀效果的检测（A）和所富集DNA的特异性分析（B） A：Western blot检测免疫沉淀后目的蛋白OsNFYA4的水平（1：不加anti-Myc抗体的免疫沉淀样品（ChIPs-）;2：加anti-Myc抗体的免疫沉淀样品（ChIPs+））;B：qRT-PCR检测免疫沉淀后所富集DNA的特异性

Fig.4 Detection of the effect of chromosome immunopreci-pitation（A）and specificity analysis of enriched DNA（B）A：The level of OsNFYA4 after immunoprecipitation detected by Western blot（1：immunoprecipitated samples（ChIPs-）without anti-Myc antibody;2：immunoprecipitated samples with anti-Myc antibody（ChIPs+））. B：Validation of the specificity of the ChIP-RP enriched DNA by qRT-PCR

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