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Somatic homologous recombination (SHR) is a major pathway of DNA double-strand break (DSB) repair, in which intact homologous regions are used as a template for the removal of lesions. Its frequency in plants is generally low, as most DSB are removed by non-homologous mechanisms in higher eukaryotes. Nevertheless, SHR frequency has been shown to increase in response to various chemical and physical agents that cause DNA damage and/or alter genome stability (reviewed in March-Díaz and Reyes, 2009). We monitor the frequency of SHR in transgenic Arabidopsis seedlings containing recombination substrates with two truncated but overlapping parts of the β-glucuronidase (GUS) reporter gene (Orel et al., 2003; Schuermann et al., 2005). Upon an SHR event, a functional version of the transgene can be restored (Figure 1A). A histochemical assay applicable to whole plantlets allows the visualization of cells in which the reporter is restored, as the encoded enzyme converts a colorless substrate into a blue compound. This type of reporter has been extensively used to identify gene products required for regulating SHR levels in plants. We analyze plants stimulated for SHR by treatments with DNA damaging agents (bleocin, mitomycin C and UV-C) and compare them to non-treated plants.

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Measuring Homologous Recombination Frequency in Arabidopsis Seedlings
拟南芥幼苗中同源重组频率的测定

植物科学 > 植物生理学 > 组织分析
作者: Marisa Rosa
Marisa RosaAffiliation: Gregor Mendel Institute of Molecular Plant Biology, Vienna, Austria
For correspondence: massrosa@berkeley.edu
Bio-protocol author page: a1272
 and Ortrun Mittelsten Scheid
Ortrun Mittelsten ScheidAffiliation: Gregor Mendel Institute of Molecular Plant Biology, Vienna, Austria
Bio-protocol author page: a1273
Vol 4, Iss 7, 4/5/2014, 3843 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1094

[Abstract] Somatic homologous recombination (SHR) is a major pathway of DNA double-strand break (DSB) repair, in which intact homologous regions are used as a template for the removal of lesions. Its frequency in plants is generally low, as most DSB are removed by non-homologous mechanisms in higher eukaryotes. Nevertheless, SHR frequency has been shown to increase in response to various chemical and physical agents that cause DNA damage and/or alter genome stability (reviewed in March-Díaz and Reyes, 2009). We monitor the frequency of SHR in transgenic Arabidopsis seedlings containing recombination substrates with two truncated but overlapping parts of the β-glucuronidase (GUS) reporter gene (Orel et al., 2003; Schuermann et al., 2005). Upon an SHR event, a functional version of the transgene can be restored (Figure 1A). A histochemical assay applicable to whole plantlets allows the visualization of cells in which the reporter is restored, as the encoded enzyme converts a colorless substrate into a blue compound. This type of reporter has been extensively used to identify gene products required for regulating SHR levels in plants. We analyze plants stimulated for SHR by treatments with DNA damaging agents (bleocin, mitomycin C and UV-C) and compare them to non-treated plants.
Keywords: Homologous recombination(同源重组), DNA repair(DNA修复), Genotoxic stress(genotoxic应力), Arabidopsis(拟南芥)

[Abstract]

Materials and Reagents

  1. Arabidopsis thaliana seeds of reporter lines IU.GUS-8 and DGU.US-1 (Figure 1) (Orel et al., 2003) [introgressed into various genetic backgrounds and genotyped for homozygosity of reporters and mutations (Rosa et al., 2013). As an example, we use wild type Columbia-0, and mutants arp6-3 and swc6-1. Both mutants lack subunits of the Arabidopsis homolog of the SWR1 complex and, additional to being sensitive to DNA damaging agents (Rosa et al., 2013), have pleiotropic developmental defects (for more information see Schuermann et al., 2005)]
  2. Sodium hypochlorite (Sigma-Aldrich, catalog number: 425044 )
  3. Tween-80 (Sigma-Aldrich, catalog number: 4780 )
  4. Sterile H2O
  5. Solid growth medium
  6. Liquid plant growth medium (same as solid growth medium but without agar)
  7. Hydroxyurea
  8. 70% ethanol
  9. Seed sterilization solution (see Recipes)
  10. Bleocin (commercial name for bleomycin) (EMD Millipore, catalog number: 203408 ) (see Recipes)
  11. Mitomycin C (Duchefa Biochemie BV, catalog number: M0133 ) (see Recipes)
  12. GUS staining solution (see Recipes)
  13. 1 M Sodium Phosphate Buffer (see Recipes)

Equipment

  1. Plastic petri dishes for plant culture (see Notes) (round 200 x 15 mm, with 20 ml of solid growth medium)
  2. Sterile hood, preferably a biological safety cabinet to avoid exposure to the genotoxins
  3. Bench top block shaker (e.g. Eppendorf, Thermomixer®)
  4. UV crosslinker (254-nm UV light bulbs, 15 watts each) (Stratagene, model: Stratalinker 2400 )
  5. Forceps
  6. Box or aluminum foil
  7. Parafilm
  8. 1.5 ml Eppendorf tubes
  9. 50 and 15 ml Falcon tubes
  10. Vacuum applicator (e.g. desiccator)
  11. Plant growth facilities with 16-h-light/8-h-dark cycles, at 21 °C
  12. Incubator at 37 °C
  13. Stereomicroscope

Procedure

  1. Wash the seeds in 1.5 ml Eppendorf tubes with 1 ml of the sterilization solution, shake in a benchtop block shaker at 300 rpm for 6 min at room temperature. Allow seeds to set in the bottom of the Eppendorf and remove supernatant. Wash with 1 ml of sterile water, 5 times for 5 min in the shaker. After the final wash, remove supernatant and dry overnight in the closed sterile hood, leaving the tubes open.
    Note: Sterilize approximately 200 seeds per genotype and treatment. All subsequent steps involving seed or plantlet manipulation should be carried in the sterile hood.
  2. Plate seeds on 200 x 15 mm petri dishes with growth medium (≈ 75 to 100 per plate and 2 plates per genotype and treatment), using flame-sterilized and cooled forceps. Stratify in the dark (in a box or wrapped in aluminum foil) at 4 °C for 2 to 4 days and transfer to the growth chambers with illumination.
    Note: Any other sterile plating techniques are suitable.
  3. Treatments
    1. Bleocin and mitomycin C: Grow plants until day 7. Add 10 ml of drug-free liquid media, or media with 0.1 μg/ml of bleocin or 10 μg/ml of mitomycin C. Seal with parafilm and return to normal growth conditions until analysis. Shaking is not necessary.
      Note: Grow until day 7 = 7 days after transfer to the growth chambers.
    2. Hydroxyurea: Plate seeds on solid growth plates, without or with 1 mM of the chemical, and grow at standard conditions until analysis.
    3. UV-C: Grow seedlings until day 4 of growth and treat with 8 kJ/m2 UV-C in a Stratalinker. Return to standard conditions until analysis.
      Note: Before the treatment sterilize the interior of the Stratalinker with 70% Ethanol and run it for 3 min with the maximum dose of UV-C. Then place plates inside and remove the lid, keeping it inside the Stratalinker. Quickly close the door, to avoid contamination, and run the desired dosage of UV-C. When the treatment is finished close the plates still inside the Stratalinker and return them to normal growth conditions.
  4. GUS histochemical assay
    1. With forceps, carefully collect the 12-day-old plants of each plate in 15 ml Falcon tubes and submerge them in 10 ml of GUS staining solution. Be careful not to break the roots, cotyledons or leafs. Vacuum infiltrate for 15 min and incubate overnight at 37 °C.
    2. De-stain the plants in 70% ethanol overnight at 37 °C. Continue to change the ethanol twice a day until most plants tissue is white.
    3. Under a stereomicroscope score the number of GUS positive blue sectors indicating recombination events per whole plant (Figure 1B). To analyze the data, generate histograms with the frequency of seedlings with different numbers of blue sectors, and the total frequency in each line and treatment (Figure 2).


    Figure 1. Principle of the assay. (A) Recombination substrates in reporter lines for SHR DGU.US-1 and IU.GUS-8 (adapted from Reference 2). These lines were designed to distinguish different somatic recombination pathways (single-strand annealing and synthesis-dependent strand annealing) upon DSB repair after expression of the endonuclease I-SceI (Orel et al., 2003; for more information about different SHR pathways see Reference 1). However, they are used here to measure SHR without I-SceI but after different exogenous DNA damage treatments. The DGU.US-1 substrate contains two fragments of the GUS ORF with homologous parts (GU, US) in direct orientation, harboring an overlap of 557 bp. In IU.GUS-8 , two fragments of the GUS ORF (“GU”, “US”; blue) in direct orientation are separated, rendering the gene not functional. A fragment of 1087 bp partially homologous to “GU” and “US”, and containing “U” is located upstream in inverted orientation. SHR events between the repeats can restore a functional gene (GUS) in different ways: in line DGU.GUS-1, the sequence in common between the two repeats is deleted, while HR between indirect repeats in IU.GUS-8 results in the restoration of the GUS gene. P: constitutive 35S promoter of cauliflower mosaic virus; T: nopaline synthase terminator; HPT: hygromycin resistance gene, BAR: phosphinothricin acetyltransferase resistance gene, GUS: β -glucuronidase gene. (B) Histochemical GUS staining allows to visualize cells in which the GUS gene was restored by SHR (from Orel et al., 2003).


    Figure 2. Examples of SHR frequency measurements. Distribution of seedlings with different numbers of blue sectors and total frequency in line IU.GUS-8. (A) Mock and (B) 1 μg/ml Bleocin treatment. Sectors/plant refers to the average number of blue sectors, corresponding to recombination events, in the analyzed seedling population. Error bars correspond to the SE. SHR frequencies in each mutant population were significantly different to their wild-type (WT) counterpart, with a P value < 0.001. Asterisks indicate the significance between treated and mock populations according to P values from unpaired t tests: ***P < 0.001 and **0.001 < P < 0.01.

Notes

  1. For all treatments, always keep the plates belonging to one experiment (mock + treatment) growing side-by-side. For example, take plates for mock and UV-C treatment out of the growth chambers at the same time.
  2. In this experiment we used Germination Media (GM) described in Reference 6 (detailed protocol available at http://www.gmi.oeaw.ac.at/research-groups/ortrun-mittelsten-scheid/research-groups/ortrun-mittelsten-scheid/gm-medium-protocol). We have not tested the use of regular MS plates but have no reason to believe the media will be a determining factor as long as the plates used in each experiment come from the same batch of media.

Recipes

  1. Seed sterilization solution
    Consisting of 5% sodium hypochlorite and 0.05% Tween-80
    Mix 500 µl of sodium hypochlorite and 5 µl of Tween-80 in 10 ml of sterile H2O
  2. Bleocin (stock 10 mg/ml)
    Dissolve 100 mg of Bleocin in 10 ml of H2O
    Make 0.2 ml aliquots and keep at -20 °C until use
    Avoid thawing/freezing cycles
    Dilute to the desired concentration in liquid media, using disposable 50 ml falcon tubes
  3. Mitomycin C (stock 0.5 mg/ml)
    Dissolve 2 mg of Mitomycin C in 4 ml of H2O immediately before use
    Dilute to the desired concentration in liquid media, using disposable 50 ml falcon tubes
  4. GUS staining solution
    Consisting of 1 mM sodium phosphate buffer (pH 7); 10 mM EDTA, 0.1% Triton-X, 100 mg/ml chloramphenicol; 2 mM potassium ferrocyanide; 2 mM potassium ferricyanide; 0.5 mg/ml X-glucuronide
    Mix:
    10 ml of 1M Sodium Phosphate Buffer Solution (pH 7) (see below)
    4 ml of 0.25 M EDTA solution pH 8
    0.5 ml 20% (w/v) Triton X-100
    0.2 ml of 50 mg/ml Chloramphenicol
    1 ml of 0.2 M Potassium Ferricyanide
    1 ml of 0.2 M Potassium Ferrocyanide
    2.5 ml 20 mg/ml X-Gluc (kept in the dark at -20 °C)
    Bring up to 100 ml with MilliQ water, filter-sterilize (0.2 micron filter) and keep at 4 °C wrapped in aluminum foil. If stored under these conditions, this solution will be stable for up to one month.
  5. 1 M Sodium Phosphate Buffer (final pH 7)
    Make 200 ml of 1 M Sodium Dihydrogen Phosphate (this is acid, about pH 4)
    Put in a beaker and gradually add to this 1 M Di-Sodium Hydrogen Phosphate (pH about 9), until the mix reaches a pH of 7 at 20 °C
    Autoclave

Acknowledgments

We thank Holger Puchta for kindly providing the recombination substrate lines and Gudrun Böhmdorfer for helpful discussions on DNA damaging agents. Work was supported by Grants GEN-AU GZ 200.140-VI/1/2006 from the Austrian Federal Ministry of Science and Research and FWF P18986-B17 from the Austrian Science Fund. This protocol was used in Rosa et al. (2013). The GUS histochemical assay was originally established by Pecinka et al. (2009).

References

  1. March-Díaz, R. and Reyes, J. C. (2009). The beauty of being a variant: H2A. Z and the SWR1 complex in plants. Mol Plant 2(4): 565-577.
  2. Masson, J. and Paszkowski, J. (1992). The culture response of Arabidopsis thaliana protoplasts is determined by the growth conditions of donor plants. Plant J 2(5): 829-833.
  3. Orel, N., Kyryk, A. and Puchta, H. (2003). Different pathways of homologous recombination are used for the repair of double-strand breaks within tandemly arranged sequences in the plant genome. Plant J 35(5): 604-612. 
  4. Rosa, M., Von Harder, M., Cigliano, R. A., Schlogelhofer, P. and Mittelsten Scheid, O. (2013). The Arabidopsis SWR1 chromatin-remodeling complex is important for DNA repair, somatic recombination, and meiosis. Plant Cell 25(6): 1990-2001.
  5. Pecinka, A., Rosa, M., Schikora, A., Berlinger, M., Hirt, H., Luschnig, C. and Mittelsten Scheid, O. (2009). Transgenerational stress memory is not a general response in Arabidopsis. PLoS One 4(4): e5202.
  6. Schuermann, D., Molinier, J., Fritsch, O. and Hohn, B. (2005). The dual nature of homologous recombination in plants. Trends Genet 21(3): 172-181. 
  7. Waterworth, W. M., Drury, G. E., Bray, C. M. and West, C. E. (2011). Repairing breaks in the plant genome: the importance of keeping it together. New Phytol 192(4): 805-822. 

材料和试剂

  1. 拟南芥种子IU.GUS-8和DGU.US-1(图1)(Orel等人,2003)的种子[渐渗入各种遗传背景和 基因分型为报告基因的纯合性和突变(Rosa等人,2013)。 例如,我们使用野生型Columbia-0和突变体 arp6-3 和 swc6-1 。 这两种突变体缺乏SWR1复合体的拟南芥属同源物的亚基,并且除了对DNA损伤剂敏感外(Rosa等人,2013),具有多向性发育缺陷( 更多信息参见Schuermann等人,2005)]
  2. 次氯酸钠(Sigma-Aldrich,目录号:425044)
  3. 吐温-80(Sigma-Aldrich,目录号:4780)
  4. 无菌H 2 O 2/b
  5. 固体培养基
  6. 液体植物生长培养基(与固体生长培养基相同,但不含琼脂)
  7. 羟基脲
  8. 70%乙醇
  9. 种子灭菌溶液(见配方)
  10. Bleocin(博来霉素的商品名)(EMD Millipore,目录号:203408)(参见Recipes)
  11. 丝裂霉素C(Duchefa Biochemie BV,目录号:M0133)(参见Recipes)
  12. GUS染色溶液(参见配方)
  13. 1 M磷酸钠缓冲液(参见配方)

设备

  1. 用于植物培养的塑料培养皿(参见注释)(圆形200×15mm,具有20ml固体生长培养基)
  2. 无菌罩,优选生物安全柜,以避免暴露于基因毒素
  3. 台式块振动器(如 Eppendorf,Thermomixer ®
  4. UV交联剂(254nm UV灯泡,每种15瓦)(Stratagene,型号:Stratalinker 2400)
  5. 镊子
  6. 箱或铝箔
  7. parafilm
  8. 1.5 ml Eppendorf管
  9. 50和15ml Falcon管
  10. 真空应用器(如干燥器)
  11. 植物生长设施,在21℃下,16小时光/8小时黑暗循环
  12. 37℃的培养箱
  13. 立体显微镜

程序

  1. 在1.5ml Eppendorf管中用1ml灭菌溶液洗涤种子,在台式块振荡器中在室温下以300rpm振摇6分钟。使种子固定在Eppendorf的底部并除去上清液。用1ml无菌水洗涤5次,在振荡器中5分钟。最后一次洗涤后,取出上清液,在封闭的无菌罩中干燥过夜,留下管子开口 注意:每个基因型和处理消毒约200粒种子。所有涉及种子或苗子操作的后续步骤应在无菌罩中进行。
  2. 使用火焰灭菌和冷却的镊子在具有生长培养基(每板约75至100个和每个基因型2个板并处理)的200×15mm培养皿上平板种子。在4℃下在黑暗中(在盒子中或包裹在铝箔中)分层2至4天,并用照明转移到生长室。
    注意:任何其他无菌电镀技术都是适合的。
  3. 治疗
    1. Bleocin和丝裂霉素C:生长植物直到第7天。加入10ml无药液体培养基或含有0.1μg/ml bleocin或10μg/ml丝裂霉素C的培养基。用石蜡膜密封并恢复正常生长条件直到分析。无需振动。
      注意:生长至第7天=转移到生长室后7天。
    2. 羟基脲:在固体生长板上铺种子,没有或含有1mM的化学物质,并在标准条件下生长直到分析。
    3. UV-C:生长幼苗直到生长的第4天,并在Stratalinker中用8kJ/m 2 UV-C处理。返回标准条件直到分析。
      注意:在治疗之前,用70%乙醇灭菌Stratalinker的内部,并且用最大剂量的UV-C运行3分钟。然后将板放在里面,取下盖子,保持它在Stratalinker内。快速关闭门,以避免污染,并运行所需剂量的UV-C。当治疗完成时,关闭板仍然在Stratalinker内,并恢复到正常的生长条件。
  4. GUS组织化学测定法
    1. 用镊子,仔细收集每个板的12天龄的植物在15毫升Falcon管中,并将其浸没在10毫升GUS染色溶液中。小心不要破坏根,子叶或叶子。真空浸润15分钟,并在37℃下孵育过夜
    2. 在70%乙醇中将植物在37℃下脱色过夜。继续每天更换乙醇两次,直到大多数植物组织是白色的。
    3. 在立体显微镜下,GUS阳性蓝色区段的数目表示每个整株植物的重组事件(图1B)。为了分析数据,生成具有不同数量的蓝色扇区的幼苗的频率的直方图,以及每行和处理中的总频率(图2)。


    图1.测定原理。(A)SHR DGU.US-1和IU.GUS-8(改编自参考文献2)的报道系中的重组底物。这些系被设计为在内切核酸酶I-SceI表达后在DSB修复后区分不同的体细胞重组途径(单链退火和合成依赖性链退火)(Orel等人,2003;关于不同SHR途径的信息参见参考文献1)。然而,它们在这里用于测量不含I-SceI的SHR,但是在不同的外源DNA损伤处理后。 DGU.US-1底物含有GUS ORF的两个片段,其具有直接取向的同源部分(GU,US),具有557bp的重叠。在IU.GUS-8中,直接取向的GUS ORF("GU","US";蓝色)的两个片段分开,使得基因不起作用。与"GU"和"US"部分同源并且含有"U"的1087bp的片段以反向方向位于上游。重复之间的SHR事件可以以不同的方式恢复功能基因(GUS):在DGU.GUS-1系中,删除两个重复之间共同的序列,而IU.GUS-8中的间接重复之间的HR导致恢复GUS基因。 P:花椰菜花叶病毒的组成型35S启动子; T:胭脂碱合酶终止子; HPT:潮霉素抗性基因,BAR:膦丝菌素乙酰转移酶抗性基因,GUS:β-葡糖醛酸糖苷酶基因。 (B)组织化学GUS染色允许观察其中GUS基因由SHR恢复的细胞(来自Orel等人,2003)。


    图2. SHR频率测量的实例。在线IU.GUS-8中具有不同数目的蓝色扇区和总频率的幼苗的分布。 (A)模拟物和(B)1μg/ml Bleocin处理。扇区/植物指在分析的幼苗群体中对应于重组事件的蓝色区段的平均数目。误差棒对应于SE。每个突变群体中的SHR频率与其野生型(WT)对应物显着不同, 0.001。星号表示根据来自未配对的试验的P值的处理的和模拟群体之间的显着性:*** P < 0.001和** 0.001 < P < 0.01。

笔记

  1. 对于所有处理,总是保持板属于一个实验(模拟+处理)并排生长。 例如,将生长室中的模拟和UV-C处理板同时取出
  2. 在该实验中,我们使用参考文献6中描述的萌发培养基(GM)(详细方案可参见http://www.gmi.oeaw.ac.at/research-groups/ortrun-mittelsten-scheid/research-groups/ortrun-mittelsten -scheid/gm-medium-protocol)。 我们没有测试过使用常规MS培养板,但没有理由相信培养基会成为决定性因素,只要每个实验中使用的培养板来自同一批培养基。

食谱

  1. 种子灭菌溶液
    由5%次氯酸钠和0.05%Tween-80组成 将500μl次氯酸钠和5μl吐温-80在10ml无菌H 2 O中混合
  2. Bleocin(原液10mg/ml)
    将100mg的Bleocin溶解在10ml H 2 O中 使0.2毫升等分试样,并保持在-20°C,直到使用
    避免解冻/冻结周期
    在液体介质中稀释至所需浓度,使用一次性50ml falcon管
  3. 丝裂霉素C(原液0.5mg/ml)
    在使用前立即将2mg丝裂霉素C溶于4ml H 2 O中
    在液体介质中稀释至所需浓度,使用一次性50ml falcon管
  4. GUS染色溶液
    由1mM磷酸钠缓冲液(pH7); 10mM EDTA,0.1%Triton-X,100mg/ml氯霉素; 2mM亚铁氰化钾; 2mM铁氰化钾; 0.5mg/ml X-葡萄糖醛酸苷 混合:
    10ml 1M磷酸钠缓冲溶液(pH7)(见下文)
    4ml 0.25M EDTA溶液pH 8
    0.5ml 20%(w/v)Triton X-100 0.2ml 50mg/ml氯霉素
    1ml 0.2M铁氰化钾
    1ml 0.2M亚铁氰化钾 2.5ml 20mg/ml X-Gluc(保存在-20℃的黑暗中) 用MilliQ水加至100ml,过滤灭菌(0.2微米过滤器),并保持在4℃,包裹在铝箔中。 如果在这些条件下储存,该溶液将稳定长达一个月
  5. 1M磷酸钠缓冲液(最终pH 7)
    制备200ml 1M磷酸二氢钠(这是酸,约pH 4) 放入烧杯中并逐渐加入到该1M磷酸二氢钠(pH约9)中,直到混合物在20℃下达到pH 7为止。
    高压灭菌器

致谢

我们感谢Holger Puchta友好地提供重组底物系和GudrunBöhmdorfer有助于讨论DNA损伤剂。工作得到来自奥地利联邦科学和研究部的Grants GEN-AU GZ 200.140-VI/1/2006和来自奥地利科学基金的FWF P18986-B17的支持。该方案用于Rosa等人(2013)。 GUS组织化学测定最初由Pecinka等人建立。 (2009)。

参考文献

  1. March-Díaz,R。和Reyes,J.C。(2009)。 作为一个变体的美丽:H2A。 Z和SWR1复合物。 Mol Plant 2(4):565-577。
  2. Masson,J。和Paszkowski,J。(1992)。 拟南芥的培养物反应原生质体由供体植物的生长条件决定。植物J 2(5):829-833。
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How to cite this protocol: Rosa, M. and Scheid, O. M. (2014). Measuring Homologous Recombination Frequency in Arabidopsis Seedlings. Bio-protocol 4(7): e1094. DOI: 10.21769/BioProtoc.1094; Full Text



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