Optimization and Application of the Chromatin Immunoprecipitation Based on Rice Protoplast

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

Abstract

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

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.

Figures/Tables 7

References 27

[1]	Mitsuda N, Ohme-Takagi M. Functional analysis of transcription factors in Arabidopsis[J]. Plant Cell Physiol, 2009, 50(7):1232-1248. doi: 10.1093/pcp/pcp075 URL
[2]	Zou YM, Wang SF, Zhou YY, et al. Transcriptional regulation of the immune receptor FLS2 controls the ontogeny of plant innate immunity[J]. Plant Cell, 2018, 30(11):2779-2794. doi: 10.1105/tpc.18.00297 URL
[3]	Bannister AJ, Kouzarides T. Basic peptides enhance protein/DNA interaction in vitro[J]. Nucleic Acids Res, 1992, 20(13):3523. pmid: 1630932
[4]	Wang MM, Reed RR. Molecular cloning of the olfactory neuronal transcription factor Olf-1 by genetic selection in yeast[J]. Nature, 1993, 364(6433):121-126. doi: 10.1038/364121a0 URL
[5]	杨立文, 刘双荣, 李玉红, 等. 植物转录因子与DNA互作研究技术[J]. 植物学报, 2020, 55(4):468-474. doi: 10.11983/CBB20057
	Yang LW, Liu SR, Li YH, et al. Methods for examining transcription factor-DNA interaction in plants[J]. Chin Bull Bot, 2020, 55(4):468-474.
[6]	Gilmour DS, Lis JT. Detecting protein-DNA interactions in vivo:distribution of RNA polymerase on specific bacterial genes[J]. Proc Natl Acad Sci USA, 1984, 81(14):4275-4279. doi: 10.1073/pnas.81.14.4275 URL
[7]	Solomon MJ, Varshavsky A. Formaldehyde-mediated DNA-protein crosslinking:a probe for in vivo chromatin structures[J]. Proc Natl Acad Sci USA, 1985, 82(19):6470-6474. doi: 10.1073/pnas.82.19.6470 URL
[8]	Mundade R, Ozer HG, Wei H, et al. Role of ChIP-seq in the discovery of transcription factor binding sites, differential gene regulation mechanism, epigenetic marks and beyond[J]. Cell Cycle, 2014, 13(18):2847-2852. doi: 10.4161/15384101.2014.949201 pmid: 25486472
[9]	Kaufmann K, Muiño JM, Østerås M, et al. Chromatin immunoprecipitation(ChIP)of plant transcription factors followed by sequencing(ChIP-SEQ)or hybridization to whole genome arrays(ChIP-CHIP)[J]. Nat Protoc, 2010, 5(3):457-472. doi: 10.1038/nprot.2009.244 pmid: 20203663
[10]	Nardini M, Gnesutta N, Donati G, et al. Sequence-specific transcription factor NF-Y displays histone-like DNA binding and H2B-like ubiquitination[J]. Cell, 2013, 152(1/2):132-143. doi: 10.1016/j.cell.2012.11.047 URL
[11]	Zhao BT, Ge LF, Liang RQ, et al. Members of miR-169 family are induced by high salinity and transiently inhibit the NF-YA transcription factor[J]. BMC Mol Biol, 2009, 10:29. doi: 10.1186/1471-2199-10-29 URL
[12]	Luan MD, Xu MY, Lu YM, et al. Expression of zma-miR169 miRNAs and their target ZmNF-YA genes in response to abiotic stress in maize leaves[J]. Gene, 2015, 555(2):178-185. doi: 10.1016/j.gene.2014.11.001 URL
[13]	Ding Q, Zeng J, He XQ. MiR169 and its target PagHAP2-6 regulated by ABA are involved in poplar cambium dormancy[J]. J Plant Physiol, 2016, 198:1-9. doi: 10.1016/j.jplph.2016.03.017 URL
[14]	Li WX, Oono Y, Zhu JH, et al. The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and posttranscriptionally to promote drought resistance[J]. Plant Cell, 2008, 20(8):2238-2251. doi: 10.1105/tpc.108.059444 URL
[15]	Zhao M, Ding H, Zhu JK, et al. Involvement of miR169 in the nitrogen-starvation responses in Arabidopsis[J]. New Phytol, 2011, 190(4):906-915. doi: 10.1111/j.1469-8137.2011.03647.x URL
[16]	Combier JP, Frugier F, de Billy F, et al. MtHAP2-1 is a key transcriptional regulator of symbiotic nodule development regulated by microRNA169 in Medicago truncatula[J]. Genes Dev, 2006, 20(22):3084-3088. doi: 10.1101/gad.402806 URL
[17]	Warpeha KM, Upadhyay S, Yeh J, et al. The GCR1, GPA1, PRN1, NF-Y signal chain mediates both blue light and abscisic acid responses in Arabidopsis[J]. Plant Physiol, 2007, 143(4):1590-1600. doi: 10.1104/pp.106.089904 URL
[18]	Xu MY, Zhang L, Li WW, et al. Stress-induced early flowering is mediated by miR169 in Arabidopsis thaliana[J]. J Exp Bot, 2013, 65(1):89-101. doi: 10.1093/jxb/ert353 URL
[19]	Para A, Li Y, Coruzzi GM. μChIP-seq for genome-wide mapping of in vivo TF-DNA interactions in Arabidopsis root protoplasts[J]. Methods Mol Biol Clifton N J, 2018, 1761:249-261.
[20]	He F, Zhang F, Sun WX, et al. A versatile vector toolkit for functional analysis of rice genes[J]. Rice(N Y), 2018, 11(1):27.
[21]	Chen HM, Zou Y, Shang YL, et al. Firefly luciferase complementation imaging assay for protein-protein interactions in plants[J]. Plant Physiol, 2008, 146(2):368-376.
[22]	Yu C, Chen YT, Cao YQ, et al. Overexpression of miR169o, an overlapping microRNA in response to both nitrogen limitation and bacterial infection, promotes nitrogen use efficiency and susceptibility to bacterial blight in rice[J]. Plant Cell Physiol, 2018, 59(6):1234-1247. doi: 10.1093/pcp/pcy060 URL
[23]	翁小煜, 周少立, 宗伟, 等. 水稻染色质免疫共沉淀[M]. Bio-protocol, 2018, Bio-101:e1010135.
	Weng XY, Zhou SL, Zong W, et al. ChIP assay in rice[M]. Bio-protocol, 2018, Bio-101:e1010135. DOI: 10.21769/BioProtoc.1010135. doi: 10.21769/BioProtoc.1010135
[24]	Lescot M, Déhais P, Thijs G, et al. PlantCARE, a database of plant Cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J]. Nucleic Acids Res, 2002, 30(1):325-327. doi: 10.1093/nar/30.1.325 URL
[25]	Baranello L, Kouzine F, Sanford S, et al. ChIP bias as a function of cross-linking time[J]. Chromosome Res, 2016, 24(2):175-181. doi: 10.1007/s10577-015-9509-1 pmid: 26685864
[26]	Ricardi MM, González RM, Iusem ND. Protocol:fine-tuning of a Chromatin Immunoprecipitation(ChIP)protocol in tomato[J]. Plant Methods, 2010, 6:11. doi: 10.1186/1746-4811-6-11 URL
[27]	董浩, 彭小薇, 王晓英, 等. 染色质免疫共沉淀技术对羊种布鲁氏菌转录调控因子MucR靶基因的筛选[J]. 中国农业大学学报, 2016, 21(4):102-106.
	Dong H, Peng XW, Wang XY, et al. Identification of the targets of MucR by chromatin immunoprecipitation in Brucella melitensis[J]. J China Agric Univ, 2016, 21(4):102-106.

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）

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）

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

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

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