植物小RNA荧光原位杂交实验方法

doi:10.11983/CBB21057

摘要/Abstract

摘要：

小RNA是对植物生长发育十分重要的一类小分子核苷酸, 在多种生命过程以及胁迫响应中发挥重要调控作用。对小RNA的定位研究有助于揭示它们的功能, 而小RNA荧光原位杂交(sRNA-FISH)是一种通过荧光检测技术对生物体内小 RNA进行定性或半定量分析的技术, 目前该技术已经在动物体内被广泛应用, 而在植物体内的应用还比较少。该文详细介绍了基于超高分辨率显微镜的sRNA-FISH的具体操作流程以及注意事项, 该技术可用于探究植物组织内小RNA的表达与定位。

关键词: 荧光原位杂交, 小RNA, 植物

Abstract:

Small RNAs are a type of small nucleotide molecules that are essential for plant growth and development, playing a key role in variety of life processes and in response to stresses. Research on the location of small RNAs could discover their functions in plants. Small RNA FISH is a qualitative or semi-quantitative analysis of small RNA in organisms by fluorescence detection technology. At present, this technology has been widely used in animals, but it is still less applied in plants. This article introduces the specific operation procedures and attentions based on ultra-high resolution microscopy that combines locked nucleic acid (LNA) probe in situ hybridization with immunofluorescence. This protocol can be used to detect the expression and localization of small RNA in plant tissues.

Key words: fluorescent in situ hybridization, small RNA, plants

胡滨滨, 薛治慧, 张翠. 植物小RNA荧光原位杂交实验方法. 植物学报, 2021, 56(3): 330-338.

Binbin Hu, Zhihui Xue, Cui Zhang. Protocols for Small RNA FISH in Plants. Chinese Bulletin of Botany, 2021, 56(3): 330-338.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB21057

https://www.chinbullbotany.com/CN/Y2021/V56/I3/330

图/表 2

图1 小RNA荧光原位杂交实验流程图 (A) 探针设计; (B) 石蜡切片; (C) 探针杂交; (D) 一抗孵育; (E) 二抗孵育; (F) 光谱拆分以及激光共聚焦显微成像

Figure 1 Outlines of small RNA fluorescent in situ hybridization (A) Probe design; (B) Paraffin sectioning; (C) Probe hybridization; (D) Primary antibody incubation; (E) Secondary antibody incubation; (F) Spectral unmixing and imaging by confocal microscope

图2 小RNA FISH效果示例 (A) miR165/166反义寡核苷酸探针; (B) 阴性对照: miR165/ 166顺义寡核苷酸探针。Bars=50 μm

Figure 2 Schematic diagram of sRNA FISH (A) Antisense probe of miR165/166; (B) Negative control: Sense probe of miR165/166. Bars=50 μm

参考文献 55

1	Baumberger N, Baulcombe DC (2005). Arabidopsis ARGONAUTE1 is an RNA slicer that selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci USA 102, 11928-11933. DOI URL
2	Bhogale S, Mahajan AS, Natarajan B, Rajabhoj M, Thulasiram HV, Banerjee AK (2014). MicroRNA156: a potential graft-transmissible microRNA that modulates plant architecture and tuberization in Solanum tuberosum ssp. andigena. Plant Physiol 164, 1011-1027. DOI URL
3	Bleckmann A, Dresselhaus T (2016). Fluorescent whole- mount RNA in situ hybridization (F-WISH) in plant germ cells and the fertilized ovule. Methods 98, 66-73. DOI PMID
4	Bruno L, Muto A, Spadafora ND, Iaria D, Chiappetta A, Van Lijsebettens M, Bitonti MB (2011). Multi-probe in situ hybridization to whole mount Arabidopsis seedlings. Int J Dev Biol 55, 197-203. DOI URL
5	Bruno L, Ronchini M, Gagliardi O, Corinti T, Chiappetta A, Gerola P, Bitonti MB (2015). Analysis of ATGUS1 and ATGUS2 in Arabidopsis root apex by a highly sensitive TSA-MISH method. Int J Dev Biol 59, 221-228. DOI URL
6	Buhtz A, Springer F, Chappell L, Baulcombe DC, Kehr J (2008). Identification and characterization of small RNAs from the phloem of Brassica napus. Plant J 53, 739-749. DOI URL
7	Carlsbecker A, Lee JY, Roberts CJ, Dettmer J, Lehesranta S, Zhou J, Lindgren O, Moreno-Risueno MA, Vatén A, Thitamadee S, Campilho A, Sebastian J, Bowman JL, Helariutta Y, Benfey PN (2010). Cell signaling by microRNA165/6 directs gene dose-dependent root cell fate. Nature 465, 316-321. DOI PMID
8	Chen XM (2004). A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303, 2022-2025. DOI URL
9	Chen XM (2009). Small RNAs and their roles in plant development. Annu Rev Cell Dev Biol 25, 21-44. DOI URL
10	Chitwood DH, Nogueira FT, Howell MD, Montogmery TA, Carrington JC, Timmermans MC (2008). Pattern formation in leaves via small RNA mobility. Dev Biol 319, 587-588.
11	Chitwood DH, Nogueira FTS, Howell MD, Montgomery TA, Carrington JC, Timmermans MCP (2009). Pattern formation via small RNA mobility. Gene Dev 23, 549-554. DOI URL
12	Fischer AH, Jacobson KA, Rose J, Zeller R (2008). Paraffin embedding tissue samples for sectioning. CSH Protoc 2008, pdb.prot4989.
13	Himanen K, Woloszynska M, Boccardi TM, De Groeve S, Nelissen H, Bruno L, Vuylsteke M, Van Lijsebettens M (2012). Histone H2b monoubiquitination is required to reach maximal transcript levels of circadian clock genes in Arabidopsis. Plant J 72, 249-260. DOI URL
14	Howell MD, Fahlgren N, Chapman EJ, Cumbie JS, Sullivan CM, Givan SA, Kasschau KD, Carrington JC (2007). Genome-wide analysis of the RNA-DEPENDENT RNA POLYMERASE6/DICER-LIKE4 pathway in Arabidop- sis reveals dependency on miRNA- and tasiRNA-directed targeting. Plant Cell 19, 926-942. PMID
15	Huang K, Baldrich P, Meyers BC, Caplan JL (2019). sRNA-FISH: versatile fluorescent in situ detection of small RNAs in plants. Plant J 98, 359-369. DOI
16	Huen AK, Rodriguez-Medina C, Ho AYY, Atkins CA, Smith PMC (2017). Long-distance movement of phospha- te starvation-responsive microRNAs in Arabidopsis. Plant Biol 19, 643-649. DOI URL
17	Javelle M, Timmermans MCP (2012). In situ localization of small RNAs in plants by using LNA probes. Nat Protoc 7, 533-541. DOI PMID
18	Jiang JM (2019). Fluorescence in situ hybridization in plants: recent developments and future applications. Chromoso- me Res 27, 153-165.
19	Jones-Rhoades MW, Bartel DP, Bartel B (2006). Micro- RNAs and their regulatory roles in plants. Annu Rev Plant Biol 57, 19-53. PMID
20	Jung JH, Park CM (2007). MIR166/ 165 genes exhibit dynamic expression patterns in regulating shoot apical meris- tem and floral development in Arabidopsis. Planta 225, 1327-1338. DOI URL
21	Kidner C, Timmermans M (2006). In situ hybridization as a tool to study the role of microRNAs in plant development. Methods Mol Biol 342, 159-179.
22	Kim VN, Han JJ, Siomi MC (2009). Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 10, 126-139.
23	Knauer S, Holt AL, Rubio-Somoza I, Tucker EJ, Hinze A, Pisch M, Javelle M, Timmermans MC, Tucker MR, Laux T (2013). A protodermal miR394 signal defines a region of stem cell competence in the Arabidopsis shoot meristem. Dev Cell 24, 125-132. DOI URL
24	Kurihara Y, Watanabe Y (2004). Arabidopsis micro-RNA biogenesis through dicer-like 1 protein functions. Proc Natl Acad Sci USA 101, 12753-12758. DOI URL
25	Lamb JC, Danilova T, Bauer MJ, Meyer JM, Holland JJ, Jensen MD, Birchler JA (2007). Single-gene detection and karyotyping using small-target fluorescence in situ hybridization on maize somatic chromosomes. Genetics 175, 1047-1058. DOI URL
26	Lee Y, Kim M, Han JJ, Yeom KH, Lee S, Baek SH, Kim VN (2004). MicroRNA genes are transcribed by RNA polymerase II. EMBO J 23, 4051-4060. DOI URL
27	Levsky JM, Singer RH (2003). Fluorescence in situ hybridization: past, present and future. J Cell Sci 116, 2833-2838. DOI URL
28	Li JJ, Yang ZY, Yu B, Liu J, Chen XM (2005). Methylation protects miRNAs and siRNAs from a 3'-end uridylation activity in Arabidopsis. Curr Biol 15, 1501-1507. DOI URL
29	Li S, Wang XT, Xu WY, Liu T, Cai CM, Chen LY, Clark CB, Ma JX (2021). Unidirectional movement of small RNAs from shoots to roots in interspecific heterografts. Nat Plants 7, 50-59. DOI URL
30	Lin SI, Chiang SF, Lin WY, Chen JW, Tseng CY, Wu PC, Chiou TJ (2008). Regulatory network of microRNA399 and PHO2 by systemic signaling. Plant Physiol 147, 732-746. DOI URL
31	Liu QL, Yao XZ, Pi LM, Wang H, Cui XF, Huang H (2009). The ARGONAUTE10 gene modulates shoot apical meris- tem maintenance and establishment of leaf polarity by repressing miR165/166 in Arabidopsis. Plant J 58, 27-40. DOI URL
32	Lu J, Tsourkas A (2009). Imaging individual microRNAs in single mammalian cells in situ. Nucleic Acids Res 37, e100. DOI URL
33	Moissiard G, Parizotto EA, Himber C, Voinnet O (2007). Transitivity in Arabidopsis can be primed, requires the redundant action of the antiviral Dicer-like 4 and Dicer-like 2, and is compromised by viral-encoded suppressor proteins. RNA 13, 1268-1278. DOI URL
34	Nodine MD, Bartel DP (2010). MicroRNAs prevent precocious gene expression and enable pattern formation during plant embryogenesis. Gene Dev 24, 2678-2692. DOI URL
35	Nogueira FTS, Chitwood DH, Madi S, Ohtsu K, Schnable PS, Scanlon MJ, Timmermans MCP (2009). Regulation of small RNA accumulation in the maize shoot apex. PLoS Genet 5, e1000320. DOI URL
36	Nogueira FTS, Madi S, Chitwood DH, Juarez MT, Timmer- mans MCP (2007). Two small regulatory RNAs establish opposing fates of a developmental axis. Gene Dev 21, 750-755. PMID
37	Ori N, Cohen AR, Etzioni A, Brand A, Yanai O, Shleizer S, Menda N, Amsellem Z, Efroni I, Pekker I, Alvarez JP, Blum E, Zamir D, Eshed Y (2007). Regulation of LANCEOLATE by miR319 is required for compound-leaf development in tomato. Nat Genet 39, 787-791. DOI URL
38	Pagliarani C, Gambino G (2019). Small RNA mobility: spread of RNA silencing effectors and its effect on developmental processes and stress adaptation in plants. Int J Mol Sci 20, 4306. DOI URL
39	Pant BD, Buhtz A, Kehr J, Scheible WR (2008). MicroRNA399 is a long-distance signal for the regulation of plant phosphate homeostasis. Plant J 53, 731-738. DOI URL
40	Pant BD, Musialak-Lange M, Nuc P, May P, Buhtz A, Kehr J, Walther D, Scheible WR (2009). Identification of nutrient-responsive Arabidopsis and rapeseed microRNAs by comprehensive real-time polymerase chain reaction profiling and small RNA sequencing. Plant Physiol 150, 1541-1555. DOI URL
41	Parizotto EA, Dunoyer P, Rahm N, Himber C, Voinnet O (2004). In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. Gene Dev 18, 2237-2242. PMID
42	Park MY, Wu G, Gonzalez-Sulser A, Vaucheret H, Poethig RS (2005). Nuclear processing and export of microRNAs in Arabidopsis. Proc Natl Acad Sci USA 102, 3691-3696. DOI URL
43	Raman S, Greb T, Peaucelle A, Blein T, Laufs P, Theres K (2008). Interplay of miR164, CUP-SHAPED COTYLEDON genes and LATERAL SUPPRESSOR controls axillary meristem formation in Arabidopsis thaliana. Plant J 55, 65-76. DOI URL
44	Rozier F, Mirabet V, Vernoux T, Das P (2014). Analysis of 3D gene expression patterns in plants using whole-mount RNA in situ hybridization. Nat Protoc 9, 2464-2475. DOI URL
45	Skopelitis DS, Hill K, Klesen S, Marco CF, von Born P, Chitwood DH, Timmermans MCP (2018). Gating of miRNA movement at defined cell-cell interfaces governs their impact as positional signals. Nat Commun 9, 3107. DOI PMID
46	Tirichine L, Andrey P, Biot E, Maurin Y, Gaudin V (2009). 3D fluorescent in situ hybridization using Arabidopsis leaf cryosections and isolated nuclei. Plant Methods 5, 11. DOI PMID
47	Vaucheret H, Vazquez F, Crété P, Bartel DP (2004). The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Gene Dev 18, 1187-1197. PMID
48	Wollmann H, Mica E, Todesco M, Long JA, Weigel D (2010). On reconciling the interactions between APETA- LA2, miR172 and AGAMOUS with the ABC model of flower development. Development 137, 3633-3642. DOI PMID
49	Woloszynska M, Le Gall S, Neyt P, Boccardi TM, Grasser M, Längst G, Aesaert S, Coussens G, Dhondt S, Van De Slijke E, Bruno L, Fung-Uceda J, Mas P, Van Montagu M, Inzé D, Himanen K, De Jaeger G, Grasser KD, Van Lijsebettens M (2019). Histone 2B monoubiquitination complex integrates transcript elongation with RNA processing at circadian clock and flowering regulators. Proc Natl Acad Sci USA 116, 8060-8069. DOI URL
50	Xue ZH, Liu LY, Zhang C (2020). Regulation of shoot apical meristem and axillary meristem development in plants. Int J Mol Sci 21, 2917. DOI URL
51	Yang WB, Schuster C, Prunet N, Dong QK, Landrein B, Wightman R, Meyerowitz EM (2020). Visualization of protein coding, long noncoding, and nuclear RNAs by fluorescence in situ hybridization in sections of shoot apical meristems and developing flowers. Plant Physiol 182, 147-158. DOI URL
52	Yang WB, Wightman R, Meyerowitz EM (2017). Cell cycle control by nuclear sequestration of CDC20 and CDH1 mRNA in plant stem cells. Mol Cell 68, 1108-1119. DOI URL
53	Yu Y, Zhang YC, Chen XM, Chen YQ (2019). Plant noncoding RNAs: hidden players in development and stress responses. Annu Rev Cell Dev Biol 35, 407-431. DOI
54	Zhang C, Fan LS, Le BH, Ye PY, Mo BX, Chen XM (2020). Regulation of ARGONAUTE10 expression enables temporal and spatial precision in axillary meristem initiation in Arabidopsis. Dev Cell 55, 603-616. DOI URL
55	Zhang C, Wang J, Wenkel S, Chandler JW, Werr W, Jiao YL (2018). Spatiotemporal control of axillary meristem formation by interacting transcriptional regulators. Development 145, dev158352.

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