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Virus-based MicroRNA Silencing
基于病毒的microRNA沉默   

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Abstract

Virus-based microRNA silencing (VbMS) is a viable and prompt method to screen and characterize the function of microRNAs (miRNAs) in plants. The Tobacco rattle virus (TRV)-based VbMS method was originally developed by the Yule Liu's group (Sha et al., 2014) using miRNA target mimic (TM) methodology. Here, we describe the TRV-based VbMS method for silencing endogenous miRNA in Nicotiana benthamiana and tomato via Agrobacterium infiltritions. For each assay, Agrobacterium cultures containing pTRV1 and specific pTRV2e derivative harboring TM fragments are mixed and infiltrated into plant tissues. Generally within 3 weeks, the target miRNAs gene will be silenced and the newly developed tissues will exhibit corresponding phenotypes.

Keywords: MicroRNA(microRNA), Plant virus(植物病毒), MicroRNA silencing(microRNA沉默), MicroRNA target mimic(microRNA靶模拟), Virus-based vector(病毒载体)

Materials and Reagents

  1. Centrifuge tubes
  2. Sterile 1 ml syringe (needle removed)
  3. Sterile bacterial culture tubes
  4. Plant materials
    Nicotiana benthamiana, tomato (cultivar Moneymaker)
    Note: seeds can be obtained from Yule Liu’s lab.
  5. Bacteria strains
    1. Escherichia coli: DH5α, ccdb survival (Thermo Fisher Scientific, InvitrogenTM, catalog number: A10460 )
    2. Agrobacterium tumefaciens: GV3101, GV2260 (alternative to GV3101)
      Note: All strains can be obtained from Yule Liu’s lab.
  6. Plasmids
    1. pTRV1 (Dong et al., 2007): a T-DNA vector containing 2 x 35 s promoter, Nos terminator and full cDNA of TRV RNA1 (from Ppk20 strain).
    2.  pTRV2e (Sha et al., 2014): a T-DNA vector containing 2 x 35 s promoter, Nos terminator and cDNA clone of TRV RNA2, of which the sub-genomic promoter of coat protein from Pea early brown virus (PEBV) (Wang et al., 1997) and a ligation independent cloning (LIC) cassette are inserted immediately downstream of the TRV CP gene.
    3. pTRV2e-GFP: GFP gene was inserted at LIC cassette into pTRV2e. This construct can be used in a control assay to show successful exogenous expression.
    4. The pTRV1 (Arabidopsis, ABRC, catalog number: CD3-1039 ) and pTRV2e (Arabidopsis, ABRC, catalog number: CD3-1866 ) vectors can be ordered at the Arabidopsis Biological Resource Center (ABRC, http://www.arabidopsis.org/).
  7. Culture Media
    1. Liquid Luria-Bertani (LB) medium
      Solid LB medium plate with 1.5% agar
      Note: LB medium is autoclaved at 120 °C for 20 min before appropriate antibiotics are added.
  8. Antibiotics
    1. Kanamycin (Sangon Biotech, USP Grade)
    2. Rifampicin (Sangon Biotech, USP Grade)
    3. Gentamicin (Sangon Biotech, USP Grade)
  9. PCR reagents
    1. EasyTaq DNA polymerase (Beijing TransGen Biotech, catalog number: AP112 )
    2. EasyPfu DNA polymerase (Beijing TransGen Biotech, catalog number: AP211 )
    3. dNTP Mix (Roche Diagnostics, catalog number: 04729706103 )
  10. Infiltration reagents
    1. Dimethyl sulfoxide, DMSO (AMRESCO, ACS grade)
    2. MgCl2 (Beijing Chemical Works, Analytical pure) (see Recipes)
    3. 2-(N-Morpholino) ethanesulfonic acid, MES (AMRESCO, Regent Grade) (see Recipes)
    4. Acetosyringone (3, 5-Dimethoxy-4-hydroxyacetophenone) (AS) [Sigma-Aldrich, Purity (HPLC)] (see Recipes)
    5. Infiltration buffer (see Recipes)

Equipment

  1. Plant growth chamber (24 °C, 16 h/8 h light/dark photoperiod, 40-80% humidity)
  2. Centrifuge
  3. PCR instrument
  4. 37 °C and 28 °C incubators with shaking

Procedure

  1. TMs designing.
    1. The TM molecules were designed empirically by adding 3-4 nucleotides into the complementary sequences between sites opposite to the 10th and 11th nt of the targeted miRNA.
      Keep the other position with base-paring to the miRNA.
      Note: Figure 1 shows an example for TM design.


    Figure 1. Example for TM design. A. DNA sequence of TM for miRNA319 (TM319). B. Base paring of TM319 and Nbe-miR319.Red letters are nucleotides inserted in to the complementary sequences between sites opposite to the 10th and 11th nt (denoted by “-“).

  2. Cloning.
    1. The miRNA TM fragment was inserted into pTRV2e at LIC cassette as described in the previous study (Sha et al., 2014).
    2. Correct constructs were screened by PCR and must be confirmed by DNA sequencing.
  3. Plasmid extraction.
    1. Correct clones were grown in 2~5 ml liquid LB medium (containing 50 µg/ml Kanamycin) at 37 °C, with shaking at 200 rpm for 16 h.
    2. Collect the bacteria and extract plasmids using Alkaline Lysis Method (Sambrook, 2001).
  4. Agrobacterium transformation.
    1. Transform pTRV1, pTRV2e or its derivatives into Agrobacterium strain GV3101 (or GV2260) respectively.
    2. Grown for 2 days on solid LB media (containing 50 µg/ml Kanamycin, 50 µg/ml Rifampicin) at 28 °C.
    3. Confirm that the Agrobacteria contain desired plasmid by PCR using specific primers and streak the correct transformants on LB plate.
  5. Preparation of Agro-infiltrates.
    1. Grow correct transformants containing pTRV1, pTRV2e or pTRV2e derivatives (Sha et al., 2014, Figure 1) in 5 ml liquid LB media respectively (containing 50 µg/ml Kanamycin, 50 µg/ml Rifampicin) in 28 °C incubator shaking at 200 rpm overnight.
    2. Collect the culture media and adjust each agrobacterium culture to OD600=1.0. Mix equal volume of agrobacterium culture (OD600=1.0) of pTRV1 and that of pTRV2e or pTRV2e derivatives together.
    3. Pellet the mixed agrobacteria by centrifuging at 3,000 x g for 5 min at room temperature.
    4. Discard the supernatant and re-suspend the pellet with equal volume of infiltration buffer (to keep OD600 ≈1.0).
    5. Incubate the re-suspended agrobacteria at room temperature for 2.5-6 h.
      Note: Inoculating agrobacteria into media for culturing should be done on clean bench and all equipment used needs to be sterile.
  6. Plant infiltration.
    1. Select 6-leaf-stage plants for VbMS assay. Infiltrate the re-suspended agrobacteria into the abaxial side of 3-4 expanded leaves (avoid the mid vein) with 1 ml needless syringe.


    Figure 2. Schematic diagram of Agrobacterium infiltration

  7. Plant growth and evaluation of miRNA silencing effects.
    1. Grow the infiltrated plants at 24 °C with a 16 h/8 h light/dark photoperiod and the light intensity is 200 μmol m-2 s-1.
      In 2-3 weeks post inoculation the target miRNA will be silenced at the whole plant level.
    2. The newly developed organs will show phenotypes of strong silencing of the corresponding miRNA. These tissues can be used for appropriate experiments.
      Note: Figure 2 shows a schematic overview of infiltration procedure. Each leaf is often injected at 2-4 sites throughout the leaf lamina, each injection site has a diameter >1 cm.

Recipes

  1. 1 M MgCl2
    20.33 g MgCl2.6H2O dissolved in 100 ml ddH2O, autoclaved by 120 °C, 20 min, stored at 4 °C.
  2. 1 M MES
    21.325 g MES dissolved in 100 ml ddH2O, sterilized via filtration through 0.22 µm membrane, stored at room temperature.
  3. 200 mM acetosyringone (AS)
    0.3924 g AS dissolved in 10 ml DMSO, stored at -20 °C as 1 ml aliquots.
  4. Infiltration buffer (10 mM MgCl2, 10 mM MES, 200 µM AS)
    Add 1 ml MgCl2 (1 M), 1 ml MES (1 M), 100 μl AS (200 mM), add ddH2O to 100 ml.

References

  1. Liu, Y., Schiff, M., Marathe, R. and Dinesh-Kumar, S. P. (2002). Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J 30(4): 415-429.
  2. Sambrook, J. (2001). Molecular cloning: A laboratory manual, third edition. Cold Spring Harbor Laboratory Press.
  3. Sha, A., Zhao, J., Yin, K., Tang, Y., Wang, Y., Wei, X., Hong, Y. and Liu, Y. (2014). Virus-based microRNA silencing in plants. Plant Physiol 164(1): 36-47.
  4. Wang, D., MacFarlane, S. A. and Maule, A. J. (1997). Viral determinants of pea early browning virus seed transmission in pea. Virology 234(1): 112-117.

简介

基于病毒的微RNA沉默(VbMS)是筛选和表征微RNA(miRNA)在植物中的功能的可行和迅速的方法。 基于烟草响铃病毒(TRV)的VbMS方法最初是由Yule Liu的小组(Sha等人,2014)使用miRNA靶模拟物TM方法开发的。 在这里,我们描述基于TRV的VbMS方法,用于通过土壤杆菌浸润物在本氏烟草和番茄中沉默内源性miRNA。 对于每个测定,将含有pTRV1的土壤杆菌培养物和含有TM片段的特异性pTRV2e衍生物混合并渗入植物组织中。 通常在3周内,靶miRNA基因将被沉默,并且新开发的组织将显示相应的表型。

关键字:microRNA, 植物病毒, microRNA沉默, microRNA靶模拟, 病毒载体

材料和试剂

  1. 离心管
  2. 无菌1 ml注射器(针头取出)
  3. 无菌细菌培养管
  4. 植物材料
    本塞姆氏烟草,番茄(品种Moneymaker)
    注意:种子可以从Yule Liu的实验室获得。
  5. 细菌菌株
    1. 大肠杆菌:DH5α,ccdb 存活(Thermo Fisher Scientific,Invitrogen TM ,目录号:A10460)
    2. 根癌农杆菌:GV3101,GV2260(GV3101的替代品)
      注意:所有菌株均可从Yule Liu的实验室获得。
  6. 质粒
    1. pTRV1(Dong等人,2007):包含2×35s的T-DNA载体 启动子,Nos终止子和TRV RNA1的完整cDNA(来自Ppk20菌株)
    2. < pTRV2e(Sha et al。,2014):含有2×35s启动子的T-DNA载体, ?Nos终止子和TRV RNA2的cDNA克隆,其中亚基因组 来自豌豆早期棕色病毒(PEBV)的外壳蛋白的启动子(Wang等人, ?1997)和连接非依赖性克隆(LIC)盒 紧接在TRV CP基因的下游
    3. pTRV2e-GFP:GFP 基因 在LIC盒中插入pTRV2e。这个结构可以用于 ?控制测定显示成功的外源表达
    4. pTRV1 (拟南芥,ABRC,目录号:CD3-1039)和pTRV2e(拟南芥, ABRC,目录号:CD3-1866)载体可以订购 拟南芥生物资源中心(ABRC,http://www.arabidopsis.org/)。
  7. 文化媒体
    1. 液体Luria-Bertani(LB)培养基
      具有1.5%琼脂的固体LB培养基平板 注意:在加入适当的抗生素之前,将LB培养基在120℃下高压灭菌20分钟。
  8. 抗生素
    1. 卡那霉素(Sangon Biotech,USP Grade)
    2. 利福平(Sangon Biotech,USP Grade)
    3. 庆大霉素(Sangon Biotech,USP Grade)
  9. PCR试剂
    1. EasyTaq DNA聚合酶(Beijing TransGen Biotech,目录号:AP112)
    2. EasyPfu DNA聚合酶(Beijing TransGen Biotech,目录号:AP211)
    3. dNTP Mix(Roche Diagnostics,目录号:04729706103)
  10. 浸润试剂
    1. 二甲基亚砜,DMSO(AMRESCO,ACS级)
    2. MgCl 2(北京化工,分析纯)(参见配方)
    3. 2-(N-吗啉代)乙磺酸,MES(AMRESCO,Regent Grade)(参见配方)
    4. 乙酰丁香酮(3,5-二甲氧基-4-羟基苯乙酮)(AS)[Sigma-Aldrich,Purity(HPLC)](参见配方)
    5. 浸润缓冲液(见配方)

设备

  1. 植物生长室(24℃,16小时/8小时光/暗光周期,40-80%湿度)
  2. 离心机
  3. PCR仪
  4. 37℃和28℃振荡孵育器

程序

  1. TM设计。
    1. 通过添加3-4个核苷酸经验地设计TM分子 进入与第10位相反的位点之间的互补序列 第11个nt的目标miRNA。
      保持其他位置与基准配对到miRNA。
      注意:图1显示了TM设计的示例。


    图1. TM设计的示例。A.Mi3的TM DNA序列(TM319)。 B. TM319和Nbe-miR319的碱基配对。红色字母是插入到与第10和第11个nt(由" - "表示)相对的位点之间的互补序列中的核苷酸。

  2. 克隆。
    1. 如先前研究中所述将miRNA TM片段插入到LIC盒中的pTRV2e中(Sha等人,2014)。
    2. 通过PCR筛选正确的构建体,并且必须通过DNA测序来确认。
  3. 质粒提取
    1. 将正确的克隆在2?5ml液体LB培养基(含有50μL)中生长 μg/ml卡那霉素)中,在200rpm振荡16小时。
    2. 收集细菌并使用碱性裂解法(Sambrook,2001)提取质粒。
  4. 土壤杆菌转化
    1. 将pTRV1,pTRV2e或其衍生物分别转化到农杆菌菌株GV3101(或GV2260)中。
    2. 在28℃下在固体LB培养基(含有50μg/ml卡那霉素,50μg/ml利福平)上生长2天。
    3. 通过PCR使用确认土壤杆菌含有所需的质粒 并在LB平板上划线正确的转化体。
  5. 农业渗透物的制备
    1. 生长包含pTRV1,pTRV2e或pTRV2e的正确转化体 衍生物(Sha等人,2014,图1)在5ml液体LB培养基中 (含50μg/ml卡那霉素,50μg/ml利福平) 在200rpm振荡培养箱过夜。
    2. 收集文化 并将每个农杆菌培养物调整至OD 600 = 1.0。混合均匀 pTRV1的土壤杆菌培养物的体积(OD <600 = 1.0)和pTRV2e的体积 或pTRV2e衍生物。
    3. 通过在室温下以3,000xg离心5分钟来对混合的农杆菌进行离心。
    4. 弃去上清液并用等体积的浸润缓冲液重悬细胞沉淀(以保持OD <600>≈1.0)。
    5. 在室温下孵育重悬的农杆菌2.5-6小时 注意:将农杆菌接种到培养基中以进行培养 在清洁工作台上,使用的所有设备都需要是无菌的。
  6. 植物渗入。
    1. 选择6叶期植物用于VbMS测定。渗透 重新悬浮的农杆菌进入3-4扩展叶的后轴侧 (避免中间静脉)用1毫升不必要的注射器。


    图2.土壤杆菌渗入
    示意图
  7. 植物生长和miRNA沉默效应的评价
    1. 在24℃下以16小时/8小时光/暗光周期生长浸润的植物,并且光强度为200μmol/m 2 -2s -1 s -1。
      在接种后2-3周,靶miRNA将在整个植物水平上沉默。
    2. 新发展的器官将显示强烈沉默的表型 的相应miRNA。这些组织可以适当使用 实验 注意:图2显示了一个示意图 渗透程序。每个叶通常在2-4个位点注射 在整个叶片中,每个注射部位具有> 1cm的直径

食谱

  1. 1 M MgCl 2
    溶解在100ml ddH 2 O中的20.33g MgCl 2·6H 2 O,通过120℃高压灭菌, 20分钟,储存在4℃
  2. 1 M MES
    21.325g溶解在100ml ddH 2 O中的MES,通过0.22μm膜过滤灭菌,在室温下贮存。
  3. 200mM乙酰丁香酮(AS)
    0.3924g AS溶解在10ml DMSO中,在-20℃下以1ml等分试样储存
  4. 渗透缓冲液(10mM MgCl 2,10mM MES,200μMAS)
    加入1ml MgCl 2(1M),1ml MES(1M),100μlAS(200mM),加入ddH 2 O至100ml。 />

参考文献

  1. Liu,Y.,Schiff,M.,Marathe,R。和Dinesh-Kumar,S.P。(2002)。 烟草Rar1,EDS1和NPR1/NIM1样基因是N介导的烟草花叶病毒抗性所必需的。 Plant J 30(4):415-429。
  2. Sambrook,J。(2001)。分子克隆:实验室手册,第三版。冷泉港实验室出版社。
  3. Sha,A.,Zhao,J.,Yin,K.,Tang,Y.,Wang,Y.,Wei,X.,Hong,Y.and Liu,Y.(2014)。 植物中基于病毒的微RNA沉默 植物生理 164(1):36-47。
  4. Wang,D.,MacFarlane,S.A.and Maule,A.J。(1997)。 豌豆早期褐变病毒种子传播在豌豆中的病毒决定因素病毒学 234(1):112-117。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zhao, J. and Liu, Y. (2016). Virus-based MicroRNA Silencing. Bio-protocol 6(2): e1714. DOI: 10.21769/BioProtoc.1714.
  2. Sha, A., Zhao, J., Yin, K., Tang, Y., Wang, Y., Wei, X., Hong, Y. and Liu, Y. (2014). Virus-based microRNA silencing in plants. Plant Physiol 164(1): 36-47.
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