适用于多种禾本科植物染色质转座酶可及性测序的提核建库方法

doi:10.3724/SP.J.1006.2023.22041

摘要/Abstract

摘要：

为建立适用于多种禾本科植物染色质转座酶可及性测序(assay for transposase accessible chromatin with high-throughput sequencing, ATAC-seq)的细胞核提取和建库体系, 本试验以水稻、小麦和结缕草的叶片及根系为研究材料, 通过控制变量试验确定最佳的细胞核提取方法: 采用液氮研磨样品, 初步提核离心速度选取1200×g, 2次ORB溶液去除细胞器等杂质, 蔗糖梯度缓冲液纯化细胞核。通过构建文库、库检及测序分析发现, 所提取的细胞核适用于染色质转座酶可及性测序的建库, 可获得高质量测序数据。本试验建立的适用于多种禾本科植物的提核体系为获取禾本科植物染色质开放区域信息以及深入研究基因表达的调控方式奠定了基础。

关键词: 禾本科, 染色质可及性, ATAC-seq, 细胞核

Abstract:

In order to establish a system for extracting nuclei and constructing library of assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) in gramineous plants, the leaves and roots of rice, wheat, and zoysia were used as the research materials. The best method to extract nuclei was determined by designing control variable experiments: grinding samples with liquid nitrogen, selecting 1200×g centrifugal stress for extracting initial nuclei, using ORB buffer twice and SCB buffer to purify nuclei. It was suggested that the extracted nuclei were suitable for the sequencing of transposase accessible chromatin and high-quality sequencing data could be obtained by testing. In summary, an integrated system suitable for gramineous plants to extract nuclei had been established. This method will lay a foundation for obtaining the information of chromatin accessible regions and the regulation of gene expression in gramineous plants.

Key words: gramineae, chromatin accessibility, ATAC-seq, nuclei

祁泽文, 黄铭涵, 张佳卉, 刘艺, 韩烈保, 何航. 适用于多种禾本科植物染色质转座酶可及性测序的提核建库方法[J]. 作物学报, 2023, 49(7): 1799-1807.

QI Ze-Wen, HUANG Ming-Han, ZHANG Jia-Hui, LIU Yi, HAN Lie-Bao, HE Hang. A method of nuclei extraction and library construction for chromatin transposase accessibility sequencing in gramineous plants[J]. Acta Agronomica Sinica, 2023, 49(7): 1799-1807.

图/表 4

图1

图2

图3

图4

参考文献 34

[1]	Tolsma T O, Hansen J C. Post-translational modifications and chromatin dynamics. Essays Biochem, 2019, 63: 89-96. doi: 10.1042/EBC20180067 pmid: 31015385
[2]	谢秋, 李才华, 王滔, 孙正怡, 郁琦, 张霆. 利用染色质开放性测序技术探讨叶酸缺乏对胚胎干细胞基因组结构变化调控影响. 生殖医学杂志, 2019, 28: 673-678.
	Xie Q, Li C H, Wang T, Sun Z Y, Yu Q, Zhang T. Exploring effect of folic acid deficiency on the regulation of genome structure changes in embryonic stem cells by using open chromatin sequencing. J Reprod Med, 2019, 63: 89-96. (in Chinese with English abstract)
[3]	吴杰, 全建平, 叶勇, 吴珍芳, 杨杰, 杨明, 郑恩琴. 染色质转座酶可及性测序研究进展. 遗传, 2020, 42: 333-346.
	Wu J, Quan J P, Ye Y, Wu Z F, Yang J, Yang M, Zheng E Q. Advances in assay for transposase-accessible chromatin with high-throughput sequencing. Hereditas, 2020, 42: 333-346. (in Chinese with English abstract)
[4]	许兰, 任立成. 染色质可及性分析的研究进展. 生物化学与生物物理进展, 2020, 49: 1462-1470.
	Xu L Ren L C. Research progress of chromatin accessibility analysis. Prog Biochem Biophys, 2020, 49: 1462-1470.
[5]	廖彬, 杨佳怡, 陈桂芳, 高运华, 王晶, 任歌. 染色质转座酶可及性测序研究与数据分析. 中国测试, 2020, 46(10): 4-10.
	Liao B, Yang J Y, Chen G F, Gao Y H, Wang J, Ren G. Research progress and data analysis of assay for transposase-accessible chromatin with high-throughput sequencing. Chin Measur Test Technol, 2020, 46(10): 4-10. (in Chinese with English abstract)
[6]	Giresi P G, Kim J, McDaniell R M, Iyer V R, Lieb J D. FAIRE (formaldehyde-assisted isolation of regulatory elements) isolates active regulatory elements from human chromatin. Genome Res, 2007, 17: 877-885. pmid: 17179217
[7]	Schones D E, Cui K, Cuddapah S, Roh T Y, Barski A, Wang Z, Wei G, Zhao K. Dynamic regulation of nucleosome positioning in the human genome. Cell, 2008, 132: 887-898. doi: 10.1016/j.cell.2008.02.022 pmid: 18329373
[8]	Sing L, Craford G E. DNase-seq: a high-resolution technique for mapping active gene regulatory elements across the genome from mammalian cells. Cold Spring Harb Protoc, 2010, (2): 5384. doi: 10.1101/pdb.prot5384. doi: 10.1101/pdb.prot5384
[9]	Buenrostro J D, Giresi P G, Zaba L C, Chang H Y, Greenleaf W G. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods, 2013, 10: 1213-1218. doi: 10.1038/nmeth.2688 pmid: 24097267
[10]	Buenrostro J D, Wu B, Chang H Y, Greenleaf W G. ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr Protoc Mol Biol, 2015, 109: 1-9.
[11]	Yan F, Powell D R, Curtis D J, Wong N C. From reads to insight: a hitchhiker’s guide to ATAC-seq data analysis. Genome Biol, 2020, 21: 1-16. doi: 10.1186/s13059-019-1906-x
[12]	Ackermann A M, Wang Z P, Schug J, Naji A, Kaestner K H. Integration of ATAC-seq and RNA-seq identifies human alpha cell and beta cell signature genes. Mol Metab, 2016, 5: 233-244. doi: S2212-8778(16)00003-X pmid: 26977395
[13]	Wang Y Q, Zhang X M, Song Q, Hou Y L, Liu J, Sun Y, Wang P C. Characterization of the chromatin accessibility in an Alzheimer’s disease (AD) mouse model. Alzheimers Res Ther, 2020, 12: 29. doi: 10.1186/s13195-020-00598-2
[14]	王祥, 殷伟, 唐云雯, 殷雪, 于蕾, 岳才军. 植物细胞核分离纯化的探讨. 科技创新与应用, 2014, (1): 37-38.
	Wang X, Yin W, Tang Y W, Yin X, Yu L, Yue C J. Study on isolation and purification of plant nuclei. Technol Innovat Appl, 2014, (1): 37-38. (in Chinese)
[15]	Lu F H, McKenzie N, Gardiner L J, Luo M C, Hall A, Bevan M W. Assay for Transposase-Accessible Chromatin (ATAC) Sequencing. 2019, https://dx.doi.org/10.17504/protocols.io.75whq7e.
[16]	刘鸿森.基于单细胞ATAC-seq技术的玉米花粉细胞核遗传重组的研究. 哈尔滨工业大学博士学位论文, 黑龙江哈尔滨, 2020.
	Liu H S.Using Single Cell ATAC-seq Method to Analyze Recombination in Maize Pollen Nuclei. PhD Dissertation of Harbin Institute of Technology, Harbin, Heilongjiang, China, 2020. (in Chinese with English abstract)
[17]	Bolger A M, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 2014, 30: 14-20.
[18]	Langmead B, Salzberg S L. Fast gapped-read alignment with Bowtie 2. Nat Methods, 2012, 9: 357-359. doi: 10.1038/nmeth.1923 pmid: 22388286
[19]	Rodesch G, Bracard S.In memoriam Professor Luc Picard (1937− 2021). Interv Neuroradiol, 2021, 27: 462-464. doi: 10.1177/15910199211026156
[20]	Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R. The sequence alignment/map format and SAMtools. Bioinformatics, 2009, 25: 2078-2079. doi: 10.1093/bioinformatics/btp352 pmid: 19505943
[21]	Robinson J T, Thorvaldsdóttir H, Winckler W, Guttman M, Lander E S, Getz G, Mesirov J P. Integrative genomics viewer. Nat Biotechnol, 2011, 29: 24-26. doi: 10.1038/nbt.1754 pmid: 21221095
[22]	Zhao L, Lin X L, Yang Y M, Bie X M, Zhang H, Chen J C, Liu X M, Wang H, Jiang J F, Fu X D, Zhang X S, Xiao J. Chromatin reprogramming and transcriptional regulation orchestrate embryogenesis in hexaploidy wheat. BioRxiv, 2022, 477188. .
[23]	Dong P F, Tu X Y, Chu P Y, Lü P T, Zhu N, Grierson D, Du B J, Li P, Zhong S. 3D chromatin architecture of large plant genomes determined by local A/B compartments. Mol Plant, 2017, 10: 1497-1509. doi: S1674-2052(17)30339-8 pmid: 29175436
[24]	Lee J H, Kim E W, Croteau D L, Bohr V R. Heterochromatin: an epigenetic point of view in aging. Exp Mol Med, 2020, 52: 1466-1474. doi: 10.1038/s12276-020-00497-4
[25]	Ranzoni A M, Tangherloni A, Berest I, Riva S G, Myers B, Strzelecka P M, Xu J, Panada E, Mohorianu I, Zaugg J B, Cvejic A. Integrative single-cell RNA-seq and ATAC-seq analysis of human developmental hematopoiesis. Cell Stem Cell, 2021, 28: 472-487. doi: 10.1016/j.stem.2020.11.015 pmid: 33352111
[26]	Liang D, Elwell A L, Aygün N, Krupa O, Wolter J M, Kyere F A, Lafferty M J, Cheek K E, Courtney K P, Yusupova M, Garrett M E, Ashley-Koch A, Crawford G E, Love M I, Torre-Ubieta L, Geschwind D H, Stein J L. Cell-type-specific effects of genetic variation on chromatin accessibility during human neuronal differentiation. Nat Neurosci, 2021, 24: 941-953. doi: 10.1038/s41593-021-00858-w pmid: 34017130
[27]	Liu L Q, Liu C Y, Quintero A, Wu L, Yuan Y, Wang M Y, Cheng M N, Leng L Z, Xu L Q, Dong G Y, Li R, Liu Y, Wei X Y, Xu J S, Chen X W, Lu H, Chen D S, Wang Q L, Zhou Q, Lin X X, Li G, Liu S, Wang Q, Wang H R, Fink J L, Gao Z L, Liu X, Hou Y, Zhu S D, Yang H M, Ye Y, Lin G, Chen F, Herrmann C, Eils R, Shang Z C, Xu X. Deconvolution of single-cell multi-omics layers reveals regulatory heterogeneity. Nat Commun, 2019, 10: 470. doi: 10.1038/s41467-018-08205-7 pmid: 30692544
[28]	Muto Y, Wilson P C, Ledru N, Wu H J, Dimke H, Waikar S S, Humphreys B D. Single cell transcriptional and chromatin accessibility profiling redefine cellular heterogeneity in the adult human kidney. Nat Commun, 2021, 12: 2190. doi: 10.1038/s41467-021-22368-w pmid: 33850129
[29]	Zhang J W, Chen L L, Xing F, Kudrna D A, Yao W, Copetti D, Mu T, Li W M, Song J M, Xie W B, Lee S H, Talag J, Shao L, An Y, Zhang C L, Ouyang Y D, Sun S, Jiao W B, Li F, Du B G, Luo M Z, Maldonado C E, Goicoechea J L, Xiong L Z, Wu C Y, Xing Y Z, Zhou D X, Yu S B, Zhao Y, Wang G W, Yu Y, Luo Y J, Zhou Z W, Hurtado B E, Danowitz A, Wing R A, Zhang Q F. Extensive sequence divergence between the reference genomes of two elite indica rice varieties Zhenshan 97 and Minghui 63. Proc Natl Acad Sci USA, 2016, 113: E5163-E5171.
[30]	陈琳, 林焱, 陈鹏飞, 王绍华, 丁艳锋. 水稻响应缺铁的韧皮部汁液蛋白质组学分析. 植物学报, 2019, 54: 194-207. doi: 10.11983/CBB18184
	Chen L, Lin Y, Chen P F, Wang S H, Ding Y F. Effect of Iron deficiency on the protein profile of rice (Oryza sativa) phloem sap. Chin Bull Bot, 2019, 54: 194-207 (in Chinese with English abstract).
[31]	Ling H Q, Zhao S C, Liu D C, Wang J Y, Sun H, Zhang C, Fan H J, Li D, Dong L L, Tao Y, Gao C, Wu H L, Li Y W, Cui Y, Guo X S, Zheng S R, Wang B, Yu K, Liang Q S, Yang W L, Lou X Y, Chen J, Feng M J, Jian J B, Zhang X F, Luo G B, Jiang Y, Liu J J, Wang Z B, Sha Y H, Zhang B R, Wu H, Tang D D, Shen Q H, Xue P Y, Zou S H, Wang X J, Liu X, Wang F M, Yang Y P, An X L, Dong Z Y, Zhang K P, Zhang X Q, Luo M C, Dvorak J, Tong Y P, Wang J, Yang H M, Li Z S, Wang D W, Zhang A M, Wang J. Draft genome of the wheat A-genome progenitor Triticum urartu. Nature, 2013, 496: 87-90. doi: 10.1038/nature11997
[32]	Jia J Z, Zhao S C, Kong X Y, Li Y R, Zhao G Y, He W M, Appels R, Pfeifer M, Tao Y, Zhang X Y, Jing R L, Zhang C, Ma Y Z, Gao L F, Gao C, Spannagl M, Mayer K F, Li D, Pan S K, Zheng F Y, Hu Q, Xia X C, Li J W, Liang Q S, Chen J, Wicker T, Gou C Y, Kuang H H, He G Y, Luo Y D, Keller B, Xia Q J, Lu P, Wang J Y, Zou H F, Zhang R Z, Xu J Y, Gao J L, Middleton C, Quan Z W, Liu G M, Liu X, He Z H, Mao L, Wang J. Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat adaptation. Nature, 2013, 496: 91-95. doi: 10.1038/nature12028
[33]	韩烈保, 祁泽文, 尹德东, 晁跃辉, 袁建波, 谢琦. 一种直接使用结缕草叶片制备原生质体的方法. 北京:CN107043736B,2021-01-29.
	Han L B, Qi Z W, Yin D D, Chao Y H, Yuan J B, Xie Q. A method for preparing protoplasts directly from Zoysia japonica leaves. Beijing: CN107043736B, 2021-01-29. (in Chinese)
[34]	许昊, 刘小亮, 胡玲, 于海礼, 黄毅, 李洪涛, 万瑛. 基于工程化转座酶的少量细胞RNA-seq文库构建的研究. 第三军医大学学报, 2018, 41: 866-870.
	Xu H, Liu X L, Hu L, Yu H L, Huang Y, Li H T, Wang Y. RNA-seq library construction for small quantity of cells with engineered transposase. Acta Acad Med Mil Tert, 2018, 41: 866-870. (in Chinese with English abstract)

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed